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Peng H, Rao Y, Shang W, Yang Y, Tan L, Liu L, Hu Z, Wang Y, Huang X, Liu H, Li M, Guo Z, Chen J, Yang Y, Wu J, Yuan W, Hu Q, Rao X. Vancomycin‐intermediate Staphylococcus aureus employs CcpA‐GlmS metabolism regulatory cascade to resist vancomycin. MEDCOMM – FUTURE MEDICINE 2024; 3. [DOI: 10.1002/mef2.70007] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Subscribe] [Scholar Register] [Received: 08/07/2024] [Accepted: 11/28/2024] [Indexed: 01/04/2025]
Abstract
AbstractVancomycin (VAN)‐intermediate Staphylococcus aureus (VISA) is a critical cause of VAN treatment failure worldwide. Multiple genetic changes are reportedly associated with VISA formation, whereas VISA strains often present common phenotypes, such as reduced autolysis and thickened cell wall. However, how mutated genes lead to VISA common phenotypes remains unclear. Here, we show a metabolism regulatory cascade (CcpA‐GlmS), whereby mutated two‐component systems (TCSs) link to the common phenotypes of VISA. We found that ccpA deletion decreased VAN resistance in VISA strains with diverse genetic backgrounds. Metabolic alteration in VISA was associated with ccpA upregulation, which was directly controlled by TCSs WalKR and GraSR. RNA‐sequencing revealed the crucial roles of CcpA in changing the carbon flow and nitrogen flux of VISA to promote VAN resistance. A gate enzyme (GlmS) that drives carbon flow to the cell wall precursor biosynthesis was upregulated in VISA. CcpA directly controlled glmS expression. Blocking CcpA sensitized VISA strains to VAN treatment in vitro and in vivo. Overall, this work uncovers a link between the formation of VISA phenotypes and commonly mutated genes. Inhibition of CcpA‐GlmS cascade is a promising strategy to restore the therapeutic efficiency of VAN against VISA infections.
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Affiliation(s)
- Huagang Peng
- Department of Microbiology, College of Basic Medical Sciences, Army Medical University Key Laboratory of Microbial Engineering under the Educational Committee in Chongqing Chongqing China
| | - Yifan Rao
- Department of Emergency Medicine, Xinqiao Hospital Army Medical University Chongqing China
| | - Weilong Shang
- Department of Microbiology, College of Basic Medical Sciences, Army Medical University Key Laboratory of Microbial Engineering under the Educational Committee in Chongqing Chongqing China
| | - Yi Yang
- Department of Microbiology, College of Basic Medical Sciences, Army Medical University Key Laboratory of Microbial Engineering under the Educational Committee in Chongqing Chongqing China
| | - Li Tan
- Department of Microbiology, College of Basic Medical Sciences, Army Medical University Key Laboratory of Microbial Engineering under the Educational Committee in Chongqing Chongqing China
| | - Lu Liu
- Department of Microbiology, College of Basic Medical Sciences, Army Medical University Key Laboratory of Microbial Engineering under the Educational Committee in Chongqing Chongqing China
| | - Zhen Hu
- Department of Microbiology, College of Basic Medical Sciences, Army Medical University Key Laboratory of Microbial Engineering under the Educational Committee in Chongqing Chongqing China
| | - Yuting Wang
- Department of Microbiology, College of Basic Medical Sciences, Army Medical University Key Laboratory of Microbial Engineering under the Educational Committee in Chongqing Chongqing China
| | - Xiaonan Huang
- Department of Microbiology, College of Basic Medical Sciences, Army Medical University Key Laboratory of Microbial Engineering under the Educational Committee in Chongqing Chongqing China
| | - He Liu
- Department of Microbiology, College of Basic Medical Sciences, Army Medical University Key Laboratory of Microbial Engineering under the Educational Committee in Chongqing Chongqing China
| | - Mengyang Li
- Department of Microbiology, School of Medicine Chongqing University Chongqing China
| | - Zuwen Guo
- Department of Microbiology, College of Basic Medical Sciences, Army Medical University Key Laboratory of Microbial Engineering under the Educational Committee in Chongqing Chongqing China
| | - Juan Chen
- Department of Pharmacy, Xinqiao Hospital Army Medical University Chongqing China
| | - Yuhua Yang
- Department of Microbiology, College of Basic Medical Sciences, Army Medical University Key Laboratory of Microbial Engineering under the Educational Committee in Chongqing Chongqing China
| | - Jianghong Wu
- Department of Microbiology, College of Basic Medical Sciences, Army Medical University Key Laboratory of Microbial Engineering under the Educational Committee in Chongqing Chongqing China
| | - Wenchang Yuan
- KingMed School of Laboratory Medicine Guangzhou Medical University Guangzhou China
| | - Qiwen Hu
- Department of Microbiology, College of Basic Medical Sciences, Army Medical University Key Laboratory of Microbial Engineering under the Educational Committee in Chongqing Chongqing China
| | - Xiancai Rao
- Department of Microbiology, College of Basic Medical Sciences, Army Medical University Key Laboratory of Microbial Engineering under the Educational Committee in Chongqing Chongqing China
- Department of Microbiology, School of Medicine Chongqing University Chongqing China
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Al Musaimi O. Lasso peptides realm: Insights and applications. Peptides 2024; 182:171317. [PMID: 39489300 DOI: 10.1016/j.peptides.2024.171317] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 08/29/2024] [Revised: 10/05/2024] [Accepted: 10/29/2024] [Indexed: 11/05/2024]
Abstract
Lasso peptides exhibit a range of bioactivities, including antiviral effects, inhibition of the glucagon receptor, blockade of the endothelin type B receptor, inhibition of myosin light chain kinase, and modulation of the atrial natriuretic factor, as well as notable antimicrobial properties. Intriguingly, lasso peptides exhibit remarkable proteolytic and thermal stability, addressing one of the key challenges that traditional peptides often face. The challenge in producing those valuable peptides remains the main hurdle in the way of producing larger quantities or even modifying them with more potent analogues. Genome mining and heterologous expression approaches have greatly facilitated the production of lasso peptides, moving beyond mere isolation techniques. This advancement not only allows for larger quantities but also enables the creation of additional analogues with improved stability and potency. This review aims to explore the unique bioactivities and stability of lasso peptides, along with recent advancements in genome mining and heterologous expression that address production challenges and open pathways for engineering potent analogues.
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Affiliation(s)
- Othman Al Musaimi
- School of Pharmacy, Newcastle University, Newcastle upon Tyne UK NE1 7RU, UK; Department of Chemical Engineering, Imperial College London, London SW7 2AZ, UK.
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Xiong Y, Wang R, Zheng J, Fang D, He P, Liu S, Lin Z, Chen X, Chen C, Shang Y, Yu Z, Liu X, Han S. Discovery of novel dihydropyrrolidone-thiadiazole compound crosstalk between the YycG/F two-component regulatory pathway and cell membrane homeostasis to combat methicillin-resistant Staphylococcus aureus. Eur J Med Chem 2024; 277:116770. [PMID: 39208742 DOI: 10.1016/j.ejmech.2024.116770] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/04/2024] [Revised: 07/30/2024] [Accepted: 08/11/2024] [Indexed: 09/04/2024]
Abstract
The rapid emergence and spread of multidrug-resistant (MDR) Gram-positive pathogens present a significant challenge to global healthcare. Methicillin-resistant Staphylococcus aureus (MRSA) is a particular concern because of its high resistance to most antibiotics. Based on our previously reported chemical structure of compound 62, a series of novel derivatives were synthesized and evaluated for their antibacterial activities. We found that some of these derivatives displayed effective antibacterial activity against Gram-positive pathogens, with minimal cytotoxicity (CC50>100 μM) and hemolytic activity (HC50>200 μM). Among these derivatives, the minimum inhibitory concentration (MIC) of 62-7c against Gram-positive bacterial isolates ranged from 6.25 to 25 μM. This derivative also exhibited significant synergistic antibacterial effects with daptomycin both in vitro and in vivo, with an ability to eradicate planktonic and persister cells of MRSA. Additionally, 62-7c inhibited biofilm formation and eradicated mature biofilms of MRSA. Mechanistic studies revealed that 62-7c inhibited the YycG kinase activity and disrupted the cell membrane by binding to cardiolipin (CL), leading to cell death. Importantly, no development of drug resistance was observed even after 20 serial passages. Furthermore, 62-7c exhibited high biosafety and potent effectiveness in combating infections in both mouse pneumonia and mouse wound models infected with MRSA. Thus, our study revealed that 62-7c has the potential to serve as a novel antibacterial agent for treating MRSA infections.
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Affiliation(s)
- Yanpeng Xiong
- College of Biotechnology and Pharmaceutical Engineering, Nanjing Tech University, Nanjing 211816, China; Department of Infectious Diseases, Shenzhen Nanshan People's Hospital, Shenzhen University Medical School, Shenzhen 518052, China
| | - Ruian Wang
- College of Biotechnology and Pharmaceutical Engineering, Nanjing Tech University, Nanjing 211816, China
| | - Jiaoyang Zheng
- College of Biotechnology and Pharmaceutical Engineering, Nanjing Tech University, Nanjing 211816, China
| | - Di Fang
- College of Biotechnology and Pharmaceutical Engineering, Nanjing Tech University, Nanjing 211816, China
| | - Peikun He
- Department of Infectious Diseases, Shenzhen Nanshan People's Hospital, Shenzhen University Medical School, Shenzhen 518052, China
| | - Shanghong Liu
- Department of Infectious Diseases, Shenzhen Nanshan People's Hospital, Shenzhen University Medical School, Shenzhen 518052, China
| | - Zhiwei Lin
- Laboratory of Respiratory Disease, People's Hospital of Yangjiang, Yangjiang 529500, China
| | - Xuecheng Chen
- College of Biotechnology and Pharmaceutical Engineering, Nanjing Tech University, Nanjing 211816, China
| | - Chengchun Chen
- Department of Infectious Diseases, Shenzhen Nanshan People's Hospital, Shenzhen University Medical School, Shenzhen 518052, China
| | - Yongpeng Shang
- Department of Infectious Diseases, Shenzhen Nanshan People's Hospital, Shenzhen University Medical School, Shenzhen 518052, China
| | - Zhijian Yu
- Department of Infectious Diseases, Shenzhen Nanshan People's Hospital, Shenzhen University Medical School, Shenzhen 518052, China.
| | - Xiaoju Liu
- Department of Infectious Diseases, Shenzhen Nanshan People's Hospital, Shenzhen University Medical School, Shenzhen 518052, China.
| | - Shiqing Han
- College of Biotechnology and Pharmaceutical Engineering, Nanjing Tech University, Nanjing 211816, China.
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Fait A, Silva SF, Abrahamsson JÅH, Ingmer H. Staphylococcus aureus response and adaptation to vancomycin. Adv Microb Physiol 2024; 85:201-258. [PMID: 39059821 DOI: 10.1016/bs.ampbs.2024.04.006] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 07/28/2024]
Abstract
Antibiotic resistance is an increasing challenge for the human pathogen Staphylococcus aureus. Methicillin-resistant S. aureus (MRSA) clones have spread globally, and a growing number display decreased susceptibility to vancomycin, the favoured antibiotic for treatment of MRSA infections. These vancomycin-intermediate S. aureus (VISA) or heterogeneous vancomycin-intermediate S. aureus (hVISA) strains arise from accumulation of a variety of point mutations, leading to cell wall thickening and reduced vancomycin binding to the cell wall building block, Lipid II, at the septum. They display only minor changes in vancomycin susceptibility, with varying tolerance between cells in a population, and therefore, they can be difficult to detect. In this review, we summarize current knowledge of VISA and hVISA. We discuss the role of genetic strain background or epistasis for VISA development and the possibility of strains being 'transient' VISA with gene expression changes mediated by, for example, VraTSR, GraXSR, or WalRK signal transduction systems, leading to temporary vancomycin tolerance. Additionally, we address collateral susceptibility to other antibiotics than vancomycin. Specifically, we estimate how mutations in rpoB, encoding the β-subunit of the RNA polymerase, affect overall protein structure and compare changes with rifampicin resistance. Ultimately, such in-depth analysis of VISA and hVISA strains in terms of genetic and transcriptional changes, as well as changes in protein structures, may pave the way for improved detection and guide antibiotic therapy by revealing strains at risk of VISA development. Such tools will be valuable for keeping vancomycin an asset also in the future.
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Affiliation(s)
- Anaëlle Fait
- Department of Veterinary and Animal Sciences, University of Copenhagen, Frederiksberg, Denmark; Department of Environmental Systems Science, ETH Zürich, Zürich, Switzerland
| | - Stephanie Fulaz Silva
- Department of Veterinary and Animal Sciences, University of Copenhagen, Frederiksberg, Denmark
| | | | - Hanne Ingmer
- Department of Veterinary and Animal Sciences, University of Copenhagen, Frederiksberg, Denmark.
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Ahator SD, Hegstad K, Lentz CS, Johannessen M. Deciphering Staphylococcus aureus-host dynamics using dual activity-based protein profiling of ATP-interacting proteins. mSystems 2024; 9:e0017924. [PMID: 38656122 PMCID: PMC11097646 DOI: 10.1128/msystems.00179-24] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/06/2024] [Accepted: 03/26/2024] [Indexed: 04/26/2024] Open
Abstract
The utilization of ATP within cells plays a fundamental role in cellular processes that are essential for the regulation of host-pathogen dynamics and the subsequent immune response. This study focuses on ATP-binding proteins to dissect the complex interplay between Staphylococcus aureus and human cells, particularly macrophages (THP-1) and keratinocytes (HaCaT), during an intracellular infection. A snapshot of the various protein activity and function is provided using a desthiobiotin-ATP probe, which targets ATP-interacting proteins. In S. aureus, we observe enrichment in pathways required for nutrient acquisition, biosynthesis and metabolism of amino acids, and energy metabolism when located inside human cells. Additionally, the direct profiling of the protein activity revealed specific adaptations of S. aureus to the keratinocytes and macrophages. Mapping the differentially activated proteins to biochemical pathways in the human cells with intracellular bacteria revealed cell-type-specific adaptations to bacterial challenges where THP-1 cells prioritized immune defenses, autophagic cell death, and inflammation. In contrast, HaCaT cells emphasized barrier integrity and immune activation. We also observe bacterial modulation of host processes and metabolic shifts. These findings offer valuable insights into the dynamics of S. aureus-host cell interactions, shedding light on modulating host immune responses to S. aureus, which could involve developing immunomodulatory therapies. IMPORTANCE This study uses a chemoproteomic approach to target active ATP-interacting proteins and examines the dynamic proteomic interactions between Staphylococcus aureus and human cell lines THP-1 and HaCaT. It uncovers the distinct responses of macrophages and keratinocytes during bacterial infection. S. aureus demonstrated a tailored response to the intracellular environment of each cell type and adaptation during exposure to professional and non-professional phagocytes. It also highlights strategies employed by S. aureus to persist within host cells. This study offers significant insights into the human cell response to S. aureus infection, illuminating the complex proteomic shifts that underlie the defense mechanisms of macrophages and keratinocytes. Notably, the study underscores the nuanced interplay between the host's metabolic reprogramming and immune strategy, suggesting potential therapeutic targets for enhancing host defense and inhibiting bacterial survival. The findings enhance our understanding of host-pathogen interactions and can inform the development of targeted therapies against S. aureus infections.
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Affiliation(s)
- Stephen Dela Ahator
- Centre for New Antibacterial Strategies (CANS) & Research Group for Host-Microbe Interactions, Department of Medical Biology, Faculty of Health Sciences, UiT–The Arctic University of Norway, Tromsø, Norway
| | - Kristin Hegstad
- Centre for New Antibacterial Strategies (CANS) & Research Group for Host-Microbe Interactions, Department of Medical Biology, Faculty of Health Sciences, UiT–The Arctic University of Norway, Tromsø, Norway
- Norwegian National Advisory Unit on Detection of Antimicrobial Resistance, Department of Microbiology and Infection Control, University Hospital of North Norway, Tromsø, Norway
| | - Christian S. Lentz
- Centre for New Antibacterial Strategies (CANS) & Research Group for Host-Microbe Interactions, Department of Medical Biology, Faculty of Health Sciences, UiT–The Arctic University of Norway, Tromsø, Norway
| | - Mona Johannessen
- Centre for New Antibacterial Strategies (CANS) & Research Group for Host-Microbe Interactions, Department of Medical Biology, Faculty of Health Sciences, UiT–The Arctic University of Norway, Tromsø, Norway
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6
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Ali L, Abdel Aziz MH. Crosstalk involving two-component systems in Staphylococcus aureus signaling networks. J Bacteriol 2024; 206:e0041823. [PMID: 38456702 PMCID: PMC11025333 DOI: 10.1128/jb.00418-23] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 03/09/2024] Open
Abstract
Staphylococcus aureus poses a serious global threat to human health due to its pathogenic nature, adaptation to environmental stress, high virulence, and the prevalence of antimicrobial resistance. The signaling network in S. aureus coordinates and integrates various internal and external inputs and stimuli to adapt and formulate a response to the environment. Two-component systems (TCSs) of S. aureus play a central role in this network where surface-expressed histidine kinases (HKs) receive and relay external signals to their cognate response regulators (RRs). Despite the purported high fidelity of signaling, crosstalk within TCSs, between HK and non-cognate RR, and between TCSs and other systems has been detected widely in bacteria. The examples of crosstalk in S. aureus are very limited, and there needs to be more understanding of its molecular recognition mechanisms, although some crosstalk can be inferred from similar bacterial systems that share structural similarities. Understanding the cellular processes mediated by this crosstalk and how it alters signaling, especially under stress conditions, may help decipher the emergence of antibiotic resistance. This review highlights examples of signaling crosstalk in bacteria in general and S. aureus in particular, as well as the effect of TCS mutations on signaling and crosstalk.
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Affiliation(s)
- Liaqat Ali
- Fisch College of Pharmacy, The University of Texas at Tyler, Tyler, Texas, USA
| | - May H. Abdel Aziz
- Fisch College of Pharmacy, The University of Texas at Tyler, Tyler, Texas, USA
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7
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Jiang JH, Cameron DR, Nethercott C, Aires-de-Sousa M, Peleg AY. Virulence attributes of successful methicillin-resistant Staphylococcus aureus lineages. Clin Microbiol Rev 2023; 36:e0014822. [PMID: 37982596 PMCID: PMC10732075 DOI: 10.1128/cmr.00148-22] [Citation(s) in RCA: 6] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/21/2023] Open
Abstract
Methicillin-resistant Staphylococcus aureus (MRSA) is a leading cause of severe and often fatal infections. MRSA epidemics have occurred in waves, whereby a previously successful lineage has been replaced by a more fit and better adapted lineage. Selection pressures in both hospital and community settings are not uniform across the globe, which has resulted in geographically distinct epidemiology. This review focuses on the mechanisms that trigger the establishment and maintenance of current, dominant MRSA lineages across the globe. While the important role of antibiotic resistance will be mentioned throughout, factors which influence the capacity of S. aureus to colonize and cause disease within a host will be the primary focus of this review. We show that while MRSA possesses a diverse arsenal of toxins including alpha-toxin, the success of a lineage involves more than just producing toxins that damage the host. Success is often attributed to the acquisition or loss of genetic elements involved in colonization and niche adaptation such as the arginine catabolic mobile element, as well as the activity of regulatory systems, and shift metabolism accordingly (e.g., the accessory genome regulator, agr). Understanding exactly how specific MRSA clones cause prolonged epidemics may reveal targets for therapies, whereby both core (e.g., the alpha toxin) and acquired virulence factors (e.g., the Panton-Valentine leukocidin) may be nullified using anti-virulence strategies.
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Affiliation(s)
- Jhih-Hang Jiang
- Department of Microbiology, Infection Program, Monash Biomedicine Discovery Institute, Monash University, Clayton, Victoria, Australia
- Department of Infectious Diseases, The Alfred Hospital and Central Clinical School, Monash University, Melbourne, Victoria, Australia
| | - David R Cameron
- Department of Biomedical Research, University of Bern, Bern, Switzerland
| | - Cara Nethercott
- Department of Microbiology, Infection Program, Monash Biomedicine Discovery Institute, Monash University, Clayton, Victoria, Australia
| | - Marta Aires-de-Sousa
- Laboratory of Molecular Genetics, Institutode Tecnologia Químicae Biológica António Xavier (ITQB-NOVA), Universidade Nova de Lisboa, Oeiras, Portugal
- Escola Superior de Saúde da Cruz Vermelha Portuguesa-Lisboa (ESSCVP-Lisboa), Lisbon, Portugal
| | - Anton Y Peleg
- Department of Microbiology, Infection Program, Monash Biomedicine Discovery Institute, Monash University, Clayton, Victoria, Australia
- Department of Infectious Diseases, The Alfred Hospital and Central Clinical School, Monash University, Melbourne, Victoria, Australia
- Centre to Impact Antimicrobial Resistance, Monash University, Clayton, Melbourne, Victoria, Australia
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Sharkey LKR, Guerillot R, Walsh CJ, Turner AM, Lee JYH, Neville SL, Klatt S, Baines SL, Pidot SJ, Rossello FJ, Seemann T, McWilliam HEG, Cho E, Carter GP, Howden BP, McDevitt CA, Hachani A, Stinear TP, Monk IR. The two-component system WalKR provides an essential link between cell wall homeostasis and DNA replication in Staphylococcus aureus. mBio 2023; 14:e0226223. [PMID: 37850732 PMCID: PMC10746227 DOI: 10.1128/mbio.02262-23] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/30/2023] [Accepted: 09/05/2023] [Indexed: 10/19/2023] Open
Abstract
IMPORTANCE The opportunistic human pathogen Staphylococcus aureus uses an array of protein sensing systems called two-component systems (TCS) to sense environmental signals and adapt its physiology in response by regulating different genes. This sensory network is key to S. aureus versatility and success as a pathogen. Here, we reveal for the first time the full extent of the regulatory network of WalKR, the only staphylococcal TCS that is indispensable for survival under laboratory conditions. We found that WalKR is a master regulator of cell growth, coordinating the expression of genes from multiple, fundamental S. aureus cellular processes, including those involved in maintaining cell wall metabolism, protein biosynthesis, nucleotide metabolism, and the initiation of DNA replication.
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Affiliation(s)
- Liam K. R. Sharkey
- Department of Microbiology and Immunology, Doherty Institute for Infection and Immunity, University of Melbourne, Melbourne, Victoria, Australia
| | - Romain Guerillot
- Department of Microbiology and Immunology, Doherty Institute for Infection and Immunity, University of Melbourne, Melbourne, Victoria, Australia
| | - Calum J. Walsh
- Department of Microbiology and Immunology, Doherty Institute for Infection and Immunity, University of Melbourne, Melbourne, Victoria, Australia
| | - Adrianna M. Turner
- Department of Microbiology and Immunology, Doherty Institute for Infection and Immunity, University of Melbourne, Melbourne, Victoria, Australia
| | - Jean Y. H. Lee
- Department of Microbiology and Immunology, Doherty Institute for Infection and Immunity, University of Melbourne, Melbourne, Victoria, Australia
| | - Stephanie L. Neville
- Department of Microbiology and Immunology, Doherty Institute for Infection and Immunity, University of Melbourne, Melbourne, Victoria, Australia
| | - Stephan Klatt
- The Florey Institute of Neuroscience and Mental Health, Melbourne Dementia Research Centre, The University of Melbourne, Parkville, Victoria, Australia
| | - Sarah L. Baines
- Department of Microbiology and Immunology, Doherty Institute for Infection and Immunity, University of Melbourne, Melbourne, Victoria, Australia
| | - Sacha J. Pidot
- Department of Microbiology and Immunology, Doherty Institute for Infection and Immunity, University of Melbourne, Melbourne, Victoria, Australia
| | - Fernando J. Rossello
- University of Melbourne Centre for Cancer Research, The University of Melbourne, Melbourne, Victoria, Australia
- Australian Regenerative Medicine Institute, Monash University, Melbourne, Victoria, Australia
| | - Torsten Seemann
- Department of Microbiology and Immunology, Doherty Institute for Infection and Immunity, University of Melbourne, Melbourne, Victoria, Australia
- Department of Microbiology and Immunology, Centre for Pathogen Genomics, Doherty Institute for Infection and Immunity, University of Melbourne, Melbourne, Victoria, Australia
| | - Hamish E. G. McWilliam
- Department of Microbiology and Immunology, Doherty Institute for Infection and Immunity, University of Melbourne, Melbourne, Victoria, Australia
| | - Ellie Cho
- Biological Optical Microscopy Platform, University of Melbourne, Melbourne, Victoria, Australia
| | - Glen P. Carter
- Department of Microbiology and Immunology, Doherty Institute for Infection and Immunity, University of Melbourne, Melbourne, Victoria, Australia
| | - Benjamin P. Howden
- Department of Microbiology and Immunology, Doherty Institute for Infection and Immunity, University of Melbourne, Melbourne, Victoria, Australia
- Department of Microbiology and Immunology, Centre for Pathogen Genomics, Doherty Institute for Infection and Immunity, University of Melbourne, Melbourne, Victoria, Australia
| | - Christopher A. McDevitt
- Department of Microbiology and Immunology, Doherty Institute for Infection and Immunity, University of Melbourne, Melbourne, Victoria, Australia
| | - Abderrahman Hachani
- Department of Microbiology and Immunology, Doherty Institute for Infection and Immunity, University of Melbourne, Melbourne, Victoria, Australia
| | - Timothy P. Stinear
- Department of Microbiology and Immunology, Doherty Institute for Infection and Immunity, University of Melbourne, Melbourne, Victoria, Australia
- Department of Microbiology and Immunology, Centre for Pathogen Genomics, Doherty Institute for Infection and Immunity, University of Melbourne, Melbourne, Victoria, Australia
| | - Ian R. Monk
- Department of Microbiology and Immunology, Doherty Institute for Infection and Immunity, University of Melbourne, Melbourne, Victoria, Australia
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9
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Zhang R, Ashford NK, Li A, Ross DH, Werth BJ, Xu L. High-throughput analysis of lipidomic phenotypes of methicillin-resistant Staphylococcus aureus by coupling in situ 96-well cultivation and HILIC-ion mobility-mass spectrometry. Anal Bioanal Chem 2023; 415:6191-6199. [PMID: 37535099 PMCID: PMC11059195 DOI: 10.1007/s00216-023-04890-6] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/21/2022] [Revised: 07/23/2023] [Accepted: 07/25/2023] [Indexed: 08/04/2023]
Abstract
Antimicrobial resistance is a major threat to human health as resistant pathogens spread globally, and the development of new antimicrobials is slow. Since many antimicrobials function by targeting cell wall and membrane components, high-throughput lipidomics for bacterial phenotyping is of high interest for researchers to unveil lipid-mediated pathways when dealing with a large number of lab-selected or clinical strains. However, current practice for lipidomic analysis requires the cultivation of bacteria on a large scale, which does not replicate the growth conditions for high-throughput bioassays that are normally carried out in 96-well plates, such as susceptibility tests, growth curve measurements, and biofilm quantitation. Analysis of bacteria grown under the same condition as other bioassays would better inform the differences in susceptibility and other biological metrics. In this work, a high-throughput method for cultivation and lipidomic analysis of antimicrobial-resistant bacteria was developed for standard 96-well plates exemplified by methicillin-resistant Staphylococcus aureus (MRSA). By combining a 30-mm liquid chromatography (LC) column with ion mobility (IM) separation, elution time could be dramatically shortened to 3.6 min for a single LC run without losing major lipid features. Peak capacity was largely rescued by the addition of the IM dimension. Through multi-linear calibration, the deviation of retention time can be limited to within 5%, making database-based automatic lipid identification feasible. This high-throughput method was further validated by characterizing the lipidomic phenotypes of antimicrobial-resistant mutants derived from the MRSA strain, W308, grown in a 96-well plate.
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Affiliation(s)
- Rutan Zhang
- Department of Medicinal Chemistry, University of Washington, Seattle, WA, 98195, USA
| | - Nate K Ashford
- Department of Pharmacy, University of Washington, Seattle, WA, 98195, USA
| | - Amy Li
- Department of Medicinal Chemistry, University of Washington, Seattle, WA, 98195, USA
| | - Dylan H Ross
- Department of Medicinal Chemistry, University of Washington, Seattle, WA, 98195, USA
- Biological Sciences Division, Pacific Northwest National Laboratory, WA, 99352, Richland, USA
| | - Brian J Werth
- Department of Pharmacy, University of Washington, Seattle, WA, 98195, USA
| | - Libin Xu
- Department of Medicinal Chemistry, University of Washington, Seattle, WA, 98195, USA.
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10
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Paredes A, Iheacho C, Smith AT. Metal Messengers: Communication in the Bacterial World through Transition-Metal-Sensing Two-Component Systems. Biochemistry 2023; 62:2339-2357. [PMID: 37539997 PMCID: PMC10530140 DOI: 10.1021/acs.biochem.3c00296] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 08/05/2023]
Abstract
Bacteria survive in highly dynamic and complex environments due, in part, to the presence of systems that allow the rapid control of gene expression in the presence of changing environmental stimuli. The crosstalk between intra- and extracellular bacterial environments is often facilitated by two-component signal transduction systems that are typically composed of a transmembrane histidine kinase and a cytosolic response regulator. Sensor histidine kinases and response regulators work in tandem with their modular domains containing highly conserved structural features to control a diverse array of genes that respond to changing environments. Bacterial two-component systems are widespread and play crucial roles in many important processes, such as motility, virulence, chemotaxis, and even transition metal homeostasis. Transition metals are essential for normal prokaryotic physiological processes, and the presence of these metal ions may also influence pathogenic virulence if their levels are appropriately controlled. To do so, bacteria use transition-metal-sensing two-component systems that bind and respond to rapid fluctuations in extracytosolic concentrations of transition metals. This perspective summarizes the structural and metal-binding features of bacterial transition-metal-sensing two-component systems and places a special emphasis on understanding how these systems are used by pathogens to establish infection in host cells and how these systems may be targeted for future therapeutic developments.
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Affiliation(s)
- Alexander Paredes
- Department of Chemistry and Biochemistry, University of Maryland, Baltimore County, Baltimore, Maryland 21250, United States
| | - Chioma Iheacho
- Department of Chemistry and Biochemistry, University of Maryland, Baltimore County, Baltimore, Maryland 21250, United States
| | - Aaron T Smith
- Department of Chemistry and Biochemistry, University of Maryland, Baltimore County, Baltimore, Maryland 21250, United States
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11
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Liu C, Zhang H, Peng X, Blackledge MS, Furlani RE, Li H, Su Z, Melander RJ, Melander C, Michalek S, Wu H. Small Molecule Attenuates Bacterial Virulence by Targeting Conserved Response Regulator. mBio 2023; 14:e0013723. [PMID: 37074183 PMCID: PMC10294662 DOI: 10.1128/mbio.00137-23] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/19/2023] [Accepted: 01/23/2023] [Indexed: 04/20/2023] Open
Abstract
Antibiotic tolerance within a biofilm community presents a serious public health challenge. Here, we report the identification of a 2-aminoimidazole derivative that inhibits biofilm formation by two pathogenic Gram-positive bacteria, Streptococcus mutans and Staphylococcus aureus. In S. mutans, the compound binds to VicR, a key response regulator, at the N-terminal receiver domain, and concurrently inhibits expression of vicR and VicR-regulated genes, including the genes that encode the key biofilm matrix producing enzymes, Gtfs. The compound inhibits S. aureus biofilm formation via binding to a Staphylococcal VicR homolog. In addition, the inhibitor effectively attenuates S. mutans virulence in a rat model of dental caries. As the compound targets bacterial biofilms and virulence through a conserved transcriptional factor, it represents a promising new class of anti-infective agents that can be explored to prevent or treat a host of bacterial infections. IMPORTANCE Antibiotic resistance is a major public health issue due to the growing lack of effective anti-infective therapeutics. New alternatives to treat and prevent biofilm-driven microbial infections, which exhibit high tolerance to clinically available antibiotics, are urgently needed. We report the identification of a small molecule that inhibits biofilm formation by two important pathogenic Gram-positive bacteria, Streptococcus mutans and Staphylococcus aureus. The small molecule selectively targets a transcriptional regulator leading to attenuation of a biofilm regulatory cascade and concurrent reduction of bacterial virulence in vivo. As the regulator is highly conserved, the finding has broad implication for the development of antivirulence therapeutics that selectively target biofilms.
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Affiliation(s)
- Chang Liu
- Department of Pediatric Dentistry, University of Alabama at Birmingham Schools of Dentistry and Medicine, Birmingham, Alabama, USA
- Department of Microbiology, University of Alabama at Birmingham Schools of Dentistry and Medicine, Birmingham, Alabama, USA
| | - Hua Zhang
- Department of Pediatric Dentistry, University of Alabama at Birmingham Schools of Dentistry and Medicine, Birmingham, Alabama, USA
- Department of Integrative Biomedical & Diagnostic Sciences, Oregon Health & Science University School of Dentistry, Portland, Oregon, USA
- Department of Microbiology, University of Alabama at Birmingham Schools of Dentistry and Medicine, Birmingham, Alabama, USA
| | - Xian Peng
- Department of Pediatric Dentistry, University of Alabama at Birmingham Schools of Dentistry and Medicine, Birmingham, Alabama, USA
- Department of Microbiology, University of Alabama at Birmingham Schools of Dentistry and Medicine, Birmingham, Alabama, USA
| | - Meghan S. Blackledge
- Department of Chemistry and Biochemistry, University of Notre Dame, Notre Dame, Indiana, USA
| | - Robert E. Furlani
- Department of Chemistry and Biochemistry, University of Notre Dame, Notre Dame, Indiana, USA
| | - Haoting Li
- Department of Chemistry and Biochemistry, University of Notre Dame, Notre Dame, Indiana, USA
| | - Zhaoming Su
- Department of Chemistry and Biochemistry, University of Notre Dame, Notre Dame, Indiana, USA
| | - Roberta J. Melander
- Department of Chemistry and Biochemistry, University of Notre Dame, Notre Dame, Indiana, USA
| | - Christian Melander
- Department of Chemistry and Biochemistry, University of Notre Dame, Notre Dame, Indiana, USA
| | - Suzanne Michalek
- Department of Integrative Biomedical & Diagnostic Sciences, Oregon Health & Science University School of Dentistry, Portland, Oregon, USA
| | - Hui Wu
- Department of Pediatric Dentistry, University of Alabama at Birmingham Schools of Dentistry and Medicine, Birmingham, Alabama, USA
- Department of Integrative Biomedical & Diagnostic Sciences, Oregon Health & Science University School of Dentistry, Portland, Oregon, USA
- Department of Microbiology, University of Alabama at Birmingham Schools of Dentistry and Medicine, Birmingham, Alabama, USA
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12
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Patel H, Rawat S. A genetic regulatory see-saw of biofilm and virulence in MRSA pathogenesis. Front Microbiol 2023; 14:1204428. [PMID: 37434702 PMCID: PMC10332168 DOI: 10.3389/fmicb.2023.1204428] [Citation(s) in RCA: 23] [Impact Index Per Article: 11.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/12/2023] [Accepted: 05/30/2023] [Indexed: 07/13/2023] Open
Abstract
Staphylococcus aureus is one of the most common opportunistic human pathogens causing several infectious diseases. Ever since the emergence of the first methicillin-resistant Staphylococcus aureus (MRSA) strain decades back, the organism has been a major cause of hospital-acquired infections (HA-MRSA). The spread of this pathogen across the community led to the emergence of a more virulent subtype of the strain, i.e., Community acquired Methicillin resistant Staphylococcus aureus (CA-MRSA). Hence, WHO has declared Staphylococcus aureus as a high-priority pathogen. MRSA pathogenesis is remarkable because of the ability of this "superbug" to form robust biofilm both in vivo and in vitro by the formation of polysaccharide intercellular adhesin (PIA), extracellular DNA (eDNA), wall teichoic acids (WTAs), and capsule (CP), which are major components that impart stability to a biofilm. On the other hand, secretion of a diverse array of virulence factors such as hemolysins, leukotoxins, enterotoxins, and Protein A regulated by agr and sae two-component systems (TCS) aids in combating host immune response. The up- and downregulation of adhesion genes involved in biofilm formation and genes responsible for synthesizing virulence factors during different stages of infection act as a genetic regulatory see-saw in the pathogenesis of MRSA. This review provides insight into the evolution and pathogenesis of MRSA infections with a focus on genetic regulation of biofilm formation and virulence factors secretion.
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Affiliation(s)
| | - Seema Rawat
- Microbiology Laboratory, School of Life Sciences, Central University of Gujarat, Gandhinagar, Gujarat, India
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13
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Shu X, Shi Y, Huang Y, Yu D, Sun B. Transcription tuned by S-nitrosylation underlies a mechanism for Staphylococcus aureus to circumvent vancomycin killing. Nat Commun 2023; 14:2318. [PMID: 37085493 PMCID: PMC10120478 DOI: 10.1038/s41467-023-37949-0] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/12/2022] [Accepted: 04/06/2023] [Indexed: 04/23/2023] Open
Abstract
Treatment of Staphylococcus aureus infections is a constant challenge due to emerging resistance to vancomycin, a last-resort drug. S-nitrosylation, the covalent attachment of a nitric oxide (NO) group to a cysteine thiol, mediates redox-based signaling for eukaryotic cellular functions. However, its role in bacteria is largely unknown. Here, proteomic analysis revealed that S-nitrosylation is a prominent growth feature of vancomycin-intermediate S. aureus. Deletion of NO synthase (NOS) or removal of S-nitrosylation from the redox-sensitive regulator MgrA or WalR resulted in thinner cell walls and increased vancomycin susceptibility, which was due to attenuated promoter binding and released repression of genes involved in cell wall metabolism. These genes failed to respond to H2O2-induced oxidation, suggesting distinct transcriptional responses to alternative modifications of the cysteine residue. Furthermore, treatment with a NOS inhibitor significantly decreased vancomycin resistance in S. aureus. This study reveals that transcriptional regulation via S-nitrosylation underlies a mechanism for NO-mediated bacterial antibiotic resistance.
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Affiliation(s)
- Xueqin Shu
- Department of Oncology, The First Affiliated Hospital, University of Science and Technology of China, Hefei, China
- Department of Life Science and Medicine, University of Science and Technology of China, Hefei, China
| | - Yingying Shi
- Department of Oncology, The First Affiliated Hospital, University of Science and Technology of China, Hefei, China
- Department of Life Science and Medicine, University of Science and Technology of China, Hefei, China
| | - Yi Huang
- Department of Oncology, The First Affiliated Hospital, University of Science and Technology of China, Hefei, China
- Department of Life Science and Medicine, University of Science and Technology of China, Hefei, China
| | - Dan Yu
- Laboratory of Dermatology, Beijing Pediatric Research Institute, Beijing Children's Hospital, Capital Medical University, Key Laboratory of Major Diseases in Children, Ministry of Education, National Center for Children's Health, Beijing, China.
| | - Baolin Sun
- Department of Oncology, The First Affiliated Hospital, University of Science and Technology of China, Hefei, China.
- Department of Life Science and Medicine, University of Science and Technology of China, Hefei, China.
- CAS Laboratory of Innate Immunity and Chronic Disease, University of Science and Technology of China, Hefei, China.
- Hefei National Laboratory for Physical Sciences at Microscale, Hefei, China.
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14
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Brogan AP, Rudner DZ. Regulation of peptidoglycan hydrolases: localization, abundance, and activity. Curr Opin Microbiol 2023; 72:102279. [PMID: 36812681 PMCID: PMC10031507 DOI: 10.1016/j.mib.2023.102279] [Citation(s) in RCA: 5] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/12/2022] [Revised: 01/17/2023] [Accepted: 01/18/2023] [Indexed: 02/22/2023]
Abstract
Most bacteria are surrounded by a cell wall composed of peptidoglycan (PG) that specifies shape and protects the cell from osmotic rupture. Growth, division, and morphogenesis are intimately linked to the synthesis of this exoskeleton but also its hydrolysis. The enzymes that cleave the PG meshwork require careful control to prevent aberrant hydrolysis and loss of envelope integrity. Bacteria employ diverse mechanisms to control the activity, localization, and abundance of these potentially autolytic enzymes. Here, we discuss four examples of how cells integrate these control mechanisms to finely tune cell wall hydrolysis. We highlight recent advances and exciting avenues for future investigation.
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Affiliation(s)
- Anna P Brogan
- Department of Microbiology, Harvard Medical School, Boston, MA 02115, USA
| | - David Z Rudner
- Department of Microbiology, Harvard Medical School, Boston, MA 02115, USA.
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15
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Comparative Genomics Identifies Novel Genetic Changes Associated with Oxacillin, Vancomycin and Daptomycin Susceptibility in ST100 Methicillin-Resistant Staphylococcus aureus. Antibiotics (Basel) 2023; 12:antibiotics12020372. [PMID: 36830286 PMCID: PMC9952151 DOI: 10.3390/antibiotics12020372] [Citation(s) in RCA: 2] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/03/2023] [Revised: 02/09/2023] [Accepted: 02/10/2023] [Indexed: 02/17/2023] Open
Abstract
Infections due to vancomycin-intermediate S. aureus (VISA) and heterogeneous VISA (hVISA) represent a serious concern due to their association with vancomycin treatment failure. However, the underlying molecular mechanism responsible for the hVISA/VISA phenotype is complex and not yet fully understood. We have previously characterized two ST100-MRSA-hVISA clinical isolates recovered before and after 40 days of vancomycin treatment (D1 and D2, respectively) and two in vitro VISA derivatives (D23C9 and D2P11), selected independently from D2 in the presence of vancomycin. This follow-up study was aimed at further characterizing these isogenic strains and obtaining their whole genome sequences to unravel changes associated with antibiotic resistance. It is interesting to note that none of these isogenic strains carry SNPs in the regulatory operons vraUTSR, walKR and/or graXRS. Nonetheless, genetic changes including SNPs, INDELs and IS256 genomic insertions/rearrangements were found both in in vivo and in vitro vancomycin-selected strains. Some were found in the downstream target genes of the aforementioned regulatory operons, which are involved in cell wall and phosphate metabolism, staphylococcal growth and biofilm formation. Some of the genetic changes reported herein have not been previously associated with vancomycin, daptomycin and/or oxacillin resistance in S. aureus.
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16
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Barbuti MD, Myrbråten IS, Morales Angeles D, Kjos M. The cell cycle of Staphylococcus aureus: An updated review. Microbiologyopen 2023; 12:e1338. [PMID: 36825883 PMCID: PMC9733580 DOI: 10.1002/mbo3.1338] [Citation(s) in RCA: 11] [Impact Index Per Article: 5.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/19/2022] [Revised: 11/29/2022] [Accepted: 11/29/2022] [Indexed: 12/13/2022] Open
Abstract
As bacteria proliferate, DNA replication, chromosome segregation, cell wall synthesis, and cytokinesis occur concomitantly and need to be tightly regulated and coordinated. Although these cell cycle processes have been studied for decades, several mechanisms remain elusive, specifically in coccus-shaped cells such as Staphylococcus aureus. In recent years, major progress has been made in our understanding of how staphylococci divide, including new, fundamental insights into the mechanisms of cell wall synthesis and division site selection. Furthermore, several novel proteins and mechanisms involved in the regulation of replication initiation or progression of the cell cycle have been identified and partially characterized. In this review, we will summarize our current understanding of the cell cycle processes in the spheroid model bacterium S. aureus, with a focus on recent advances in the understanding of how these processes are regulated.
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Affiliation(s)
- Maria D. Barbuti
- Faculty of Chemistry, Biotechnology and Food ScienceNorwegian University of Life Sciences (NMBU)ÅsNorway
| | - Ine S. Myrbråten
- Faculty of Chemistry, Biotechnology and Food ScienceNorwegian University of Life Sciences (NMBU)ÅsNorway
| | - Danae Morales Angeles
- Faculty of Chemistry, Biotechnology and Food ScienceNorwegian University of Life Sciences (NMBU)ÅsNorway
| | - Morten Kjos
- Faculty of Chemistry, Biotechnology and Food ScienceNorwegian University of Life Sciences (NMBU)ÅsNorway
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17
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Schulz LM, Rothe P, Halbedel S, Gründling A, Rismondo J. Imbalance of peptidoglycan biosynthesis alters the cell surface charge of Listeria monocytogenes. Cell Surf 2022; 8:100085. [PMID: 36304571 PMCID: PMC9593813 DOI: 10.1016/j.tcsw.2022.100085] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/05/2022] [Revised: 10/19/2022] [Accepted: 10/19/2022] [Indexed: 02/09/2023] Open
Abstract
The bacterial cell wall is composed of a thick layer of peptidoglycan and cell wall polymers, which are either embedded in the membrane or linked to the peptidoglycan backbone and referred to as lipoteichoic acid (LTA) and wall teichoic acid (WTA), respectively. Modifications of the peptidoglycan or WTA backbone can alter the susceptibility of the bacterial cell towards cationic antimicrobials and lysozyme. The human pathogen Listeria monocytogenes is intrinsically resistant towards lysozyme, mainly due to deacetylation and O-acetylation of the peptidoglycan backbone via PgdA and OatA. Recent studies identified additional factors, which contribute to the lysozyme resistance of this pathogen. One of these is the predicted ABC transporter, EslABC. An eslB mutant is hyper-sensitive towards lysozyme, likely due to the production of thinner and less O-acetylated peptidoglycan. Using a suppressor screen, we show here that suppression of eslB phenotypes could be achieved by enhancing peptidoglycan biosynthesis, reducing peptidoglycan hydrolysis or alterations in WTA biosynthesis and modification. The lack of EslB also leads to a higher negative surface charge, which likely stimulates the activity of peptidoglycan hydrolases and lysozyme. Based on our results, we hypothesize that the portion of cell surface exposed WTA is increased in the eslB mutant due to the thinner peptidoglycan layer and that latter one could be caused by an impairment in UDP-N-acetylglucosamine (UDP-GlcNAc) production or distribution.
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Affiliation(s)
- Lisa Maria Schulz
- Department of General Microbiology, Institute of Microbiology and Genetics, GZMB, Georg-August University Göttingen, Grisebachstr. 8, 37077 Göttingen, Germany
| | - Patricia Rothe
- FG11, Division of Enteropathogenic Bacteria and Legionella, Robert Koch Institute, Burgstraße 37, 38855 Wernigerode, Germany
| | - Sven Halbedel
- FG11, Division of Enteropathogenic Bacteria and Legionella, Robert Koch Institute, Burgstraße 37, 38855 Wernigerode, Germany
- Institute for Medical Microbiology and Hospital Hygiene, Otto von Guericke University Magdeburg, Leipziger Straße 44, 39120 Magdeburg, Germany
| | - Angelika Gründling
- Section of Molecular Microbiology and Medical Research Council Centre for Molecular Bacteriology and Infection, Imperial College London, London SW7 2AZ, United Kingdom
| | - Jeanine Rismondo
- Department of General Microbiology, Institute of Microbiology and Genetics, GZMB, Georg-August University Göttingen, Grisebachstr. 8, 37077 Göttingen, Germany
- Section of Molecular Microbiology and Medical Research Council Centre for Molecular Bacteriology and Infection, Imperial College London, London SW7 2AZ, United Kingdom
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18
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Zhang R, Polenakovik H, Barreras Beltran IA, Waalkes A, Salipante SJ, Xu L, Werth BJ. Emergence of Dalbavancin, Vancomycin, and Daptomycin Nonsusceptible Staphylococcus aureus in a Patient Treated With Dalbavancin: Case Report and Isolate Characterization. Clin Infect Dis 2022; 75:1641-1644. [PMID: 35510938 PMCID: PMC10200325 DOI: 10.1093/cid/ciac341] [Citation(s) in RCA: 17] [Impact Index Per Article: 5.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/23/2022] [Indexed: 12/29/2022] Open
Abstract
A patient with end-stage renal disease received 2 doses of dalbavancin for methicillin-resistant Staphylococcus aureus (MRSA) arteriovenous fistula infection and presented 5 weeks later with infective endocarditis secondary to vancomycin, daptomycin, and dalbavancin nonsusceptible MRSA. Resistance was associated with walK and scrA mutations, reduced long-chain lipid content, and reduced membrane fluidity.
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Affiliation(s)
- Rutan Zhang
- Department of Medicinal Chemistry, University of Washington School of Pharmacy, Seattle, Washington, USA
| | - Hari Polenakovik
- Veterans Affairs Medical Center, Dayton, Ohio, USA
- Department of Medicine, Wright State University Boonshoft School of Medicine, Dayton, Ohio, USA
| | | | - Adam Waalkes
- Department of Laboratory Medicine, University of Washington, Seattle, Washington, USA
| | - Stephen J Salipante
- Department of Laboratory Medicine, University of Washington, Seattle, Washington, USA
| | - Libin Xu
- Department of Medicinal Chemistry, University of Washington School of Pharmacy, Seattle, Washington, USA
| | - Brian J Werth
- Department of Pharmacy, University of Washington School of Pharmacy, Seattle, Washington, USA
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19
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Pendleton A, Yeo WS, Alqahtani S, DiMaggio DA, Stone CJ, Li Z, Singh VK, Montgomery CP, Bae T, Brinsmade SR. Regulation of the Sae Two-Component System by Branched-Chain Fatty Acids in Staphylococcus aureus. mBio 2022; 13:e0147222. [PMID: 36135382 PMCID: PMC9600363 DOI: 10.1128/mbio.01472-22] [Citation(s) in RCA: 7] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/20/2022] [Accepted: 09/01/2022] [Indexed: 11/29/2022] Open
Abstract
Staphylococcus aureus is a ubiquitous Gram-positive bacterium and an opportunistic human pathogen. S. aureus pathogenesis relies on a complex network of regulatory factors that adjust gene expression. Two important factors in this network are CodY, a repressor protein responsive to nutrient availability, and the SaeRS two-component system (TCS), which responds to neutrophil-produced factors. Our previous work revealed that CodY regulates the secretion of many toxins indirectly via Sae through an unknown mechanism. We report that disruption of codY results in increased levels of phosphorylated SaeR (SaeR~P) and that codY mutant cell membranes contain a higher percentage of branched-chain fatty acids (BCFAs) than do wild-type membranes, prompting us to hypothesize that changes to membrane composition modulate the activity of the SaeS sensor kinase. Disrupting the lpdA gene encoding dihydrolipoyl dehydrogenase, which is critical for BCFA synthesis, significantly reduced the abundance of SaeR, phosphorylated SaeR, and BCFAs in the membrane, resulting in reduced toxin production and attenuated virulence. Lower SaeR levels could be explained in part by reduced stability. Sae activity in the lpdA mutant could be complemented genetically and chemically with exogenous short- or full-length BCFAs. Intriguingly, lack of lpdA also alters the activity of other TCSs, suggesting a specific BCFA requirement managing the basal activity of multiple TCSs. These results reveal a novel method of posttranscriptional virulence regulation via BCFA synthesis, potentially linking CodY activity to multiple virulence regulators in S. aureus. IMPORTANCE Two-component systems (TCSs) are an essential way that bacteria sense and respond to their environment. These systems are usually composed of a membrane-bound histidine kinase that phosphorylates a cytoplasmic response regulator. Because most of the histidine kinases are embedded in the membrane, lipids can allosterically regulate the activity of these sensors. In this study, we reveal that branched-chain fatty acids (BCFAs) are required for the activation of multiple TCSs in Staphylococcus aureus. Using both genetic and biochemical data, we show that the activity of the virulence activator SaeS and the phosphorylation of its response regulator SaeR are reduced in a branched-chain keto-acid dehydrogenase complex mutant and that defects in BCFA synthesis have far-reaching consequences for exotoxin secretion and virulence. Finally, we show that mutation of the global nutritional regulator CodY alters BCFA content in the membrane, revealing a potential mechanism of posttranscriptional regulation of the Sae system by CodY.
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Affiliation(s)
| | - Won-Sik Yeo
- Department of Biology, Georgetown University, Washington, DC, USA
| | - Shahad Alqahtani
- Department of Biology, Georgetown University, Washington, DC, USA
| | | | - Carl J. Stone
- Department of Biology, Georgetown University, Washington, DC, USA
| | - Zhaotao Li
- Center for Microbial Pathogenesis, Abigail Wexner Institute at Nationwide Children’s Hospital, Columbus, Ohio, USA
| | - Vineet K. Singh
- Department of Microbiology and Immunology, A.T. Still University of Health Sciences, Kirksville, Missouri, USA
| | - Christopher P. Montgomery
- Center for Microbial Pathogenesis, Abigail Wexner Institute at Nationwide Children’s Hospital, Columbus, Ohio, USA
- Department of Pediatrics, College of Medicine, The Ohio State University, Columbus, Ohio, USA
| | - Taeok Bae
- Department of Microbiology and Immunology, Indiana University School of Medicine-Northwest, Gary, Indiana, USA
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20
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Mutation in the Two-Component System Regulator YycH Leads to Daptomycin Tolerance in Methicillin-Resistant Staphylococcus aureus upon Evolution with a Population Bottleneck. Microbiol Spectr 2022; 10:e0168722. [PMID: 35913149 PMCID: PMC9431245 DOI: 10.1128/spectrum.01687-22] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/20/2022] Open
Abstract
Adaptive laboratory evolution (ALE) is a useful tool to study the evolution of antibiotic tolerance in bacterial populations under diverse environmental conditions. The role of population bottlenecks in the evolution of tolerance has been investigated in Escherichia coli, but not in a more clinically relevant pathogen, methicillin-resistant Staphylococcus aureus (MRSA). In this study, we used ALE to evolve MRSA under repetitive daptomycin treatment and incorporated population bottlenecks following antibiotic exposure. We observed that the populations finally attained a tolerance mutation in the yycH gene after 2 weeks of evolution with population bottlenecks, and additional mutations in yycI and several other genes further increased the tolerance level. The tolerant populations also became resistant to another glycopeptide antibiotic, vancomycin. Through proteomics, we showed that yycH and yycI mutations led to the loss of function of the proteins and downregulated the WalKR two-component system and the downstream players, including the autolysin Atl and amidase Sle1, which are important for cell wall metabolism. Overall, our study offers new insights into the evolution of daptomycin tolerance under population bottlenecking conditions, which are commonly faced by pathogens during infection; the study also identified new mutations conferring daptomycin tolerance and revealed the proteome alterations in the evolved tolerant populations. IMPORTANCE Although population bottlenecks are known to influence the evolutionary dynamics of microbial populations, how such bottlenecks affect the evolution of tolerance to antibiotics in a clinically relevant methicillin-resistant S. aureus (MRSA) pathogen are still unclear. Here, we performed in vitro evolution of MRSA under cyclic daptomycin treatment and applied population bottlenecks following the treatment. We showed that under these experimental conditions, MRSA populations finally attained mutations in yycH, yycI, and several other genes that led to daptomycin tolerance. The discovered yycH and yycI mutations caused early termination of the genes and loss of function of the proteins, and they subsequently downregulated the expression of proteins controlled by the WalKR two-component system, such as Atl and Sle1. In addition, we compared our proteomics data with multiple studies on distinct daptomycin-tolerant MRSA mutants to identify proteins with a consistent expression pattern that could serve as biological markers for daptomycin tolerance in MRSA.
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21
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Chen H, Yu C, Wu H, Li G, Li C, Hong W, Yang X, Wang H, You X. Recent Advances in Histidine Kinase-Targeted Antimicrobial Agents. Front Chem 2022; 10:866392. [PMID: 35860627 PMCID: PMC9289397 DOI: 10.3389/fchem.2022.866392] [Citation(s) in RCA: 16] [Impact Index Per Article: 5.3] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/31/2022] [Accepted: 05/16/2022] [Indexed: 11/15/2022] Open
Abstract
The prevalence of antimicrobial-resistant pathogens significantly limited the number of effective antibiotics available clinically, which urgently requires new drug targets to screen, design, and develop novel antibacterial drugs. Two-component system (TCS), which is comprised of a histidine kinase (HK) and a response regulator (RR), is a common mechanism whereby bacteria can sense a range of stimuli and make an appropriate adaptive response. HKs as the sensor part of the bacterial TCS can regulate various processes such as growth, vitality, antibiotic resistance, and virulence, and have been considered as a promising target for antibacterial drugs. In the current review, we highlighted the structural basis and functional importance of bacterial TCS especially HKs as a target in the discovery of new antimicrobials, and summarize the latest research progress of small-molecule HK-inhibitors as potential novel antimicrobial drugs reported in the past decade.
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Affiliation(s)
- Hongtong Chen
- Laboratory of Pharmacology/Beijing Key Laboratory of Antimicrobial Agents, Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, China
| | - Chengqi Yu
- School of Basic Medical Science, Capital Medical University, Beijing, China
| | - Han Wu
- School of Pharmacy, Minzu University of China, Beijing, China
- Key Laboratory of Ethnomedicine (Minzu University of China), Ministry of Education, Beijing, China
| | - Guoqing Li
- Laboratory of Pharmacology/Beijing Key Laboratory of Antimicrobial Agents, Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, China
| | - Congran Li
- Laboratory of Pharmacology/Beijing Key Laboratory of Antimicrobial Agents, Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, China
| | - Wei Hong
- Beijing Institute of Collaborative Innovation, Beijing, China
| | - Xinyi Yang
- Laboratory of Pharmacology/Beijing Key Laboratory of Antimicrobial Agents, Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, China
| | - Hao Wang
- School of Pharmacy, Minzu University of China, Beijing, China
- Key Laboratory of Ethnomedicine (Minzu University of China), Ministry of Education, Beijing, China
- Institute of National Security, Minzu University of China, Beijing, China
| | - Xuefu You
- Laboratory of Pharmacology/Beijing Key Laboratory of Antimicrobial Agents, Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, China
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22
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Menard G, Silard C, Suriray M, Rouillon A, Augagneur Y. Thirty Years of sRNA-Mediated Regulation in Staphylococcus aureus: From Initial Discoveries to In Vivo Biological Implications. Int J Mol Sci 2022; 23:ijms23137346. [PMID: 35806357 PMCID: PMC9266662 DOI: 10.3390/ijms23137346] [Citation(s) in RCA: 11] [Impact Index Per Article: 3.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/25/2022] [Revised: 06/20/2022] [Accepted: 06/28/2022] [Indexed: 01/27/2023] Open
Abstract
Staphylococcus aureus is a widespread livestock and human pathogen that colonizes diverse microenvironments within its host. Its adaptation to the environmental conditions encountered within humans relies on coordinated gene expression. This requires a sophisticated regulatory network, among which regulatory RNAs (usually called sRNAs) have emerged as key players over the last 30 years. In S. aureus, sRNAs regulate target genes at the post-transcriptional level through base–pair interactions. The functional characterization of a subset revealed that they participate in all biological processes, including virulence, metabolic adaptation, and antibiotic resistance. In this review, we report 30 years of S. aureus sRNA studies, from their discovery to the in-depth characterizations of some of them. We also discuss their actual in vivo contribution, which is still lagging behind, and their place within the complex regulatory network. These shall be key aspects to consider in order to clearly uncover their in vivo biological functions.
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Affiliation(s)
- Guillaume Menard
- CHU Rennes, INSERM, BRM (Bacterial Regulatory RNAs and Medicine), SB2H (Service de Bactériologie Hygiène-Hospitalière), University Rennes, UMR_S 1230, F-35000 Rennes, France; (G.M.); (M.S.)
| | - Chloé Silard
- INSERM, BRM (Bacterial Regulatory RNAs and Medicine), University Rennes, UMR_S 1230, F-35000 Rennes, France; (C.S.); (A.R.)
| | - Marie Suriray
- CHU Rennes, INSERM, BRM (Bacterial Regulatory RNAs and Medicine), SB2H (Service de Bactériologie Hygiène-Hospitalière), University Rennes, UMR_S 1230, F-35000 Rennes, France; (G.M.); (M.S.)
| | - Astrid Rouillon
- INSERM, BRM (Bacterial Regulatory RNAs and Medicine), University Rennes, UMR_S 1230, F-35000 Rennes, France; (C.S.); (A.R.)
| | - Yoann Augagneur
- INSERM, BRM (Bacterial Regulatory RNAs and Medicine), University Rennes, UMR_S 1230, F-35000 Rennes, France; (C.S.); (A.R.)
- Correspondence: ; Tel.: +33-223234631
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Wang M, Buist G, van Dijl JM. Staphylococcus aureus cell wall maintenance - the multifaceted roles of peptidoglycan hydrolases in bacterial growth, fitness, and virulence. FEMS Microbiol Rev 2022; 46:6604383. [PMID: 35675307 PMCID: PMC9616470 DOI: 10.1093/femsre/fuac025] [Citation(s) in RCA: 30] [Impact Index Per Article: 10.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/08/2021] [Revised: 04/22/2022] [Accepted: 05/25/2022] [Indexed: 01/07/2023] Open
Abstract
Staphylococcus aureus is an important human and livestock pathogen that is well-protected against environmental insults by a thick cell wall. Accordingly, the wall is a major target of present-day antimicrobial therapy. Unfortunately, S. aureus has mastered the art of antimicrobial resistance, as underscored by the global spread of methicillin-resistant S. aureus (MRSA). The major cell wall component is peptidoglycan. Importantly, the peptidoglycan network is not only vital for cell wall function, but it also represents a bacterial Achilles' heel. In particular, this network is continuously opened by no less than 18 different peptidoglycan hydrolases (PGHs) encoded by the S. aureus core genome, which facilitate bacterial growth and division. This focuses attention on the specific functions executed by these enzymes, their subcellular localization, their control at the transcriptional and post-transcriptional levels, their contributions to staphylococcal virulence and their overall importance in bacterial homeostasis. As highlighted in the present review, our understanding of the different aspects of PGH function in S. aureus has been substantially increased over recent years. This is important because it opens up new possibilities to exploit PGHs as innovative targets for next-generation antimicrobials, passive or active immunization strategies, or even to engineer them into effective antimicrobial agents.
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Affiliation(s)
- Min Wang
- Department of Medical Microbiology, University of Groningen, University Medical Center Groningen, Hanzeplein 1, PO Box 30001, 9700 RB Groningen, the Netherlands
| | | | - Jan Maarten van Dijl
- Corresponding author: Department of Medical Microbiology, University of Groningen, University Medical Center Groningen, Hanzeplein 1, P.O. box 30001, HPC EB80, 9700 RB Groningen, the Netherlands, Tel. +31-50-3615187; Fax. +31-50-3619105; E-mail:
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The WalRK Two-Component System Is Essential for Proper Cell Envelope Biogenesis in Clostridioides difficile. J Bacteriol 2022; 204:e0012122. [PMID: 35575581 PMCID: PMC9210968 DOI: 10.1128/jb.00121-22] [Citation(s) in RCA: 13] [Impact Index Per Article: 4.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/20/2022] Open
Abstract
The WalR-WalK two-component regulatory system (TCS) is found in all Firmicutes, in which it regulates the expression of multiple genes required for remodeling the cell envelope during growth and division. Unlike most TCSs, WalRK is essential for viability, so it has attracted interest as a potential antibiotic target. In this study, we used overexpression of WalR and CRISPR interference to investigate the Wal system of Clostridioides difficile, a major cause of hospital-associated diarrhea in high-income countries. We confirmed that the wal operon is essential and identified morphological defects and cell lysis as the major terminal phenotypes of altered wal expression. We also used transcriptome sequencing (RNA-seq) to identify over 150 genes whose expression changes in response to WalR levels. This gene set is enriched in cell envelope genes and includes genes encoding several predicted PG hydrolases and proteins that could regulate PG hydrolase activity. A distinct feature of the C. difficile cell envelope is the presence of an S-layer, and we found that WalR affects expression of several genes which encode S-layer proteins. An unexpected finding was that some Wal-associated phenotypic defects were inverted in comparison to what has been reported for other Firmicutes. For example, downregulation of Wal signaling caused C. difficile cells to become longer rather than shorter, as in Bacillus subtilis. Likewise, downregulation of Wal rendered C. difficile more sensitive to vancomycin, whereas reduced Wal activity is linked to increased vancomycin resistance in Staphylococcus aureus. IMPORTANCE The WalRK two-component system (TCS) is essential for coordinating synthesis and turnover of peptidoglycan in Firmicutes. We investigated the WalRK TCS in Clostridioides difficile, an important bacterial pathogen with an atypical cell envelope. We confirmed that WalRK is essential and regulates cell envelope biogenesis, although several of the phenotypic changes we observed were opposite to what has been reported for other Firmicutes. We also identified over 150 genes whose expression is controlled either directly or indirectly by WalR. Overall, our findings provide a foundation for future investigations of an important regulatory system and potential antibiotic target in C. difficile.
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Natural transformation allows transfer of SCCmec-mediated methicillin resistance in Staphylococcus aureus biofilms. Nat Commun 2022; 13:2477. [PMID: 35513365 PMCID: PMC9072672 DOI: 10.1038/s41467-022-29877-2] [Citation(s) in RCA: 38] [Impact Index Per Article: 12.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/23/2021] [Accepted: 03/16/2022] [Indexed: 12/30/2022] Open
Abstract
SCCmec is a large mobile genetic element that includes the mecA gene and confers resistance to β-lactam antibiotics in methicillin-resistant Staphylococcus aureus (MRSA). There is evidence that SCCmec disseminates among staphylococci, but the transfer mechanisms are unclear. Here, we show that two-component systems mediate the upregulation of natural competence genes in S. aureus under biofilm growth conditions, and this enhances the efficiency of natural transformation. We observe SCCmec transfer via natural transformation from MRSA, and from methicillin-resistant coagulase-negative staphylococci, to methicillin-sensitive S. aureus. The process requires the SCCmec recombinase genes ccrAB, and the stability of the transferred SCCmec varies depending on SCCmec types and recipients. Our results suggest that natural transformation plays a role in the transfer of SCCmec and possibly other mobile genetic elements in S. aureus biofilms. SCCmec is a large mobile genetic element that confers resistance to β-lactam antibiotics in methicillin-resistant Staphylococcus aureus. Here, the authors show that biofilm growth conditions enhance the efficiency of natural transformation in S. aureus and allow the transfer of SCCmec to methicillin-sensitive strains.
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26
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Haubrich BA, Nayyab S, Gallati M, Hernandez J, Williams C, Whitman A, Zimmerman T, Li Q, Chen Y, Zhou CZ, Basu A, Reid CW. Inhibition of Streptococcus pneumoniae growth by masarimycin. MICROBIOLOGY (READING, ENGLAND) 2022; 168. [PMID: 35467499 DOI: 10.1099/mic.0.001182] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 11/18/2022]
Abstract
Despite renewed interest, development of chemical biology methods to study peptidoglycan metabolism has lagged in comparison to the glycobiology field in general. To address this, a panel of diamides were screened against the Gram-positive bacterium Streptococcus pneumoniae to identify inhibitors of bacterial growth. The screen identified the diamide masarimycin as a bacteriostatic inhibitor of S. pneumoniae growth with an MIC of 8 µM. The diamide inhibited detergent-induced autolysis in a concentration-dependent manner, indicating perturbation of peptidoglycan degradation as the mode-of-action. Cell based screening of masarimycin against a panel of autolysin mutants, identified a higher MIC against a ΔlytB strain lacking an endo-N-acetylglucosaminidase involved in cell division. Subsequent biochemical and phenotypic analyses suggested that the higher MIC was due to an indirect interaction with LytB. Further analysis of changes to the cell surface in masarimycin treated cells identified the overexpression of several moonlighting proteins, including elongation factor Tu which is implicated in regulating cell shape. Checkerboard assays using masarimycin in concert with additional antibiotics identified an antagonistic relationship with the cell wall targeting antibiotic fosfomycin, which further supports a cell wall mode-of-action.
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Affiliation(s)
- Brad A Haubrich
- Center for Health and Behavioral Sciences, Department of Science and Technology, Bryant University, 1150 Douglas Pike, Smithfield, RI 02917, USA.,Department of Basic Sciences, Touro University Nevada, College of Osteopathic Medicine, Henderson, NV 89014, USA
| | - Saman Nayyab
- Center for Health and Behavioral Sciences, Department of Science and Technology, Bryant University, 1150 Douglas Pike, Smithfield, RI 02917, USA.,Amherst Department of Molecular and Cellular Biology, University of Massachusetts, 230 Stockbridge Rd Amherst, MA, USA
| | - Mika Gallati
- Center for Health and Behavioral Sciences, Department of Science and Technology, Bryant University, 1150 Douglas Pike, Smithfield, RI 02917, USA
| | - Jazmeen Hernandez
- Center for Health and Behavioral Sciences, Department of Science and Technology, Bryant University, 1150 Douglas Pike, Smithfield, RI 02917, USA
| | - Caroline Williams
- Center for Health and Behavioral Sciences, Department of Science and Technology, Bryant University, 1150 Douglas Pike, Smithfield, RI 02917, USA
| | - Andrew Whitman
- Center for Health and Behavioral Sciences, Department of Science and Technology, Bryant University, 1150 Douglas Pike, Smithfield, RI 02917, USA
| | - Tahl Zimmerman
- Department of Family and Consumer Sciences, North Carolina A&T State University, Greensboro, NC, USA
| | - Qiong Li
- School of Life Sciences, University of Science and Technology of China, Hefei, Anhui, 230027, PR China
| | - Yuxing Chen
- School of Life Sciences, University of Science and Technology of China, Hefei, Anhui, 230027, PR China
| | - Cong-Zhao Zhou
- School of Life Sciences, University of Science and Technology of China, Hefei, Anhui, 230027, PR China
| | - Amit Basu
- Department of Chemistry, Brown University, Providence, RI, USA
| | - Christopher W Reid
- Center for Health and Behavioral Sciences, Department of Science and Technology, Bryant University, 1150 Douglas Pike, Smithfield, RI 02917, USA
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Bleul L, Francois P, Wolz C. Two-Component Systems of S. aureus: Signaling and Sensing Mechanisms. Genes (Basel) 2021; 13:34. [PMID: 35052374 PMCID: PMC8774646 DOI: 10.3390/genes13010034] [Citation(s) in RCA: 34] [Impact Index Per Article: 8.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/21/2021] [Revised: 12/21/2021] [Accepted: 12/22/2021] [Indexed: 12/18/2022] Open
Abstract
Staphylococcus aureus encodes 16 two-component systems (TCSs) that enable the bacteria to sense and respond to changing environmental conditions. Considering the function of these TCSs in bacterial survival and their potential role as drug targets, it is important to understand the exact mechanisms underlying signal perception. The differences between the sensing of appropriate signals and the transcriptional activation of the TCS system are often not well described, and the signaling mechanisms are only partially understood. Here, we review present insights into which signals are sensed by histidine kinases in S. aureus to promote appropriate gene expression in response to diverse environmental challenges.
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Affiliation(s)
- Lisa Bleul
- Interfaculty Institute of Microbiology and Infection Medicine, University of Tübingen, 72076 Tubingen, Germany;
- Cluster of Excellence EXC 2124 “Controlling Microbes to Fight Infections”, University of Tübingen, Elfriede-Aulhorn-Str. 6, 72076 Tubingen, Germany
| | - Patrice Francois
- Genomic Research Laboratory, Infectious Diseases Service, University Hospitals of Geneva University Medical Center, Michel Servet 1, CH-1211 Geneva, Switzerland;
| | - Christiane Wolz
- Interfaculty Institute of Microbiology and Infection Medicine, University of Tübingen, 72076 Tubingen, Germany;
- Cluster of Excellence EXC 2124 “Controlling Microbes to Fight Infections”, University of Tübingen, Elfriede-Aulhorn-Str. 6, 72076 Tubingen, Germany
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28
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Wu S, Zhang J, Peng Q, Liu Y, Lei L, Zhang H. The Role of Staphylococcus aureus YycFG in Gene Regulation, Biofilm Organization and Drug Resistance. Antibiotics (Basel) 2021; 10:antibiotics10121555. [PMID: 34943766 PMCID: PMC8698359 DOI: 10.3390/antibiotics10121555] [Citation(s) in RCA: 18] [Impact Index Per Article: 4.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/10/2021] [Revised: 12/09/2021] [Accepted: 12/16/2021] [Indexed: 02/05/2023] Open
Abstract
Antibiotic resistance is a serious global health concern that may have significant social and financial consequences. Methicillin-resistant Staphylococcus aureus (MRSA) infection is responsible for substantial morbidity and leads to the death of 21.8% of infected patients annually. A lack of novel antibiotics has prompted the exploration of therapies targeting bacterial virulence mechanisms. The two-component signal transduction system (TCS) enables microbial cells to regulate gene expression and the subsequent metabolic processes that occur due to environmental changes. The YycFG TCS in S. aureus is essential for bacterial viability, the regulation of cell membrane metabolism, cell wall synthesis and biofilm formation. However, the role of YycFG-associated biofilm organization in S. aureus antimicrobial drug resistance and gene regulation has not been discussed in detail. We reviewed the main molecules involved in YycFG-associated cell wall biosynthesis, biofilm development and polysaccharide intercellular adhesin (PIA) accumulation. Two YycFG-associated regulatory mechanisms, accessory gene regulator (agr) and staphylococcal accessory regulator (SarA), were also discussed. We highlighted the importance of biofilm formation in the development of antimicrobial drug resistance in S. aureus infections. Data revealed that inhibition of the YycFG pathway reduced PIA production, biofilm formation and bacterial pathogenicity, which provides a potential target for the management of MRSA-induced infections.
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Affiliation(s)
- Shizhou Wu
- Department of Orthopedics, West China Hospital, Sichuan University, Chengdu 610041, China; (S.W.); (J.Z.); (Q.P.)
| | - Junqi Zhang
- Department of Orthopedics, West China Hospital, Sichuan University, Chengdu 610041, China; (S.W.); (J.Z.); (Q.P.)
| | - Qi Peng
- Department of Orthopedics, West China Hospital, Sichuan University, Chengdu 610041, China; (S.W.); (J.Z.); (Q.P.)
| | - Yunjie Liu
- West China School of Public Health, Sichuan University, Chengdu 610041, China;
| | - Lei Lei
- West China Hospital of Stomatology, Sichuan University, Chengdu 610041, China
- Correspondence: (L.L.); (H.Z.)
| | - Hui Zhang
- Department of Orthopedics, West China Hospital, Sichuan University, Chengdu 610041, China; (S.W.); (J.Z.); (Q.P.)
- Correspondence: (L.L.); (H.Z.)
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Demonstration of the role of cell wall homeostasis in Staphylococcus aureus growth and the action of bactericidal antibiotics. Proc Natl Acad Sci U S A 2021; 118:2106022118. [PMID: 34716264 PMCID: PMC8612353 DOI: 10.1073/pnas.2106022118] [Citation(s) in RCA: 31] [Impact Index Per Article: 7.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/29/2021] [Accepted: 09/02/2021] [Indexed: 12/29/2022] Open
Abstract
Bacterial cell wall peptidoglycan is essential, maintaining both cellular integrity and morphology, in the face of internal turgor pressure. Peptidoglycan synthesis is important, as it is targeted by cell wall antibiotics, including methicillin and vancomycin. Here, we have used the major human pathogen Staphylococcus aureus to elucidate both the cell wall dynamic processes essential for growth (life) and the bactericidal effects of cell wall antibiotics (death) based on the principle of coordinated peptidoglycan synthesis and hydrolysis. The death of S. aureus due to depletion of the essential, two-component and positive regulatory system for peptidoglycan hydrolase activity (WalKR) is prevented by addition of otherwise bactericidal cell wall antibiotics, resulting in stasis. In contrast, cell wall antibiotics kill via the activity of peptidoglycan hydrolases in the absence of concomitant synthesis. Both methicillin and vancomycin treatment lead to the appearance of perforating holes throughout the cell wall due to peptidoglycan hydrolases. Methicillin alone also results in plasmolysis and misshapen septa with the involvement of the major peptidoglycan hydrolase Atl, a process that is inhibited by vancomycin. The bactericidal effect of vancomycin involves the peptidoglycan hydrolase SagB. In the presence of cell wall antibiotics, the inhibition of peptidoglycan hydrolase activity using the inhibitor complestatin results in reduced killing, while, conversely, the deregulation of hydrolase activity via loss of wall teichoic acids increases the death rate. For S. aureus, the independent regulation of cell wall synthesis and hydrolysis can lead to cell growth, death, or stasis, with implications for the development of new control regimes for this important pathogen.
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30
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Pardoux R, Dolla A, Aubert C. Metal-containing PAS/GAF domains in bacterial sensors. Coord Chem Rev 2021. [DOI: 10.1016/j.ccr.2021.214000] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/09/2022]
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31
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Wang W, Sun B. VraCP regulates cell wall metabolism and antibiotic resistance in vancomycin-intermediate Staphylococcus aureus strain Mu50. J Antimicrob Chemother 2021; 76:1712-1723. [PMID: 33948657 PMCID: PMC8212773 DOI: 10.1093/jac/dkab113] [Citation(s) in RCA: 7] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/08/2020] [Accepted: 03/13/2021] [Indexed: 01/29/2023] Open
Abstract
Objectives Vancomycin-intermediate Staphylococcus aureus (VISA) is increasingly being reported. Previous studies have shown that vraC and vraP may be involved in vancomycin resistance, although the molecular mechanism remains elusive. Methods The vraC (SAV0577), vraP (SAV0578) and vraCP mutants were constructed in Mu50 by allelic replacement. Some common VISA phenotypes were assessed in mutants, such as, susceptibility to the cell wall-associated antibiotics, cell wall thickness, autolysis activity and growth rate. RT-qPCR was performed to reveal the differential genes associated with these phenotypes. The binding abilities of VraC and VraCP to the promoters of target genes were determined by electrophoretic mobility shift assay (EMSA). Results VraP forms a stable complex with VraC to preserve their own stability. The vraC, vraP and vraCP mutants exhibited increased susceptibility to the cell wall-associated antibiotics and thinner cell walls compared with the WT strain. Consistent with these phenotypes, RT-qPCR revealed downregulated transcription of glyS, sgtB, ddl and alr2, which are involved in cell wall biosynthesis. Moreover, the transcription of cell wall hydrolysis genes, including sceD, lytM and isaA, was significantly downregulated, supporting the finding that mutants exhibited reduced autolysis rates. EMSA confirmed that both VraC and VraCP can directly bind to the sceD, lytM and isaA promoter regions containing the consensus sequence (5′-TTGTAAN2AN3TGTAA-3′), which is crucial for the binding of VraCP with target genes. GFP-reporter assays further revealed VraC and VraCP can enhance promoter activity of sceD to positively regulate its expression. Conclusions vraCP plays a significant role in cell wall metabolism and antibiotic resistance in Mu50.
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Affiliation(s)
- Wanying Wang
- Department of Oncology, The First Affiliated Hospital, University of Science and Technology of China, Hefei, People's Republic of China
| | - Baolin Sun
- Department of Oncology, The First Affiliated Hospital, University of Science and Technology of China, Hefei, People's Republic of China
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Wu S, Liu Y, Lei L, Zhang H. Antisense yycG modulates the susceptibility of Staphylococcus aureus to hydrogen peroxide via the sarA. BMC Microbiol 2021; 21:160. [PMID: 34053439 PMCID: PMC8165985 DOI: 10.1186/s12866-021-02218-x] [Citation(s) in RCA: 7] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/21/2020] [Accepted: 05/05/2021] [Indexed: 02/08/2023] Open
Abstract
Background The infectious pathogen Staphylococcus aureus (S. aureus) is primarily associated with osteomyelitis. Hydrogen peroxide drainage is an effective antimicrobial treatment that has been adopted to combat S. aureus infections. Previous investigations have indicated that the antisense RNA (asRNA) strategy negatively modulates S. aureus YycFG TCS, and it significantly disrupts biofilm formation. However, the effects of the antisense yycG RNA (ASyycG) strategy on the susceptibility of biofilm-producing S. aureus to hydrogen peroxide and the mechanisms underlying this effect have not been elucidated to date. Results Overexpression of ASyycG inhibited the transcription of biofilm formation-related genes, including sarA and icaA. Additionally, the CFU counts and the live bacterial ratios of ASyycG biofilm-producing S. aureus treated with H2O2 were notably reduced across the groups. Notably, the predicted promoter regions of the sarA and icaA genes were directly regulated by YycF. Conclusions ASyycG was observed to sensitize biofilm-producing S. aureus to H2O2 intervention synergistically via the sarA and thus may represent a supplementary strategy for managing osteomyelitis. However, future in-depth studies should attempt to replicate our findings in animal models, such as the rat osteomyelitis model. Supplementary Information The online version contains supplementary material available at 10.1186/s12866-021-02218-x.
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Affiliation(s)
- Shizhou Wu
- Department of Orthopedics, West China Hospital, Sichuan University, No.37 Guoxue Alley, Sichuan, 610041, Chengdu, P.R. China
| | - Yunjie Liu
- West China School of Public Health, Sichuan University, Chengdu, China
| | - Lei Lei
- Department of Preventive Dentistry, Hospital of Stomatology, State Key Laboratory of Oral Diseases,, Sichuan University, NO.14 Third Section, Renmin South Road, Sichuan, 610041, Chengdu, P.R. China.
| | - Hui Zhang
- Department of Orthopedics, West China Hospital, Sichuan University, No.37 Guoxue Alley, Sichuan, 610041, Chengdu, P.R. China.
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Apt (Adenine Phosphoribosyltransferase) Mutation in Laboratory-Selected Vancomycin-Intermediate Staphylococcus aureus. Antibiotics (Basel) 2021; 10:antibiotics10050583. [PMID: 34069103 PMCID: PMC8170892 DOI: 10.3390/antibiotics10050583] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/25/2021] [Revised: 04/19/2021] [Accepted: 04/27/2021] [Indexed: 11/16/2022] Open
Abstract
Comparative genomic sequencing of laboratory-derived vancomycin-intermediate Staphylococcusaureus (VISA) (MM66-3 and MM66-4) revealed unique mutations in both MM66-3 (in apt and ssaA6), and MM66-4 (in apt and walK), compared to hetero-VISA parent strain MM66. Transcriptional profiling revealed that both MM66 VISA shared 79 upregulated genes and eight downregulated genes. Of these, 30.4% of the upregulated genes were associated with the cell envelope, whereas 75% of the downregulated genes were associated with virulence. In concordance with mutations and transcriptome alterations, both VISA strains demonstrated reduced autolysis, reduced growth in the presence of salt and reduced virulence factor activity. In addition to mutations in genes linked to cell wall metabolism (ssaA6 and walK), the same mutation in apt which encodes adenine phosphoribosyltransferase, was confirmed in both MM66 VISA. Apt plays a role in both adenine metabolism and accumulation and both MM66 VISA grew better than MM66 in the presence of adenine or 2-fluoroadenine indicating a reduction in the accumulation of these growth inhibiting compounds in the VISA strains. MM66 apt mutants isolated via 2-fluoroadenine selection also demonstrated reduced susceptibility to the cell wall lytic dye Congo red and vancomycin. Finding that apt mutations contribute to reduced vancomycin susceptibility once again suggests a role for altered purine metabolism in a VISA mechanism.
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Buchad H, Nair M. The small RNA SprX regulates the autolysin regulator WalR in Staphylococcus aureus. Microbiol Res 2021; 250:126785. [PMID: 34000511 DOI: 10.1016/j.micres.2021.126785] [Citation(s) in RCA: 13] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/18/2020] [Revised: 04/29/2021] [Accepted: 04/30/2021] [Indexed: 10/21/2022]
Abstract
Pathogenesis of Staphylococcus aureus is attributed to its remarkable adaptation to changes in the environment, mediated by the arsenal of virulence factors, which are regulated by intricate mechanisms that include small RNAs (sRNAs) as important regulatory molecules. The sRNA SprX was previously described to be involved in the regulation of S. aureus pathogenicity, by modifying the expression of surface-associated clumping factor B and the secreted delta haemolysin. This study describes the regulation by SprX, of expression of multiple autolysins, which play an essential role in cell wall metabolism and function as important virulence factors that facilitate adhesion, internalization, and immune evasion during S. aureus colonization and pathogenesis. SprX acts by positively regulating the expression of autolysin regulator WalR. Overexpression of SprX resulted in differential regulation of autolysins IsaA, and LytM, while WalR levels were unchanged. SprX knockdown strain exhibited down-regulation of multiple autolytic bands corresponding to the major autolysin AtlA and its process intermediates in cell wall degradation zymography, and 0.2 to 0.1 fold reduction of lytM, atlA, isaA, and walR transcripts in qRT-PCRs. Down-regulation of SprX resulted in altered phenotype with high cell aggregation as analyzed by SEM, decrease in biofilm formation and higher resistance to Triton X-100-induced lysis, all of which indicate that SprX is essential for expression of autolysins. A putative RNA-RNA interaction was indicated in silico between SprX and walR mRNA and further confirmed by in vitro RNA-RNA interaction in electrophoretic mobility shift assays. These findings elucidate a new mechanism in which SprX modulates the S. aureus pathogenicity by regulating the regulator of autolysins in cell wall metabolism and as virulence factors.
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Affiliation(s)
- Hasmatbanu Buchad
- Department of Microbiology and Biotechnology Centre, The Maharaja Sayajirao University of Baroda, Vadodara, Gujarat, 390002, India.
| | - Mrinalini Nair
- Department of Microbiology and Biotechnology Centre, The Maharaja Sayajirao University of Baroda, Vadodara, Gujarat, 390002, India.
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Pei X, Liu M, Zhou H, Fan H. Screening for phagocytosis resistance-related genes via a transposon mutant library of Streptococcus suis serotype 2. Virulence 2021; 11:825-838. [PMID: 32614642 PMCID: PMC7567436 DOI: 10.1080/21505594.2020.1782088] [Citation(s) in RCA: 5] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/22/2022] Open
Abstract
Streptococcus suis serotype 2 (SS2) is a serious zoonotic pathogen which causes symptoms of streptococcal toxic shock syndrome (STSS) and septicemia; these symptoms suggest that SS2 may have evade innate immunity. Phagocytosis is an important innate immunity process where phagocytosed pathogens are killed by lysosome enzymes, reactive oxygen, and nitrogen species, and acidic environments in macrophages following engulfment. A previously constructed mutant SS2 library was screened, revealing 13 mutant strains with decreased phagocytic resistance. Through inverse PCR, the transposon insertion sites were determined. Through bioinformatic analysis, the 13 disrupted genes were identified as Cps2F, 3 genes belonging to ABC transporters, WalR, TehB, rpiA, S-transferase encoding gene, prs, HsdM, GNAT family N-acetyltransferase encoding gene, proB, and upstream region of DnaK. Except for the capsular polysaccharide biosynthesis associated Cps2F, the other genes had not been linked to a role in anti-phagocytosis. The survival ability in macrophages and whole blood of randomly picked mutant strains were significantly impaired compared with wild-type ZY05719. The virulence of the mutant strains was also attenuated in a mouse infection model. In the WalR mutant, the transcription of HP1065 decreased significantly compared with wild-type strain, indicating WalR might regulated HP1065 expression and contribute to the anti-phagocytosis of SS2. In conclusion, we identified 13 genes that influenced the phagocytosis resistant ability of SS2, and many of these genes have not been reported to be associated with resistance to phagocytosis. Our work provides novel insight into resistance to phagocytosis, and furthers our understanding of the pathogenesis mechanism of SS2.
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Affiliation(s)
- Xiaomeng Pei
- MOE Joint International Research Laboratory of Animal Health and Food Safety, College of Veterinary Medicine, Nanjing Agricultural University , Nanjing, China
| | - Mingxing Liu
- MOE Joint International Research Laboratory of Animal Health and Food Safety, College of Veterinary Medicine, Nanjing Agricultural University , Nanjing, China
| | - Hong Zhou
- MOE Joint International Research Laboratory of Animal Health and Food Safety, College of Veterinary Medicine, Nanjing Agricultural University , Nanjing, China
| | - Hongjie Fan
- MOE Joint International Research Laboratory of Animal Health and Food Safety, College of Veterinary Medicine, Nanjing Agricultural University , Nanjing, China.,Jiangsu Co-innovation Center for the Prevention and Control of Important Animal Infectious Diseases and Zoonoses, Yangzhou University , Yangzhou, China
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Zhu J, Liu B, Shu X, Sun B. A novel mutation of walK confers vancomycin-intermediate resistance in methicillin-susceptible Staphylococcus aureus. Int J Med Microbiol 2021; 311:151473. [PMID: 33445057 DOI: 10.1016/j.ijmm.2021.151473] [Citation(s) in RCA: 11] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/11/2020] [Revised: 12/24/2020] [Accepted: 01/04/2021] [Indexed: 10/22/2022] Open
Abstract
With the treatment failure by vancomycin and poor clinical outcomes, the emergence and spread of vancomycin intermediate-resistant Staphylococcus aureus (VISA) has raised more concerns in recent years. While most VISA strains are isolated from methicillin-resistant S. aureus (MRSA), the mechanism underlying the generation of VISA from methicillin-susceptible S. aureus (MSSA) is still largely unknown. Here, we identified a total of 10 mutations in 9 genes through comparative genome analysis from laboratory-derived VISA strain. We verified the role of a novel mutation of WalK (I237T) and our results further indicated that the introduction of WalK (I237T) by allelic replacement can confer vancomycin resistance in MSSA with common VISA characteristics, including thickened cell walls, reduced autolysis, and attenuated virulence. Consistent with these phenotypes, real-time quantitative reverse transcription-PCR revealed the altered expression of several genes associated with cell wall metabolism and virulence control. In addition, electrophoretic mobility shift assay indicated that WalR can directly bind to the promoter regions of oatA, sle1, and mgt, fluorescence-based promoter activity and β-galactosidase assays revealed WalK (I237T) can alter promoter activities of oatA, mgt, and sle1, thus regulating genes expression. These findings broaden our understanding of the regulatory network by WalKR system and decipher the molecular mechanisms of developmental VISA resistance in MSSA with point mutations.
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Affiliation(s)
- Jiade Zhu
- Department of Oncology, The First Affiliated Hospital, CAS Key Laboratory of Innate Immunity and Chronic Disease, and Division of Life Sciences and Medicine, University of Science and Technology of China, Anhui, 230027, China
| | - Banghui Liu
- Department of Oncology, The First Affiliated Hospital, CAS Key Laboratory of Innate Immunity and Chronic Disease, and Division of Life Sciences and Medicine, University of Science and Technology of China, Anhui, 230027, China
| | - Xueqin Shu
- Department of Oncology, The First Affiliated Hospital, CAS Key Laboratory of Innate Immunity and Chronic Disease, and Division of Life Sciences and Medicine, University of Science and Technology of China, Anhui, 230027, China
| | - Baolin Sun
- Department of Oncology, The First Affiliated Hospital, CAS Key Laboratory of Innate Immunity and Chronic Disease, and Division of Life Sciences and Medicine, University of Science and Technology of China, Anhui, 230027, China.
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Progress Overview of Bacterial Two-Component Regulatory Systems as Potential Targets for Antimicrobial Chemotherapy. Antibiotics (Basel) 2020; 9:antibiotics9100635. [PMID: 32977461 PMCID: PMC7598275 DOI: 10.3390/antibiotics9100635] [Citation(s) in RCA: 60] [Impact Index Per Article: 12.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/26/2020] [Revised: 09/14/2020] [Accepted: 09/21/2020] [Indexed: 12/18/2022] Open
Abstract
Bacteria adapt to changes in their environment using a mechanism known as the two-component regulatory system (TCS) (also called “two-component signal transduction system” or “two-component system”). It comprises a pair of at least two proteins, namely the sensor kinase and the response regulator. The former senses external stimuli while the latter alters the expression profile of bacterial genes for survival and adaptation. Although the first TCS was discovered and characterized in a non-pathogenic laboratory strain of Escherichia coli, it has been recognized that all bacteria, including pathogens, use this mechanism. Some TCSs are essential for cell growth and fitness, while others are associated with the induction of virulence and drug resistance/tolerance. Therefore, the TCS is proposed as a potential target for antimicrobial chemotherapy. This concept is based on the inhibition of bacterial growth with the substances acting like conventional antibiotics in some cases. Alternatively, TCS targeting may reduce the burden of bacterial virulence and drug resistance/tolerance, without causing cell death. Therefore, this approach may aid in the development of antimicrobial therapeutic strategies for refractory infections caused by multi-drug resistant (MDR) pathogens. Herein, we review the progress of TCS inhibitors based on natural and synthetic compounds.
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Werth BJ, Ashford NK, Penewit K, Waalkes A, Holmes EA, Ross DH, Shen T, Hines KM, Salipante SJ, Xu L. Dalbavancin exposure in vitro selects for dalbavancin-non-susceptible and vancomycin-intermediate strains of methicillin-resistant Staphylococcus aureus. Clin Microbiol Infect 2020; 27:910.e1-910.e8. [PMID: 32866650 DOI: 10.1016/j.cmi.2020.08.025] [Citation(s) in RCA: 19] [Impact Index Per Article: 3.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/08/2020] [Revised: 08/13/2020] [Accepted: 08/21/2020] [Indexed: 12/11/2022]
Abstract
OBJECTIVES Dalbavancin is a lipoglycopeptide active against methicillin-resistant Staphylococcus aureus (MRSA). Its long half-life (8.5-16 days) allows for once-weekly or single-dose treatments but could prolong the mutant selection window, promoting resistance and cross-resistance to related antimicrobials such as vancomycin. The objective of this study was to evaluate the capacity of post-distributional pharmacokinetic exposures of dalbavancin to select for resistance and cross-resistance in MRSA. METHODS We simulated average, post-distributional exposures of single-dose (1500 mg) dalbavancin (fCmax 9.9 μg/mL, β-elimination t1/2 204 h) in an in vitro pharmacokinetic/pharmacodynamic (PK/PD) model for 28 days (672 h) against five MRSA strains and one methicillin-susceptible strain (MSSA). Samples were collected at least daily, and surviving colonies were enumerated and screened for resistance on drug-free and dalbavancin-supplemented medium respectively. Isolates from resistance screening plates were subjected to whole-genome sequencing (WGS) and susceptibly testing against dalbavancin, vancomycin, daptomycin, and six β-lactams with varying penicillin-binding protein (PBP) affinities. RESULTS Dalbavancin was bactericidal against most strains for days 1-4 before regrowth of less susceptible subpopulations occurred. Isolates with eight-fold increases in dalbavancin MIC were detected as early as day 4 but increased 64-128-fold in all models by day 28. Vancomycin and daptomycin MICs increased 4-16-fold, exceeding the susceptibly breakpoints for both antibiotics; β-lactam MICs generally decreased by two-to eight-fold, suggesting a dalbavancin-β-lactam seesaw effect, but increased by eight-fold or more in certain isolates. Resistant isolates carried mutations in a variety of genes, most commonly walKR, apt, stp1, and atl. CONCLUSIONS In our in vitro system, post-distributional dalbavancin exposures selected for stable mutants with reduced susceptibility to dalbavancin, vancomycin, and daptomycin, and generally increased susceptibility to β-lactams in all strains of MRSA tested. The clinical significance of these findings remains unclear, but created an opportunity to genotype a unique collection of dalbavancin-resistant strains for the first time. Mutations involved genes previously associated with vancomycin intermediate susceptibility and daptomycin non-susceptibility, most commonly walKR-associated genes.
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Affiliation(s)
- Brian J Werth
- Department of Pharmacy, School of Pharmacy, University of Washington, Seattle, WA, USA.
| | - Nathaniel K Ashford
- Department of Pharmacy, School of Pharmacy, University of Washington, Seattle, WA, USA
| | - Kelsi Penewit
- Department of Laboratory Medicine, School of Pharmacy, University of Washington, Seattle, WA, USA
| | - Adam Waalkes
- Department of Laboratory Medicine, School of Pharmacy, University of Washington, Seattle, WA, USA
| | - Elizabeth A Holmes
- Department of Laboratory Medicine, School of Pharmacy, University of Washington, Seattle, WA, USA
| | - Dylan H Ross
- Department of Medicinal Chemistry, School of Pharmacy, University of Washington, Seattle, WA, USA
| | - Tianwei Shen
- Department of Medicinal Chemistry, School of Pharmacy, University of Washington, Seattle, WA, USA
| | - Kelly M Hines
- Department of Medicinal Chemistry, School of Pharmacy, University of Washington, Seattle, WA, USA; University of Georgia, Department of Chemistry, Athens, GA, USA
| | - Stephen J Salipante
- Department of Laboratory Medicine, School of Pharmacy, University of Washington, Seattle, WA, USA
| | - Libin Xu
- Department of Medicinal Chemistry, School of Pharmacy, University of Washington, Seattle, WA, USA
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Cafiso V, Stracquadanio S, Lo Verde F, De Guidi I, Zega A, Pigola G, Stefani S. Genomic and Long-Term Transcriptomic Imprints Related to the Daptomycin Mechanism of Action Occurring in Daptomycin- and Methicillin-Resistant Staphylococcus aureus Under Daptomycin Exposure. Front Microbiol 2020; 11:1893. [PMID: 32922373 PMCID: PMC7456847 DOI: 10.3389/fmicb.2020.01893] [Citation(s) in RCA: 8] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/09/2019] [Accepted: 07/20/2020] [Indexed: 01/06/2023] Open
Abstract
Daptomycin (DAP) is one of the last-resort treatments for heterogeneous vancomycin-intermediate Staphylococcus aureus (hVISA) and vancomycin-intermediate S. aureus (VISA) infections. DAP resistance (DAP-R) is multifactorial and mainly related to cell-envelope modifications caused by single-nucleotide polymorphisms and/or modulation mechanisms of transcription emerging as result of a self-defense process in response to DAP exposure. Nevertheless, the role of these adaptations remains unclear. We aim to investigate the comparative genomics and late post-exponential growth-phase transcriptomics of two DAP-resistant/DAP-susceptible (DAPR/S) methicillin-resistant S. aureus (MRSA) clinical strain pairs to focalize the genomic and long-term transcriptomic fingerprinting and adaptations related to the DAP mechanism of action acquired in vivo under DAP pressure using Illumina whole-genome sequencing (WGS), RNA-seq, bioinformatics, and real-time qPCR validation. Comparative genomics revealed that membrane protein and transcriptional regulator coding genes emerged as shared functional coding-gene clusters harboring mutational events related to the DAP-R onset in a strain-dependent manner. Pairwise transcriptomic enrichment analysis highlighted common and strain pair-dependent Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways, whereas DAPR/S double-pair cross-filtering returned 53 differentially expressed genes (DEGs). A multifactorial long-term transcriptomic-network characterized DAPR MRSA includes alterations in (i) peptidoglycan biosynthesis, cell division, and cell-membrane (CM) organization genes, as well as a cidB/lytS autolysin genes; (ii) ldh2 involved in fermentative metabolism; (iii) CM-potential perturbation genes; and (iv) oxidative and heat/cold stress response-related genes. Moreover, a D-alanyl–D-alanine decrease in cell-wall muropeptide characterized DAP/glycopeptide cross-reduced susceptibility mechanisms in DAPR MRSA. Our data provide a snapshot of DAPR MRSA genomic and long-term transcriptome signatures related to the DAP mechanism of action (MOA) evidencing that a complex network of genomic changes and transcriptomic adaptations is required to acquire DAP-R.
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Affiliation(s)
- Viviana Cafiso
- Department of Biomedical and Biotechnological Sciences, University of Catania, Catania, Italy
| | - Stefano Stracquadanio
- Department of Biomedical and Biotechnological Sciences, University of Catania, Catania, Italy
| | - Flavia Lo Verde
- Department of Biomedical and Biotechnological Sciences, University of Catania, Catania, Italy
| | - Irene De Guidi
- Department of Biomedical and Biotechnological Sciences, University of Catania, Catania, Italy
| | - Alessandra Zega
- Department of Biomedical and Biotechnological Sciences, University of Catania, Catania, Italy
| | - Giuseppe Pigola
- Department of Clinical and Experimental Medicine, University of Catania, Catania, Italy
| | - Stefania Stefani
- Department of Biomedical and Biotechnological Sciences, University of Catania, Catania, Italy
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Gajdiss M, Monk IR, Bertsche U, Kienemund J, Funk T, Dietrich A, Hort M, Sib E, Stinear TP, Bierbaum G. YycH and YycI Regulate Expression of Staphylococcus aureus Autolysins by Activation of WalRK Phosphorylation. Microorganisms 2020; 8:microorganisms8060870. [PMID: 32526915 PMCID: PMC7355866 DOI: 10.3390/microorganisms8060870] [Citation(s) in RCA: 25] [Impact Index Per Article: 5.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/12/2020] [Revised: 06/05/2020] [Accepted: 06/07/2020] [Indexed: 11/16/2022] Open
Abstract
Staphylococcus aureus is a facultative pathogen that can encode numerous antibiotic resistance and immune evasion genes and can cause severe infections. Reduced susceptibility to last resort antibiotics such as vancomycin and daptomycin is often associated with mutations in walRK, an essential two-component regulatory system (TCS). This study focuses on the WalK accessory membrane proteins YycH and YycI and their influence on WalRK phosphorylation. Depletion of YycH and YycI by antisense RNA caused an impaired autolysis, indicating a positive regulatory function on WalK as has been previously described. Phosphorylation assays with full-length recombinant proteins in phospholipid liposomes showed that YycH and YycI stimulate WalK activity and that both regulatory proteins are needed for full activation of the WalK kinase. This was validated in vivo through examining the phosphorylation status of WalR using Phos-tag SDS-PAGE with a yycHI deletion mutant exhibiting reduced levels of phosphorylated WalR. In the yycHI knockdown strain, muropeptide composition of the cell wall was not affected, however, the wall teichoic acid content was increased. In conclusion, a direct modulation of WalRK phosphorylation activity by the accessory proteins YycH and YycI is reported both in vitro and in vivo. Taken together, our results show that YycH and YycI are important in the direct regulation of WalRK-dependent cell wall metabolism.
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Affiliation(s)
- Mike Gajdiss
- Institute of Medical Microbiology, Immunology and Parasitology, University Hospital Bonn, 53125 Bonn, Germany; (M.G.); (J.K.); (T.F.); (A.D.); (M.H.); (E.S.)
| | - Ian R. Monk
- Department of Microbiology and Immunology, Doherty Institute for Infection and Immunity, University of Melbourne, Melbourne, VIC 3010, Australia; (I.R.M.); (T.P.S.)
| | - Ute Bertsche
- Department of Infection Biology, University of Tuebingen, 72076 Tuebingen, Germany;
| | - Janina Kienemund
- Institute of Medical Microbiology, Immunology and Parasitology, University Hospital Bonn, 53125 Bonn, Germany; (M.G.); (J.K.); (T.F.); (A.D.); (M.H.); (E.S.)
| | - Tanja Funk
- Institute of Medical Microbiology, Immunology and Parasitology, University Hospital Bonn, 53125 Bonn, Germany; (M.G.); (J.K.); (T.F.); (A.D.); (M.H.); (E.S.)
| | - Alina Dietrich
- Institute of Medical Microbiology, Immunology and Parasitology, University Hospital Bonn, 53125 Bonn, Germany; (M.G.); (J.K.); (T.F.); (A.D.); (M.H.); (E.S.)
| | - Michael Hort
- Institute of Medical Microbiology, Immunology and Parasitology, University Hospital Bonn, 53125 Bonn, Germany; (M.G.); (J.K.); (T.F.); (A.D.); (M.H.); (E.S.)
| | - Esther Sib
- Institute of Medical Microbiology, Immunology and Parasitology, University Hospital Bonn, 53125 Bonn, Germany; (M.G.); (J.K.); (T.F.); (A.D.); (M.H.); (E.S.)
| | - Timothy P. Stinear
- Department of Microbiology and Immunology, Doherty Institute for Infection and Immunity, University of Melbourne, Melbourne, VIC 3010, Australia; (I.R.M.); (T.P.S.)
| | - Gabriele Bierbaum
- Institute of Medical Microbiology, Immunology and Parasitology, University Hospital Bonn, 53125 Bonn, Germany; (M.G.); (J.K.); (T.F.); (A.D.); (M.H.); (E.S.)
- Correspondence:
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Multi-targeted Antisense Oligonucleotide Delivery by a Framework Nucleic Acid for Inhibiting Biofilm Formation and Virulence. NANO-MICRO LETTERS 2020; 12:74. [PMID: 34138282 PMCID: PMC7770702 DOI: 10.1007/s40820-020-0409-3] [Citation(s) in RCA: 44] [Impact Index Per Article: 8.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 12/24/2019] [Accepted: 02/12/2020] [Indexed: 02/05/2023]
Abstract
A framework nucleic acid delivery system was developed through self-assembly, which can deliver antisense oligonucleotides against multiple targets in bacterial cells. The ASOs-tFNAs (750 nM) was found to simultaneously inhibit the expression of gtfBCD, gbpB, and ftf, and significantly reduce the extracellular polysaccharide synthesis and biofilm thickness. Biofilm formation is responsible for numerous chronic infections and represents a serious health challenge. Bacteria and the extracellular polysaccharides (EPS) cause biofilms to become adherent, toxic, resistant to antibiotics, and ultimately difficult to remove. Inhibition of EPS synthesis can prevent the formation of bacterial biofilms, reduce their robustness, and promote removal. Here, we have developed a framework nucleic acid delivery system with a tetrahedral configuration. It can easily access bacterial cells and functions by delivering antisense oligonucleotides that target specific genes. We designed antisense oligonucleotide sequences with multiple targets based on conserved regions of the VicK protein-binding site. Once delivered to bacterial cells, they significantly decreased EPS synthesis and biofilm thickness. Compared to existing approaches, this system is highly efficacious because it simultaneously reduces the expression of all targeted genes (gtfBCD, gbpB, ftf). We demonstrate a novel nucleic acid-based nanomaterial with multi-targeted inhibition that has great potential for the treatment of chronic infections caused by biofilms.![]()
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A Kayvirus Distant Homolog of Staphylococcal Virulence Determinants and VISA Biomarker Is a Phage Lytic Enzyme. Viruses 2020; 12:v12030292. [PMID: 32156046 PMCID: PMC7150955 DOI: 10.3390/v12030292] [Citation(s) in RCA: 7] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/22/2019] [Revised: 03/02/2020] [Accepted: 03/04/2020] [Indexed: 01/07/2023] Open
Abstract
Staphylococcal bacteriophages of the Kayvirus genus are candidates for therapeutic applications. One of their proteins, Tgl, is slightly similar to two staphylococcal virulence factors, secreted autolysins of lytic transglycosylase motifs IsaA and SceD. We show that Tgl is a lytic enzyme secreted by the bacterial transport system and localizes to cell peripheries like IsaA and SceD. It causes lysis of E. coli cells expressing the cloned tgl gene, but could be overproduced when depleted of signal peptide. S. aureus cells producing Tgl lysed in the presence of nisin, which mimics the action of phage holin. In vitro, Tgl protein was able to destroy S. aureus cell walls. The production of Tgl decreased S. aureus tolerance to vancomycin, unlike the production of SceD, which is associated with decreased sensitivity to vancomycin. In the genomes of kayviruses, the tgl gene is located a few genes away from the lysK gene, encoding the major endolysin. While lysK is a late phage gene, tgl can be transcribed by a host RNA polymerase, like phage early genes. Taken together, our data indicate that tgl belongs to the kayvirus lytic module and encodes an additional endolysin that can act in concert with LysK in cell lysis.
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Macori G, Bellio A, Bianchi DM, Chiesa F, Gallina S, Romano A, Zuccon F, Cabrera-Rubio R, Cauquil A, Merda D, Auvray F, Decastelli L. Genome-Wide Profiling of Enterotoxigenic Staphylococcus aureus Strains Used for the Production of Naturally Contaminated Cheeses. Genes (Basel) 2019; 11:E33. [PMID: 31892220 PMCID: PMC7016664 DOI: 10.3390/genes11010033] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/09/2019] [Revised: 12/19/2019] [Accepted: 12/22/2019] [Indexed: 12/12/2022] Open
Abstract
Staphylococcus aureus is a major human pathogen and an important cause of livestock infections. More than 20 staphylococcal enterotoxins with emetic activity can be produced by specific strains responsible for staphylococcal food poisoning, one of the most common food-borne diseases. Whole genome sequencing provides a comprehensive view of the genome structure and gene content that have largely been applied in outbreak investigations and genomic comparisons. In this study, six enterotoxigenic S. aureus strains were characterised using a combination of molecular, phenotypical and computational methods. The genomes were analysed for the presence of virulence factors (VFs), where we identified 110 genes and classified them into five categories: adherence (n = 31), exoenzymes (n = 28), genes involved in host immune system evasion (n = 7); iron uptake regulatory system (n = 8); secretion machinery factors and toxins' genes (n = 36), and 39 genes coding for transcriptional regulators related to staphylococcal VFs. Each group of VFs revealed correlations among the six enterotoxigenic strains, and further analysis revealed their accessory genomic content, including mobile genetic elements. The plasmids pLUH02 and pSK67 were detected in the strain ProNaCC1 and ProNaCC7, respectively, carrying out the genes sed, ser, and selj. The genes carried out by prophages were detected in the strain ProNaCC2 (see), ProNaCC4, and ProNaCC7 (both positive for sea). The strain ProNaCC5 resulted positive for the genes seg, sei, sem, sen, seo grouped in an exotoxin gene cluster, and the strain ProNaCC6 resulted positive for seh, a transposon-associated gene. The six strains were used for the production of naturally contaminated cheeses which were tested with the European Screening Method for staphylococcal enterotoxins. The results obtained from the analysis of toxins produced in cheese, combined with the genomic features represent a portrait of the strains that can be used for the production of staphylococcal enterotoxin-positive cheese as reference material.
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Affiliation(s)
- Guerrino Macori
- National Reference Laboratory for Coagulase-Positive Staphylococci including Staphylococcus aureus, Istituto Zooprofilattico Sperimentale del Piemonte, Liguria e Valle d’Aosta, Via Bologna 148, 10154 Torino, Italy; (A.B.); (D.M.B.); (S.G.); (A.R.); (F.Z.); (L.D.)
| | - Alberto Bellio
- National Reference Laboratory for Coagulase-Positive Staphylococci including Staphylococcus aureus, Istituto Zooprofilattico Sperimentale del Piemonte, Liguria e Valle d’Aosta, Via Bologna 148, 10154 Torino, Italy; (A.B.); (D.M.B.); (S.G.); (A.R.); (F.Z.); (L.D.)
| | - Daniela Manila Bianchi
- National Reference Laboratory for Coagulase-Positive Staphylococci including Staphylococcus aureus, Istituto Zooprofilattico Sperimentale del Piemonte, Liguria e Valle d’Aosta, Via Bologna 148, 10154 Torino, Italy; (A.B.); (D.M.B.); (S.G.); (A.R.); (F.Z.); (L.D.)
| | - Francesco Chiesa
- Dipartimento di Scienze Veterinarie, Università di Torino, 10095 Grugliasco, Italy;
| | - Silvia Gallina
- National Reference Laboratory for Coagulase-Positive Staphylococci including Staphylococcus aureus, Istituto Zooprofilattico Sperimentale del Piemonte, Liguria e Valle d’Aosta, Via Bologna 148, 10154 Torino, Italy; (A.B.); (D.M.B.); (S.G.); (A.R.); (F.Z.); (L.D.)
| | - Angelo Romano
- National Reference Laboratory for Coagulase-Positive Staphylococci including Staphylococcus aureus, Istituto Zooprofilattico Sperimentale del Piemonte, Liguria e Valle d’Aosta, Via Bologna 148, 10154 Torino, Italy; (A.B.); (D.M.B.); (S.G.); (A.R.); (F.Z.); (L.D.)
| | - Fabio Zuccon
- National Reference Laboratory for Coagulase-Positive Staphylococci including Staphylococcus aureus, Istituto Zooprofilattico Sperimentale del Piemonte, Liguria e Valle d’Aosta, Via Bologna 148, 10154 Torino, Italy; (A.B.); (D.M.B.); (S.G.); (A.R.); (F.Z.); (L.D.)
| | - Raúl Cabrera-Rubio
- Teagasc Food Research Centre, Moorepark, Fermoy, P61 C996, Ireland-APC Microbiome Ireland, University College Cork, T12YT20 Cork, Ireland;
| | - Alexandra Cauquil
- European Laboratory for Coagulase-Positive Staphylococci including Staphylococcus aureus, Laboratory for Food Safety, ANSES, Université Paris-Est, F-94700 Maisons-Alfort, France; (A.C.); (D.M.); (F.A.)
| | - Déborah Merda
- European Laboratory for Coagulase-Positive Staphylococci including Staphylococcus aureus, Laboratory for Food Safety, ANSES, Université Paris-Est, F-94700 Maisons-Alfort, France; (A.C.); (D.M.); (F.A.)
| | - Fréderic Auvray
- European Laboratory for Coagulase-Positive Staphylococci including Staphylococcus aureus, Laboratory for Food Safety, ANSES, Université Paris-Est, F-94700 Maisons-Alfort, France; (A.C.); (D.M.); (F.A.)
| | - Lucia Decastelli
- National Reference Laboratory for Coagulase-Positive Staphylococci including Staphylococcus aureus, Istituto Zooprofilattico Sperimentale del Piemonte, Liguria e Valle d’Aosta, Via Bologna 148, 10154 Torino, Italy; (A.B.); (D.M.B.); (S.G.); (A.R.); (F.Z.); (L.D.)
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Dobihal GS, Brunet YR, Flores-Kim J, Rudner DZ. Homeostatic control of cell wall hydrolysis by the WalRK two-component signaling pathway in Bacillus subtilis. eLife 2019; 8:52088. [PMID: 31808740 PMCID: PMC7299342 DOI: 10.7554/elife.52088] [Citation(s) in RCA: 46] [Impact Index Per Article: 7.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/21/2019] [Accepted: 12/05/2019] [Indexed: 12/31/2022] Open
Abstract
Bacterial cells are encased in a peptidoglycan (PG) exoskeleton that protects them from osmotic lysis and specifies their distinct shapes. Cell wall hydrolases are required to enlarge this covalently closed macromolecule during growth, but how these autolytic enzymes are regulated remains poorly understood. Bacillus subtilis encodes two functionally redundant D,L-endopeptidases (CwlO and LytE) that cleave peptide crosslinks to allow expansion of the PG meshwork during growth. Here, we provide evidence that the essential and broadly conserved WalR-WalK two component regulatory system continuously monitors changes in the activity of these hydrolases by sensing the cleavage products generated by these enzymes and modulating their levels and activity in response. The WalR-WalK pathway is conserved among many Gram-positive pathogens where it controls transcription of distinct sets of PG hydrolases. Cell wall remodeling in these bacteria may be subject to homeostatic control mechanisms similar to the one reported here.
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Affiliation(s)
| | - Yannick R Brunet
- Department of Microbiology, Harvard Medical School, Boston, United States
| | - Josué Flores-Kim
- Department of Microbiology, Harvard Medical School, Boston, United States
| | - David Z Rudner
- Department of Microbiology, Harvard Medical School, Boston, United States
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45
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Wu X, Song Q, Han A. Interacting proteins of the essential two-component system YycFG in Bacillus subtilis. J Basic Microbiol 2019; 59:950-959. [PMID: 31339578 DOI: 10.1002/jobm.201800701] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/13/2018] [Revised: 06/14/2019] [Accepted: 06/24/2019] [Indexed: 11/06/2022]
Abstract
Two-component signal transduction systems (TCSs) play a major role in adaption and survival of microorganisms in a dynamic and sometimes dangerous environment. YycFG is an essential TCS for many Gram-positive bacteria, such as Bacillus subtilis, which regulates many important biological processes. However, its functional essentiality remains largely unknown. Here, we report several YycFG interacting proteins through coimmunoprecipitation (Co-IP) and mass spectrometry (MS) analyses. We engineered the B. subtilis genome by a knock-in approach to express YycG with a C-terminal Flag and YycF with an N-terminal HA tag. Immunoprecipitated fractions using anti-Flag or anti-HA agarose were subjected to MS analyses. A total of 41 YycG interacting proteins and four YycF interacting proteins were identified, most of which are involved in cellular metabolic processes, including cell wall synthesis and modification. The interactions of YycG with AsnB and FabL, as examples, were further validated in vitro. This study provided a clue that YycFG may be directly involved in regulation of bacterial central metabolic pathways.
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Affiliation(s)
- Xuanang Wu
- State Key Laboratory for Cellular Stress Biology, School of Life Sciences, Xiamen University, Xiang'an, Xiamen, China
| | - Qi Song
- State Key Laboratory for Cellular Stress Biology, School of Life Sciences, Xiamen University, Xiang'an, Xiamen, China
| | - Aidong Han
- State Key Laboratory for Cellular Stress Biology, School of Life Sciences, Xiamen University, Xiang'an, Xiamen, China
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46
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Monk IR, Shaikh N, Begg SL, Gajdiss M, Sharkey LKR, Lee JYH, Pidot SJ, Seemann T, Kuiper M, Winnen B, Hvorup R, Collins BM, Bierbaum G, Udagedara SR, Morey JR, Pulyani N, Howden BP, Maher MJ, McDevitt CA, King GF, Stinear TP. Zinc-binding to the cytoplasmic PAS domain regulates the essential WalK histidine kinase of Staphylococcus aureus. Nat Commun 2019; 10:3067. [PMID: 31296851 PMCID: PMC6624279 DOI: 10.1038/s41467-019-10932-4] [Citation(s) in RCA: 36] [Impact Index Per Article: 6.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/30/2018] [Accepted: 06/05/2019] [Indexed: 01/23/2023] Open
Abstract
WalKR (YycFG) is the only essential two-component regulator in the human pathogen Staphylococcus aureus. WalKR regulates peptidoglycan synthesis, but this function alone does not explain its essentiality. Here, to further understand WalKR function, we investigate a suppressor mutant that arose when WalKR activity was impaired; a histidine to tyrosine substitution (H271Y) in the cytoplasmic Per-Arnt-Sim (PASCYT) domain of the histidine kinase WalK. Introducing the WalKH271Y mutation into wild-type S. aureus activates the WalKR regulon. Structural analyses of the WalK PASCYT domain reveal a metal-binding site, in which a zinc ion (Zn2+) is tetrahedrally-coordinated by four amino acids including H271. The WalKH271Y mutation abrogates metal binding, increasing WalK kinase activity and WalR phosphorylation. Thus, Zn2+-binding negatively regulates WalKR. Promoter-reporter experiments using S. aureus confirm Zn2+ sensing by this system. Identification of a metal ligand recognized by the WalKR system broadens our understanding of this critical S. aureus regulon.
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Affiliation(s)
- Ian R Monk
- Department of Microbiology and Immunology, Doherty Institute for Infection and Immunity, University of Melbourne, Melbourne, VIC, 3000, Australia.
| | - Nausad Shaikh
- Institute for Molecular Bioscience, The University of Queensland, St Lucia, QLD, 4067, Australia
| | - Stephanie L Begg
- Department of Microbiology and Immunology, Doherty Institute for Infection and Immunity, University of Melbourne, Melbourne, VIC, 3000, Australia
| | - Mike Gajdiss
- University Clinics of Bonn, Institute of Medical Microbiology, Immunology and Parasitology, 53127, Bonn, Germany
| | - Liam K R Sharkey
- Department of Microbiology and Immunology, Doherty Institute for Infection and Immunity, University of Melbourne, Melbourne, VIC, 3000, Australia
| | - Jean Y H Lee
- Department of Microbiology and Immunology, Doherty Institute for Infection and Immunity, University of Melbourne, Melbourne, VIC, 3000, Australia
| | - Sacha J Pidot
- Department of Microbiology and Immunology, Doherty Institute for Infection and Immunity, University of Melbourne, Melbourne, VIC, 3000, Australia
| | - Torsten Seemann
- Department of Microbiology and Immunology, Doherty Institute for Infection and Immunity, University of Melbourne, Melbourne, VIC, 3000, Australia.,Melbourne Bioinformatics, University of Melbourne, Melbourne, VIC, 3000, Australia
| | | | | | - Rikki Hvorup
- Institute for Molecular Bioscience, The University of Queensland, St Lucia, QLD, 4067, Australia
| | - Brett M Collins
- Institute for Molecular Bioscience, The University of Queensland, St Lucia, QLD, 4067, Australia
| | - Gabriele Bierbaum
- University Clinics of Bonn, Institute of Medical Microbiology, Immunology and Parasitology, 53127, Bonn, Germany
| | - Saumya R Udagedara
- Department of Biochemistry and Genetics, La Trobe Institute for Molecular Science, La Trobe University, Melbourne, VIC, 3086, Australia
| | - Jacqueline R Morey
- Department of Molecular and Biomedical Sciences, School of Biological Sciences, The University of Adelaide, Adelaide, SA, 5005, Australia
| | - Neha Pulyani
- Department of Biochemistry and Genetics, La Trobe Institute for Molecular Science, La Trobe University, Melbourne, VIC, 3086, Australia
| | - Benjamin P Howden
- Department of Microbiology and Immunology, Doherty Institute for Infection and Immunity, University of Melbourne, Melbourne, VIC, 3000, Australia
| | - Megan J Maher
- Department of Biochemistry and Genetics, La Trobe Institute for Molecular Science, La Trobe University, Melbourne, VIC, 3086, Australia
| | - Christopher A McDevitt
- Department of Microbiology and Immunology, Doherty Institute for Infection and Immunity, University of Melbourne, Melbourne, VIC, 3000, Australia.,Department of Molecular and Biomedical Sciences, School of Biological Sciences, The University of Adelaide, Adelaide, SA, 5005, Australia
| | - Glenn F King
- Institute for Molecular Bioscience, The University of Queensland, St Lucia, QLD, 4067, Australia
| | - Timothy P Stinear
- Department of Microbiology and Immunology, Doherty Institute for Infection and Immunity, University of Melbourne, Melbourne, VIC, 3000, Australia.
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47
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Tan S, Ludwig KC, Müller A, Schneider T, Nodwell JR. The Lasso Peptide Siamycin-I Targets Lipid II at the Gram-Positive Cell Surface. ACS Chem Biol 2019; 14:966-974. [PMID: 31026131 DOI: 10.1021/acschembio.9b00157] [Citation(s) in RCA: 34] [Impact Index Per Article: 5.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/29/2022]
Abstract
Ribosomally synthesized post-translationally modified peptides (RiPPs) are a diverse class of biologically active molecules produced by many environmental bacteria. While thousands of these compounds have been identified, mostly through genome mining, a relatively small number has been investigated at the molecular level. One less understood class of RiPPs is the lasso peptides. These are 20-25 amino acid residue compounds bearing an N-terminal macrocyclic ring and a C-terminal tail that is threaded through the ring. We have carried out a detailed investigation on the mechanism of action of the siamycin-I lasso peptide. We demonstrate that siamycin-I interacts with lipid II, the central building block of the major cell wall component peptidoglycan, which is readily accessible on the outside of the cell. This interaction compromises cell wall biosynthesis in a manner that activates the liaI stress response. Additionally, resistance to siamycin-I can be brought about by mutations in the essential WalKR two-component system that causes thickening of the cell wall. Siamycin-I is the first lasso peptide that has been shown to inhibit cell wall biosynthesis.
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Affiliation(s)
- Stephanie Tan
- Department of Biochemistry, MaRS Discovery District, University of Toronto, 661 University Avenue, Toronto, Ontario, Canada M5G 1M1
| | - Kevin C. Ludwig
- Institute for Pharmaceutical Microbiology, University of Bonn, Meckenheimer Allee 168, 53115 Bonn, Germany
| | - Anna Müller
- Institute for Pharmaceutical Microbiology, University of Bonn, Meckenheimer Allee 168, 53115 Bonn, Germany
- German Centre for Infection Research (DZIF), partner site Bonn-Cologne, Bonn, Germany
| | - Tanja Schneider
- Institute for Pharmaceutical Microbiology, University of Bonn, Meckenheimer Allee 168, 53115 Bonn, Germany
- German Centre for Infection Research (DZIF), partner site Bonn-Cologne, Bonn, Germany
| | - Justin R. Nodwell
- Department of Biochemistry, MaRS Discovery District, University of Toronto, 661 University Avenue, Toronto, Ontario, Canada M5G 1M1
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48
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Maudsdotter L, Ushijima Y, Morikawa K. Fitness of Spontaneous Rifampicin-Resistant Staphylococcus aureus Isolates in a Biofilm Environment. Front Microbiol 2019; 10:988. [PMID: 31134027 PMCID: PMC6514104 DOI: 10.3389/fmicb.2019.00988] [Citation(s) in RCA: 7] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/11/2019] [Accepted: 04/18/2019] [Indexed: 11/25/2022] Open
Abstract
Biofilms of S. aureus accumulate cells resistant to the antibiotic rifampicin. We show here that the accumulation of rifampicin resistant mutants (RifR) in biofilms is not equable but rather is a local event, suggesting that the growth of a few locally emerged mutants is responsible for this. Competition assays demonstrated that, compared to wild-type bacteria, the isolated RifR mutants have a growth advantage in biofilms, but not in planktonic culture. To gain insight into the mechanism of the growth advantage, we tested the involvement of the two-component systems (TCS) that sense and respond to environmental changes. We found that a deletion of SrrAB or NreBC has a drastic effect on the growth advantage of RifR mutants, suggesting the importance of oxygen/respiration responses. All six of the RifR isolates tested showed increased resistance to at least one of the common stresses found in the biofilm environment (i.e., oxidative, nitric acid, and organic acid stress). The RifR mutants also had a growth advantage in a biofilm flow model, which highlights the physiological relevance of our findings.
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Affiliation(s)
- Lisa Maudsdotter
- Department of Biomedical Science, University of Tsukuba, Tsukuba, Japan
| | - Yuri Ushijima
- Department of Biomedical Science, University of Tsukuba, Tsukuba, Japan
| | - Kazuya Morikawa
- Department of Biomedical Science, University of Tsukuba, Tsukuba, Japan
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49
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Wu S, Liu Y, Lei L, Zhang H. Antisense yycG Regulation of Antibiotic Sensitivity of Methicillin-Resistant Staphylococcus aureus in Chronic Osteomyelitis. Surg Infect (Larchmt) 2019; 20:472-479. [PMID: 31038392 DOI: 10.1089/sur.2019.016] [Citation(s) in RCA: 12] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/05/2023] Open
Abstract
Background: Methicillin-resistant Staphylococcus aureus (MRSA) is an urgent medical problem in osteomyelitis. The YycFG two-component regulatory system (TCS) allows bacteria to adapt rapidly to physical, chemical, and biological stresses. The recombinant plasmid shuttle vector was used to overexpress an antisense RNA (asRNA) to inhibit target gene expression by sequence-specific double-stranded RNA complex degradation. In the current study, antisense yycG RNA (ASyycG)-overexpression MRSA clinical isolates were constructed. Methods: Bacterial growth was monitored, and biofilm biomass was determined by crystal violet microtiter assay. Quantitative reverse transcription polymerase chain reaction analysis was used to identify expression of yycF/G/H and icaA/D in MRSA and ASyycG strains. The expression of YycG protein was quantified by Western blot assays. The antibiotic resistance of ASyycG strains was compared with that of the MRSA strains. Results: The ASyycG strains showed a decrease in growth rate compared with the MRSA strains. Of note, overexpression of ASyycG led to a reduction in biofilm formation and adhesion force. ASyycG strains had decreased expressions of the yycF/G/H and icaA/D. Furthermore, Western blot data showed that expression of the YycG protein decreased by 40% in ASyycG strains compared with MRSA strains. In addition, the effect of yycG asRNA improved the susceptibility of ASyycG strains to cefoxitin. Conclusions: The ASyycG strains inhibited biofilm organization and increased antibiotic sensitivity, which may be attributed to altered intracellular polysaccharide construction.
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Affiliation(s)
- Shizhou Wu
- 1Department of Orthopedics, West China Hospital, West China Medical School, Sichuan University, Chengdu, China.,2State Key Laboratory of Oral Diseases, West China Hospital of Stomatology, Sichuan University, Chengdu, Sichuan, China
| | - Yunjie Liu
- 3West China School of Public Health, Sichuan University, Chengdu, Sichuan, China
| | - Lei Lei
- 2State Key Laboratory of Oral Diseases, West China Hospital of Stomatology, Sichuan University, Chengdu, Sichuan, China
| | - Hui Zhang
- 1Department of Orthopedics, West China Hospital, West China Medical School, Sichuan University, Chengdu, China
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50
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Peng H, Rao Y, Yuan W, Zheng Y, Shang W, Hu Z, Yang Y, Tan L, Xiong K, Li S, Zhu J, Hu X, Hu Q, Rao X. Reconstruction of the Vancomycin-Susceptible Staphylococcus aureus Phenotype From a Vancomycin-Intermediate S. aureus XN108. Front Microbiol 2018; 9:2955. [PMID: 30546356 PMCID: PMC6279853 DOI: 10.3389/fmicb.2018.02955] [Citation(s) in RCA: 8] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/15/2018] [Accepted: 11/16/2018] [Indexed: 11/17/2022] Open
Abstract
The emergence of vancomycin-intermediate Staphylococcus aureus (VISA) has raised healthcare concerns worldwide. VISA is often associated with multiple genetic changes. However, the relative contributions of these changes to VISA phenotypes are incompletely defined. We have characterized VISA XN108 with vancomycin MIC of 12 μg/ml. Genome comparison revealed that WalK(S221P), GraS(T136I), and RpoB(H481N) mutations possibly contributed to the VISA phenotype of XN108. In this study, the above mutations were stepwise cured, and the phenotypes between XN108 and its derivates were compared. We constructed four isogenic mutant strains, XN108-WalK(P221S) (termed as K65), XN108-GraS(I136T) (termed as S65), XN108-RpoB(N481H) (termed as B65), and XN108-WalK(P221S)/GraS(I136T) (termed as KS65), using the allelic replacement experiments with the native alleles derived from a vancomycin-susceptible S. aureus isolate DP65. Antimicrobial susceptibility test revealed K65 and S65 exhibited decreased vancomycin resistance, whereas B65 revealed negligibly differed when compared with the wild-type XN108. Sequentially introducing WalK(P221S) and GraS(I136T) completely converted XN108 into a VSSA phenotype. Transmission electronic microscopy and autolysis determination demonstrated that cell wall thickening and decreasing autolysis were associated with the change of vancomycin resistance levels. Compared with XN108, K65 exhibited 577 differentially expressed genes (DEGs), whereas KS65 presented 555 DEGs. Of those DEGs, 390 were common in K65 and KS65, including those upregulated genes responsible for citrate cycle and bacterial autolysis, and the downregulated genes involved in peptidoglycan biosynthesis and teichoic acid modification. In conclusion, a VSSA phenotype could be completely reconstituted from a VISA strain XN108. WalK(S221P) and GraS(T136I) mutations may work synergistically in conferring vancomycin resistance in XN108.
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Affiliation(s)
- Huagang Peng
- Department of Microbiology, College of Basic Medical Sciences, Key Laboratory of Microbial Engineering Under the Educational Committee in Chongqing, Army Medical University (Third Military Medical University), Chongqing, China
| | - Yifan Rao
- Institute of Modern Biopharmaceuticals, School of Life Sciences, Southwest University, Chongqing, China
| | - Wenchang Yuan
- Department of Clinical Laboratory, The Fifth Affiliated Hospital of Guangzhou Medical University, Guangzhou, China
| | - Ying Zheng
- Department of Microbiology, College of Basic Medical Sciences, Key Laboratory of Microbial Engineering Under the Educational Committee in Chongqing, Army Medical University (Third Military Medical University), Chongqing, China
| | - Weilong Shang
- Department of Microbiology, College of Basic Medical Sciences, Key Laboratory of Microbial Engineering Under the Educational Committee in Chongqing, Army Medical University (Third Military Medical University), Chongqing, China
| | - Zhen Hu
- Department of Microbiology, College of Basic Medical Sciences, Key Laboratory of Microbial Engineering Under the Educational Committee in Chongqing, Army Medical University (Third Military Medical University), Chongqing, China
| | - Yi Yang
- Department of Microbiology, College of Basic Medical Sciences, Key Laboratory of Microbial Engineering Under the Educational Committee in Chongqing, Army Medical University (Third Military Medical University), Chongqing, China
| | - Li Tan
- Department of Microbiology, College of Basic Medical Sciences, Key Laboratory of Microbial Engineering Under the Educational Committee in Chongqing, Army Medical University (Third Military Medical University), Chongqing, China
| | - Kun Xiong
- Department of Microbiology, College of Basic Medical Sciences, Key Laboratory of Microbial Engineering Under the Educational Committee in Chongqing, Army Medical University (Third Military Medical University), Chongqing, China
| | - Shu Li
- Department of Microbiology, College of Basic Medical Sciences, Key Laboratory of Microbial Engineering Under the Educational Committee in Chongqing, Army Medical University (Third Military Medical University), Chongqing, China
| | - Junmin Zhu
- Department of Microbiology, College of Basic Medical Sciences, Key Laboratory of Microbial Engineering Under the Educational Committee in Chongqing, Army Medical University (Third Military Medical University), Chongqing, China
| | - Xiaomei Hu
- Department of Microbiology, College of Basic Medical Sciences, Key Laboratory of Microbial Engineering Under the Educational Committee in Chongqing, Army Medical University (Third Military Medical University), Chongqing, China
| | - Qiwen Hu
- Department of Microbiology, College of Basic Medical Sciences, Key Laboratory of Microbial Engineering Under the Educational Committee in Chongqing, Army Medical University (Third Military Medical University), Chongqing, China
| | - Xiancai Rao
- Department of Microbiology, College of Basic Medical Sciences, Key Laboratory of Microbial Engineering Under the Educational Committee in Chongqing, Army Medical University (Third Military Medical University), Chongqing, China
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