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Geng J, Long J, Hu Q, Liu M, Ge A, Du Y, Zhang T, Jin Y, Yang H, Chen S, Duan G. Current status of cyclopropane fatty acids on bacterial cell membranes characteristics and physiological functions. Microb Pathog 2025; 200:107295. [PMID: 39805345 DOI: 10.1016/j.micpath.2025.107295] [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: 05/12/2024] [Revised: 01/04/2025] [Accepted: 01/10/2025] [Indexed: 01/16/2025]
Abstract
Wide-ranging sophisticated physiological activities of cell membranes are associated with changes in fatty acid structure and composition. The cfa gene is a core regulator of cell membrane fatty acid cyclopropanation reaction. Its encoded cyclopropane fatty acid synthase (CFA synthase) catalyzes the binding of unsaturated fatty acid (UFA) to methylene groups, which undergoes cyclopropanation modification to produce cyclopropane fatty acids (CFAs). Compelling evidence suggests a large role for the cfa gene and CFAs in bacterial adaptive responses. This review provides an overview of the relationship of CFAs with bacterial cell membrane properties and physiological functions, including the roles of cell membrane fluidity, stability, and permeability to protons, bacterial growth, acid resistance, and especially in bacterial antibiotic resistance and pathogenicity. The dysregulation and inhibition of the cfa gene may serve as potential therapeutic targets against bacterial drug resistance and pathogenicity. Therefore, elucidating the biological function of CFAs during the stationary growth phase therefore provides invaluable insights into the bacterial pathogenesis and the development of novel antimicrobial agents.
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Affiliation(s)
- Juan Geng
- Department of Epidemiology and Health Statistics, College of Public Health, Zhengzhou University, Zhengzhou, China
| | - Jinzhao Long
- Department of Epidemiology and Health Statistics, College of Public Health, Zhengzhou University, Zhengzhou, China
| | - Quanman Hu
- Department of Epidemiology and Health Statistics, College of Public Health, Zhengzhou University, Zhengzhou, China
| | - Mengyue Liu
- Department of Epidemiology and Health Statistics, College of Public Health, Zhengzhou University, Zhengzhou, China
| | - Anmin Ge
- Department of Epidemiology and Health Statistics, College of Public Health, Zhengzhou University, Zhengzhou, China; Penglai Center for Disease Control and Prevention, Yantai, China
| | - Yazhe Du
- Department of Epidemiology and Health Statistics, College of Public Health, Zhengzhou University, Zhengzhou, China
| | - Teng Zhang
- Department of Epidemiology and Health Statistics, College of Public Health, Zhengzhou University, Zhengzhou, China
| | - Yuefei Jin
- Department of Epidemiology and Health Statistics, College of Public Health, Zhengzhou University, Zhengzhou, China
| | - Haiyan Yang
- Department of Epidemiology and Health Statistics, College of Public Health, Zhengzhou University, Zhengzhou, China
| | - Shuaiyin Chen
- Department of Epidemiology and Health Statistics, College of Public Health, Zhengzhou University, Zhengzhou, China.
| | - Guangcai Duan
- Department of Epidemiology and Health Statistics, College of Public Health, Zhengzhou University, Zhengzhou, China
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Ku RH, Lu HF, Li LH, Yeh TY, Lin YT, Yang TC. Roles of the rpoEc-chrR-chrA operon in superoxide tolerance and β-lactam susceptibility of Stenotrophomonas maltophilia. Front Cell Infect Microbiol 2025; 15:1492008. [PMID: 39967789 PMCID: PMC11832516 DOI: 10.3389/fcimb.2025.1492008] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/06/2024] [Accepted: 01/17/2025] [Indexed: 02/20/2025] Open
Abstract
Introduction The rpoE-chrR pair is a regulatory system used by photosynthetic microorganisms to overcome singlet oxygen stress. rpoE and chrR encode the sigma factor σE and anti-sigma factor ChrR, respectively. Stenotrophomonas maltophilia, an opportunistic pathogen, is a multidrug-resistant gram-negative bacterium. Although it is not a photosynthetic microorganism, a rpoE-chrR homolog (smlt2377-smlt2378) was found in the S. maltophilia genome. In this study, we aimed to assess the significance of σEc-ChrR pair in oxidative stress alleviation and antibiotic susceptibility of S. maltophilia KJ. Methods Reverse transcription-polymerase chain reaction was used to validate the presence of operon. The contribution of rpoEc-chrR-chrA operon to oxidative stress alleviation and antibiotic susceptibility was evaluated using mutant constructs and stress-tolerance assays. RNA-seq transcriptome assay of wild-type KJ, KJΔChrR (chrR mutant), and KJΔChrRΔRpoEc (chrR/rpoEc double mutant) was performed to reveal the σEc regulon. Results The rpoEc-chrR pair and downstream chrA formed an operon. Inactivation of chrR upregulated the expression of rpoEc-chrR-chrA operon in an σEc- and ChrA-dependent manner. σEc activation contributed to superoxide tolerance and increased β-lactam susceptibility but did not affect the tolerance to singlet oxygen and hydrogen peroxide. Transcriptome analysis revealed that expression of the nine-gene cluster, smlt2375-smlt2367, was significantly upregulated in KJΔChrR and reverted to the wild-type level in KJΔChrRΔRpoEc. smlt2375-smlt2367 cluster was located upstream of the rpoEc-chrR-chrA operon and divergently transcribed, seeming to be involved in membrane lipid modification. Deletion of smlt2375-smlt2367 cluster from the chromosome of KJΔChrR reverted the superoxide tolerance and β-lactam susceptibility to the wild-type level. Discussion The rpoEc-chrR pair of S. maltophilia was involved in superoxide tolerance and β-lactam susceptibility. Notably, a novel regulatory circuit involving rpoEc-chrR-chrA operon and smlt2375-smlt2367 cluster was revealed.
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Affiliation(s)
- Ren-Hsuan Ku
- Department of Biotechnology and Laboratory Science in Medicine, National Yang Ming Chiao Tung University, Taipei, Taiwan
| | - Hsu-Feng Lu
- Department of Medical Laboratory Science and Biotechnology, Asia University, Taichung, Taiwan
| | - Li-Hua Li
- Department of Pathology and Laboratory Medicine, Taipei Veterans General Hospital, Taipei, Taiwan
- School of Medical Laboratory Science and Biotechnology, College of Medical Science and Technology, Taipei Medical University, Taipei, Taiwan
| | - Ting-Yu Yeh
- Department of Biotechnology and Laboratory Science in Medicine, National Yang Ming Chiao Tung University, Taipei, Taiwan
| | - Yi-Tsung Lin
- Division of Infectious Diseases, Department of Medicine, Taipei Veterans General Hospital, Taipei, Taiwan
- School of Medicine, National Yang Ming Chiao Tung University, Taipei, Taiwan
| | - Tsuey-Ching Yang
- Department of Biotechnology and Laboratory Science in Medicine, National Yang Ming Chiao Tung University, Taipei, Taiwan
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Bang S, Shin YH, Park SM, Deng L, Williamson RT, Graham DB, Xavier RJ, Clardy J. Unusual Phospholipids from Morganella morganii Linked to Depression. J Am Chem Soc 2025; 147:2998-3002. [PMID: 39818770 PMCID: PMC11783507 DOI: 10.1021/jacs.4c15158] [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] [Received: 10/28/2024] [Revised: 01/13/2025] [Accepted: 01/14/2025] [Indexed: 01/19/2025]
Abstract
A multifactorial association study detected a probable causal connection between the prevalence of Morganella morganii in the gut microbiome and the incidence of major depressive disorder (MDD) in the human host. A bioassay-guided fractionation approach identified bacterially produced metabolites that induced pro-inflammatory immune responses. The metabolites are unusual phospholipids that resemble conventional cardiolipins, in which diethanolamine (DEA) replaces the central glycerol. These molecular chimeras of endogenous metabolites from phospholipid biosynthetic pathways and the industrially produced micropollutant DEA activate TLR2/TLR1 receptors and induce the production of pro-inflammatory cytokines, especially IL-6. Their activity in conventional immunomodulatory assays largely parallels that of immunogenic cardiolipins with conventional structures. The molecular mechanism connecting these chimeric cardiolipins to MDD is supported by other studies and has implications for conditions other than MDD.
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Affiliation(s)
- Sunghee Bang
- Department
of Biological Chemistry and Molecular Pharmacology, Harvard Medical School and Blavatnik Institute, Boston, Massachusetts 02115, United States
| | - Yern-Hyerk Shin
- Department
of Biological Chemistry and Molecular Pharmacology, Harvard Medical School and Blavatnik Institute, Boston, Massachusetts 02115, United States
| | - Sung-Moo Park
- Broad
Institute of MIT and Harvard, Cambridge, Massachusetts 02142, United States
- Department
of Molecular Biology and Center for the Study of Inflammatory Bowel
Disease, Massachusetts General Hospital, Boston, Massachusetts 02114, United States
| | - Lei Deng
- Broad
Institute of MIT and Harvard, Cambridge, Massachusetts 02142, United States
- Department
of Molecular Biology and Center for the Study of Inflammatory Bowel
Disease, Massachusetts General Hospital, Boston, Massachusetts 02114, United States
| | - R. Thomas Williamson
- Department
of Chemistry and Biochemistry, University
of North Carolina Wilmington, Wilmington, North Carolina 28409, United States
| | - Daniel B. Graham
- Broad
Institute of MIT and Harvard, Cambridge, Massachusetts 02142, United States
- Department
of Molecular Biology and Center for the Study of Inflammatory Bowel
Disease, Massachusetts General Hospital, Boston, Massachusetts 02114, United States
| | - Ramnik J. Xavier
- Broad
Institute of MIT and Harvard, Cambridge, Massachusetts 02142, United States
- Department
of Molecular Biology and Center for the Study of Inflammatory Bowel
Disease, Massachusetts General Hospital, Boston, Massachusetts 02114, United States
| | - Jon Clardy
- Department
of Biological Chemistry and Molecular Pharmacology, Harvard Medical School and Blavatnik Institute, Boston, Massachusetts 02115, United States
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Hu W, Huo X, Ma T, Li Z, Yang T, Yang H, Feng S. Insights into the role of cyclopropane fatty acid synthase (CfaS) from extreme acidophile in bacterial defense against environmental acid stress. Extremophiles 2024; 29:1. [PMID: 39549088 DOI: 10.1007/s00792-024-01368-w] [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: 05/15/2024] [Accepted: 10/26/2024] [Indexed: 11/18/2024]
Abstract
The cell membrane remodeling mediated by cyclopropane fatty acid synthase (CfaS) plays a crucial role in microbial physiological processes resisting various environmental stressors, including acid. Herein, we found a relatively high proportion (24.8%-28.3%) of cyclopropane fatty acid (CFA) Cy-19:0 in the cell membrane of a newly isolated extreme acidophile, Acidithiobacillus caldus CCTCC AB 2019256, under extreme acid stress. Overexpression of the CfaS encoding gene cfaS2 in Escherichia coli conferred enhanced acid resistance. GC-MS analysis revealed a 3.52-fold increase in the relative proportion of Cy-19:0 in the cell membrane of the overexpression strain compared to the control. Correspondingly, membrane fluidity, permeability and cell surface hydrophobicity were reduced to varying degrees. Additionally, HPLC analysis indicated that the overexpression strain had 1.54-, 1.42-, 1.85-, 1.20- and 1.05-fold higher levels of intracellular glutamic acid, arginine, aspartic acid, methionine and alanine, respectively, compared to the control. Overall, our findings shed light on the role of CfaS derived from extreme acidophile in bacterial defense against environmental acid stress, potentially facilitating its application in the design and development of industrial microbial chassis cells for organic acid production.
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Affiliation(s)
- Wenbo Hu
- School of Life Sciences, Henan Institute of Science and Technology, Xinxiang, Henan, China
| | - Xingyu Huo
- The Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, 1800 Lihu Road, Wuxi, People's Republic of China
| | - Tengfei Ma
- School of Life Sciences, Henan Institute of Science and Technology, Xinxiang, Henan, China
| | - Zhigang Li
- School of Life Sciences, Henan Institute of Science and Technology, Xinxiang, Henan, China
| | - Tianyou Yang
- School of Life Sciences, Henan Institute of Science and Technology, Xinxiang, Henan, China
| | - Hailin Yang
- The Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, 1800 Lihu Road, Wuxi, People's Republic of China
- Key Laboratory of Carbohydrate Chemistry and Biotechnology, Ministry of Education, Jiangnan University, 1800 Lihu Road, Wuxi, People's Republic of China
| | - Shoushuai Feng
- The Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, 1800 Lihu Road, Wuxi, People's Republic of China.
- Key Laboratory of Carbohydrate Chemistry and Biotechnology, Ministry of Education, Jiangnan University, 1800 Lihu Road, Wuxi, People's Republic of China.
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Krzyżek P, Migdał P, Tusiewicz K, Zawadzki M, Szpot P. Subinhibitory concentrations of antibiotics affect development and parameters of Helicobacter pylori biofilm. Front Pharmacol 2024; 15:1477317. [PMID: 39469629 PMCID: PMC11513322 DOI: 10.3389/fphar.2024.1477317] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/07/2024] [Accepted: 09/27/2024] [Indexed: 10/30/2024] Open
Abstract
Introduction Helicobacter pylori causes chronic gastric diseases in nearly 50% of people around the world. It is suggested that biofilm formation has a pronounced effect on the dynamic resistance spread and recurrence of these infections. Methods To mimic the scenario of therapeutic ineffectiveness, we investigated the impact of sub-minimal inhibitory concentrations (sub-MICs) of antibiotics on the development and parameters of biofilms produced by clinical H. pylori strains. Results We observed that constant exposure of planktonic forms to metronidazole or levofloxacin stimulated the speed of autoaggregation and the amount of extracellular matrix, resulting in increased dimensions of the developed biofilms. Contrary to this, continuous exposure to clarithromycin negatively affected a number of biofilm-related reactions and led to the biofilm-weakening effect. Through assessing the membrane fatty acid profiles of antibiotic-exposed cells, we confirmed that metronidazole and levofloxacin induced a biofilm-like phenotype, while clarithromycin kept bacteria in a planktonic form. Discussion Our results suggest that sub-MICs of antibiotics affect the biochemical and biophysical properties of the developing biofilm of H. pylori strains and may impact the effectiveness of antibiotic treatment.
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Affiliation(s)
- Paweł Krzyżek
- Department of Microbiology, Faculty of Medicine, Wroclaw Medical University, Wroclaw, Poland
| | - Paweł Migdał
- Department of Bees Breeding, Institute of Animal Husbandry, Wroclaw University of Environmental and Life Sciences, Wroclaw, Poland
| | - Kaja Tusiewicz
- Department of Forensic Medicine, Wroclaw Medical University, Wroclaw, Poland
| | - Marcin Zawadzki
- Department of Social Sciences and Infectious Diseases, Faculty of Medicine, Wroclaw University of Science and Technology, Wroclaw, Poland
| | - Paweł Szpot
- Department of Forensic Medicine, Wroclaw Medical University, Wroclaw, Poland
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Ezeduru V, Shao ARQ, Venegas FA, McKay G, Rich J, Nguyen D, Thibodeaux CJ. Defining the functional properties of cyclopropane fatty acid synthase from Pseudomonas aeruginosa PAO1. J Biol Chem 2024; 300:107618. [PMID: 39095026 PMCID: PMC11387697 DOI: 10.1016/j.jbc.2024.107618] [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: 06/26/2024] [Revised: 07/20/2024] [Accepted: 07/22/2024] [Indexed: 08/04/2024] Open
Abstract
Cyclopropane fatty acid synthases (CFAS) catalyze the conversion of unsaturated fatty acids to cyclopropane fatty acids (CFAs) within bacterial membranes. This modification alters the biophysical properties of membranes and has been correlated with virulence in several human pathogens. Despite the central role played by CFAS enzymes in regulating bacterial stress responses, the mechanistic properties of the CFAS enzyme family and the consequences of CFA biosynthesis remain largely uncharacterized in most bacteria. We report the first characterization of the CFAS enzyme from Pseudomonas aeruginosa (PA), an opportunistic human pathogen with complex membrane biology that is frequently associated with antimicrobial resistance and high tolerance to various external stressors. We demonstrate that CFAs are produced by a single enzyme in PA and that cfas gene expression is upregulated during the transition to stationary phase and in response to oxidative stress. Analysis of PA lipid extracts reveal a massive increase in CFA production as PA cells enter stationary phase and help define the optimal membrane composition for in vitro assays. The purified PA-CFAS enzyme forms a stable homodimer and preferentially modifies phosphatidylglycerol lipid substrates and membranes with a higher content of unsaturated acyl chains. Bioinformatic analysis across bacterial phyla shows highly divergent amino acid sequences within the lipid-binding domain of CFAS enzymes, perhaps suggesting distinct membrane-binding properties among different orthologs. This work lays an important foundation for further characterization of CFAS in P. aeruginosa and for examining the functional differences between CFAS enzymes from different bacteria.
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Affiliation(s)
- Vivian Ezeduru
- Department of Chemistry, McGill University, Montreal, Quebec, Canada
| | - Annie R Q Shao
- Department of Chemistry, McGill University, Montreal, Quebec, Canada
| | - Felipe A Venegas
- Department of Chemistry, McGill University, Montreal, Quebec, Canada
| | - Geoffrey McKay
- Research Institute of the McGill University Health Center, McGill University, Montreal, Quebec, Canada
| | - Jacquelyn Rich
- Research Institute of the McGill University Health Center, McGill University, Montreal, Quebec, Canada; Department of Microbiology and Immunology, McGill University, Montreal, Quebec, Canada
| | - Dao Nguyen
- Research Institute of the McGill University Health Center, McGill University, Montreal, Quebec, Canada; Department of Microbiology and Immunology, McGill University, Montreal, Quebec, Canada; Department of Medicine, McGill University, Montreal, Quebec, Canada
| | - Christopher J Thibodeaux
- Department of Chemistry, McGill University, Montreal, Quebec, Canada; Centre de Recherche en Biologie Structurale, McGill University, Montreal, Quebec, Canada.
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Kloula Ben Ghorbal S, Dhaya I, Ouzari IH, Chatti A. Cyclopropanation and membrane unsaturation improve antibiotic resistance of swarmer Pseudomonas and its sod mutants exposed to radiations, in vitro and in silico approch. World J Microbiol Biotechnol 2024; 40:243. [PMID: 38869625 DOI: 10.1007/s11274-024-04033-8] [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: 02/16/2024] [Accepted: 05/22/2024] [Indexed: 06/14/2024]
Abstract
It was known that UVc irradiation increases the reactive oxygen species' (ROS) levels in bacteria hence the intervention of antioxidant enzymes and causes also changes in fatty acids (FAs) composition enabling bacteria to face antibiotics. Here, we intended to elucidate an interrelationship between SOD and susceptibility to antibiotics by studying FA membrane composition of UVc-treated P. aeruginosa PAO1 and its isogenic mutants (sodM, sodB and sod MB) membrane, after treatment with antibiotics. Swarmer mutants defective in genes encoding superoxide dismutase were pre-exposed to UVc radiations and then tested by disk diffusion method for their contribution to antibiotic tolerance in comparison with the P. aeruginosa wild type (WT). Moreover, fatty acid composition of untreated and UVc-treated WT and sod mutants was examined by Gaz chromatography and correlated to antibiotic resistance. Firstly, it has been demonstrated that after UVc exposure, swarmer WT strain, sodM and sodB mutants remain resistant to polymixin B, a membrane target antibiotic, through membrane unsaturation supported by the intervention of Mn-SOD after short UVc exposure and cyclopropanation of unsaturated FAs supported by the action of Fe-SOD after longer UVc exposure. However, resistance for ciprofloxacin is correlated with increase in saturated FAs. This correlation has been confirmed by a molecular docking approach showing that biotin carboxylase, involved in the initial stage of FA biosynthesis, exhibits a high affinity for ciprofloxacin. This investigation has explored the correlation of antibiotic resistance with FA content of swarmer P.aeruginosa pre-exposed to UVc radiations, confirmed to be antibiotic target dependant.
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Affiliation(s)
- Salma Kloula Ben Ghorbal
- Laboratoire de Traitement des Eaux Usées, Centre de Recherches et Technologies des Eaux Usées, Technopole Borj Cedria, BP 273, 8020, Soliman, Tunisia.
| | - Ibtihel Dhaya
- LR18ES03- Laboratory of Neurophysiology Cellular Physiopathology and Biomolecules Valorization, University of Tunis El Manar, Tunis, Tunisia
| | - Imene-Hadda Ouzari
- Laboratoire des Microorganismes et Biomolécules Actives, Faculté des Sciences de Tunis, Physiques et Naturelles de Tunis, University Tunis El Manar, Tunis, Tunisia
| | - Abdelwaheb Chatti
- Laboratoire de Traitement des Eaux Usées, Centre de Recherches et Technologies des Eaux Usées, Technopole Borj Cedria, BP 273, 8020, Soliman, Tunisia
- Unite de Biochimie des Lipides et Interactions des Macromolécules en Biologie (03/UR/0902), Laboratoire de Biochimie et Biologie Moléculaire, Faculté des Sciences de Bizerte, Zarzouna, 7021, Bizerte, Tunisia
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McClain MS, Boeglin WE, Algood HMS, Brash AR. Fatty acids of Helicobacter pylori lipoproteins CagT and Lpp20. Microbiol Spectr 2024; 12:e0047024. [PMID: 38501821 PMCID: PMC11064636 DOI: 10.1128/spectrum.00470-24] [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: 02/24/2024] [Accepted: 03/04/2024] [Indexed: 03/20/2024] Open
Abstract
Bacterial lipoproteins are post-translationally modified by the addition of acyl chains that anchor the protein to bacterial membranes. This modification includes two ester-linked and one amide-linked acyl chain on lipoproteins from Gram-negative bacteria. Helicobacter pylori lipoproteins have important functions in pathogenesis (including delivering the CagA oncoprotein to mammalian cells) and are recognized by host innate and adaptive immune systems. The number and variety of acyl chains on lipoproteins impact the innate immune response through Toll-like receptor 2. The acyl chains added to lipoproteins are derived from membrane phospholipids. H. pylori membrane phospholipids have previously been shown to consist primarily of C14:0 and C19:0 cyclopropane-containing acyl chains. However, the acyl composition of H. pylori lipoproteins has not been determined. In this study, we characterized the acyl composition of two representative H. pylori lipoproteins, Lpp20 and CagT. Fatty acid methyl esters were prepared from both purified lipoproteins and analyzed by gas chromatography-mass spectrometry. For comparison, we also analyzed H. pylori phospholipids. Consistent with previous studies, we observed that the H. pylori phospholipids contain primarily C14:0 and C19:0 cyclopropane-containing fatty acids. In contrast, both the ester-linked and amide-linked fatty acids found in H. pylori lipoproteins were observed to be almost exclusively C16:0 and C18:0. A discrepancy between the acyl composition of membrane phospholipids and lipoproteins as reported here for H. pylori has been previously reported in other bacteria including Borrelia and Brucella. We discuss possible mechanisms.IMPORTANCEColonization of the stomach by Helicobacter pylori is an important risk factor in the development of gastric cancer, the third leading cause of cancer-related death worldwide. H. pylori persists in the stomach despite an immune response against the bacteria. Recognition of lipoproteins by TLR2 contributes to the innate immune response to H. pylori. However, the role of H. pylori lipoproteins in bacterial persistence is poorly understood. As the host response to lipoproteins depends on the acyl chain content, defining the acyl composition of H. pylori lipoproteins is an important step in characterizing how lipoproteins contribute to persistence.
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Affiliation(s)
- Mark S. McClain
- Department of Medicine, Vanderbilt University School of Medicine, Nashville, Tennessee, USA
- Vanderbilt Institute for Infection Immunology and Inflammation, Vanderbilt University Medical Center, Nashville, Tennessee, USA
| | - William E. Boeglin
- Department of Pharmacology, Vanderbilt University School of Medicine, Nashville, Tennessee, USA
| | - Holly M. Scott Algood
- Department of Medicine, Vanderbilt University School of Medicine, Nashville, Tennessee, USA
- Vanderbilt Institute for Infection Immunology and Inflammation, Vanderbilt University Medical Center, Nashville, Tennessee, USA
- Department of Pathology, Microbiology, and Immunology, Vanderbilt University School of Medicine, Nashville, Tennessee, USA
- Vanderbilt Center for Immunobiology, Vanderbilt Medical Center, Nashville, Tennessee, USA
- Veterans Affairs Tennessee Valley Healthcare System, Nashville, Tennessee, USA
| | - Alan R. Brash
- Department of Pharmacology, Vanderbilt University School of Medicine, Nashville, Tennessee, USA
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Lee TH, Charchar P, Separovic F, Reid GE, Yarovsky I, Aguilar MI. The intricate link between membrane lipid structure and composition and membrane structural properties in bacterial membranes. Chem Sci 2024; 15:3408-3427. [PMID: 38455013 PMCID: PMC10915831 DOI: 10.1039/d3sc04523d] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/27/2023] [Accepted: 01/26/2024] [Indexed: 03/09/2024] Open
Abstract
It is now evident that the cell manipulates lipid composition to regulate different processes such as membrane protein insertion, assembly and function. Moreover, changes in membrane structure and properties, lipid homeostasis during growth and differentiation with associated changes in cell size and shape, and responses to external stress have been related to drug resistance across mammalian species and a range of microorganisms. While it is well known that the biomembrane is a fluid self-assembled nanostructure, the link between the lipid components and the structural properties of the lipid bilayer are not well understood. This perspective aims to address this topic with a view to a more detailed understanding of the factors that regulate bilayer structure and flexibility. We describe a selection of recent studies that address the dynamic nature of bacterial lipid diversity and membrane properties in response to stress conditions. This emerging area has important implications for a broad range of cellular processes and may open new avenues of drug design for selective cell targeting.
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Affiliation(s)
- Tzong-Hsien Lee
- Department of Biochemistry and Molecular Biology, Monash University Clayton VIC 3800 Australia
| | - Patrick Charchar
- School of Engineering, RMIT University Melbourne Victoria 3001 Australia
| | - Frances Separovic
- School of Chemistry, Bio21 Molecular Science and Biotechnology Institute, University of Melbourne VIC 3010 Australia
| | - Gavin E Reid
- School of Chemistry, Bio21 Molecular Science and Biotechnology Institute, University of Melbourne VIC 3010 Australia
- Department of Biochemistry and Pharmacology, University of Melbourne Parkville VIC 3010 Australia
| | - Irene Yarovsky
- School of Engineering, RMIT University Melbourne Victoria 3001 Australia
| | - Marie-Isabel Aguilar
- Department of Biochemistry and Molecular Biology, Monash University Clayton VIC 3800 Australia
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Ostroumova OS, Efimova SS. Lipid-Centric Approaches in Combating Infectious Diseases: Antibacterials, Antifungals and Antivirals with Lipid-Associated Mechanisms of Action. Antibiotics (Basel) 2023; 12:1716. [PMID: 38136750 PMCID: PMC10741038 DOI: 10.3390/antibiotics12121716] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/31/2023] [Revised: 12/05/2023] [Accepted: 12/08/2023] [Indexed: 12/24/2023] Open
Abstract
One of the global challenges of the 21st century is the increase in mortality from infectious diseases against the backdrop of the spread of antibiotic-resistant pathogenic microorganisms. In this regard, it is worth targeting antibacterials towards the membranes of pathogens that are quite conservative and not amenable to elimination. This review is an attempt to critically analyze the possibilities of targeting antimicrobial agents towards enzymes involved in pathogen lipid biosynthesis or towards bacterial, fungal, and viral lipid membranes, to increase the permeability via pore formation and to modulate the membranes' properties in a manner that makes them incompatible with the pathogen's life cycle. This review discusses the advantages and disadvantages of each approach in the search for highly effective but nontoxic antimicrobial agents. Examples of compounds with a proven molecular mechanism of action are presented, and the types of the most promising pharmacophores for further research and the improvement of the characteristics of antibiotics are discussed. The strategies that pathogens use for survival in terms of modulating the lipid composition and physical properties of the membrane, achieving a balance between resistance to antibiotics and the ability to facilitate all necessary transport and signaling processes, are also considered.
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Affiliation(s)
- Olga S. Ostroumova
- Laboratory of Membrane and Ion Channel Modeling, Institute of Cytology, Russian Academy of Sciences, Tikhoretsky Ave. 4, St. Petersburg 194064, Russia;
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Pan Z, Guo J, Zhong Y, Fan L, Su Y. Gentamicin resistance to Escherichia coli related to fatty acid metabolism based on transcriptome analysis. Can J Microbiol 2023; 69:328-338. [PMID: 37224563 DOI: 10.1139/cjm-2023-0036] [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: 05/26/2023]
Abstract
Antibiotic overuse and misuse have promoted the emergence and spread of antibiotic-resistant bacteria. Increasing bacterial resistance to antibiotics is a major healthcare problem, necessitating elucidation of antibiotic resistance mechanisms. In this study, we explored the mechanism of gentamicin resistance by comparing the transcriptomes of antibiotic-sensitive and -resistant Escherichia coli. A total of 410 differentially expressed genes were identified, of which 233 (56.83%) were up-regulated and 177 (43.17%) were down-regulated in the resistant strain compared with the sensitive strain. Gene Ontology (GO) analysis classifies differential gene expression into three main categories: biological processes, cellular components, and molecular functions. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis indicated that the up-regulated genes were enriched in eight metabolic pathways, including fatty acid metabolism, which suggests that fatty acid metabolism may be involved in the development of gentamicin resistance in E. coli. This was demonstrated by measuring the acetyl-CoA carboxylase activity, plays a fundamental role in fatty acid metabolism, was increased in gentamicin-resistant E. coli. Treatment of fatty acid synthesis inhibitor, triclosan, promoted gentamicin-mediated killing efficacy to antibiotic-resistant bacteria. We also found that exogenous addition of oleic acid, which involved in fatty acid metabolism, reduced E. coli sensitivity to gentamicin. Overall, our results provide insight into the molecular mechanism of gentamicin resistance development in E. coli.
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Affiliation(s)
- Zhiyu Pan
- Department of Cell Biology & Institute of Biomedicine, National Engineering Research Center of Genetic Medicine, MOE Key Laboratory of Tumor Molecular Biology, Guangdong Provincial Key Laboratory of Bioengineering Medicine, College of Life Science and Technology, Jinan University, Guangzhou, 510632, China
| | - Juan Guo
- Department of Cell Biology & Institute of Biomedicine, National Engineering Research Center of Genetic Medicine, MOE Key Laboratory of Tumor Molecular Biology, Guangdong Provincial Key Laboratory of Bioengineering Medicine, College of Life Science and Technology, Jinan University, Guangzhou, 510632, China
| | - Yilin Zhong
- Department of Cell Biology & Institute of Biomedicine, National Engineering Research Center of Genetic Medicine, MOE Key Laboratory of Tumor Molecular Biology, Guangdong Provincial Key Laboratory of Bioengineering Medicine, College of Life Science and Technology, Jinan University, Guangzhou, 510632, China
| | - Lvyuan Fan
- Department of Cell Biology & Institute of Biomedicine, National Engineering Research Center of Genetic Medicine, MOE Key Laboratory of Tumor Molecular Biology, Guangdong Provincial Key Laboratory of Bioengineering Medicine, College of Life Science and Technology, Jinan University, Guangzhou, 510632, China
| | - Yubin Su
- Department of Cell Biology & Institute of Biomedicine, National Engineering Research Center of Genetic Medicine, MOE Key Laboratory of Tumor Molecular Biology, Guangdong Provincial Key Laboratory of Bioengineering Medicine, College of Life Science and Technology, Jinan University, Guangzhou, 510632, China
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12
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Zhu X, Guo Z, Wang N, Liu J, Zuo Y, Li K, Song C, Song Y, Gong C, Xu X, Yuan F, Zhang L. Environmental stress stimulates microbial activities as indicated by cyclopropane fatty acid enhancement. THE SCIENCE OF THE TOTAL ENVIRONMENT 2023; 873:162338. [PMID: 36813189 DOI: 10.1016/j.scitotenv.2023.162338] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 11/01/2022] [Revised: 01/24/2023] [Accepted: 02/15/2023] [Indexed: 06/18/2023]
Abstract
Soil microbial responses to environmental stress remain a critical question in microbial ecology. The content of cyclopropane fatty acid (CFA) in cytomembrane has been widely used to evaluate environmental stress on microorganisms. Here, we used CFA to investigate the ecological suitability of microbial communities and found a stimulating impact of CFA on microbial activities during wetland reclamation in Sanjiang Plain, Northeastern China. The seasonality of environmental stress resulted in the fluctuation of CFA content in the soil, which suppressed microbial activities due to nutrient loss upon wetland reclamation. After land conversion, the aggravation of temperature stress to microbes increased the CFA content by 5 % (autumn) to 163 % (winter), which led to the suppression of microbial activities by 7 %-47 %. By contrast, the warmer soil temperature and permeability decreased the CFA content by 3 % to 41 % and consequently aggravated the microbial reduction by 15 %-72 % in spring and summer. Complex microbial communities of 1300 CFA-produced species were identified using a sequencing approach, suggesting that soil nutrients dominated the differentiation in these microbial community structures. Further analysis with structural equation modeling highlighted the important function of CFA content to environmental stress and the stimulating influence of CFA induced by environmental stress on microbial activities. Our study shows the biological mechanisms of seasonal CFA content for microbial adaption to environmental stress under wetland reclamation. It advances our knowledge of microbial physiology affecting soil element cycling caused by anthropogenic activities.
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Affiliation(s)
- Xinhao Zhu
- Key Laboratory of Wetland Ecology and Environment, Northeast Institute of Geography and Agroecology, Chinese Academy of Sciences, Changchun 130102, Jilin, China; Biology Department, San Diego State University, San Diego, CA 92182, USA
| | - Ziyu Guo
- Key Laboratory of Wetland Ecology and Environment, Northeast Institute of Geography and Agroecology, Chinese Academy of Sciences, Changchun 130102, Jilin, China; University of Chinese Academy of Sciences, Beijing 100049, China
| | - Nannan Wang
- Key Laboratory of Wetland Ecology and Environment, Northeast Institute of Geography and Agroecology, Chinese Academy of Sciences, Changchun 130102, Jilin, China
| | - Jianzhao Liu
- Key Laboratory of Wetland Ecology and Environment, Northeast Institute of Geography and Agroecology, Chinese Academy of Sciences, Changchun 130102, Jilin, China; University of Chinese Academy of Sciences, Beijing 100049, China
| | - Yunjiang Zuo
- Key Laboratory of Wetland Ecology and Environment, Northeast Institute of Geography and Agroecology, Chinese Academy of Sciences, Changchun 130102, Jilin, China; University of Chinese Academy of Sciences, Beijing 100049, China
| | - Kexin Li
- Key Laboratory of Wetland Ecology and Environment, Northeast Institute of Geography and Agroecology, Chinese Academy of Sciences, Changchun 130102, Jilin, China; University of Chinese Academy of Sciences, Beijing 100049, China
| | - Changchun Song
- Key Laboratory of Wetland Ecology and Environment, Northeast Institute of Geography and Agroecology, Chinese Academy of Sciences, Changchun 130102, Jilin, China
| | - Yanyu Song
- Key Laboratory of Wetland Ecology and Environment, Northeast Institute of Geography and Agroecology, Chinese Academy of Sciences, Changchun 130102, Jilin, China
| | - Chao Gong
- Key Laboratory of Wetland Ecology and Environment, Northeast Institute of Geography and Agroecology, Chinese Academy of Sciences, Changchun 130102, Jilin, China
| | - Xiaofeng Xu
- Biology Department, San Diego State University, San Diego, CA 92182, USA
| | - Fenghui Yuan
- Key Laboratory of Wetland Ecology and Environment, Northeast Institute of Geography and Agroecology, Chinese Academy of Sciences, Changchun 130102, Jilin, China; Department of Soil, Water, and Climate, University of Minnesota, Saint Paul, MN 55108, USA.
| | - Lihua Zhang
- Key Laboratory of Ecology and Environment in Minority Areas, Minzu University of China, Beijing 100081, China.
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13
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Li XH, Xu JY, Wang X, Liao LJ, Huang L, Huang YQ, Zhang ZF. BanXiaXieXin decoction treating gastritis mice with drug-resistant Helicobacter pylori and its mechanism. World J Gastroenterol 2023; 29:2818-2835. [PMID: 37274067 PMCID: PMC10237109 DOI: 10.3748/wjg.v29.i18.2818] [Citation(s) in RCA: 8] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 03/02/2023] [Revised: 03/16/2023] [Accepted: 04/13/2023] [Indexed: 05/11/2023] Open
Abstract
BACKGROUND Helicobacter pylori (H. pylori) is the main pathogen that causes a variety of upper digestive diseases. The drug resistance rate of H. pylori is increasingly higher, and the eradication rate is increasingly lower. The antimicrobial resistance of H. pylori is an urgent global problem. It has been confirmed that Banxia Xiexin decoction (BXXXT) demonstrates the effects of treating gastrointestinal diseases, inhibiting H. pylori and protecting gastric mucosa. The purpose of the present study is to further explore the therapeutic effects of BXXXT on drug-resistant H. pylori.
AIM To confirm that BXXXT demonstrates therapeutical effects in vivo and in vitro on gastritis mice with drug-resistant H. pylori and explain its mechanism to provide an experimental basis for promoting the application of BXXXT.
METHODS The aqueous extract of BXXXT was gained by water decocting method. The inhibitory effect of the aqueous extract on H. pylori was detected by dilution in vitro; drug-resistant H. pylori cells were used to build an acute gastritis model in vivo. Thereafter, the model mice were treated with the aqueous extract of BXXXT. The amount of H. pylori colonization, the repair of gastric mucosal damage, changes of inflammatory factors, apoptosis, etc., were assessed. In terms of mechanism exploration, the main medicinal compositions of BXXXT aqueous extract and the synergistic bacteriostatic effects they had demonstrated were analyzed using mass spectrometry; the immune function of peripheral blood cells such as CD3+ T and CD4+ T of mice with gastritis before and after treatment with BXXXT aqueous extract was detected using a flow cytometry; the H. pylori transcriptome and proteome after treatment with BXXXT aqueous extract were detected. Differently expressed genes were screened and verification was performed thereon with knockout expression.
RESULTS The minimum inhibitory concentration of BXXXT aqueous extract against H. pylori was 256-512 μg/mL. A dose of 28 mg/kg BXXXT aqueous extract treatment produced better therapeutical effects than the standard triple therapy did; the BXXXT aqueous extract have at least 11 ingredients inhibiting H. pylori, including berberine, quercetin, baicalin, luteolin, gallic acid, rosmarinic acid, aloe emodin, etc., of which berberine, aloe emodin, luteolin and gallic acid have a synergistic effect; BXXXT aqueous extract was found to stimulate the expressions of CD3+ T and CD4+ T and increase the number of CD4+ T/CD8+ T in gastritis mice; the detection of transcriptome and proteome, quantitative polymerase chain reaction, Western blotting and knockout verification revealed that the main targets of BXXXT aqueous extract are CFAs related to urea enzymes, and CagA, VacA, etc.
CONCLUSION BXXXT aqueous extract could demonstrate good therapeutic effects on drug-resistance H. pylori in vitro and in vivo and its mechanism comes down to the synergistic or additional antibacterial effects of berberine, emodin and luteolin, the main components of the extract; the extract could activate the immune function and enhance bactericidal effects; BXXXT aqueous extract, with main targets of BXXXT aqueous extract related to urease, virulence factors, etc., could reduce the urease and virulence of H. pylori, weaken its colonization, and reduce its inflammatory damage to the gastric mucosa.
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Affiliation(s)
- Xiao-Hua Li
- Duate School, Guangxi Medical University, Nanning 5330021, Guangxi Zhuang Autonomous Region, China
| | - Jia-Yin Xu
- Guangxi Technology Innovation Cooperation Base of Prevention and Control Pathogenic Microbes with Drug Resistance, Youjiang Medical University for Nationalities, Baise 533000, Guangxi Zhuang Autonomous Region, China
| | - Xue Wang
- Guangxi Technology Innovation Cooperation Base of Prevention and Control Pathogenic Microbes with Drug Resistance, Youjiang Medical University for Nationalities, Baise 533000, Guangxi Zhuang Autonomous Region, China
| | - Li-Juan Liao
- Guangxi Technology Innovation Cooperation Base of Prevention and Control Pathogenic Microbes with Drug Resistance, Youjiang Medical University for Nationalities, Baise 533000, Guangxi Zhuang Autonomous Region, China
| | - Liang Huang
- Key Laboratory of the Prevention and Treatment of Drug Resistant Microbial Infecting, Youjiang Medical University for Nationalities, Baise 533000, Guangxi Zhuang Autonomous Region, China
| | - Yan-Qiang Huang
- Guangxi Technology Innovation Cooperation Base of Prevention and Control Pathogenic Microbes with Drug Resistance, Youjiang Medical University for Nationalities, Baise 533000, Guangxi Zhuang Autonomous Region, China
| | - Zeng-Feng Zhang
- Department of Microbiology, Guangxi Medical University, Nanning 530021, Guangxi Zhuang Autonomous Region, China
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Lee HS, Lee HJ, Kim B, Kim SH, Cho DH, Jung HJ, Bhatia SK, Choi KY, Kim W, Lee J, Lee SH, Yang YH. Inhibition of Cyclopropane Fatty Acid Synthesis in the Membrane of Halophilic Halomonas socia CKY01 by Kanamycin. BIOTECHNOL BIOPROC E 2022. [DOI: 10.1007/s12257-022-0086-9] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/09/2022]
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15
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Advances in the Structural Biology, Mechanism, and Physiology of Cyclopropane Fatty Acid Modifications of Bacterial Membranes. Microbiol Mol Biol Rev 2022; 86:e0001322. [PMID: 35435731 PMCID: PMC9199407 DOI: 10.1128/mmbr.00013-22] [Citation(s) in RCA: 11] [Impact Index Per Article: 3.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/20/2022] Open
Abstract
Cyclopropane fatty acid (CFA) synthase catalyzes a remarkable reaction. The
cis
double bonds of unsaturated fatty acyl chains of phospholipid bilayers are converted to cyclopropane rings by transfer of a methylene moiety from S-adenosyl-L-methionine (SAM).
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16
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Shao S, Zhang Y, Yin K, Zhang Y, Wei L, Wang Q. FabR senses long-chain unsaturated fatty acids to control virulence in pathogen Edwardsiella piscicida. Mol Microbiol 2022; 117:737-753. [PMID: 34932231 DOI: 10.1111/mmi.14869] [Citation(s) in RCA: 3] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/23/2021] [Revised: 12/11/2021] [Accepted: 12/18/2021] [Indexed: 11/28/2022]
Abstract
Long-chain unsaturated fatty acids (UFAs) can serve as nutrient sources or building blocks for bacterial membranes. However, little is known about how UFAs may be incorporated into the virulence programs of pathogens. A previous investigation identified FabR as a positive regulator of virulence gene expression in Edwardsiella piscicida. Here, chromatin immunoprecipitation-sequencing coupled with RNA-seq analyses revealed that 10 genes were under the direct control of FabR, including fabA, fabB, and cfa, which modulate the composition of UFAs. The binding of FabR to its target DNA was facilitated by oleoyl-CoA and inhibited by stearoyl-CoA. In addition, analyses of enzyme mobility shift assay and DNase I footprinting with wild-type and a null mutant (F131A) of FabR demonstrated crucial roles of FabR in binding to the promoters of fabA, fabB, and cfa. Moreover, FabR also binds to the promoter region of the virulence regulator esrB for its activation, facilitating the expression of the type III secretion system (T3SS) in response to UFAs. Furthermore, FabR coordinated with RpoS to modulate the expression of T3SS. Collectively, our results elucidate the molecular machinery of FabR regulating bacterial fatty acid composition and virulence in enteric pathogens, further expanding our knowledge of its crucial role in host-pathogen interactions.
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Affiliation(s)
- Shuai Shao
- State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, Shanghai, China
| | - Yi Zhang
- State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, Shanghai, China
| | - Kaiyu Yin
- State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, Shanghai, China
| | - Yuanxing Zhang
- Southern Marine Science and Engineering Guangdong Laboratory (Zhuhai), Zhuhai, China
- Shanghai Engineering Research Center of Maricultured Animal Vaccines, Shanghai, China
| | - Lifan Wei
- State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, Shanghai, China
- Department of Endodontics and Operative Dentistry, Ninth People's Hospital, College of Stomatology, Shanghai Jiao Tong University School of Medicine, Shanghai, China
| | - Qiyao Wang
- State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, Shanghai, China
- Shanghai Engineering Research Center of Maricultured Animal Vaccines, Shanghai, China
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17
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Multi-Omic Analysis to Characterize Metabolic Adaptation of the E. coli Lipidome in Response to Environmental Stress. Metabolites 2022; 12:metabo12020171. [PMID: 35208246 PMCID: PMC8880424 DOI: 10.3390/metabo12020171] [Citation(s) in RCA: 8] [Impact Index Per Article: 2.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/17/2022] [Revised: 02/08/2022] [Accepted: 02/09/2022] [Indexed: 12/17/2022] Open
Abstract
As an adaptive survival response to exogenous stress, bacteria undergo dynamic remodelling of their lipid metabolism pathways to alter the composition of their cellular membranes. Here, using Escherichia coli as a well characterised model system, we report the development and application of a ‘multi-omics’ strategy for comprehensive quantitative analysis of the temporal changes in the lipidome and proteome profiles that occur under exponential growth phase versus stationary growth phase conditions i.e., nutrient depletion stress. Lipidome analysis performed using ‘shotgun’ direct infusion-based ultra-high resolution accurate mass spectrometry revealed a quantitative decrease in total lipid content under stationary growth phase conditions, along with a significant increase in the mol% composition of total cardiolipin, and an increase in ‘odd-numbered’ acyl-chain length containing glycerophospholipids. The inclusion of field asymmetry ion mobility spectrometry was shown to enable the enrichment and improved depth of coverage of low-abundance cardiolipins, while ultraviolet photodissociation-tandem mass spectrometry facilitated more complete lipid structural characterisation compared with conventional collision-induced dissociation, including unambiguous assignment of the odd-numbered acyl-chains as containing cyclopropyl modifications. Proteome analysis using data-dependent acquisition nano-liquid chromatography mass spectrometry and tandem mass spectrometry analysis identified 83% of the predicted E. coli lipid metabolism enzymes, which enabled the temporal dependence associated with the expression of key enzymes responsible for the observed adaptive lipid metabolism to be determined, including those involved in phospholipid metabolism (e.g., ClsB and Cfa), fatty acid synthesis (e.g., FabH) and degradation (e.g., FadA/B,D,E,I,J and M), and proteins involved in the oxidative stress response resulting from the generation of reactive oxygen species during β-oxidation or lipid degradation.
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18
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Helicobacter pylori FabX contains a [4Fe-4S] cluster essential for unsaturated fatty acid synthesis. Nat Commun 2021; 12:6932. [PMID: 34836944 PMCID: PMC8626469 DOI: 10.1038/s41467-021-27148-0] [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: 07/15/2021] [Accepted: 10/29/2021] [Indexed: 11/08/2022] Open
Abstract
Unsaturated fatty acids (UFAs) are essential for functional membrane phospholipids in most bacteria. The bifunctional dehydrogenase/isomerase FabX is an essential UFA biosynthesis enzyme in the widespread human pathogen Helicobacter pylori, a bacterium etiologically related to 95% of gastric cancers. Here, we present the crystal structures of FabX alone and in complexes with an octanoyl-acyl carrier protein (ACP) substrate or with holo-ACP. FabX belongs to the nitronate monooxygenase (NMO) flavoprotein family but contains an atypical [4Fe-4S] cluster absent in all other family members characterized to date. FabX binds ACP via its positively charged α7 helix that interacts with the negatively charged α2 and α3 helices of ACP. We demonstrate that the [4Fe-4S] cluster potentiates FMN oxidation during dehydrogenase catalysis, generating superoxide from an oxygen molecule that is locked in an oxyanion hole between the FMN and the active site residue His182. Both the [4Fe-4S] and FMN cofactors are essential for UFA synthesis, and the superoxide is subsequently excreted by H. pylori as a major resource of peroxide which may contribute to its pathogenic function in the corrosion of gastric mucosa.
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Cyclopropane Fatty Acids are Important for Salmonella enterica serovar Typhimurium Virulence. Infect Immun 2021; 90:e0047921. [PMID: 34662213 DOI: 10.1128/iai.00479-21] [Citation(s) in RCA: 6] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/20/2022] Open
Abstract
A variety of eubacteria, plants and protozoa can modify membrane lipids by cyclopropanation, which is reported to modulate membrane permeability and fluidity. The ability to cyclopropanate membrane lipids has been associated with resistance to oxidative stress in Mycobacterium tuberculosis, organic solvent stress in Escherichia coli, and acid stress in E. coli and Salmonella. In bacteria, the cfa gene encoding cyclopropane fatty acid (CFA) synthase is induced during the stationary phase of growth. In the present study we constructed a cfa mutant of Salmonella enterica serovar Typhimurium 14028s (S. Typhimurium) and determined the contribution of CFA-modified lipids to stress resistance and virulence in mice. Cyclopropane fatty acid content was quantified in wild-type and cfa mutant S. Typhimurium. CFA levels in a cfa mutant were greatly reduced compared to wild-type, indicating that CFA synthase is the major enzyme responsible for cyclopropane modification of lipids in Salmonella. S. Typhimurium cfa mutants were more sensitive to extreme acid pH, the protonophore CCCP, and hydrogen peroxide, compared to wild-type. In addition, cfa mutants exhibited reduced viability in murine macrophages and could be rescued by addition of the NADPH phagocyte oxidase inhibitor diphenyleneiodonium (DPI) chloride. S. Typhimurium lacking cfa was also attenuated for virulence in mice. These observations indicate that CFA modification of lipids makes an important contribution to Salmonella virulence.
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20
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Effects of a Δ-9-fatty acid desaturase and a cyclopropane-fatty acid synthase from the novel psychrophile Pseudomonas sp. B14-6 on bacterial membrane properties. J Ind Microbiol Biotechnol 2020; 47:1045-1057. [PMID: 33259029 DOI: 10.1007/s10295-020-02333-0] [Citation(s) in RCA: 15] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/09/2020] [Accepted: 11/17/2020] [Indexed: 10/22/2022]
Abstract
Psychrophilic bacteria, living at low and mild temperatures, can contribute significantly to our understanding of microbial responses to temperature, markedly occurring in the bacterial membrane. Here, a newly isolated strain, Pseudomonas sp. B14-6, was found to dynamically change its unsaturated fatty acid and cyclic fatty acid content depending on temperature which was revealed by phospholipid fatty acid (PLFA) analysis. Genome sequencing yielded the sequences of the genes Δ-9-fatty acid desaturase (desA) and cyclopropane-fatty acid-acyl-phospholipid synthase (cfa). Overexpression of desA in Escherichia coli led to an increase in the levels of unsaturated fatty acids, resulting in decreased membrane hydrophobicity and increased fluidity. Cfa proteins from different species were all found to promote bacterial growth, despite their sequence diversity. In conclusion, PLFA analysis and genome sequencing unraveled the temperature-related behavior of Pseudomonas sp. B14-6 and the functions of two membrane-related enzymes. Our results shed new light on temperature-dependent microbial behaviors and might allow to predict the consequences of global warming on microbial communities.
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21
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Han C, Xia K, Yang J, Zhang H, DeLisa MP, Liang X. Investigation of lipid profile in Acetobacter pasteurianus Ab3 against acetic acid stress during vinegar production. Extremophiles 2020; 24:909-922. [PMID: 33026498 DOI: 10.1007/s00792-020-01204-x] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/02/2020] [Accepted: 09/21/2020] [Indexed: 10/23/2022]
Abstract
Elucidation of the acetic acid resistance (AAR) mechanisms of Acetobacter pasteurianus is significant for vinegar production. In this study, cell membrane lipid profile of A. pasteurianus Ab3 was investigated by gas chromatography-mass spectrometer (GC-MS) and high performance liquid chromatography-electrospray ionization (HPLC-ESI) combined with high resolution accurate mass/mass spectrometry (HRAM/MS). We observed that cell remodeled the membrane physical state by decreasing the ratio of saturated fatty acids (SFAs)/unsaturated fatty acids (UFAs), and increasing the chain length of fatty acids (FAs) and the content of cyclopropane FAs in response to extreme acid stress. Noticeably, the content of octadecadienoic acid (C18:2) elevated remarkably. Moreover, a continuous reduction in cell membrane fluidity and a "V-type" variance in permeability were discovered. The content of glycerophospholipid and ceramide increased significantly in cells harvested from culture with acidity of 75 g/L and 95 g/L compared to that with acidity of 30 g/L. Among the identified lipid species, the content of phosphatidylcholine (e.g. PC 19:0/18:2 and 19:1/18:0), ceramide (e.g. Cer d18:0/16:1 and d18:0/16:1 + O), and dimethylphosphatidylethanolamine (e.g. dMePE 19:1/16:1) increased notably with increasing acidity. Collectively, these findings refresh our current understanding of the AAR mechanisms in A. pasteurianus Ab3, and should direct future strain breeding and vinegar fermentation.
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Affiliation(s)
- Chengcheng Han
- Department of Biochemical Engineering, School of Food Science and Biotechnology, Zhejiang Gongshang University, Hangzhou, 310018, China
| | - Kai Xia
- Department of Biochemical Engineering, School of Food Science and Biotechnology, Zhejiang Gongshang University, Hangzhou, 310018, China
| | - Jieqiong Yang
- Department of Biochemical Engineering, School of Food Science and Biotechnology, Zhejiang Gongshang University, Hangzhou, 310018, China
| | - Hong Zhang
- Department of Biochemical Engineering, School of Food Science and Biotechnology, Zhejiang Gongshang University, Hangzhou, 310018, China
| | - Matthew P DeLisa
- Robert Frederick Smith School of Chemical and Biomolecular Engineering, Cornell University, 120 Olin Hall, Ithaca, NY, 14853, USA
| | - Xinle Liang
- Department of Biochemical Engineering, School of Food Science and Biotechnology, Zhejiang Gongshang University, Hangzhou, 310018, China.
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22
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Randolph CE, Blanksby SJ, McLuckey SA. Enhancing detection and characterization of lipids using charge manipulation in electrospray ionization-tandem mass spectrometry. Chem Phys Lipids 2020; 232:104970. [PMID: 32890498 PMCID: PMC7606777 DOI: 10.1016/j.chemphyslip.2020.104970] [Citation(s) in RCA: 17] [Impact Index Per Article: 3.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/15/2020] [Revised: 08/25/2020] [Accepted: 08/31/2020] [Indexed: 12/14/2022]
Abstract
Heightened awareness regarding the implication of disturbances in lipid metabolism with respect to prevalent human-related pathologies demands analytical techniques that provide unambiguous structural characterization and accurate quantitation of lipids in complex biological samples. The diversity in molecular structures of lipids along with their wide range of concentrations in biological matrices present formidable analytical challenges. Modern mass spectrometry (MS) offers an unprecedented level of analytical power in lipid analysis, as many advancements in the field of lipidomics have been facilitated through novel applications of and developments in electrospray ionization tandem mass spectrometry (ESI-MS/MS). ESI allows for the formation of intact lipid ions with little to no fragmentation and has become widely used in contemporary lipidomics experiments due to its sensitivity, reproducibility, and compatibility with condensed-phase modes of separation, such as liquid chromatography (LC). Owing to variations in lipid functional groups, ESI enables partial chemical separation of the lipidome, yet the preferred ion-type is not always formed, impacting lipid detection, characterization, and quantitation. Moreover, conventional ESI-MS/MS approaches often fail to expose diverse subtle structural features like the sites of unsaturation in fatty acyl constituents or acyl chain regiochemistry along the glycerol backbone, representing a significant challenge for ESI-MS/MS. To overcome these shortcomings, various charge manipulation strategies, including charge-switching, have been developed to transform ion-type and charge state, with aims of increasing sensitivity and selectivity of ESI-MS/MS approaches. Importantly, charge manipulation approaches afford enhanced ionization efficiency, improved mixture analysis performance, and access to informative fragmentation channels. Herein, we present a critical review of the current suite of solution-based and gas-phase strategies for the manipulation of lipid ion charge and type relevant to ESI-MS/MS.
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Affiliation(s)
- Caitlin E Randolph
- Department of Chemistry, Purdue University, West Lafayette, Indiana 47907-2084, USA
| | - Stephen J Blanksby
- Central Analytical Research Facility, Institute for Future Environments, Queensland University of Technology, Brisbane, QLD 4000, Australia
| | - Scott A McLuckey
- Department of Chemistry, Purdue University, West Lafayette, Indiana 47907-2084, USA.
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23
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Abstract
The original strategies developed by Helicobacter pylori to persistently colonise its host and to deregulate its cellular functions make this bacterium an outstanding model to study host-pathogen interaction and the mechanisms responsible for bacterial-induced carcinogenesis. During the last year, significant results were obtained on the role of bacterial factors essential for gastric colonisation such as spiral shape maintenance, orientation through chemotaxis and the formation of bacteria clonal population islands inside the gastric glands. Particularities of the H pylori cell surface, a structure important for immune escape, were demonstrated. New insights in the bacterial stress response revealed the importance of DNA methylation-mediated regulation. Further findings were reported on H pylori components that mediate natural transformation and mechanisms of bacterial DNA horizontal transfer which maintain a high level of H pylori genetic variability. Within-host evolution was found to be niche-specific and probably associated with physiological differences between the antral and oxyntic gastric mucosa. In addition, with the progress of CryoEM, high-resolution structures of the major virulence factors, VacA and CagT4SS, were obtained. The use of gastric organoid models fostered research revealing, preferential accumulation of bacteria at the site of injury during infection. Several studies further characterised the role of CagA in the oncogenic properties of H pylori, identifying the activation of novel CagA-dependent pathways, leading to the promotion of genetic instabilities, epithelial-to-mesenchymal transition and finally carcinogenesis. Recent studies also highlight that microRNA-mediated regulation and epigenetic modifications, through DNA methylation, are key events in the H pylori-induced tumorigenesis process.
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Affiliation(s)
- Milica Denic
- Département de Microbiologie, Institut Pasteur, UMR CNRS 2001, Unité Pathogenèse de Helicobacter, Paris, France.,Sorbonne Paris Cité, Cellule Pasteur, Université Paris Diderot, Paris, France
| | - Eliette Touati
- Département de Microbiologie, Institut Pasteur, UMR CNRS 2001, Unité Pathogenèse de Helicobacter, Paris, France
| | - Hilde De Reuse
- Département de Microbiologie, Institut Pasteur, UMR CNRS 2001, Unité Pathogenèse de Helicobacter, Paris, France
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24
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Cai Y, Wang C, Chen Z, Xu Z, Li H, Li W, Sun Y. Transporters HP0939, HP0497, and HP0471 participate in intrinsic multidrug resistance and biofilm formation in Helicobacter pylori by enhancing drug efflux. Helicobacter 2020; 25:e12715. [PMID: 32548895 DOI: 10.1111/hel.12715] [Citation(s) in RCA: 17] [Impact Index Per Article: 3.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 03/05/2020] [Revised: 05/17/2020] [Accepted: 05/26/2020] [Indexed: 02/06/2023]
Abstract
BACKGROUND The multidrug resistance of Helicobacter pylori is becoming an increasingly serious issue. It is therefore necessary to study the mechanism of multidrug resistance of H pylori. We have previously identified that the HP0939, HP0497, and HP0471 transporters affect the efflux of drugs from H pylori. As efflux pumps participate in bacterial multidrug resistance and biofilm formation, we hypothesized that these transporters could be involved in the multidrug resistance and biofilm formation of H pylori. MATERIALS AND METHODS We therefore constructed three knockout strains, Δhp0939, Δhp0497, and Δhp0471, and three high-expression strains, Hp0939he , Hp0497he , and Hp0471he , using the wild-type (WT) 26 695 strain of H pylori as the template. The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of wild strains, knockout strains, and high-expression strains to amoxicillin, metronidazole, and other antibiotics were measured. The efflux capacity of high-expression strains and wild strains was compared by Hoechst 33 342 accumulation assay. RESULTS Determination of the MIC and MBC of the antibiotics revealed that the knockout strains were more sensitive to antibiotics, while the high-expression strains were less sensitive to antibiotics, compared to the WT. The ability of the high-expression strains to efflux drugs was significantly higher than that of the WT. We also induced H pylori to form biofilms, and observed that the knockout strains could barely form biofilms and were more sensitive to several antibiotics, compared to the WT. The mRNA expression of hp0939, hp0497, and hp0471 in the clinically sensitive and multidrug-resistant strains was determined, and it was found that these genes were highly expressed in the multidrug-resistant strains that were isolated from the clinics. CONCLUSIONS In this study, we found three transporters involved in intrinsic multidrug resistance of H pylori.
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Affiliation(s)
- Yuying Cai
- Department of Microbiology, Key Laboratory of Medical Microbiology and Parasitology, Guizhou Medical University, Guiyang, China.,Institute of Pathogen Biology, School of Basic Medical Sciences, Shandong University, Jinan, China
| | - Caixia Wang
- Department of Microbiology, Key Laboratory of Medical Microbiology and Parasitology, Guizhou Medical University, Guiyang, China.,Institute of Pathogen Biology, School of Basic Medical Sciences, Shandong University, Jinan, China
| | - Zhenghong Chen
- Department of Microbiology, Key Laboratory of Medical Microbiology and Parasitology, Guizhou Medical University, Guiyang, China
| | - Zhengzheng Xu
- Department of Microbiology, Key Laboratory for Experimental Teratology of Ministry of Education, Key Laboratory of Infection and Immunology of Shandong Province, School of Basic Medicine, Shandong University, Jinan, China
| | - Huanjie Li
- Department of Microbiology, Key Laboratory for Experimental Teratology of Ministry of Education, Key Laboratory of Infection and Immunology of Shandong Province, School of Basic Medicine, Shandong University, Jinan, China
| | - Wenjuan Li
- Department of Microbiology, Key Laboratory for Experimental Teratology of Ministry of Education, Key Laboratory of Infection and Immunology of Shandong Province, School of Basic Medicine, Shandong University, Jinan, China
| | - Yundong Sun
- Department of Microbiology, Key Laboratory for Experimental Teratology of Ministry of Education, Key Laboratory of Infection and Immunology of Shandong Province, School of Basic Medicine, Shandong University, Jinan, China
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25
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Luiz de Freitas L, Aparecida Dos Santos CI, Carneiro DG, Dantas Vanetti MC. Nisin and acid resistance in Salmonella is enhanced by N-dodecanoyl-homoserine lactone. Microb Pathog 2020; 147:104320. [PMID: 32534181 DOI: 10.1016/j.micpath.2020.104320] [Citation(s) in RCA: 11] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/21/2020] [Revised: 06/02/2020] [Accepted: 06/03/2020] [Indexed: 01/22/2023]
Abstract
Salmonella is a foodborne pathogen that can develop resistance to different stresses, which is essential for successful infection of the host. Some genes directly related to acid resistance are also involved in cationic peptide resistance in Gram-negative bacteria and could be under the control of quorum sensing (QS) mediated by autoinducer 1, known as acyl-homoserine lactone. Here, we investigated the influence of autoinducer 1, N-dodecanoyl-homoserine lactone (C12-HSL) on the resistance of Salmonella enterica subspecies enterica serovar Enteritidis to nisin and acid stress. Salmonella cells growing in anaerobic tryptic soy agar (TSB) at a pH of 7.0 for 7 h were submitted to acid stress at a pH of 4.5 in the presence and absence of nisin and were either supplemented or not with C12-HSL. Viable cell counts, gene expression, membrane charge alterations, fatty acid composition, and intracellular content leakage were observed. The autoinducer C12-HSL increased nisin resistance and survival at a pH of 4.5 in Salmonella. Also, C12-HSL increased the expression of the genes, phoP, phoQ, pmrA, and pmrB, which are involved with antimicrobial and acid resistance. The positive charge on the cell surface and concentration of cyclopropane fatty acid of the cellular membrane were increased in the presence of C12-HSL under acidic conditions, whereas membrane fluidity decreased. The loss of K+ and NADPH, promoted by nisin, was reduced in the presence of C12-HSL at a pH of 4.5. Taken together, these findings suggest that quorum sensing plays an important role in enhanced nisin and acid resistance in Salmonella.
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Affiliation(s)
- Leonardo Luiz de Freitas
- Department of Microbiology, Universidade Federal de Viçosa (UFV), Av. Peter Henry Rolfs, S/N, Viçosa, MG, Brazil
| | | | - Deisy Guimarães Carneiro
- Department of Microbiology, Universidade Federal de Viçosa (UFV), Av. Peter Henry Rolfs, S/N, Viçosa, MG, Brazil
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26
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Zeng Q, Yang Q, Jia J, Bi H. A Moraxella Virulence Factor Catalyzes an Essential Esterase Reaction of Biotin Biosynthesis. Front Microbiol 2020; 11:148. [PMID: 32117167 PMCID: PMC7026016 DOI: 10.3389/fmicb.2020.00148] [Citation(s) in RCA: 9] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/04/2019] [Accepted: 01/22/2020] [Indexed: 11/13/2022] Open
Abstract
Pimeloyl-acyl carrier protein (ACP) methyl ester esterase catalyzes the last biosynthetic step of the pimelate moiety of biotin, a key intermediate in biotin biosynthesis. The paradigm pimeloyl-ACP methyl ester esterase is the BioH protein of Escherichia coli that hydrolyses the ester bond of pimeloyl-ACP methyl ester. Biotin synthesis in E. coli also requires the function of the malonyl-ACP methyltransferase gene (bioC) to employ a methylation strategy to allow elongation of a temporarily disguised malonate moiety to a pimelate moiety by the fatty acid synthetic enzymes. However, bioinformatics analyses of the extant bacterial genomes showed that bioH is absent in many bioC-containing bacteria. The genome of the Gram-negative bacterium, Moraxella catarrhalis lacks a gene encoding a homolog of any of the six known pimeloyl-ACP methyl ester esterase isozymes suggesting that this organism encodes a novel pimeloyl-ACP methyl ester esterase isoform. We report that this is the case. The gene encoding the new isoform, called btsA, was isolated by complementation of an E. coli bioH deletion strain. The requirement of BtsA for the biotin biosynthesis in M. catarrhalis was confirmed by a biotin auxotrophic phenotype caused by deletion of btsA in vivo and a reconstituted in vitro desthiobiotin synthesis system. Purified BtsA was shown to cleave the physiological substrate pimeloyl-ACP methyl ester to pimeloyl-ACP by use of a Ser117-His254-Asp287 catalytic triad. The lack of sequence alignment with other isozymes together with phylogenetic analyses revealed BtsA as a new class of pimeloyl-ACP methyl ester esterase. The involvement of BtsA in M. catarrhalis virulence was confirmed by the defect of bacterial invasion to lung epithelial cells and survival within macrophages in the ΔbtsA strains. Identification of the new esterase gene btsA exclusive in Moraxella species that links biotin biosynthesis to bacterial virulence, can reveal a new valuable target for development of drugs against M. catarrhalis.
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Affiliation(s)
- Qi Zeng
- Jiangsu Key Laboratory of Pathogen Biology, Department of Pathogen Biology, Nanjing Medical University, Nanjing, China
| | - Qi Yang
- Jiangsu Key Laboratory of Pathogen Biology, Department of Pathogen Biology, Nanjing Medical University, Nanjing, China
| | - Jia Jia
- Jiangsu Key Laboratory of Pathogen Biology, Department of Pathogen Biology, Nanjing Medical University, Nanjing, China
| | - Hongkai Bi
- Jiangsu Key Laboratory of Pathogen Biology, Department of Pathogen Biology, Nanjing Medical University, Nanjing, China
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