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Corrales D, Alcántara C, Zúñiga M, Monedero V. Ppx1 putative exopolyphosphatase is essential for polyphosphate accumulation in Lacticaseibacillus paracasei. Appl Environ Microbiol 2024; 90:e0229023. [PMID: 38619267 PMCID: PMC11107151 DOI: 10.1128/aem.02290-23] [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: 12/22/2023] [Accepted: 03/27/2024] [Indexed: 04/16/2024] Open
Abstract
The linear polymer polyphosphate (poly-P) is present across all three domains of life and serves diverse physiological functions. The enzyme polyphosphate kinase (Ppk) is responsible for poly-P synthesis, whereas poly-P degradation is carried out by the enzyme exopolyphosphatase (Ppx). In many Lactobacillaceae, the Ppk-encoding gene (ppk) is found clustered together with two genes encoding putative exopolyphosphatases (ppx1 and ppx2) each having different domain compositions, with the gene order ppx1-ppk-ppx2. However, the specific function of these ppx genes remains unexplored. An in-frame deletion of ppx1 in Lacticaseibacillus paracasei BL23 resulted in bacteria unable to accumulate poly-P, whereas the disruption of ppx2 did not affect poly-P synthesis. The expression of ppk was not altered in the Δppx1 strain, and poly-P synthesis in this strain was only restored by expressing ppx1 in trans. Moreover, no poly-P synthesis was observed when ppk was expressed from a plasmid in the Δppx1 strain. Purified Ppx2 exhibited in vitro exopolyphosphatase activity, whereas no in vitro enzymatic activity could be demonstrated for Ppx1. This observation corresponds with the absence in Ppx1 of conserved motifs essential for catalysis found in characterized exopolyphosphatases. Furthermore, assays with purified Ppk and Ppx1 evidenced that Ppx1 enhanced Ppk activity. These results demonstrate that Ppx1 is essential for poly-P synthesis in Lc. paracasei and have unveiled, for the first time, an unexpected role of Ppx1 exopolyphosphatase in poly-P synthesis.IMPORTANCEPoly-P is a pivotal molecular player in bacteria, participating in a diverse array of processes ranging from stress resilience to pathogenesis while also serving as a functional component in probiotic bacteria. The synthesis of poly-P is tightly regulated, but the underlying mechanisms remain incompletely elucidated. Our study sheds light on the distinctive role played by the two exopolyphosphatases (Ppx) found in the Lactobacillaceae bacterial group, of relevance in food and health. This particular group is noteworthy for possessing two Ppx enzymes, supposedly involved in poly-P degradation. Remarkably, our investigation uncovers an unprecedented function of Ppx1 in Lacticaseibacillus paracasei, where its absence leads to the total cessation of poly-P synthesis, paralleling the impact observed upon eliminating the poly-P forming enzyme, poly-P kinase. Unlike the anticipated role as a conventional exopolyphosphatase, Ppx1 demonstrates an unexpected function. Our results added a layer of complexity to our understanding of poly-P dynamics in bacteria.
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Affiliation(s)
- Daniela Corrales
- Instituto de Agroquímica y Tecnología de Alimentos (IATA-CSIC), Paterna, Spain
| | - Cristina Alcántara
- Instituto de Agroquímica y Tecnología de Alimentos (IATA-CSIC), Paterna, Spain
| | - Manuel Zúñiga
- Instituto de Agroquímica y Tecnología de Alimentos (IATA-CSIC), Paterna, Spain
| | - Vicente Monedero
- Instituto de Agroquímica y Tecnología de Alimentos (IATA-CSIC), Paterna, Spain
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Molina-Pardines C, Haro-Moreno JM, López-Pérez M. Phosphate-related genomic islands as drivers of environmental adaptation in the streamlined marine alphaproteobacterial HIMB59. mSystems 2023; 8:e0089823. [PMID: 38054740 PMCID: PMC10734472 DOI: 10.1128/msystems.00898-23] [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: 08/28/2023] [Accepted: 10/17/2023] [Indexed: 12/07/2023] Open
Abstract
IMPORTANCE These results shed light on the evolutionary strategies of microbes with streamlined genomes to adapt and survive in the oligotrophic conditions that dominate the surface waters of the global ocean. At the individual level, these microbes have been subjected to evolutionary constraints that have led to a more efficient use of nutrients, removing non-essential genes named as "streamlining theory." However, at the population level, they conserve a highly diverse gene pool in flexible genomic islands resulting in polyclonal populations on the same genomic background as an evolutionary response to environmental pressures. Localization of these islands at equivalent positions in the genome facilitates horizontal transfer between clonal lineages. This high level of environmental genomic heterogeneity could explain their cosmopolitan distribution. In the case of the order HIMB59 within the class Alphaproteobacteria, two factors exert evolutionary pressure and determine this intraspecific diversity: phages and the concentration of P in the environment.
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Affiliation(s)
- Carmen Molina-Pardines
- Evolutionary Genomics Group, División de Microbiología, Universidad Miguel Hernández, San Juan, Alicante, Spain
| | - Jose M. Haro-Moreno
- Evolutionary Genomics Group, División de Microbiología, Universidad Miguel Hernández, San Juan, Alicante, Spain
| | - Mario López-Pérez
- Evolutionary Genomics Group, División de Microbiología, Universidad Miguel Hernández, San Juan, Alicante, Spain
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Pirttiniemi A, Adeshara K, Happonen N, Einarsdottir E, Katayama S, Salmenkari H, Hörkkö S, Kere J, Groop PH, Lehto M. Long-chain polyphosphates inhibit type I interferon signaling and augment LPS-induced cytokine secretion in human leukocytes. J Leukoc Biol 2023; 114:250-265. [PMID: 37224571 DOI: 10.1093/jleuko/qiad058] [Citation(s) in RCA: 2] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/23/2022] [Revised: 04/20/2023] [Accepted: 05/12/2023] [Indexed: 05/26/2023] Open
Abstract
Inorganic polyphosphates are evolutionarily conserved bioactive phosphate polymers found as various chain lengths in all living organisms. In mammals, polyphosphates play a vital role in the regulation of cellular metabolism, coagulation, and inflammation. Long-chain polyphosphates are found along with endotoxins in pathogenic gram-negative bacteria and can participate in bacterial virulence. We aimed to investigate whether exogenously administered polyphosphates modulate human leukocyte function in vitro by treating the cells with 3 different chain lengths of polyphosphates (P14, P100, and P700). The long-chain polyphosphates, P700, had a remarkable capacity to downregulate type I interferon signaling dose dependently in THP1-Dual cells while only a slight elevation could be observed in the NF-κB pathway with the highest dose of P700. P700 treatment decreased lipopolysaccharide-induced IFNβ transcription and secretion, reduced STAT1 phosphorylation, and downregulated subsequent interferon-stimulated gene expression in primary human peripheral blood mononuclear cells. P700 also augmented lipopolysaccharide-induced secretion of IL-1α, IL-1β, IL-4, IL-5, IL-10, and IFNγ. Furthermore, P700 has previously been reported to increase the phosphorylation of several intracellular signaling mediators, such as AKT, mTOR, ERK, p38, GSK3α/β, HSP27, and JNK pathway components, which was supported by our findings. Taken together, these observations demonstrate the extensive modulatory effects P700 has on cytokine signaling and the inhibitory effects specifically targeted to type I interferon signaling in human leukocytes.
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Affiliation(s)
- Anniina Pirttiniemi
- Folkhälsan Institute of Genetics, Folkhälsan Research Center, Biomedicum, Haartmaninkatu 8, 00290 Helsinki, Finland
- Department of Nephrology, University of Helsinki and Helsinki University Hospital, Haartmaninkatu 4, 00290 Helsinki, Finland
- Clinical and Molecular Metabolism, Faculty of Medicine Research Programs, University of Helsinki, Biomedicum, Haartmaninkatu 8, 00290 Helsinki, Finland
| | - Krishna Adeshara
- Folkhälsan Institute of Genetics, Folkhälsan Research Center, Biomedicum, Haartmaninkatu 8, 00290 Helsinki, Finland
- Department of Nephrology, University of Helsinki and Helsinki University Hospital, Haartmaninkatu 4, 00290 Helsinki, Finland
- Clinical and Molecular Metabolism, Faculty of Medicine Research Programs, University of Helsinki, Biomedicum, Haartmaninkatu 8, 00290 Helsinki, Finland
| | - Natalie Happonen
- Medical Microbiology and Immunology, Research Unit of Biomedicine, University of Oulu, Aapistie 5A, 90220 Oulu, Finland
- Medical Research Center Oulu, Oulu University Hospital and University of Oulu, Aapistie 5A, 90220 Oulu, Finland
- Nordlab, Oulu University Hospital, Kajaanintie 50, 90220 Oulu, Finland
| | - Elisabet Einarsdottir
- Science for Life Laboratory, Department of Gene Technology, KTH-Royal Institute of Technology, Tomtebodavägen 23A, 17165 Solna, Sweden
| | - Shintaro Katayama
- Folkhälsan Institute of Genetics, Folkhälsan Research Center, Biomedicum, Haartmaninkatu 8, 00290 Helsinki, Finland
- Stem Cells and Metabolism Research Program, University of Helsinki, Biomedicum, Haartmaninkatu 8, 00290 Helsinki, Finland
- Department of Biosciences and Nutrition, Karolinska Institutet, Neo, Blickagången 16, Flemingsberg, SE-14183 Huddinge, Sweden
| | - Hanne Salmenkari
- Folkhälsan Institute of Genetics, Folkhälsan Research Center, Biomedicum, Haartmaninkatu 8, 00290 Helsinki, Finland
- Department of Nephrology, University of Helsinki and Helsinki University Hospital, Haartmaninkatu 4, 00290 Helsinki, Finland
- Clinical and Molecular Metabolism, Faculty of Medicine Research Programs, University of Helsinki, Biomedicum, Haartmaninkatu 8, 00290 Helsinki, Finland
| | - Sohvi Hörkkö
- Medical Microbiology and Immunology, Research Unit of Biomedicine, University of Oulu, Aapistie 5A, 90220 Oulu, Finland
- Medical Research Center Oulu, Oulu University Hospital and University of Oulu, Aapistie 5A, 90220 Oulu, Finland
| | - Juha Kere
- Folkhälsan Institute of Genetics, Folkhälsan Research Center, Biomedicum, Haartmaninkatu 8, 00290 Helsinki, Finland
- Stem Cells and Metabolism Research Program, University of Helsinki, Biomedicum, Haartmaninkatu 8, 00290 Helsinki, Finland
- Department of Biosciences and Nutrition, Karolinska Institutet, Neo, Blickagången 16, Flemingsberg, SE-14183 Huddinge, Sweden
| | - Per-Henrik Groop
- Folkhälsan Institute of Genetics, Folkhälsan Research Center, Biomedicum, Haartmaninkatu 8, 00290 Helsinki, Finland
- Department of Nephrology, University of Helsinki and Helsinki University Hospital, Haartmaninkatu 4, 00290 Helsinki, Finland
- Clinical and Molecular Metabolism, Faculty of Medicine Research Programs, University of Helsinki, Biomedicum, Haartmaninkatu 8, 00290 Helsinki, Finland
- Department of Diabetes, Central Clinical School, Monash University, Alfred Centre, 99 Commercial Road, Melbourne 3004, VIC, Australia
| | - Markku Lehto
- Folkhälsan Institute of Genetics, Folkhälsan Research Center, Biomedicum, Haartmaninkatu 8, 00290 Helsinki, Finland
- Department of Nephrology, University of Helsinki and Helsinki University Hospital, Haartmaninkatu 4, 00290 Helsinki, Finland
- Clinical and Molecular Metabolism, Faculty of Medicine Research Programs, University of Helsinki, Biomedicum, Haartmaninkatu 8, 00290 Helsinki, Finland
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Exopolyphosphatases PPX1 and PPX2 from Mycobacterium tuberculosis regulate dormancy response and pathogenesis. Microb Pathog 2022; 173:105885. [DOI: 10.1016/j.micpath.2022.105885] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/31/2022] [Revised: 11/12/2022] [Accepted: 11/12/2022] [Indexed: 11/21/2022]
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Functional Genetic Diversity and Plant Growth Promoting Potential of Polyphosphate Accumulating Bacteria in Soil. Microbiol Spectr 2022; 10:e0034521. [PMID: 35196785 PMCID: PMC8865437 DOI: 10.1128/spectrum.00345-21] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/27/2022] Open
Abstract
Polyphosphate (polyP) accumulation is an important trait of microorganisms. Implication of polyP accumulating bacteria (PAB) in enhanced biological phosphate removal, heavy metal sequestration, and dissolution of dental enamel is well studied. Phosphorous (P) accumulated within microbial biomass also regulates labile P in soil; however, abundance and diversity of the PAB in soil is still unexplored. Present study investigated the genetic and functional diversity of PAB in rhizosphere soil. Here, we report the abundance of Pseudomonas spp. as high PAB in soil, suggesting their contribution to global P cycling. Additional subset analysis of functional genes i.e., polyphosphate kinase (ppk) and exopolyphosphatase (ppx) in all PAB, indicates their significance in bacterial growth and metabolism. Distribution of functional genes in phylogenetic tree represent a more biologically realistic discrimination for the two genes. Distribution of ppx gene disclosed its phylogenetic conservation at species level, however, clustering of ppk gene of similar species in different clades illustrated its environmental condition mediated modifications. Selected PAB showed tolerance to abiotic stress and strong correlation with plant growth promotary (PGP) traits viz. phosphate solubilization, auxin and siderophore production. Interaction of PAB with A. thaliana enhanced the growth and phosphate status of the plant under salinity stress, suggestive of their importance in P cycling and stress alleviation. IMPORTANCE Study discovered the abundance of Pseudomonas genera as a high phosphate accumulator in soil. The presence of functional genes (polyphosphate kinase [ppk] and exopolyphosphatase [ppx]) in all PAB depicts their importance in polyphosphate metabolism in bacteria. Genetic and functional diversity reveals conservation of the ppx gene at species level. Furthermore, we found a positive correlation between PAB and plant growth promotary traits, stress tolerance, and salinity stress alleviation in A. thaliana.
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Zhang A, Lu Z, Xu Y, Qi T, Li W, Zhang L, Cui Z. The structure of exopolyphosphatase (PPX) from Porphyromonas gingivalis in complex with substrate analogs and magnesium ions reveals the basis for polyphosphate processivity. J Struct Biol 2021; 213:107767. [PMID: 34214602 DOI: 10.1016/j.jsb.2021.107767] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/07/2021] [Revised: 06/25/2021] [Accepted: 06/25/2021] [Indexed: 10/21/2022]
Abstract
The enzymes exopolyphosphatase/guanosine pentaphosphate phosphohydrolase (PPX/GppA) play important roles in the bacterial stringent response. PPX degrades inorganic polyphosphate (polyP), a polymer composed of a few to hundreds of phosphate residues supporting cell survival in the stationary phase. The crystal structure of PPX from Porphyromonas gingivalis (PgPPX) in complex with catalytic magnesium ions and several sulfate ions was solved. PgPPX contained two domains and represented a "closed" configuration. Four sulfate ions forming a linear dispersed chain were observed in the aqueduct of the PPX dimer, which the long polyP chain most likely occupied. The side chain of R255 stretched into the cavity where polyP could be located, obstructing the entrance of larger substrates such as NTP and NDP. This study provided the first view into the structure of the PPX/GppA homolog in complex with magnesium ions and substrate analogs and explained how PgPPX implemented its functionality.
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Affiliation(s)
- Aili Zhang
- Food and Pharmacy College, Xuchang University, China; Key Laboratory of Biomarker Based Rapid-detection Technology for Food Safety of Henan Province, Xuchang University, China
| | - Zuokun Lu
- Food and Pharmacy College, Xuchang University, China; Key Laboratory of Biomarker Based Rapid-detection Technology for Food Safety of Henan Province, Xuchang University, China.
| | - Yanhao Xu
- Food and Pharmacy College, Xuchang University, China
| | - Tiantian Qi
- Food and Pharmacy College, Xuchang University, China
| | - Wenwen Li
- Food and Pharmacy College, Xuchang University, China
| | - Liang Zhang
- Food and Pharmacy College, Xuchang University, China; Key Laboratory of Biomarker Based Rapid-detection Technology for Food Safety of Henan Province, Xuchang University, China
| | - Zhaohui Cui
- Food and Pharmacy College, Xuchang University, China; Key Laboratory of Biomarker Based Rapid-detection Technology for Food Safety of Henan Province, Xuchang University, China
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7
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Harita D, Kanie K, Kimura Y. Enzymatic properties of Myxococcus xanthus exopolyphosphatases mxPpx1 and mxPpx2. BIOCHIMICA ET BIOPHYSICA ACTA-PROTEINS AND PROTEOMICS 2021; 1869:140660. [PMID: 33857634 DOI: 10.1016/j.bbapap.2021.140660] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Subscribe] [Scholar Register] [Received: 11/23/2020] [Revised: 03/02/2021] [Accepted: 04/10/2021] [Indexed: 11/18/2022]
Abstract
Myxococcus xanthus possesses two exopolyphosphatases, mxPpx1 and mxPpx2, which belong to the family of Ppx/GppA phosphatases; however, their catalytic properties have not been described. mxPpx1 and mxPpx2 contain 311 and 505 amino acid residues, respectively; mxPpx2 has an additional C-terminal region, which corresponds to the metal-dependent HDc phosphohydrolase domain. mxPpx1 mainly hydrolyzed short-chain polyPs (polyP3 and polyP4), whereas mxPpx2 preferred long-chain polyP60-70 and polyP700-1000. mxPpx2 was activated by 25-50 mM KCl, but mxPpx1 did not significantly depend on K+. In addition, mxPpx1 and mxPpx2 showed weak hydrolysis of ATP and GTP in the absence of K+, and mxPpx2 could also hydrolyze guanosine pentaphosphate (pppGpp) in the presence of K+. The exopolyphosphatase activity of mxPpx1 toward polyP3 was inhibited by polyP700-1000 and that of mxPpx2 toward polyP60-70 and polyP700-1000, by pyrophosphate. To clarify the function of the mxPpx2 C-terminal domain, it was fused to mxPpx1 (mxPpx1-2C) and deleted from mxPpx2 (mxPpx2∆C). Compared to wild-type mxPpx2, mxPpx2∆C had significantly reduced exopolyphosphatase activity toward long-chain polyPs (by 90%), whereas that toward polyP3 and polyP4 was much less affected; furthermore, the phosphohydrolase activity toward pppGpp, ATP, and GTP was also decreased (by 30-75%). In contrast, mxPpx1-2C had increased hydrolytic activity compared to mxPpx1. Furthermore, mxPpx2∆C lost the requirement for K+ characteristic for the wild-type enzyme, whereas mxPpx1-2C acquired it. These results suggest that the C-terminal domain of mxPpx2 is necessary for its maximum hydrolytic activity, especially toward long-chain polyPs, and defines mxPpx2 dependency on K+ for activation.
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Affiliation(s)
- Daiki Harita
- Department of Applied Biological Science, Faculty of Agriculture, Kagawa University, Miki-cho, Kagawa, Japan
| | - Kousei Kanie
- Department of Applied Biological Science, Faculty of Agriculture, Kagawa University, Miki-cho, Kagawa, Japan
| | - Yoshio Kimura
- Department of Applied Biological Science, Faculty of Agriculture, Kagawa University, Miki-cho, Kagawa, Japan.
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8
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Denoncourt A, Downey M. Model systems for studying polyphosphate biology: a focus on microorganisms. Curr Genet 2021; 67:331-346. [PMID: 33420907 DOI: 10.1007/s00294-020-01148-x] [Citation(s) in RCA: 26] [Impact Index Per Article: 6.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/24/2020] [Revised: 12/08/2020] [Accepted: 12/14/2020] [Indexed: 12/19/2022]
Abstract
Polyphosphates (polyP) are polymers of inorganic phosphates joined by high-energy bonds to form long chains. These chains are present in all forms of life but were once disregarded as 'molecular fossils'. PolyP has gained attention in recent years following new links to diverse biological roles ranging from energy storage to cell signaling. PolyP research in humans and other higher eukaryotes is limited by a lack of suitable tools and awaits the identification of enzymatic players that would enable more comprehensive studies. Therefore, many of the most important insights have come from single-cell model systems. Here, we review determinants of polyP metabolism, regulation, and function in major microbial systems, including bacteria, fungi, protozoa, and algae. We highlight key similarities and differences that may aid in our understanding of how polyP impacts cell physiology at a molecular level.
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Affiliation(s)
- Alix Denoncourt
- Department of Cellular and Molecular Medicine, University of Ottawa, Ottawa, K1H 8M5, Canada.,Ottawa Institute of Systems Biology, Ottawa, K1H 8M5, Canada
| | - Michael Downey
- Department of Cellular and Molecular Medicine, University of Ottawa, Ottawa, K1H 8M5, Canada. .,Ottawa Institute of Systems Biology, Ottawa, K1H 8M5, Canada.
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Zhang J, Wang L, Shi L, Chen X, Chen C, Hong Z, Cao Y, Zhao L. Survival strategy of Cronobacter sakazakii against ampicillin pressure: Induction of the viable but nonculturable state. Int J Food Microbiol 2020; 334:108819. [DOI: 10.1016/j.ijfoodmicro.2020.108819] [Citation(s) in RCA: 8] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/23/2020] [Revised: 07/27/2020] [Accepted: 08/05/2020] [Indexed: 12/18/2022]
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10
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Rakshit D, Dasgupta S, Das B, Bhadra RK. Functional Insights Into the Role of gppA in (p)ppGpp Metabolism of Vibrio cholerae. Front Microbiol 2020; 11:564644. [PMID: 33117311 PMCID: PMC7552824 DOI: 10.3389/fmicb.2020.564644] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/22/2020] [Accepted: 09/08/2020] [Indexed: 11/13/2022] Open
Abstract
The stringent response, an adaptive response to nutrient limitation and exposure to xenobiotics in bacteria, is mediated by two intracellular signaling molecules, pppGpp and ppGpp, together called (p)ppGpp. The cellular level of (p)ppGpp in bacterial cells is controlled by the Rel/Spo family of proteins. In the cholera pathogen, Vibrio cholerae, (p)ppGpp metabolism is regulated by the products of at least three genes relA, spoT, and relV. In this study, we identify and characterize the function of the guanosine-5′-triphosphate 3′-diphosphate pyrophosphatase A (GppA) encoding gene gppA of V. cholerae. Genomic analysis indicates that the gppA locus is conserved in vibrios and organized as a bicistronic operon along with the rhlB gene. We engineered the genome of V. cholerae to develop different mutants devoid of GppA and/or other phosphate metabolic enzymes. Our findings indicate that in V. cholerae, GppA plays an important role in the conversion of pppGpp to ppGpp during amino acid deprivation but not during glucose starvation. Quantitative analyses of the gppA transcript level reveal its differential expression pattern at different growth phases and starvation conditions. It has been observed that the GppA deficiency during amino acid starvation condition could be complemented by overexpressing the exopolyphosphatase coding gene ppx of V. cholerae. By deletion analysis, we further demonstrate that the amino and carboxy terminal sequences flanking the Ppx-GppA motif of the GppA protein of V. cholerae are also important for its enzymatic function.
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Affiliation(s)
- Dipayan Rakshit
- Infectious Diseases and Immunology Division, CSIR-Indian Institute of Chemical Biology, Kolkata, India
| | - Shreya Dasgupta
- Infectious Diseases and Immunology Division, CSIR-Indian Institute of Chemical Biology, Kolkata, India
| | - Bhabatosh Das
- Molecular Genetics Laboratory, Infection and Immunology Division, Translational Health Science and Technology Institute, NCR Biotech Science Cluster, Haryana, India
| | - Rupak K Bhadra
- Infectious Diseases and Immunology Division, CSIR-Indian Institute of Chemical Biology, Kolkata, India
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Daly I, Jellali S, Mehri I, Reis MAM, Freitas EB, Oehmen A, Chatti A. Phosphorus and ammonium removal characteristics from aqueous solutions by a newly isolated plant growth-promoting bacterium. ENVIRONMENTAL TECHNOLOGY 2020; 41:2603-2617. [PMID: 30689524 DOI: 10.1080/09593330.2019.1575917] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 09/06/2018] [Accepted: 01/19/2019] [Indexed: 06/09/2023]
Abstract
An indigenous plant growth-promoting bacterium isolated from Peganum Harmala rhizosphere in the arid ecosystem was found to solubilize and accumulate phosphates. This isolate was identified as Pseudomonas sp. (PHR6) by partial 16S rRNA gene sequence analysis. Controlled batch experiments on nutrients removal by this isolate in mineral medium showed relatively high efficiencies after 24 h of aerobic incubation with average values of 117.59 and 335.38 mg gVSS-1 for phosphorus (P-PO4) and nitrogen (N-NH4), respectively. Furthermore, the strain performed heterotrophic nitrification ranging from 48.81% to 84.24% of the total removed nitrogen. On the other hand, the experimental results showed that a short idle period (24 h) significantly enhanced P accumulation (up to 95%) and N assimilation (up to 50%) of the total removed amounts. However, long idle period (20 days) revealed firstly aerobic phosphorous release phase succeeded by another removal one within 24 h of incubation. Overall, the idle treatment enhances P removal efficiency from the mineral liquid medium without significant effects on N-NH4 removal performance. The isolated strain showed also significant nutrient removal ability from synthetic wastewater providing an accumulated fraction of 98% from the total removed phosphorus amount. This study highlights the potential contribution of the selected rhizobacterium PHR6 to both environmental nutrient recycling and pollution control especially regarding phosphorus.
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Affiliation(s)
- Imen Daly
- Wastewaters and Environment Laboratory (LabEauE), Water Research and Technologies Center, Technopark of Borj-Cedria, Soliman, Tunisia
- Faculty of Sciences of Tunis, Tunis El Manar University, El-Manar II, Tunisia
| | - Salah Jellali
- Wastewaters and Environment Laboratory (LabEauE), Water Research and Technologies Center, Technopark of Borj-Cedria, Soliman, Tunisia
| | - Ines Mehri
- Laboratory of Treatment and Valorization of Water Rejects (LTVRH), Water Research and Technologies Center, Technopark of Borj-Cedria, Soliman, Tunisia
| | - Maria A M Reis
- Faculty of Sciences and Technology, Chemistry Department, FCT-UNL, Caparica, Portugal
| | - Elisabete B Freitas
- Faculty of Sciences and Technology, Chemistry Department, FCT-UNL, Caparica, Portugal
| | - Adrian Oehmen
- Faculty of Sciences and Technology, Chemistry Department, FCT-UNL, Caparica, Portugal
| | - Abdelwaheb Chatti
- Laboratory of Treatment and Valorization of Water Rejects (LTVRH), Water Research and Technologies Center, Technopark of Borj-Cedria, Soliman, Tunisia
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12
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Song H, Dharmasena MN, Wang C, Shaw GX, Cherry S, Tropea JE, Jin DJ, Ji X. Structure and activity of PPX/GppA homologs from Escherichia coli and Helicobacter pylori. FEBS J 2020; 287:1865-1885. [PMID: 31679177 PMCID: PMC8189163 DOI: 10.1111/febs.15120] [Citation(s) in RCA: 18] [Impact Index Per Article: 3.6] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/28/2019] [Revised: 08/22/2019] [Accepted: 11/01/2019] [Indexed: 01/12/2023]
Abstract
Rapid adaptation to environmental changes is crucial for bacterial survival. Almost all bacteria possess a conserved stringent response system to prompt transcriptional and metabolic responses toward stress. The adaptive process relies on alarmones, guanosine pentaphosphate (pppGpp), and tetraphosphate (ppGpp), to regulate global gene expression. The ppGpp is more potent than pppGpp in the regulatory activity, and pppGpp phosphohydrolase (GppA) plays a key role in (p)ppGpp homeostasis. Sharing a similar domain structure, GppA is indistinguishable from exopolyphosphatase (PPX), which mediates the metabolism of cellular inorganic polyphosphate. Here, our phylogenetic analysis of PPX/GppA homologs in bacteria shows a wide distribution with several distinct subfamilies, and our structural and functional analysis of Escherichia coli GppA and Helicobacter pylori PPX/GppA reveals unique properties of each homolog. These results explain how each homolog possesses its distinct functionality.
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Affiliation(s)
- He Song
- Macromolecular Crystallography Laboratory, National Cancer Institute, Frederick, MD, USA
| | | | - Chao Wang
- Macromolecular Crystallography Laboratory, National Cancer Institute, Frederick, MD, USA
| | - Gary X Shaw
- Macromolecular Crystallography Laboratory, National Cancer Institute, Frederick, MD, USA
| | - Scott Cherry
- Macromolecular Crystallography Laboratory, National Cancer Institute, Frederick, MD, USA
| | - Joseph E Tropea
- Macromolecular Crystallography Laboratory, National Cancer Institute, Frederick, MD, USA
| | - Ding J Jin
- RNA Biology Laboratory, National Cancer Institute, Frederick, MD, USA
| | - Xinhua Ji
- Macromolecular Crystallography Laboratory, National Cancer Institute, Frederick, MD, USA
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13
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Phosphate Transporter PstSCAB of Campylobacter jejuni Is a Critical Determinant of Lactate-Dependent Growth and Colonization in Chickens. J Bacteriol 2020; 202:JB.00716-19. [PMID: 31932316 DOI: 10.1128/jb.00716-19] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/21/2019] [Accepted: 12/17/2019] [Indexed: 12/17/2022] Open
Abstract
Campylobacter jejuni causes acute gastroenteritis worldwide and is transmitted primarily through poultry, in which it is often a commensal member of the intestinal microbiota. Previous transcriptome sequencing (RNA-Seq) experiment showed that transcripts from an operon encoding a high-affinity phosphate transporter (PstSCAB) of C. jejuni were among the most abundant when the bacterium was grown in chickens. Elevated levels of the pstSCAB mRNA were also identified in an RNA-Seq experiment from human infection studies. In this study, we explore the role of PstSCAB in the biology and colonization potential of C. jejuni Our results demonstrate that cells lacking PstSCAB survive poorly in stationary phase, in nutrient-limiting media, and under osmotic conditions reflective of those in the chicken. Polyphosphate levels in the mutant cells were elevated at stationary phase, consistent with alterations in expression of polyphosphate metabolism genes. The mutant strain was highly attenuated for colonization of newly hatched chicks, with levels of bacteria at several orders of magnitude below wild-type levels. Mutant and wild type grew similarly in complex media, but the pstS::kan mutant exhibited a significant growth defect in minimal medium supplemented with l-lactate, postulated as a carbon source in vivo Poor growth in lactate correlated with diminished expression of acetogenesis pathway genes previously demonstrated as important for colonizing chickens. The phosphate transport system is thus essential for diverse aspects of C. jejuni physiology and in vivo fitness and survival.IMPORTANCE Campylobacter jejuni causes millions of human gastrointestinal infections annually, with poultry a major source of infection. Due to the emergence of multidrug resistance in C. jejuni, there is need to identify alternative ways to control this pathogen. Genes encoding the high-affinity phosphate transporter PstSCAB are highly expressed by C. jejuni in chickens and humans. In this study, we address the role of PstSCAB on chicken colonization and other C. jejuni phenotypes. PstSCAB is required for colonization in chicken, metabolism and survival under different stress responses, and during growth on lactate, a potential growth substrate in chickens. Our study highlights that PstSCAB may be an effective target to develop mechanisms for controlling bacterial burden in both chicken and human.
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14
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Tiwari P, Gosain TP, Singh M, Sankhe GD, Arora G, Kidwai S, Agarwal S, Chugh S, Saini DK, Singh R. Inorganic polyphosphate accumulation suppresses the dormancy response and virulence in Mycobacterium tuberculosis. J Biol Chem 2019; 294:10819-10832. [PMID: 31113860 DOI: 10.1074/jbc.ra119.008370] [Citation(s) in RCA: 11] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/13/2019] [Revised: 05/07/2019] [Indexed: 11/06/2022] Open
Abstract
Stringent response pathways involving inorganic polyphosphate (PolyP) play an essential role in bacterial stress adaptation and virulence. The intracellular levels of PolyP are modulated by the activities of polyphosphate kinase-1 (PPK1), polyphosphate kinase-2 (PPK2), and exopolyphosphatases (PPXs). The genome of Mycobacterium tuberculosis encodes two functional PPXs, and simultaneous deletion of ppx1 and ppx2 results in a defect in biofilm formation. We demonstrate here that these PPXs cumulatively contribute to the ability of M. tuberculosis to survive in nutrient-limiting, low-oxygen growth conditions and also in macrophages. Characterization of single (Δppx2) and double knockout (dkppx) strains of M. tuberculosis indicated that PPX-mediated PolyP degradation is essential for establishing bacterial infection in guinea pigs. RNA-Seq-based transcriptional profiling revealed that relative to the parental strain, the expression levels of DosR regulon-regulated dormancy genes were significantly reduced in the dkppx mutant strain. In concordance, we also provide evidence that PolyP inhibits the autophosphorylation activities associated with DosT and DosS sensor kinases. The results in this study uncover that enzymes involved in PolyP homeostasis play a critical role in M. tuberculosis physiology and virulence and are attractive targets for developing more effective therapeutic interventions.
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Affiliation(s)
- Prabhakar Tiwari
- Tuberculosis Research Laboratory, Vaccine and Infectious Disease Research Centre, Translational Health Science and Technology Institute, Haryana 121001, India and
| | - Tannu Priya Gosain
- Tuberculosis Research Laboratory, Vaccine and Infectious Disease Research Centre, Translational Health Science and Technology Institute, Haryana 121001, India and
| | - Mamta Singh
- Tuberculosis Research Laboratory, Vaccine and Infectious Disease Research Centre, Translational Health Science and Technology Institute, Haryana 121001, India and
| | | | - Garima Arora
- Tuberculosis Research Laboratory, Vaccine and Infectious Disease Research Centre, Translational Health Science and Technology Institute, Haryana 121001, India and
| | - Saqib Kidwai
- Tuberculosis Research Laboratory, Vaccine and Infectious Disease Research Centre, Translational Health Science and Technology Institute, Haryana 121001, India and
| | - Sakshi Agarwal
- Tuberculosis Research Laboratory, Vaccine and Infectious Disease Research Centre, Translational Health Science and Technology Institute, Haryana 121001, India and
| | - Saurabh Chugh
- Tuberculosis Research Laboratory, Vaccine and Infectious Disease Research Centre, Translational Health Science and Technology Institute, Haryana 121001, India and
| | - Deepak K Saini
- Centre for BioSystems Science and Engineering and; Department of Molecular Reproduction, Development, and Genetics, Biological Sciences Building, Indian Institute of Science, Bangalore, Karnataka 560012, India
| | - Ramandeep Singh
- Tuberculosis Research Laboratory, Vaccine and Infectious Disease Research Centre, Translational Health Science and Technology Institute, Haryana 121001, India and.
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15
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Khan MM, Chattagul S, Tran BQ, Freiberg JA, Nita-Lazar A, Shirtliff ME, Sermswan RW, Ernst RK, Goodlett DR. Temporal proteomic profiling reveals changes that support Burkholderia biofilms. Pathog Dis 2019; 77:ftz005. [PMID: 30759239 PMCID: PMC6482045 DOI: 10.1093/femspd/ftz005] [Citation(s) in RCA: 9] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/27/2018] [Accepted: 02/12/2019] [Indexed: 12/14/2022] Open
Abstract
Melioidosis associated with opportunistic pathogen Burkholderia pseudomallei imparts a huge medical burden in Southeast Asia and Australia. At present there is no available human vaccine that protects against B. pseudomallei infection and antibiotic treatments are limited particularly for drug-resistant strains and bacteria in biofilm forms. Biofilm forming bacteria exhibit phenotypic features drastically different to their planktonic states, often exhibiting a diminished response to antimicrobial therapies. Our earlier work on global profiling of bacterial biofilms using transcriptomics and proteomics revealed transcript-decoupled protein abundance in bacterial biofilms. Here we employed reverse phase liquid chromatography tandem mass spectrometry (LC-MS/MS) to deduce temporal proteomic differences in planktonic and biofilm forms of Burkholderia thailandensis, which is weakly surrogate model of pathogenic B. pseudomallei as sharing a key element in genomic similarity. The proteomic analysis of B. thailandensis in biofilm versus planktonic states revealed that proteome changes support biofilm survival through decreased abundance of metabolic proteins while increased abundance of stress-related proteins. Interestingly, the protein abundance including for the transcription protein TEX, outer periplasmic TolB protein, and the exopolyphosphatase reveal adaption in bacterial biofilms that facilitate antibiotic tolerance through a non-specific mechanism. The present proteomics study of B. thailandensis biofilms provides a global snapshot of protein abundance differences and antimicrobial sensitivities in planktonic and sessile bacteria.
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Affiliation(s)
- Mohd M Khan
- University of Maryland School of Medicine, Baltimore, MD 21201, USA
- Laboratory of Immune System Biology (LISB), National Institute of Allergy and Infectious Diseases, National Institutes of Health (NIH), Bethesda, MD 20814, USA
| | - Supaksorn Chattagul
- Melioidosis Research Center, Khon Kaen University, Khon Kaen 40002, Thailand
- Department of Biochemistry, Faculty of Medicine, Khon Kaen University, Khon Kaen 40002, Thailand
- Department of Microbial Pathogenesis, University of Maryland School of Dentistry, Baltimore, MD 21201, USA
| | - Bao Q Tran
- Department of Pharmaceutical Sciences, University of Maryland School of Pharmacy, Baltimore, MD 21201, USA
| | - Jeffrey A Freiberg
- Department of Microbial Pathogenesis, University of Maryland School of Dentistry, Baltimore, MD 21201, USA
| | - Aleksandra Nita-Lazar
- Laboratory of Immune System Biology (LISB), National Institute of Allergy and Infectious Diseases, National Institutes of Health (NIH), Bethesda, MD 20814, USA
| | - Mark E Shirtliff
- Department of Microbial Pathogenesis, University of Maryland School of Dentistry, Baltimore, MD 21201, USA
| | - Rasana W Sermswan
- Melioidosis Research Center, Khon Kaen University, Khon Kaen 40002, Thailand
- Department of Biochemistry, Faculty of Medicine, Khon Kaen University, Khon Kaen 40002, Thailand
| | - Robert K Ernst
- Department of Microbial Pathogenesis, University of Maryland School of Dentistry, Baltimore, MD 21201, USA
| | - David R Goodlett
- Department of Microbial Pathogenesis, University of Maryland School of Dentistry, Baltimore, MD 21201, USA
- International Centre for Cancer Vaccine Science, University of Gdansk, 80-308 Gdańsk, Poland
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16
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Mechanisms of Bacterial Tolerance and Persistence in the Gastrointestinal and Respiratory Environments. Clin Microbiol Rev 2018; 31:31/4/e00023-18. [PMID: 30068737 DOI: 10.1128/cmr.00023-18] [Citation(s) in RCA: 115] [Impact Index Per Article: 16.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/15/2023] Open
Abstract
Pathogens that infect the gastrointestinal and respiratory tracts are subjected to intense pressure due to the environmental conditions of the surroundings. This pressure has led to the development of mechanisms of bacterial tolerance or persistence which enable microorganisms to survive in these locations. In this review, we analyze the general stress response (RpoS mediated), reactive oxygen species (ROS) tolerance, energy metabolism, drug efflux pumps, SOS response, quorum sensing (QS) bacterial communication, (p)ppGpp signaling, and toxin-antitoxin (TA) systems of pathogens, such as Escherichia coli, Salmonella spp., Vibrio spp., Helicobacter spp., Campylobacter jejuni, Enterococcus spp., Shigella spp., Yersinia spp., and Clostridium difficile, all of which inhabit the gastrointestinal tract. The following respiratory tract pathogens are also considered: Staphylococcus aureus, Pseudomonas aeruginosa, Acinetobacter baumannii, Burkholderia cenocepacia, and Mycobacterium tuberculosis Knowledge of the molecular mechanisms regulating the bacterial tolerance and persistence phenotypes is essential in the fight against multiresistant pathogens, as it will enable the identification of new targets for developing innovative anti-infective treatments.
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17
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Murch AL, Skipp PJ, Roach PL, Oyston PCF. Whole genome transcriptomics reveals global effects including up-regulation of Francisella pathogenicity island gene expression during active stringent response in the highly virulent Francisella tularensis subsp. tularensis SCHU S4. MICROBIOLOGY-SGM 2017; 163:1664-1679. [PMID: 29034854 PMCID: PMC5845702 DOI: 10.1099/mic.0.000550] [Citation(s) in RCA: 11] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [Key Words] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Indexed: 01/08/2023]
Abstract
During conditions of nutrient limitation bacteria undergo a series of global gene expression changes to survive conditions of amino acid and fatty acid starvation. Rapid reallocation of cellular resources is brought about by gene expression changes coordinated by the signalling nucleotides' guanosine tetraphosphate or pentaphosphate, collectively termed (p)ppGpp and is known as the stringent response. The stringent response has been implicated in bacterial virulence, with elevated (p)ppGpp levels being associated with increased virulence gene expression. This has been observed in the highly pathogenic Francisella tularensis sub spp. tularensis SCHU S4, the causative agent of tularaemia. Here, we aimed to artificially induce the stringent response by culturing F. tularensis in the presence of the amino acid analogue l-serine hydroxamate. Serine hydroxamate competitively inhibits tRNAser aminoacylation, causing an accumulation of uncharged tRNA. The uncharged tRNA enters the A site on the translating bacterial ribosome and causes ribosome stalling, in turn stimulating the production of (p)ppGpp and activation of the stringent response. Using the essential virulence gene iglC, which is encoded on the Francisella pathogenicity island (FPI) as a marker of active stringent response, we optimized the culture conditions required for the investigation of virulence gene expression under conditions of nutrient limitation. We subsequently used whole genome RNA-seq to show how F. tularensis alters gene expression on a global scale during active stringent response. Key findings included up-regulation of genes involved in virulence, stress responses and metabolism, and down-regulation of genes involved in metabolite transport and cell division. F. tularensis is a highly virulent intracellular pathogen capable of causing debilitating or fatal disease at extremely low infectious doses. However, virulence mechanisms are still poorly understood. The stringent response is widely recognized as a diverse and complex bacterial stress response implicated in virulence. This work describes the global gene expression profile of F. tularensis SCHU S4 under active stringent response for the first time. Herein we provide evidence for an association of active stringent response with FPI virulence gene expression. Our results further the understanding of the molecular basis of virulence and regulation thereof in F. tularensis. These results also support research into genes involved in (p)ppGpp production and polyphosphate biosynthesis and their applicability as targets for novel antimicrobials.
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Affiliation(s)
- Amber L Murch
- CBR Division, Defence Science and Technology Laboratory, Salisbury, UK
| | - Paul J Skipp
- School of Chemistry, University of Southampton, Southampton, UK
| | - Peter L Roach
- School of Chemistry, University of Southampton, Southampton, UK
| | - Petra C F Oyston
- CBR Division, Defence Science and Technology Laboratory, Salisbury, UK
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18
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Girbau C, Martinez-Malaxetxebarria I, Muruaga G, Carmona S, Alonso R, Fernandez-Astorga A. Study of Biofilm Formation Ability of Foodborne Arcobacter butzleri under Different Conditions. J Food Prot 2017; 80:758-762. [PMID: 28358260 DOI: 10.4315/0362-028x.jfp-16-505] [Citation(s) in RCA: 14] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/17/2016] [Accepted: 01/10/2017] [Indexed: 11/11/2022]
Abstract
The transmission of Arcobacter butzleri, an emerging food- and waterborne pathogen, is possibly favored by its ability to adhere to abiotic surfaces. In this study, we assessed the biofilm formation ability of 42 A. butzleri isolates recovered from different food products. Overall, nine isolates (21.4%) were able to adhere to polystyrene. Among them, a chicken-derived isolate was classified as strongly adherent. Based on the chi-square test, no relation was found between the adhesive abilities of the isolates and their source (P > 0.05). An aerobic atmosphere enhanced the adhesion ability of the majority of the adherent isolates (66.7%), because when tested in microaerobic conditions, a t test indicated that only three isolates increased their biofilm formation ability significantly (P < 0.05). In addition, seven (77.8%) of these nine isolates were able to adhere to glass surfaces, and viable cells were recovered from all the stainless steel coupons tested. Therefore, our results confirm the biofilm formation ability of A. butzleri, which may be influenced by the incubation atmosphere and the abiotic surface.
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Affiliation(s)
- Cecilia Girbau
- Department of Immunology, Microbiology and Parasitology, Faculty of Pharmacy, University of the Basque Country (UPV/EHU), C/Paseo de la Universidad 7, 01006, Vitoria-Gasteiz, Spain (ORCID: http://orcid.org/0000-0002-6576-6707 [I.M.-M.])
| | - Irati Martinez-Malaxetxebarria
- Department of Immunology, Microbiology and Parasitology, Faculty of Pharmacy, University of the Basque Country (UPV/EHU), C/Paseo de la Universidad 7, 01006, Vitoria-Gasteiz, Spain (ORCID: http://orcid.org/0000-0002-6576-6707 [I.M.-M.])
| | - Gorka Muruaga
- Department of Immunology, Microbiology and Parasitology, Faculty of Pharmacy, University of the Basque Country (UPV/EHU), C/Paseo de la Universidad 7, 01006, Vitoria-Gasteiz, Spain (ORCID: http://orcid.org/0000-0002-6576-6707 [I.M.-M.])
| | - Sandra Carmona
- Department of Immunology, Microbiology and Parasitology, Faculty of Pharmacy, University of the Basque Country (UPV/EHU), C/Paseo de la Universidad 7, 01006, Vitoria-Gasteiz, Spain (ORCID: http://orcid.org/0000-0002-6576-6707 [I.M.-M.])
| | - Rodrigo Alonso
- Department of Immunology, Microbiology and Parasitology, Faculty of Pharmacy, University of the Basque Country (UPV/EHU), C/Paseo de la Universidad 7, 01006, Vitoria-Gasteiz, Spain (ORCID: http://orcid.org/0000-0002-6576-6707 [I.M.-M.])
| | - Aurora Fernandez-Astorga
- Department of Immunology, Microbiology and Parasitology, Faculty of Pharmacy, University of the Basque Country (UPV/EHU), C/Paseo de la Universidad 7, 01006, Vitoria-Gasteiz, Spain (ORCID: http://orcid.org/0000-0002-6576-6707 [I.M.-M.])
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19
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Stringent Response Factors PPX1 and PPK2 Play an Important Role in Mycobacterium tuberculosis Metabolism, Biofilm Formation, and Sensitivity to Isoniazid In Vivo. Antimicrob Agents Chemother 2016; 60:6460-6470. [PMID: 27527086 DOI: 10.1128/aac.01139-16] [Citation(s) in RCA: 29] [Impact Index Per Article: 3.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/27/2016] [Accepted: 08/09/2016] [Indexed: 01/30/2023] Open
Abstract
Mycobacterium tuberculosis remains a global health threat largely due to the lengthy duration of curative antibiotic treatment, contributing to medical nonadherence and the emergence of drug resistance. This prolonged therapy is likely due to the presence of M. tuberculosis persisters, which exhibit antibiotic tolerance. Inorganic polyphosphate [poly(P)] is a key regulatory molecule in the M. tuberculosis stringent response mediating antibiotic tolerance. The polyphosphate kinase PPK1 is responsible for poly(P) synthesis in M. tuberculosis, while the exopolyphosphatases PPX1 and PPX2 and the GTP synthase PPK2 are responsible for poly(P) hydrolysis. In the present study, we show by liquid chromatography-tandem mass spectrometry that poly(P)-accumulating M. tuberculosis mutant strains deficient in ppx1 or ppk2 had significantly lower intracellular levels of glycerol-3-phosphate (G3P) and 1-deoxy-xylulose-5-phosphate. Real-time PCR revealed decreased expression of genes in the G3P synthesis pathway in each mutant. The ppx1-deficient mutant also showed a significant accumulation of metabolites in the tricarboxylic acid cycle, as well as altered arginine and NADH metabolism. Each poly(P)-accumulating strain showed defective biofilm formation, while deficiency of ppk2 was associated with increased sensitivity to plumbagin and meropenem and deficiency of ppx1 led to enhanced susceptibility to clofazimine. A DNA vaccine expressing ppx1 and ppk2, together with two other members of the M. tuberculosis stringent response, M. tuberculosis rel and sigE, did not show protective activity against aerosol challenge with M. tuberculosis, but vaccine-induced immunity enhanced the killing activity of isoniazid in a murine model of chronic tuberculosis. In summary, poly(P)-regulating factors of the M. tuberculosis stringent response play an important role in M. tuberculosis metabolism, biofilm formation, and antibiotic sensitivity in vivo.
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20
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Kumar A, Gangaiah D, Torrelles JB, Rajashekara G. Polyphosphate and associated enzymes as global regulators of stress response and virulence in Campylobacter jejuni. World J Gastroenterol 2016; 22:7402-7414. [PMID: 27672264 PMCID: PMC5011657 DOI: 10.3748/wjg.v22.i33.7402] [Citation(s) in RCA: 17] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 03/26/2016] [Revised: 06/03/2016] [Accepted: 07/21/2016] [Indexed: 02/06/2023] Open
Abstract
Campylobacter jejuni (C. jejuni), a Gram-negative microaerophilic bacterium, is a predominant cause of bacterial foodborne gastroenteritis in humans worldwide. Despite its importance as a major foodborne pathogen, our understanding of the molecular mechanisms underlying C. jejuni stress survival and pathogenesis is limited. Inorganic polyphosphate (poly P) has been shown to play significant roles in bacterial resistance to stress and virulence in many pathogenic bacteria. C. jejuni contains the complete repertoire of enzymes required for poly P metabolism. Recent work in our laboratory and others have demonstrated that poly P controls a plethora of C. jejuni properties that impact its ability to survive in the environment as well as to colonize/infect mammalian hosts. This review article summarizes the current literature on the role of poly P in C. jejuni stress survival and virulence and discusses on how poly P-related enzymes can be exploited for therapeutic/prevention purposes. Additionally, the review article identifies potential areas for future investigation that would enhance our understanding of the role of poly P in C. jejuni and other bacteria, which ultimately would facilitate design of effective therapeutic/preventive strategies to reduce not only the burden of C. jejuni-caused foodborne infections but also of other bacterial infections in humans.
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21
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Kumar A, Drozd M, Pina-Mimbela R, Xu X, Helmy YA, Antwi J, Fuchs JR, Nislow C, Templeton J, Blackall PJ, Rajashekara G. Novel Anti-Campylobacter Compounds Identified Using High Throughput Screening of a Pre-selected Enriched Small Molecules Library. Front Microbiol 2016; 7:405. [PMID: 27092106 PMCID: PMC4821856 DOI: 10.3389/fmicb.2016.00405] [Citation(s) in RCA: 22] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/28/2015] [Accepted: 03/14/2016] [Indexed: 12/12/2022] Open
Abstract
Campylobacter is a leading cause of foodborne bacterial gastroenteritis worldwide and infections can be fatal. The emergence of antibiotic-resistant Campylobacter spp. necessitates the development of new antimicrobials. We identified novel anti-Campylobacter small molecule inhibitors using a high throughput growth inhibition assay. To expedite screening, we made use of a "bioactive" library of 4182 compounds that we have previously shown to be active against diverse microbes. Screening for growth inhibition of Campylobacter jejuni, identified 781 compounds that were either bactericidal or bacteriostatic at a concentration of 200 μM. Seventy nine of the bactericidal compounds were prioritized for secondary screening based on their physico-chemical properties. Based on the minimum inhibitory concentration against a diverse range of C. jejuni and a lack of effect on gut microbes, we selected 12 compounds. No resistance was observed to any of these 12 lead compounds when C. jejuni was cultured with lethal or sub-lethal concentrations suggesting that C. jejuni is less likely to develop resistance to these compounds. Top 12 compounds also possessed low cytotoxicity to human intestinal epithelial cells (Caco-2 cells) and no hemolytic activity against sheep red blood cells. Next, these 12 compounds were evaluated for ability to clear C. jejuni in vitro. A total of 10 compounds had an anti-C. jejuni effect in Caco-2 cells with some effective even at 25 μM concentrations. These novel 12 compounds belong to five established antimicrobial chemical classes; piperazines, aryl amines, piperidines, sulfonamide, and pyridazinone. Exploitation of analogs of these chemical classes may provide Campylobacter specific drugs that can be applied in both human and animal medicine.
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Affiliation(s)
- Anand Kumar
- Food Animal Health Research Program, Department of Veterinary Preventive Medicine, The Ohio State UniversityWooster, OH, USA; Poultry CRC, University of New EnglandArmidale, NSW, Australia
| | - Mary Drozd
- Food Animal Health Research Program, Department of Veterinary Preventive Medicine, The Ohio State UniversityWooster, OH, USA; Poultry CRC, University of New EnglandArmidale, NSW, Australia
| | - Ruby Pina-Mimbela
- Food Animal Health Research Program, Department of Veterinary Preventive Medicine, The Ohio State University Wooster, OH, USA
| | - Xiulan Xu
- Department of Plant Pathology, Ohio Agricultural Research and Development Center, The Ohio State University Wooster, OH, USA
| | - Yosra A Helmy
- Food Animal Health Research Program, Department of Veterinary Preventive Medicine, The Ohio State University Wooster, OH, USA
| | - Janet Antwi
- College of Pharmacy, The Ohio State University Columbus, OH, USA
| | - James R Fuchs
- College of Pharmacy, The Ohio State University Columbus, OH, USA
| | - Corey Nislow
- Pharmaceutical Sciences, The University of British Columbia Vancouver, BC, Canada
| | - Jillian Templeton
- Department of Agriculture and Fisheries, EcoSciences Precinct Dutton Park, QLD, Australia
| | - Patrick J Blackall
- Poultry CRC, University of New EnglandArmidale, NSW, Australia; Queensland Alliance for Agriculture and Food Innovation, The University of Queensland, EcoSciences PrecinctDutton Park, QLD, Australia
| | - Gireesh Rajashekara
- Food Animal Health Research Program, Department of Veterinary Preventive Medicine, The Ohio State University Wooster, OH, USA
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22
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Zhang A, Guo E, Qian L, Tang NY, Watt RM, Bartlam M. Purification, crystallization and X-ray crystallographic analysis of a putative exopolyphosphatase from Zymomonas mobilis. ACTA CRYSTALLOGRAPHICA SECTION F-STRUCTURAL BIOLOGY COMMUNICATIONS 2016; 72:172-8. [PMID: 26919520 DOI: 10.1107/s2053230x16000753] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Subscribe] [Scholar Register] [Received: 11/04/2015] [Accepted: 01/14/2016] [Indexed: 11/11/2022]
Abstract
Exopolyphosphatase (PPX) enzymes degrade inorganic polyphosphate (poly-P), which is essential for the survival of microbial cells in response to external stresses. In this study, a putative exopolyphosphatase from Zymomonas mobilis (ZmPPX) was crystallized. Crystals of the wild-type enzyme diffracted to 3.3 Å resolution and could not be optimized further. The truncation of 29 amino acids from the N-terminus resulted in crystals that diffracted to 1.8 Å resolution. The crystals belonged to space group C2, with unit-cell parameters a = 122.0, b = 47.1, c = 89.5 Å, α = γ = 90, β = 124.5°. An active-site mutant that crystallized in the same space group and with similar unit-cell parameters diffracted to 1.56 Å resolution. One molecule was identified per asymmetric unit. Analytical ultracentrifugation confirmed that ZmPPX forms a dimer in solution. It was confirmed that ZmPPX possesses exopolyphosphatase activity against a synthetic poly-P substrate.
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Affiliation(s)
- Aili Zhang
- College of Life Sciences, Nankai University, People's Republic of China
| | - Erhong Guo
- College of Life Sciences, Nankai University, People's Republic of China
| | - Lanfang Qian
- College of Life Sciences, Nankai University, People's Republic of China
| | - Nga-Yeung Tang
- Faculty of Dentistry, University of Hong Kong, Hong Kong
| | - Rory M Watt
- Faculty of Dentistry, University of Hong Kong, Hong Kong
| | - Mark Bartlam
- College of Life Sciences, Nankai University, People's Republic of China
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23
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Polyphosphate kinases modulate Campylobacter jejuni outer membrane constituents and alter its capacity to invade and survive in intestinal epithelial cells in vitro. Emerg Microbes Infect 2015; 4:e77. [PMID: 26714783 PMCID: PMC4715166 DOI: 10.1038/emi.2015.77] [Citation(s) in RCA: 10] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/06/2015] [Revised: 09/28/2015] [Accepted: 10/01/2015] [Indexed: 12/12/2022]
Abstract
Campylobacter jejuni is the most prevalent cause of bacterial gastroenteritis worldwide. Polyphosphate kinases 1 and 2 (PPK1 and PPK2) regulate several cellular processes, including the biosynthesis of the bacterial cell wall. Despite their importance, whether PPK1 and PPK2 modulate the composition of C. jejuni outer membrane constituents (OMCs) and consequently impact its interaction with host cells remains unknown. Our comparative analysis between C. jejuni wild type, Δppk1, and Δppk2 strains showed qualitative and quantitative differences in the total OMC composition among these strains. Importantly, these OMC variations observed on the C. jejuni polyphosphate kinase mutants are directly related to their capacity to invade, survive, and alter the immune response of intestinal epithelial cells in vitro. Specifically, sub-fractionation of the C. jejuni OMC indicated that OMC proteins are uniquely associated with bacterial invasion, whereas C. jejuni OMC proteins, lipids, and lipoglycans are all associated with C. jejuni intracellular survival. This study provides new insights regarding the function of polyphosphate kinases and their role in C. jejuni infection.
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24
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Chandrashekhar K, Kassem II, Nislow C, Gangaiah D, Candelero-Rueda RA, Rajashekara G. Transcriptome analysis of Campylobacter jejuni polyphosphate kinase (ppk1 and ppk2) mutants. Virulence 2015; 6:814-8. [PMID: 26537695 DOI: 10.1080/21505594.2015.1104449] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/12/2022] Open
Affiliation(s)
- Kshipra Chandrashekhar
- a Food Animal Health Research Program; Ohio Agricultural Research and Development Center; Department of Veterinary Preventive Medicine ; The Ohio State University ; Wooster , OH , USA
| | - Issmat I Kassem
- a Food Animal Health Research Program; Ohio Agricultural Research and Development Center; Department of Veterinary Preventive Medicine ; The Ohio State University ; Wooster , OH , USA
| | - Corey Nislow
- b Department of Pharmaceutical Sciences ; University of British Columbia ; Vancouver , BC Canada
| | - Dharanesh Gangaiah
- a Food Animal Health Research Program; Ohio Agricultural Research and Development Center; Department of Veterinary Preventive Medicine ; The Ohio State University ; Wooster , OH , USA
| | - Rosario A Candelero-Rueda
- a Food Animal Health Research Program; Ohio Agricultural Research and Development Center; Department of Veterinary Preventive Medicine ; The Ohio State University ; Wooster , OH , USA
| | - Gireesh Rajashekara
- a Food Animal Health Research Program; Ohio Agricultural Research and Development Center; Department of Veterinary Preventive Medicine ; The Ohio State University ; Wooster , OH , USA
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25
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Pascoe B, Méric G, Murray S, Yahara K, Mageiros L, Bowen R, Jones NH, Jeeves RE, Lappin-Scott HM, Asakura H, Sheppard SK. Enhanced biofilm formation and multi-host transmission evolve from divergent genetic backgrounds in Campylobacter jejuni. Environ Microbiol 2015; 17:4779-89. [PMID: 26373338 PMCID: PMC4862030 DOI: 10.1111/1462-2920.13051] [Citation(s) in RCA: 58] [Impact Index Per Article: 5.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/07/2015] [Revised: 09/07/2015] [Accepted: 09/08/2015] [Indexed: 02/01/2023]
Abstract
Multicellular biofilms are an ancient bacterial adaptation that offers a protective environment for survival in hostile habitats. In microaerophilic organisms such as Campylobacter, biofilms play a key role in transmission to humans as the bacteria are exposed to atmospheric oxygen concentrations when leaving the reservoir host gut. Genetic determinants of biofilm formation differ between species, but little is known about how strains of the same species achieve the biofilm phenotype with different genetic backgrounds. Our approach combines genome‐wide association studies with traditional microbiology techniques to investigate the genetic basis of biofilm formation in 102 Campylobacter jejuni isolates. We quantified biofilm formation among the isolates and identified hotspots of genetic variation in homologous sequences that correspond to variation in biofilm phenotypes. Thirteen genes demonstrated a statistically robust association including those involved in adhesion, motility, glycosylation, capsule production and oxidative stress. The genes associated with biofilm formation were different in the host generalist ST‐21 and ST‐45 clonal complexes, which are frequently isolated from multiple host species and clinical samples. This suggests the evolution of enhanced biofilm from different genetic backgrounds and a possible role in colonization of multiple hosts and transmission to humans.
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Affiliation(s)
- Ben Pascoe
- College of Medicine, Institute of Life Science, Swansea University, Swansea, UK.,MRC CLIMB Consortium, Institute of Life Science, Swansea University, Swansea, UK
| | - Guillaume Méric
- College of Medicine, Institute of Life Science, Swansea University, Swansea, UK
| | - Susan Murray
- College of Medicine, Institute of Life Science, Swansea University, Swansea, UK
| | - Koji Yahara
- College of Medicine, Institute of Life Science, Swansea University, Swansea, UK.,Institute of Medical Science, University of Tokyo, Tokyo, Japan
| | - Leonardos Mageiros
- College of Medicine, Institute of Life Science, Swansea University, Swansea, UK
| | - Ryan Bowen
- College of Medicine, Institute of Life Science, Swansea University, Swansea, UK
| | - Nathan H Jones
- College of Medicine, Institute of Life Science, Swansea University, Swansea, UK
| | - Rose E Jeeves
- College of Medicine, Institute of Life Science, Swansea University, Swansea, UK
| | | | - Hiroshi Asakura
- Division of Biomedical Food Research, National Institute of Health Sciences, Tokyo, Japan
| | - Samuel K Sheppard
- College of Medicine, Institute of Life Science, Swansea University, Swansea, UK.,MRC CLIMB Consortium, Institute of Life Science, Swansea University, Swansea, UK.,Department of Zoology, University of Oxford, Oxford, UK
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26
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Affiliation(s)
- Byeonghwa Jeon
- School of Public Health; University of Alberta; Edmonton, AB Canada
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