Characterization of an Extracytoplasmic Chaperone Spy in Protecting Salmonella against Reactive Oxygen/Nitrogen Species
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Antimicrobial actions of reactive oxygen/nitrogen species (ROS/RNS) derived from products of NADPH oxidase and inducible nitric oxide (NO) synthase in host phagocytes inactivate various bacterial macromolecules. To cope with these cytotoxic radicals, pathogenic bacteria have evolved to conserve systems necessary for detoxifying ROS/RNS and repairing damages caused by their actions. In response to these stresses, bacteria also induce expression of molecular chaperones to aid in ameliorating protein misfolding. In this study, we explored the function of a newly identified chaperone Spy, that is localized exclusively in the periplasm when bacteria exposed to conditions causing spheroplast formation, in the resistance of Salmonella Typhimurium to ROS/RNS. A spy deletion mutant was constructed in S. Typhimurium by a PCR-mediated method of one-step gene inactivation with λ Red recombinase, and subjected to ROS/RNS stresses. The spy mutant Salmonella showed a modest decrease in growth rate in NO-producing cultures, and no detectable difference of growth rate in H2O2 containing cultures, compared with that of wild type Salmonella. Quantitative RT-PCR analysis showed that spy mRNA levels were similar regardless of both stresses, but were increased considerably in Salmonella mutants lacking the flavohemoglobin Hmp, which are incapable of NO detoxification, and lacking an alternative sigma factor RpoS, conferring hypersusceptibility to H2O2. Results demonstrate that Spy expression can be induced under extreme conditions of both stresses, and suggest that the protein may have supportive roles in maintaining proteostasis in the periplasm where various chaperones may act in concert with Spy, thereby protecting bacteria against toxicities of ROS/RNS.
Abstract
Antimicrobial actions of reactive oxygen/nitrogen species (ROS/RNS) derived from products of NADPH oxidase and inducible nitric oxide (NO) synthase in host phagocytes inactivate various bacterial macromolecules. To cope with these cytotoxic radicals, pathogenic bacteria have evolved to conserve systems necessary for detoxifying ROS/RNS and repairing damages caused by their actions. In response to these stresses, bacteria also induce expression of molecular chaperones to aid in ameliorating protein misfolding. In this study, we explored the function of a newly identified chaperone Spy, that is localized exclusively in the periplasm when bacteria exposed to conditions causing spheroplast formation, in the resistance of Salmonella Typhimurium to ROS/RNS. A spy deletion mutant was constructed in S. Typhimurium by a PCR-mediated method of one-step gene inactivation with λ Red recombinase, and subjected to ROS/RNS stresses. The spy mutant Salmonella showed a modest decrease in growth rate in NO-producing cultures, and no detectable difference of growth rate in H2O2 containing cultures, compared with that of wild type Salmonella. Quantitative RT-PCR analysis showed that spy mRNA levels were similar regardless of both stresses, but were increased considerably in Salmonella mutants lacking the flavohemoglobin Hmp, which are incapable of NO detoxification, and lacking an alternative sigma factor RpoS, conferring hypersusceptibility to H2O2. Results demonstrate that Spy expression can be induced under extreme conditions of both stresses, and suggest that the protein may have supportive roles in maintaining proteostasis in the periplasm where various chaperones may act in concert with Spy, thereby protecting bacteria against toxicities of ROS/RNS.
참고문헌 (33)
- Grassl GA, "Pathogenesis of enteric Salmonella infections", Curr Opin Gastroenterol, 24, pp.22~26 (2008)
- Maloy S, "salmonella.org" (1999), San Diego State University
- Hurley D, "Salmonella-host interactions-modulation of the host innate immune system", Front Immunol, 5, pp.481~ (2014)
- Fang FC, "Antimicrobial reactive oxygen and nitrogen species : concepts and controversies", Nature reviews Microbiology, 2, pp.820~832 (2004)
- Vazquez-Torres A, "Oxygen-dependent anti-Salmonella activity of macrophages", Trends Microbiol, 9, pp.29~33 (2001)
- Fang FC, "Virulent Salmonella typhimurium has two periplasmic Cu, Zn-superoxide dismutases", Proc Natl Acad Sci U S A, 96, pp.7502~7507 (1999)
- Hebrard M, "Redundant hydrogen peroxide scavengers contribute to Salmonella virulence and oxidative stress resistance", J Bacteriol, 191, pp.4605~4614 (2009)
- Bang IS, "Maintenance of nitric oxide and redox homeostasis by the salmonella flavohemoglobin hmp", The Journal of biological chemistry, 281, pp.28039~28047 (2006)
- superoxide dismutases SodCI and SodCII in intracellular bacteria : correlation with their relative contribution to pathogenicity, "Mol Microbiol", 2002, 147~156
- Forrester MT, "Protection from nitrosative stress : a central role for microbial flavohemoglobin", Free Radic Biol Med, 52, pp.1620~1633 (2012)
- Dong T, "Role of RpoS in virulence of pathogens", Infect Immun, 78, pp.887~897 (2010)
- Karlinsey JE, "The NsrR regulon in nitrosative stress resistance of Salmonella enterica serovar Typhimurium", Mol Microbiol, 85, pp.1179~1193 (2012)
- Shah DH, "RNA sequencing reveals differences between the global transcriptomes of Salmonella enterica serovar enteritidis strains with high and low pathogenicities", Appl Environ Microbiol, 80, pp.896~906 (2014)
- Lahiri A, "The LysR-type transcriptional regulator Hrg counteracts phagocyte oxidative burst and imparts survival advantage to Salmonella enterica serovar Typhimurium", Microbiology, 154, pp.2837~2846 (2008)
- Veinger L, "The small heat-shock protein IbpB from Escherichia coli stabilizes stress-denatured proteins for subsequent refolding by a multichaperone network", J Biol Chem, 273, pp.11032~11037 (1998)
- Fredriksson A, "Defense against protein carbonylation by DnaK", DnaJ and proteases of the heat shock regulon, 2005, pp.187, 4207~4213
- Takaya A, "The DnaK", DnaJ chaperone machinery of Salmonella enterica serovar Typhimurium is essential for invasion of epithelial cells and survival within macrophages, leading to systemic infection, 2004, pp.72, 1364~1373
- Quan S, "Genetic selection designed to stabilize proteins uncovers a chaperone called Spy", Nat Struct Mol Biol, 18, pp.262~269 (2011)
- Datsenko KA, "One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products", Proc Natl Acad Sci U S A, 97, pp.6640~6645 (2000)
- Chiang SM, "Regulators of oxidative stress response genes in Escherichia coli and their functional conservation in bacteria", Arch Biochem Biophys, 525, pp.161~169 (2012)
- Buchmeier NA, "Induction of Salmonella stress proteins upon infection of macrophages", Science, 248, pp.730~732 (1990)
- Slonczewski JL, "Microbiology: An Evolving Science" (2009), W. W. Norton & Company, Inc
- Pugsley AP, "Getting out : protein traffic in prokaryotes", Mol Microbiol, 52, pp.3~11 (2004)
- Ramos JL, "Responses of Gram-negative bacteria to certain environmental stressors", Curr Opin Microbiol, 4, pp.166~171 (2001)
- Rowley G, "Pushing the envelope : extracytoplasmic stress responses in bacterial pathogens", Nature reviews Microbiology, 4, pp.383~394 (2006)
- Henderson B, "Stress wars : the direct role of host and bacterial molecular chaperones in bacterial infection", Infect Immun, 74, pp.3693~3706 (2006)
- Burkinshaw BJ, "Assembly and structure of the T3SS", Biochim Biophys Acta, 1843, pp.1649~1663 (2014)
- Hagenmaier S, "A new periplasmic protein of Escherichia coli which is synthesized in spheroplasts but not in intact cells", J Bacteriol, 179, pp.2073~2076 (1997)
- Raivio TL, "Tethering of CpxP to the inner membrane prevents spheroplast induction of the cpx envelope stress response", Mol Microbiol, 37, pp.1186~1197 (2000)
- Appia-Ayme C, "ZraP is a periplasmic molecular chaperone and a repressor of the zinc-responsive two-component regulator ZraSR", Biochem J, 442, pp.85~93 (2012)
- Bang IS, "Alternative sigma factor interactions in Salmonella : sigma and sigma promote antioxidant defences by enhancing sigma levels", Mol Microbiol, 56, pp.811~823 (2005)
- Testerman TL, "The alternative sigma factor sigmaE controls antioxidant defences required for Salmonella virulence and stationary-phase survival", Mol Microbiol, 43, pp.771~782 (2002)
- Lee IS, "The stationary-phase sigma factor sigma S(RpoS)is required for a sustained acid tolerance response in virulent Salmonella typhimurium", Mol Microbiol, 17, pp.155~167 (1995)