Stringent response governs the oxidative stress resistance and virulence of Francisella tularensis

Autoři: Zhuo Ma aff001;  Kayla King aff001;  Maha Alqahtani aff002;  Madeline Worden aff001;  Parthasarathy Muthuraman aff001;  Christopher L. Cioffi aff001;  Chandra Shekhar Bakshi aff002;  Meenakshi Malik aff001
Působiště autorů: Department of Basic and Clinical Sciences, Albany College of Pharmacy and Health Sciences, Albany, New York, United States of America aff001;  Department of Microbiology and Immunology, New York Medical College, Valhalla, New York, United States of America aff002
Vyšlo v časopise: PLoS ONE 14(10)
Kategorie: Research Article
doi: 10.1371/journal.pone.0224094


Francisella tularensis is a Gram-negative bacterium responsible for causing tularemia in the northern hemisphere. F. tularensis has long been developed as a biological weapon due to its ability to cause severe illness upon inhalation of as few as ten organisms and, based on its potential to be used as a bioterror agent is now classified as a Tier 1 Category A select agent by the CDC. The stringent response facilitates bacterial survival under nutritionally challenging starvation conditions. The hallmark of stringent response is the accumulation of the effector molecules ppGpp and (p)ppGpp known as stress alarmones. The relA and spoT gene products generate alarmones in several Gram-negative bacterial pathogens. RelA is a ribosome-associated ppGpp synthetase that gets activated under amino acid starvation conditions whereas, SpoT is a bifunctional enzyme with both ppGpp synthetase and ppGpp hydrolase activities. Francisella encodes a monofunctional RelA and a bifunctional SpoT enzyme. Previous studies have demonstrated that stringent response under nutritional stresses increases expression of virulence-associated genes encoded on Francisella Pathogenicity Island. This study investigated how stringent response governs the oxidative stress response of F. tularensis. We demonstrate that RelA/SpoT-mediated ppGpp production alters global gene transcriptional profile of F. tularensis in the presence of oxidative stress. The lack of stringent response in relA/spoT gene deletion mutants of F. tularensis makes bacteria more susceptible to oxidants, attenuates survival in macrophages, and virulence in mice. This work is an important step forward towards understanding the complex regulatory network underlying the oxidative stress response of F. tularensis.

Klíčová slova:

Antioxidants – Gene expression – Gene regulation – Oxidative stress – Regulator genes – Transcriptional control – Francisella tularensis – Francisella


1. Pechous RD, McCarthy TR, Zahrt TC. Working toward the future: insights into Francisella tularensis pathogenesis and vaccine development. Microbiol Mol Biol Rev. 2009;73: 684–711. doi: 10.1128/MMBR.00028-09 19946137

2. Eisen L. Francisella tularensis : Biology, Pathogenicity, Epidemiology, and Biodefense. Emerg Infect Dis. 2007;13: 1973–1973. doi: 10.3201/eid1312.071169

3. Hirschmann JV. From Squirrels to Biological Weapons: The Early History of Tularemia. Am J Med Sci. 2018;356: 319–328. doi: 10.1016/j.amjms.2018.06.006 30146078

4. Kingry LC, Petersen JM. Comparative review of Francisella tularensis and Francisella novicida. Front Cell Infect Microbiol. 2014;4: 35. doi: 10.3389/fcimb.2014.00035 24660164

5. Santic M, Molmeret M, Klose KE, Abu Kwaik Y. Francisella tularensis travels a novel, twisted road within macrophages. Trends in Microbiology. 2006. pp. 37–44. doi: 10.1016/j.tim.2005.11.008 16356719

6. Hall JD, Woolard MD, Gunn BM, Craven RR, Taft-Benz S, Frelinger JA, et al. Infected-host-cell repertoire and cellular response in the lung following inhalation of Francisella tularensis Schu S4, LVS, or U112. Infect Immun. 2008; doi: 10.1128/IAI.01176-08 18852251

7. Bakshi CS, Malik M, Regan K, Melendez JA, Metzger DW, Pavlov VM, et al. Superoxide dismutase B gene (sodB)-deficient mutants of Francisella tularensis demonstrate hypersensitivity to oxidative stress and attenuated virulence. J Bacteriol. Center for Immunology and Microbial Disease, Albany Medical College, 47 New Scotland Avenue, Albany, NY 12208–3479, USA; 2006;188: 6443–6448. doi: 10.1128/JB.00266-06 16923916

8. Bakshi CS, Mahawar M, Melendez JA, Sellati TJ, Malik M, Metzger DW, et al. Identification of Francisella tularensis Live Vaccine Strain CuZn Superoxide Dismutase as Critical for Resistance to Extracellularly Generated Reactive Oxygen Species. J Bacteriol. Center for Immunology and Microbial Disease, MC 151, Albany Medical College, 47 New Scotland Ave., Albany, NY 12208, USA; 2009;191: 6447–6456. doi: 10.1128/JB.00534-09 19684141

9. Lindgren H, Shen H, Zingmark C, Golovliov I, Conlan W, Sjöstedt A. Resistance of Francisella tularensis strains against reactive nitrogen and oxygen species with special reference to the role of KatG. Infect Immun. Department of Clinical Microbiology, Clinical Bacteriology, Umea University, SE-901 85 Umea, Sweden.; 2007;75: 1303–1309. doi: 10.1128/IAI.01717-06 17210667

10. Alharbi A, Rabadi SM, Alqahtani M, Marghani D, Worden M, Ma Z, et al. Role of peroxiredoxin of the AhpC/TSA family in antioxidant defense mechanisms of Francisella tularensis. PLoS One. 2019; doi: 10.1371/journal.pone.0213699 30870480

11. Malik M, Bakshi CS, Jen Y, Rabadi SM, Catlett S V., Ma Z, et al. Elucidation of a mechanism of oxidative stress regulation in Francisella tularensis live vaccine strain. Mol Microbiol. 2016;101: 856–878. doi: 10.1111/mmi.13426 27205902

12. Clemens DL, Lee BY, Horwitz MA. Francisella tularensis enters macrophages via a novel process involving pseudopod loops. Infect Immun. Division of Infectious Diseases, Dept. of Medicine, UCLA School of Medicine, CHS 37–121, 10833 LeConte Ave., Los Angeles, CA 90095–1688, USA.; 2005;73: 5892–5902. doi: 10.1128/IAI.73.9.5892-5902.2005 16113308

13. Ma Z, Banik S, Rane H, Mora VT, Rabadi SM, Doyle CR, et al. EmrA1 membrane fusion protein of F rancisella tularensis LVS is required for resistance to oxidative stress, intramacrophage survival and virulence in mice. Mol Microbiol. Department of Basic and Social Sciences, Albany College of Pharmacy and Health Sciences, Albany, New York, USA; 2014;91: 976–995. doi: 10.1111/mmi.12509 24397487

14. Wehrly TD, Chong A, Virtaneva K, Sturdevant DE, Child R, Edwards JA, et al. Intracellular biology and virulence determinants of Francisella tularensis revealed by transcriptional profiling inside macrophages. Cell Microbiol. 2009;11: 1128–1150. doi: 10.1111/j.1462-5822.2009.01316.x 19388904

15. Potrykus K, Cashel M. (p)ppGpp: Still Magical? Annu Rev Microbiol. 2008; doi: 10.1146/annurev.micro.62.081307.162903 18454629

16. Cashel M, Potrykus K. Stringent Response. Brenner’s Encyclopedia of Genetics: Second Edition. 2013. doi: 10.1016/B978-0-12-374984-0.01486-8

17. Dean RE, Ireland PM, Jordan JE, Titball RW, Oyston PCF. RelA regulates virulence and intracellular survival of Francisella novicida. Microbiology. 2009;155: 4104–4113. doi: 10.1099/mic.0.031021-0 19762448

18. Charity JC, Blalock LT, Costante-Hamm MM, Kasper DL, Dove SL. Small molecule control of virulence gene expression in Francisella tularensis. PLoS Pathog. 2009;5: e1000641. doi: 10.1371/journal.ppat.1000641 19876386

19. Faron M, Fletcher JR, Rasmussen JA, Long ME, Allen L-AH, Jones BD. The Francisella tularensis migR, trmE, and cphA Genes Contribute to F. tularensis Pathogenicity Island Gene Regulation and Intracellular Growth by Modulation of the Stress Alarmone ppGpp. Infect Immun. 2013; doi: 10.1128/iai.00073-13 23716606

20. 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. 2017;163: 1664–1679. doi: 10.1099/mic.0.000550 29034854

21. Lovullo ED, Sherrill LA, Pavelka MS. Improved shuttle vectors for Francisella tularensis genetics: Research Letter. FEMS Microbiol Lett. 2009;291: 95–102. doi: 10.1111/j.1574-6968.2008.01440.x 19067747

22. Alqahtani M, Ma Z, Ketkar H, Suresh RV, Malik M, Bakshi CS. Characterization of a Unique Outer Membrane Protein Required for Oxidative Stress Resistance and Virulence of Francisella tularensis. J Bacteriol. 2018; JB.00693-17. doi: 10.1128/JB.00693-17 29378894

23. Malik M, Bakshi CS, Jen Y, Rabadi SM, Catlett S V., Ma Z, et al. Elucidation of a mechanism of oxidative stress regulation in Francisella tularensis live vaccine strain. Mol Microbiol. 2016;101: 856–878. doi: 10.1111/mmi.13426 27205902

24. Lauriano CM, Barker JR, Yoon S-S, Nano FE, Arulanandam BP, Hassett DJ, et al. MglA regulates transcription of virulence factors necessary for Francisella tularensis intraamoebae and intramacrophage survival. Proc Natl Acad Sci U S A. 2004;101: 4246–9. doi: 10.1073/pnas.0307690101 15010524

25. Battesti A, Bouveret E. Acyl carrier protein/SpoT interaction, the switch linking SpoT-dependent stress response to fatty acid metabolism. Mol Microbiol. 2006; doi: 10.1111/j.1365-2958.2006.05442.x 17078815

26. Battesti A, Bouveret E. Bacteria possessing two RelA/SpoT-like proteins have evolved a specific stringent response involving The acyl carrier protein-SpoT interaction. J Bacteriol. 2009; doi: 10.1128/JB.01195-08 18996989

27. Kim J-S, Liu L, Fitzsimmons LF, Wang Y, Crawford MA, Mastrogiovanni M, et al. DksA–DnaJ redox interactions provide a signal for the activation of bacterial RNA polymerase. Proc Natl Acad Sci. 2018; doi: 10.1073/pnas.1813572115 30429329

28. Guina T, Radulovic D, Bahrami AJ, Bolton DL, Rohmer L, Jones-Isaac KA, et al. MglA regulates Francisella tularensis subsp. novicida (Francisella novicida) response to starvation and oxidative stress. J Bacteriol. 2007;189: 6580–6586. doi: 10.1128/JB.00809-07 17644593

29. Brotcke A, Weiss DS, Kim CC, Chain P, Malfatti S, Garcia E, et al. Identification of MglA-regulated genes reveals novel virulence factors in Francisella tularensis. Infect Immun. 2006;74: 6642–6655. doi: 10.1128/IAI.01250-06 17000729

30. Brotcke A, Monack DM. Identification of fevR, a novel regulator of virulence gene expression in Francisella novicida. Infect Immun. 2008;76: 3473–3480. doi: 10.1128/IAI.00430-08 18559431

31. Gaca AO, Abranches J, Kajfasz JK, Lemos JA. Global transcriptional analysis of the stringent response in Enterococcus faecalis. Microbiology. 2012;158: 1994–2004. doi: 10.1099/mic.0.060236-0 22653948

32. Reiß S, Pané-Farré J, Fuchs S, François P, Liebeke M, Schrenzel J, et al. Global Analysis of the Staphylococcus aureus Response to Mupirocin. Antimicrob Agents Chemother. 2012;56: 787–804. doi: 10.1128/AAC.05363-11 22106209

33. Kazmierczak KM, Wayne KJ, Rechtsteiner A, Winkler ME. Roles of rel Spn in stringent response, global regulation and virulence of serotype 2 Streptococcus pneumoniae D39. Mol Microbiol. 2009;72: 590–611. doi: 10.1111/j.1365-2958.2009.06669.x 19426208

34. Nascimento MM, Lemos JA, Abranches J, Lin VK, Burne RA. Role of RelA of Streptococcus mutans in Global Control of Gene Expression. J Bacteriol. 2008;190: 28–36. doi: 10.1128/JB.01395-07 17951382

35. Nguyen D, Joshi-Datar A, Lepine F, Bauerle E, Olakanmi O, Beer K, et al. Active starvation responses mediate antibiotic tolerance in biofilms and nutrient-limited bacteria. Science (80-). 2011; doi: 10.1126/science.1211037 22096200

36. Kohanski MA, Dwyer DJ, Hayete B, Lawrence CA, Collins JJ. A Common Mechanism of Cellular Death Induced by Bactericidal Antibiotics. Cell. 2007;130: 797–810. doi: 10.1016/j.cell.2007.06.049 17803904

37. Khakimova M, Ahlgren HG, Harrison JJ, English AM, Nguyen D. The stringent response controls catalases in Pseudomonas aeruginosa and is required for hydrogen peroxide and antibiotic tolerance. J Bacteriol. 2013; doi: 10.1128/JB.02061-12 23457248

38. Martins D, McKay G, Sampathkumar G, Khakimova M, English AM, Nguyen D. Superoxide dismutase activity confers (p)ppGpp-mediated antibiotic tolerance to stationary-phase Pseudomonas aeruginosa. Proc Natl Acad Sci. 2018;115: 9797–9802. doi: 10.1073/pnas.1804525115 30201715

39. Potamitou A, Neubauer P, Holmgren A, Vlamis-Gardikas A. Expression of Escherichia coli Glutaredoxin 2 Is Mainly Regulated by ppGpp and ς S. J Biol Chem. 2002;277: 17775–17780. doi: 10.1074/jbc.M201306200 11889138

Článek vyšel v časopise


2019 Číslo 10