Skip to content
1887

Microbiology Society logo Microbiology

Volume 171, Issue 4

Transcription factors DksA and PsrA are synergistic contributors to virulence in protozoa Open Access

Abstract

The environmental bacterium , an intracellular parasite of free-living freshwater protozoa as well as an opportunistic human pathogen, has a biphasic lifestyle. The switch from the vegetative replicative form to the environmentally resilient transmissive phase form is governed by a complex stringent response-based regulatory network that includes RNA polymerase co-factor DksA. Here, we report that, through a dysfunctional DksA mutation (DksA1), a synergistic interplay was discovered between DksA and transcription regulator PsrA using the protozoan infection model. Surprisingly, expression of PsrA partially rescued the growth defect of a strain. Whilst expression of DksA expectantly could fully rescue the growth defect of the strain, it could also surprisingly rescue the growth defect of a Δ strain. Conversely, the severe intracellular growth defect of a Δ strain could be rescued by expression of DksA and DksA1, but not PsrA. phenotypic assays show that either DksA or DksA1 was required for extended culturability of bacterial cells, but normal cell morphology and pigmentation required DksA only. Comparative structural modelling predicts that the DksA1 mutation affects the coordination of Mg into the active site of RNAP, compromising transcription efficiency. Taken together, we propose that PsrA transcriptionally assists DksA in the expression of select transmissive phase traits. Additionally, evidence suggests that the long-chain fatty acid metabolic response is mediated by PsrA together with DksA, inferring a novel regulatory link to the stringent response pathway.

  • Received:
  • Accepted:
  • Published Online:
Keyword(s): biphasic lifestyle , host-microbe interaction , Legionella pneumophila , regulatory network , stringent response and transcription factor
Funding
This study was supported by the:
  • Natural Sciences and Engineering Research Council of Canada (Award 2018-04968)
    • Principle Award Recipient: GerdPrehna
  • Natural Sciences and Engineering Research Council of Canada (Award 2017-04970)
    • Principle Award Recipient: Teresade Kievit
  • Natural Sciences and Engineering Research Council of Canada (Award 2019-05490)
    • Principle Award Recipient: AnnKaren C Brassinga
© 2025 The Authors
  • This is an open-access article distributed under the terms of the Creative Commons Attribution License. This article was made open access via a Publish and Read agreement between the Microbiology Society and the corresponding author’s institution.
Loading

Article metrics loading...

/content/journal/micro/10.1099/mic.0.001551
2025-04-15
2025-04-29
Loading full text...

Full text loading...

/deliver/fulltext/micro/171/4/mic001551.html?itemId=/content/journal/micro/10.1099/mic.0.001551&mimeType=html&fmt=ahah

References

  1. Rowbotham TJ. Current views on the relationships between amoebae, legionellae and man. Isr J Med Sci 1986; 22:678–689 [PubMed]
     [Google Scholar]
  2. Steinert M, Hentschel U, Hacker J. Legionella pneumophila: an aquatic microbe goes astray. FEMS Microbiol Rev 2002; 26:149–162 [View Article] [PubMed]
     [Google Scholar]
  3. Newton HJ, Ang DKY, van Driel IR, Hartland EL. Molecular pathogenesis of infections caused by Legionella pneumophila. Clin Microbiol Rev 2010; 23:274–298 [View Article] [PubMed]
     [Google Scholar]
  4. Oliva G, Sahr T, Buchrieser C. The life cycle of L. pneumophila: ecllular differentiation Is linked to virulence and metabolism. Front Cell Infect Microbiol 2018; 8:3 [View Article] [PubMed]
     [Google Scholar]
  5. Mondino S, Schmidt S, Buchrieser C. Molecular mimicry: a paradigm of host-microbe coevolution Illustrated by Legionella. mBio 2020; 11:e01201-20 [View Article] [PubMed]
     [Google Scholar]
  6. Finsel I, Hilbi H. Formation of a pathogen vacuole according to Legionella pneumophila: how to kill one bird with many stones. Cell Microbiol 2015; 17:935–950 [View Article] [PubMed]
     [Google Scholar]
  7. Burstein D, Amaro F, Zusman T, Lifshitz Z, Cohen O et al. Genomic analysis of 38 Legionella species identifies large and diverse effector repertoires. Nat Genet 2016; 48:167–175 [View Article] [PubMed]
     [Google Scholar]
  8. Gomez-Valero L, Rusniok C, Carson D, Mondino S, Pérez-Cobas AE et al. More than 18,000 effectors in the Legionella genus genome provide multiple, independent combinations for replication in human cells. Proc Natl Acad Sci U S A 2019; 116:2265–2273 [View Article] [PubMed]
     [Google Scholar]
  9. Tilney LG, Harb OS, Connelly PS, Robinson CG, Roy CR. How the parasitic bacterium Legionella pneumophila modifies its phagosome and transforms it into rough ER: implications for conversion of plasma membrane to the ER membrane. J Cell Sci 2001; 114:4637–4650 [View Article] [PubMed]
     [Google Scholar]
  10. Kagan JC, Roy CR. Legionella phagosomes intercept vesicular traffic from endoplasmic reticulum exit sites. Nat Cell Biol 2002; 4:945–954 [View Article] [PubMed]
     [Google Scholar]
  11. Isberg RR, O’Connor TJ, Heidtman M. The Legionella pneumophila replication vacuole: making a cosy niche inside host cells. Nat Rev Microbiol 2009; 7:13–24 [View Article] [PubMed]
     [Google Scholar]
  12. Molofsky AB, Swanson MS. Differentiate to thrive: lessons from the Legionella pneumophila life cycle. Mol Microbiol 2004; 53:29–40 [View Article] [PubMed]
     [Google Scholar]
  13. Robertson P, Abdelhady H, Garduño RA. The many forms of a pleomorphic bacterial pathogen-the developmental network of Legionella pneumophila. Front Microbiol 2014; 5:670 [View Article] [PubMed]
     [Google Scholar]
  14. Zusman T, Gal-Mor O, Segal G. Characterization of a Legionella pneumophila relA insertion mutant and toles of RelA and RpoS in virulence gene expression. J Bacteriol 2002; 184:67–75 [View Article] [PubMed]
     [Google Scholar]
  15. Edwards RL, Dalebroux ZD, Swanson MS. Legionella pneumophila couples fatty acid flux to microbial differentiation and virulence. Mol Microbiol 2009; 71:1190–1204 [View Article] [PubMed]
     [Google Scholar]
  16. Dalebroux ZD, Yagi BF, Sahr T, Buchrieser C, Swanson MS. Distinct roles of ppGpp and DksA in Legionella pneumophila differentiation. Mol Microbiol 2010; 76:200–219 [View Article] [PubMed]
     [Google Scholar]
  17. Hammer BK, Tateda ES, Swanson MS. A two-component regulator induces the transmission phenotype of stationary-phase Legionella pneumophila. Mol Microbiol 2002; 44:107–118 [View Article] [PubMed]
     [Google Scholar]
  18. Rasis M, Segal G. The LetA-RsmYZ-CsrA regulatory cascade, together with RpoS and PmrA, post-transcriptionally regulates stationary phase activation of Legionella pneumophila Icm/Dot effectors. Mol Microbiol 2009; 72:995–1010 [View Article] [PubMed]
     [Google Scholar]
  19. Sahr T, Brüggemann H, Jules M, Lomma M, Albert-Weissenberger C et al. Two small ncRNAs jointly govern virulence and transmission in Legionella pneumophila. Mol Microbiol 2009; 72:741–762 [View Article] [PubMed]
     [Google Scholar]
  20. Nevo O, Zusman T, Rasis M, Lifshitz Z, Segal G. Identification of Legionella pneumophila effectors regulated by the letAS-RsmYZ-CsrA regulatory cascade, many of which modulate vesicular trafficking. J Bacteriol 2014; 196:681–692 [View Article] [PubMed]
     [Google Scholar]
  21. Häuslein I, Sahr T, Escoll P, Klausner N, Eisenreich W et al. Legionella pneumophila CsrA regulates a metabolic switch from amino acid to glycerolipid metabolism. Open Biol 2017; 7:170149 [View Article] [PubMed]
     [Google Scholar]
  22. Sahr T, Rusniok C, Impens F, Oliva G, Sismeiro O et al. The Legionella pneumophila genome evolved to accommodate multiple regulatory mechanisms controlled by the CsrA-system. PLoS Genet 2017; 13:e1006629 [View Article] [PubMed]
     [Google Scholar]
  23. Feldheim YS, Zusman T, Speiser Y, Segal G. The Legionella pneumophila CpxRA two-component regulatory system: new insights into CpxR’s function as a dual regulator and its connection to the effectors regulatory network. Mol Microbiol 2016; 99:1059–1079 [View Article] [PubMed]
     [Google Scholar]
  24. Tanner JR, Li L, Faucher SP, Brassinga AKC. The CpxRA two-component system contributes to Legionella pneumophila virulence. Mol Microbiol 2016; 100:1017–1038 [View Article] [PubMed]
     [Google Scholar]
  25. Zusman T, Aloni G, Halperin E, Kotzer H, Degtyar E et al. The response regulator PmrA is a major regulator of the icm/dot type IV secretion system in Legionella pneumophila and Coxiella burnetii. Mol Microbiol 2007; 63:1508–1523 [View Article] [PubMed]
     [Google Scholar]
  26. Al-Khodor S, Kalachikov S, Morozova I, Price CT, Abu Kwaik Y. The PmrA/PmrB two-component system of Legionella pneumophila Is a global regulator required for intracellular replication within macrophages and protozoa. Infect Immun 2009; 77:374–386 [View Article] [PubMed]
     [Google Scholar]
  27. Tiaden A, Spirig T, Weber SS, Brüggemann H, Bosshard R et al. The Legionella pneumophila response regulator LqsR promotes host cell interactions as an element of the virulence regulatory network controlled by RpoS and LetA. Cell Microbiol 2007; 9:2903–2920 [View Article] [PubMed]
     [Google Scholar]
  28. Tiaden A, Spirig T, Carranza P, Brüggemann H, Riedel K et al. Synergistic contribution of the Legionella pneumophila lqs genes to pathogen-host interactions. J Bacteriol 2008; 190:7532–7547 [View Article] [PubMed]
     [Google Scholar]
  29. Schell U, Kessler A, Hilbi H. Phosphorylation signalling through the Legionella quorum sensing histidine kinases LqsS and LqsT converges on the response regulator LqsR. Mol Microbiol 2014; 92:1039–1055 [View Article] [PubMed]
     [Google Scholar]
  30. Personnic N, Striednig B, Hilbi H. Legionella quorum sensing and its role in pathogen-host interactions. Curr Opin Microbiol 2018; 41:29–35 [View Article] [PubMed]
     [Google Scholar]
  31. Morash MG, Brassinga AKC, Warthan M, Gourabathini P, Garduño RA et al. Reciprocal expression of integration host factor and HU in the developmental cycle and infectivity of Legionella pneumophila. Appl Environ Microbiol 2009; 75:1826–1837 [View Article] [PubMed]
     [Google Scholar]
  32. Pitre CAJ, Tanner JR, Patel P, Brassinga AKC. Regulatory control of temporally expressed integration host factor (IHF) in Legionella pneumophila. Microbiology 2013; 159:475–492 [View Article] [PubMed]
     [Google Scholar]
  33. Tanner JR, Patel PG, Hellinga JR, Donald LJ, Jimenez C et al. Legionella pneumophila OxyR Is a redundant transcriptional regulator that contributes to expression control of the two-component CpxRA system. J Bacteriol 2017; 199:e00690-16 [View Article] [PubMed]
     [Google Scholar]
  34. Graham CI, Patel PG, Tanner JR, Hellinga J, MacMartin TL et al. Autorepressor PsrA is required for optimal Legionella pneumophila growth in Acanthamoeba castellanii protozoa. Mol Microbiol 2021; 116:624–647 [View Article] [PubMed]
     [Google Scholar]
  35. Hales LM, Shuman HA. The Legionella pneumophila rpoS gene is required for growth within Acanthamoeba castellanii. J Bacteriol 1999; 181:4879–4889 [View Article] [PubMed]
     [Google Scholar]
  36. Bachman MA, Swanson MS. RpoS co-operates with other factors to induce Legionella pneumophila virulence in the stationary phase. Mol Microbiol 2001; 40:1201–1214 [View Article] [PubMed]
     [Google Scholar]
  37. Abu-Zant A, Asare R, Graham JE, Abu Kwaik Y. Role for RpoS but not RelA of Legionella pneumophila in modulation of phagosome biogenesis and adaptation to the phagosomal microenvironment. Infect Immun 2006; 74:3021–3026 [View Article] [PubMed]
     [Google Scholar]
  38. Hovel-Miner G, Pampou S, Faucher SP, Clarke M, Morozova I et al. SigmaS controls multiple pathways associated with intracellular multiplication of Legionella pneumophila. J Bacteriol 2009; 191:2461–2473 [View Article] [PubMed]
     [Google Scholar]
  39. Perederina A, Svetlov V, Vassylyeva MN, Tahirov TH, Yokoyama S et al. Regulation through the secondary channel--structural framework for ppGpp-DksA synergism during transcription. Cell 2004; 118:297–309 [View Article] [PubMed]
     [Google Scholar]
  40. Ross W, Vrentas CE, Sanchez-Vazquez P, Gaal T, Gourse RL. The magic spot: a ppGpp binding site on E. coli RNA polymerase responsible for regulation of transcription initiation. Mol Cell 2013; 50:420–429 [View Article] [PubMed]
     [Google Scholar]
  41. Ross W, Sanchez-Vazquez P, Chen AY, Lee J-H, Burgos HL et al. PpGpp binding to a site at the RNAP-dksa interface accounts for its dramatic effects on transcription initiation during the stringent response. Mol Cell 2016; 62:811–823 [View Article] [PubMed]
     [Google Scholar]
  42. Myers AR, Thistle DP, Ross W, Gourse RL. Guanosine tetraphosphate has a similar affinity for each of its two binding sites on Escherichia coli RNA polymerase. Front Microbiol 2020; 11:587098 [View Article] [PubMed]
     [Google Scholar]
  43. Chatterji D, Fujita N, Ishihama A. The mediator for stringent control, ppGpp, binds to the beta-subunit of Escherichia coli RNA polymerase. Genes Cells 1998; 3:279–287 [View Article] [PubMed]
     [Google Scholar]
  44. Lemke JJ, Sanchez-Vazquez P, Burgos HL, Hedberg G, Ross W et al. Direct regulation of Escherichia coli ribosomal protein promoters by the transcription factors ppgpp and dksa. Proc Natl Acad Sci USA 2011; 108:5712–5717 [View Article] [PubMed]
     [Google Scholar]
  45. Gummesson B, Lovmar M, Nyström T. A proximal promoter element required for positive transcriptional control by guanosine tetraphosphate and DksA protein during the stringent response. J Biol Chem 2013; 288:21055–21064 [View Article] [PubMed]
     [Google Scholar]
  46. Molodtsov V, Sineva E, Zhang L, Huang X, Cashel M et al. Allosteric effector ppGpp potentiates the inhibition of transcript initiation by DksA. Mol Cell 2018; 69:828–839 [View Article] [PubMed]
     [Google Scholar]
  47. Travis BA, Schumacher MA. Diverse molecular mechanisms of transcription regulation by the bacterial alarmone ppGpp. Mol Microbiol 2022; 117:252–260 [View Article] [PubMed]
     [Google Scholar]
  48. Magnusson LU, Gummesson B, Joksimović P, Farewell A, Nyström T. Identical, independent, and opposing roles of ppGpp and DksA in Escherichia coli. . J Bacteriol 2007; 189:5193–5202 [View Article] [PubMed]
     [Google Scholar]
  49. Aberg A, Shingler V, Balsalobre C. Regulation of the fimB promoter: a case of differential regulation by ppGpp and DksA in vivo. Mol Microbiol 2008; 67:1223–1241 [View Article] [PubMed]
     [Google Scholar]
  50. Potrykus K, Cashel M. (p)ppGpp: still magical?. Annu Rev Microbiol 2008; 62:35–51 [View Article] [PubMed]
     [Google Scholar]
  51. Aberg A, Fernández-Vázquez J, Cabrer-Panes JD, Sánchez A, Balsalobre C. Similar and divergent effects of ppGpp and DksA deficiencies on transcription in Escherichia coli. J Bacteriol 2009; 191:3226–3236 [View Article] [PubMed]
     [Google Scholar]
  52. Vinella D, Potrykus K, Murphy H, Cashel M. Effects on growth by changes of the balance between GreA, GreB, and DksA suggest mutual competition and functional redundancy in Escherichia coli. J Bacteriol 2012; 194:261–273 [View Article] [PubMed]
     [Google Scholar]
  53. Gourse RL, Chen AY, Gopalkrishnan S, Sanchez-Vazquez P, Myers A et al. Transcriptional responses to ppGpp and DksA. Annu Rev Microbiol 2018; 72:163–184 [View Article] [PubMed]
     [Google Scholar]
  54. Pettersen EF, Goddard TD, Huang CC, Meng EC, Couch GS et al. UCSF ChimeraX: Structure visualization for researchers, educators, and developers. Protein Sci 2021; 30:70–82 [View Article] [PubMed]
     [Google Scholar]
  55. Landau M, Mayrose I, Rosenberg Y, Glaser F, Martz E et al. ConSurf 2005: the projection of evolutionary conservation scores of residues on protein structures. Nucleic Acids Res 2005; 33:W299–302 [View Article] [PubMed]
     [Google Scholar]
  56. Ashkenazy H, Abadi S, Martz E, Chay O, Mayrose I et al. ConSurf 2016: an improved methodology to estimate and visualize evolutionary conservation in macromolecules. Nucleic Acids Res 2016; 44:W344–50 [View Article] [PubMed]
     [Google Scholar]
  57. Feeley JC, Gibson RJ, Gorman GW, Langford NC, Rasheed JK et al. Charcoal-yeast extract agar: primary isolation medium for Legionella pneumophila. J Clin Microbiol 1979; 10:437–441 [View Article]
     [Google Scholar]
  58. Rao C, Benhabib H, Ensminger AW. Phylogenetic reconstruction of the Legionella pneumophila Philadelphia-1 laboratory strains through comparative genomics. PLoS One 2013; 8:e64129 [View Article] [PubMed]
     [Google Scholar]
  59. Wiater LA, Sadosky AB, Shuman HA. Mutagenesis of Legionella pneumophila using Tn903 dlllacZ: identification of a growth-phase-regulated pigmentation gene. Mol Microbiol 1994; 11:641–653 [View Article] [PubMed]
     [Google Scholar]
  60. Jumper J, Evans R, Pritzel A, Green T, Figurnov M et al. Highly accurate protein structure prediction with AlphaFold. Nature 2021; 596:583–589 [View Article] [PubMed]
     [Google Scholar]
  61. Skolnick J, Gao M, Zhou H, Singh S. AlphaFold 2: why it works and its implications for understanding the relationships of protein sequence, structure, and function. J Chem Inf Model 2021; 61:4827–4831 [View Article] [PubMed]
     [Google Scholar]
  62. Parshin A, Shiver AL, Lee J, Ozerova M, Schneidman-Duhovny D et al. DksA regulates RNA polymerase in Escherichia coli through a network of interactions in the secondary channel that includes Sequence Insertion 1. Proc Natl Acad Sci USA 2015; 112:E6862–71 [View Article] [PubMed]
     [Google Scholar]
  63. Kang Y, Nguyen DT, Son MS, Hoang TT. The Pseudomonas aeruginosa PsrA responds to long-chain fatty acid signals to regulate the fadBA5 beta-oxidation operon. Microbiology 2008; 154:1584–1598 [View Article] [PubMed]
     [Google Scholar]
  64. Fonseca MV, Swanson MS. Nutrient salvaging and metabolism by the intracellular pathogen Legionella pneumophila. Front Cell Infect Microbiol 2014; 4:12 [View Article] [PubMed]
     [Google Scholar]
  65. Graham CI, MacMartin TL, de Kievit TR, Brassinga AKC. Molecular regulation of virulence in Legionella pneumophila. Mol Microbiol 2024; 121:167–195 [View Article] [PubMed]
     [Google Scholar]
  66. McNealy TL, Forsbach-Birk V, Shi C, Marre R. The Hfq homolog in Legionella pneumophila demonstrates regulation by LetA and RpoS and interacts with the global regulator CsrA. J Bacteriol 2005; 187:1527–1532 [View Article] [PubMed]
     [Google Scholar]
  67. Sahr T, Rusniok C, Dervins-Ravault D, Sismeiro O, Coppee J-Y et al. Deep sequencing defines the transcriptional map of L. pneumophila and identifies growth phase-dependent regulated ncRNAs implicated in virulence. RNA Biol 2012; 9:503–519 [View Article] [PubMed]
     [Google Scholar]
  68. Byrne B, Swanson MS. Expression of Legionella pneumophila virulence traits in response to growth conditions. Infect Immun 1998; 66:3029–3034 [View Article] [PubMed]
     [Google Scholar]
  69. Anderson SE, Vadia SE, McKelvy J, Levin PA. The transcription factor DksA exerts opposing effects on cell division depending on the presence of ppGpp. mBio 2023; 14:e0242523 [View Article] [PubMed]
     [Google Scholar]
  70. Steinert M, Engelhard H, Flügel M, Wintermeyer E, Hacker J. The Lly protein protects Legionella pneumophila from light but does not directly influence its intracellular survival in Hartmannella vermiformis. Appl Environ Microbiol 1995; 61:2428–2430 [View Article] [PubMed]
     [Google Scholar]
  71. Chatfield CH, Cianciotto NP. The secreted pyomelanin pigment of Legionella pneumophila confers ferric reductase activity. Infect Immun 2007; 75:4062–4070 [View Article] [PubMed]
     [Google Scholar]
  72. Levin TC, Goldspiel BP, Malik HS. Density-dependent resistance protects Legionella pneumophila from its own antimicrobial metabolite, HGA. Elife 2019; 8:e46086 [View Article] [PubMed]
     [Google Scholar]
  73. Bryan A, Abbott ZD, Swanson MS. Constructing unmarked gene deletions in Legionella pneumophila. Methods Mol Biol 2013; 954:197–212 [View Article] [PubMed]
     [Google Scholar]
  74. Berger KH, Isberg RR. Two distinct defects in intracellular growth complemented by a single genetic locus in Legionella pneumophila. Mol Microbiol 1993; 7:7–19 [View Article] [PubMed]
     [Google Scholar]
  75. Sexton JA, Pinkner JS, Roth R, Heuser JE, Hultgren SJ et al. The Legionella pneumophila PilT homologue DotB exhibits ATPase activity that is critical for intracellular growth. J Bacteriol 2004; 186:1658–1666 [View Article] [PubMed]
     [Google Scholar]
  76. Merriam JJ, Mathur R, Maxfield-Boumil R, Isberg RR. Analysis of the Legionella pneumophila fliI gene: intracellular growth of a defined mutant defective for flagellum biosynthesis. Infect Immun 1997; 65:2497–2501 [View Article] [PubMed]
     [Google Scholar]
/content/journal/micro/10.1099/mic.0.001551
Loading
Transcription factors DksA and PsrA are synergistic contributors to Legionella pneumophila virulence in Acanthamoeba castellanii protozoa
Microbiology 171, 001551 (2025); https://doi.org/10.1099/mic.0.001551
/content/journal/micro/10.1099/mic.0.001551
/content/journal/micro/10.1099/mic.0.001551
Loading

Data & Media loading...

Supplements

Supplementary material 1

PDF

Most cited Most Cited RSS feed

This is a required field
Please enter a valid email address
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error