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Molecular Analysis of Bacterial Pathogens

John Davies
Professor John Davies

We are particularly interested in how bacterial pathogens alter their cell surface during the multiple stages that are involved in the infectious process. This might involve genetic processes such as phase and antigenic variation. It also inevitably involves changes at the transcriptional level, where the bacteria switch genes on or off in response to environmental cues. The laboratory has been involved in many projects to determine the complete nucleotide sequence of the genomes of different bacterial pathogens. This subsequently allows a “genome-wide” approach to determine which genes, of all those present in a bacterial pathogen, are expressed at a particular stage of infection.

Historically, the Davies laboratory has worked mainly on Neisseria gonorrhoeae, the causative agent of the sexually transmitted disease, gonorrhoea.

Neisseria gonorrhoeae, commonly called the gonococcus.

Neisseria gonorrhoeae (commonly called gonococcus)

However, in recent years we have been involved in projects involving other bacterial pathogens:

  • Neisseria meningitidis. Closely related to N. gonorrhoeae, but the causative agent of meningococcal meningitis and septicaemia. Much of this work is in collaboration with ex-lab member Charlene Kahler, now at the University of Western Australia.

  • Staphylococcus aureus. We are investigating the genetic and transcriptional changes that accompany the development of intermediate levels of vancomycin resistance in multi-resistant strains of Staphylococcus aureus. This project is a collaboration with ex-lab members Tim Stinear at the University of Melbourne and Ben Howden  at Austin Health.

Selected research projects

Regulation of the pilE gene in Neisseria gonorrhoeae

The pilE gene in Neisseria gonorrhoeae encodes the subunit of the major, surface expressed virulence factor, type 4 pili. Understanding the regulatory processes associated with expression of this gene provides insight into the progression of the infection as well as the mechanism of transcriptional control in prokaryotes. The control of expression of pilE is complex and the aim of this project is to elucidate the mechanisms associated with this expression.

Regulation of integration host factor (IHF) in Neisseria gonorrhoeae and Neisseria meningitidis

IHF is a heterodimeric DNA binding protein containing IhfA and IhfB subunits. Upon binding of IHF to a specific DNA sequence, a bend of up to 160° is induced in the DNA. Binding of IHF can effect gene expression and it has also been shown to have roles in DNA replication and site specific recombination. In Neisseria the role of this protein is of interest as it may potentially regulate expression of genes encoding virulence factors. Putative IHF binding sites are frequently being identified in Neisseria and IHF has been implicated in the regulation of pilE, siaA and siaD (encoding virulence factors) and ihfA in Neisseria gonorrhoeae, and nadA in Neisseria meningitidis. It is for this reason we are interested in how expression of IHF is regulated in Neisseria. It is known that IHF binds upstream of the ihfA gene in Ng suggesting autoregulation. We are mapping the promoters and other regulatory elements upstream of the ihfA and ihfB genes, and comparing the overall mode of regulation with that elucidated for Escherichia coli.

Regulation of genes involved in pilus biogenesis in Neisseria gonorrhoeae and Neisseria meningitidis.

The type 4 pili produced by the pathogenic Neisseria species consist mainly of the PilE pilin protein. However it is known that multiple gene products are needed to assemble this cell surface structure. We are investigating the regulation of these additional genes, and whether there is coordinated control of their expression. Upstream of many of the genes concerned is a genetic element which contains an IHF binding site. As IHF controls expression of pilE, there is the possibility that it may also act as a master regulator of pilus biogenesis.

Mechanisms of low-level vancomycin resistance in Staphylococcus aureus

Staphylococcus aureus remains an important human pathogen, responsible for skin infections, as well as more invasive diseases such as endocarditis and osteomyelitis. Increasingly, Staphylococcus aureus has developed antimicrobial resistance which has limited the availability of agents to treat serious infections caused by this organism. The recent emergence of low-level vancomycin resistance in S. aureus (so called vancomycin intermediate S. aureus [VISA}, and heterogenous-VISA [hVISA]) has severely compromised the ability of clinicians to treat infections caused by such strains. We are investigating the mechanisms of low-level vancomycin resistance in S. aureus. During previous clinical studies we collected a number of clinical isolate pairs of vancomycin susceptible and low-level resistant S. aureus, where resistance developed during persistent bacteraemia in a number of patients. These multiple pairs of apparently isogenic clinical strains provide a unique opportunity to determine the mechanisms of resistance in multiple pairs of strains. DNA microarray transcriptional analysis has revealed multiple consistent changes across a number of clinical pairs, suggesting an important role in the development of resistance. The same changes were not found in pairs of clinical isolates where patients had persistent bacteraemia but did not develop phenotypic vancomycin resistance. In addition to the microarray analysis, we have recently completed the whole genome sequencing of several pairs of isolates, which will provide further insight into the genetic changes leading to low-level vancomycin resistance.

Selected recent publications

  • Stinear, T.P., D.C. Olden, P.D.R. Johnson, J.K. Davies and M.L. Grayson. 2001. Enterococcal vanB resistance locus in anaerobic bacteria in human faeces. Lancet 357: 855-856.

  • Kahler, C.M., L.E. Martin, Y.-L. Tzeng, Y.K. Miller, K. Sharkey, D.S. Stephens and J.K. Davies. 2001. Polymorphisms in the pilin glycosylation locus of Neisseria meningitidis expressing class II pili. Infect. Immun. 69: 3597-3604.

  • Jenkin, G.A., T.P. Stinear, P.D.R. Johnson and J.K.Davies. (2003). Subtractive hybridisation reveals a type I polyketide synthase locus specific to Mycobacterium ulcerans. J. Bacteriol. 185: 6870-6882.

  • Stinear, T.P., A. Mve-Obiang, P.L.C. Small, W. Frigui, M.J. Pryor, R. Brosch, G.A. Jenkin, P.D.R. Johnson, J.K. Davies, R.E. Lee, S. Adusumilli, T. Garnier, S.F. Haydock, P.F. Leadlay and S.T. Cole. (2004). Giant plasmid-encoded polyketide synthases produce the macrolide toxin of Mycobacterium ulcerans. Proc Natl Acad Sci USA 101: 1345-1349.

  • Snyder, L.A.S., J.K. Davies and N.J. Saunders. (2004). Microarray genomotyping of key experimental strains of Neisseria gonorrhoeae reveals gene complement diversity and seven new neisserial genes associated with minimal mobile elements. BMC Genomics 5: 23.

  • Tzeng, Y.-L., A. Datta, K. Ambrose, M. Lo, J.K. Davies, R.W. Carlson, D.S. Stephens and C.M. Kahler. (2004). The MisR/MisS two-component regulatory system influences inner core structure and immunotype of lipooligosaccharide in Neisseria meningitidis. J Biol Chem 279: 35053-35062.

  • Laskos, L., Ryan, C.S., Fyfe, J.A.M., and J.K. Davies. (2004) In Neisseria gonorrhoeae the RpoH mediated stress response is regulated at the level of activity. J. Bacteriol. 186: 8443-8452.

  • Snyder, L.A.S., J.K. Davies, C.S. Ryan and N.J. Saunders. (2005). Comparative overview of the genomic and genetic differences between the pathogenic Neisseria strains and species. Plasmid 54: 191–218.

  • Gunesekere, I. C., C.M.  Kahler, C. S. Ryan, L. A. S Snyder, N. J Saunders, J. I. Rood and J. K. Davies. (2006). Ecf, an alternative sigma factor from Neisseria gonorrhoeae, controls expression of msrAB, which encodes methionine sulfoxide reductase. J. Bacteriol. 188: 3463-3469.

  • Stinear, T.P., S. Pidot, W. Frigui, G. Meurice, G. Reysett, T. Garnier, C. Bouchier, L. Ma, M. Tichit, J.L. Porter, J. Ryan, P.D.R. Johnson, J.K. Davies, G.A. Jenkin, T. Seemann, P.L.C. Small, F. Laval, M. Daffe, J. Parkhill and S.T. Cole. (2007). Recent adaptation to a changed environment inferred from the genome of Mycobacterium ulcerans, the causative agent of Buruli ulcer. Genome Res. 17: 192-200.

  • Han, X., R.M. Kennan, D. Parker, J.K. Davies and J.I. Rood. (2007). Type IV fimbrial biogenesis is required for protease secretion and natural transformation in Dichelobacter nodosus. J. Bacteriol., 189: 5022-5033.

  • Mantena, R.K.R., O.L.C. Wijburg, C. Vindurampulle, V.R. Bennett-Wood, A. Walduck, G.R. Drummond, J.K. Davies, R.M. Robins-Browne and R.A. Strugnell. (2008). Reactive oxygen species are the major antibacterials against Salmonella Typhimurium purine auxotrophs in the phagosome of RAW 264.7 cells. Cell. Microbiol. 10: 1058-1073.

  • Picardeau, M., D.M. Bulach, C. Bouchier, R.L. Zuerner, N. Zidane, P.J. Wilson, S. Creno, E.S. Kuczek, S. Bommezzadri, J.C. Davis, A. McGrath, M.J. Johnson, C. Boursaux-Eude, T. Seemann, Z. Rouy, R.L. Coppel, J.I. Rood, A. Lajus, J.K. Davies, C. Médigue and B. Adler. (2008). Genome Sequence of the Saprophyte Leptospira biflexa Provides Insights into the Evolution of Leptospira and the Pathogenesis of Leptospirosis. PLoS ONE 3:e1607.

  • Howden, B.P., D.J. Smith, A. Mansell, P.D.R. Johnson, P.B. Ward, T.P. Stinear and J.K. Davies. (2008). Different bacterial gene expression patterns and attenuated host immune responses are associated with the evolution of low-level vancomycin resistance during persistent methicillin-resistant Staphylococcus aureus bacteraemia. BMC Microbiology 8:39.

  • Stinear, T.P., T. Seemann, P.F. Harrison, G.A. Jenkin, J.K. Davies, P.D.R. Johnson, Z. Abdellah, C. Arrowsmith, T. Chillingworth, C. Churcher, K. Clarke, A. Cronin, P. Davis, I. Goodhead, N. Holroyd, K. Jagels, A. Lord, S. Moule, K. Mungall, H. Norbertczak, M.A. Quail, E. Rabbinowitsch, D. Walker, B. White, S. Whitehead, P.L.C. Small, R. Brosch, L. Ramakrishnan, M.A. Fischbach, J. Parkhill and S.T. Cole. (2008). Insights from the complete genome sequence of Mycobacterium marinum on the evolution of Mycobacterium tuberculosis. Genome Research 18: 729-741.

  • Han X., R.M. Kennan, J.K. Davies, L.A. Reddacliff, O.P. Dhungyel, R.J. Whittington, L. Turnbull, C.B. Whitchurch, and J.I. Rood. (2008). Twitching Motility is Essential for Virulence in Dichelobacter nodosus. J. Bacteriol. 190: 3323-3335.

  • Howden, B.P., T.P. Stinear, D. Allen, P.D.R. Johnson, P.B. Ward and J.K. Davies. (2008). Genomics reveals a point mutation in the two-component sensor gene graS that leads to vancomycin-intermediate resistance in clinical Staphylococcus aureus. Antimicrob. Agents Chemother. 52: 3755-3762.

  • Pidot, S.J., H. Hong, T. Seemann, J.L. Porter, M.J. Yip, A. Men, M. Johnson, P. Wilson, J.K. Davies, P.F. Leadlay and T.P. Stinear. (2008). Deciphering the genetic basis for polyketide variation among mycobacteria producing mycolactones. BMC Genomics 9:462

  • Hill, S., and J. Davies. (2009). Pilin gene variation in Neisseria gonorrhoeae: reassessing the old paradigms. FEMS Microbiol. Rev. 33: 521-530.

  • Tobias, N.J., T. Seeman, S.J. Pidot, J.L. Porter, L. Marsollier, E. Marion, F. Letournel, T. Zakir, J. Azuolas, J.R. Wallace, H. Hong, J.K. Davies, B.P. Howden, P.D.R. Johnson, G.A. Jenkin and T.P. Stinear. (2009). Mycolactone gene expression is controlled by strong SigA-like promoters with utility in studies of Mycobacterium ulcerans and Buruli ulcer. PLoS Negl. Trop. Dis. 3:e553.

  • Davies, J.K. (2010). Chapter 3: Genomics and recombination. In C. Genco and L. Wetzler (eds.), Neisseria – molecular mechanisms of pathogenesis, p. 41-51. Horizon Scientific Press, Norwich, UK.

  • Kaparakis, M., L. Turnbull, L. Carneiro, S. Firth, H.A. Coleman, H.C. Parkington, L. Le Bourhis, A. Karrar, J. Viala, J. Mak, M.L. Hutton, J.K. Davies, P.J. Crack, P.J. Hertzog, D.J. Philpott, S.E. Girardin, C.B. Whitchurch and R.L. Ferrero. (2010). Bacterial membrane vesicles deliver peptidoglycan to NOD1 in epithelial cells. Cellular Microbiol. 12: 372-385.

  • Howden, B.P., J.K. Davies, P.D.R. Johnson, T.P. Stinear, and M.L. Grayson. (2010). Reduced Vancomycin Susceptibility in Staphylococcus aureus, Including Vancomycin-Intermediate and Heterogenous Vancomycin-Intermediate Staphylococcus aureus: Mechanisms of Resistance, Laboratory Detection, and Clinical Implications. Clin. Microbiol. Rev. 23: 99-139.