Molecular Analysis of Bacterial Pathogens

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. We are using custom- built DNA microarrays for this transcriptional profiling. The laboratory has led the establishment of a facility at Monash that enables us to design, manufacture and analyse data from DNA microarrays.

Portion of a DNA microarray

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

Neisseria gonorrhoeae (commonly called gonococcus)

Selected research projects

Regulation of the pilE gene in Neisseria gonorrhoeae (Catherine Ryan)

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. We are using many techniques including mutational analysis, molecular modelling using known X-ray crystal structures, in vitro transcription, DNaseI footprinting and atomic force microscopy to test a hypothesis we have for the mode of regulation of this gene. The figure below depicts our current model for how pilE may be regulated in Neisseria gonorrhoeae. It represents a previously undescribed mode of regulation and may be used to explain regulation of genes in other systems.

Model constructed by our colaborator Dr James Whisstock

Model constructed by our colaborator Dr James Whisstock

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

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.

Genes regulated by IHF in Neisseria gonorrhoeae and Neisseria meningitidis (Sally Turner and Shauna Lyons-Schindler).

As outlined above, IHF has been implicated in the regulation of a number of important genes in Neisseria. Due to the potential for IHF involvement in regulation of virulence factors other than pili, we have investigated the extent of the IHF regulon in Neisseria. To date, research into this area has been difficult due to the inability to generate non-lethal IHF mutants. Following the successful isolation of an ihfA mutant in Neisseria meningitidis by our laboratory, our research now focuses upon the initial use of microarray technology to identify genes with altered expression profiles in the ihfA mutant and wildtype strains. The promoter regions of these genes will then be investigated, with the aim to demonstrate the direct involvement of IHF in their expression. We hope to identify a number of genes that are directly regulated by IHF, thus adding valuable information to the understanding of the role of IHF in Neisserial gene regulation.

Mechanisms of low-level vancomycin resistance in Staphylococcus aureus (Benjamin Howden)

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. Our collaborators have previously demonstrated an association between low-level resistance and vancomycin treatment failure.

Our group is currently 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 one pair of isolates, which will provide further insight into the genetic changes leading to low-level vancomycin resistance.

Selected recent publications

• Stinear, T., J.K. Davies, G.A. Jenkin, J.A. Hayman, F. Oppedisano and P.D.R. Johnson. 2000. Identification of Mycobacterium ulcerans in the environment from regions in southeast Australia in which it is endemic with sequence capture- PCR . Appl. Environ. Microbiol. 66: 3206-3213.
• Stinear, T.P., G.A. Jenkin, P.D.R. Johnson and J.K. Davies. 2000. A comparative genetic analysis of Mycobacterium ulcerans and Mycobacterium marinum reveals evidence of recent genetic divergence. J. Bacteriol. 182: 6322-6330.
• 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.
• Evans, M., J.K. Davies, G. Sundqvist and D.Figdor. 2002. Mechanisms involved in the resistance of Enterococcus faecalis to calcium hydroxide. Int. Endodont. J. 35: 221-228.
• Figdor, D., J.K. Davies and G. Sundqvist. 2003. Starvation survival, growth and recovery of Enterococcus faecalis in human serum. Oral Microbiol. Immunol. 18: 234-239.
• 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.
• Gunesekere, I.C., C.M. Kahler, D.R. Powell, L.A.S. Snyder, N.J. Saunders, J.I. Rood, and J.K. Davies. (2006). Comparison of the RpoH-dependent regulon and general stress response in Neisseria gonorrhoeae. J. Bacteriol. 188: 4769-4776.
• Howden, B.P., P.D. Johnson, P.B. Ward, T.P. Stinear and J.K. Davies. (2006). Isolates with low-level vancomycin resistance associated with persistent methicillin-resistant Staphylococcus aureus bacteremia. Antimicrob. Agents Chemother. 50: 3039-3047.
• 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
• McKean, S.C., J.K. Davies and R.J. Moore. (2007). Expression of phospholipase D, the major virulence factor of Corynebacterium pseudotuberculosis, is regulated by multiple environmental factors and plays a role in macrophage death. Microbiology 153: 2203-2211.