Bacterial Pathogenesis

Kadaba Sriprakash Staff
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Lab Head: Associate Professor Kadaba Sriprakash

BACKGROUND
Streptococcus pyogenes, commonly known as Group A Streptococcus (GAS), is a bacterial pathogen that infects the throat and skin and is able to cause a wide range of diseases in humans. These range from relatively benign pharyngitis (commonly known as 'strep throat') to more serious and potentially fatal diseases such as acute post-streptococcal glomerulonephritis (APSGN), rheumatic heart disease and invasive diseases. The World Heath Organization estimates that in any one year 600 million new pharyngitis cases occur and another 18.1 million individuals suffer with a serious GAS disease. More than half a million people may die from GAS diseases in any one year. Many of these deaths occur in developing countries.

In Australia, the Indigenous population suffers disproportionately from GAS related diseases. The aim of research in the Bacterial Pathogenesis Laboratory is to acquire a greater understanding of the pathogenesis of Streptococcus. This may lead to development of novel preventive strategies. Currently our major research initiatives are:

1. The molecular epidemiology of group A streptococcus.
2. The role of streptococcal virulence factors in pathogenesis.
3. Novel vaccine strategies to combat GAS infection.
4. Rapid diagnosis of streptococcal infection.

1. THE MOLECULAR EPIDEMIOLOGY OF STREPTOCOCCI
a) Lateral genetic transfer and streptococcal disease
The group C and group G streptococci (GGS) are close genetic relatives of GAS. Traditionally recognised as commensal organisms, these organisms are emerging as significant human pathogens. Despite high rates of rheumatic fever, GAS is rarely isolated from the throats of individuals in the Northern Territory. However GGS is. Our laboratory has recently demonstrated that DNA can be transferred between group A streptococcus and closely related group G streptococcus. This transfer is mediated by bacteriophages, the equivalent of a bacterial virus. We are investigating how commonly this occurs, and fully characterising these bacteriophages. Such information may help us to understand the rates at which Streptococci undergo large scale genetic drift and help us to predict the emergence of new patho-varaities from commensal organisms.

b) Microbial community dynamics
Many bacteria, some commensal and some disease causing can inhabit the throat. We are developing a molecular approach to identifying all major species in these bacterial communities. This involves using an approach called T-RFLP which distinguishes species on the basis of differences in the size of relatively conserved 16s RNA sequences. Once developed, we will use this tool to investigate whether the presence of different bacteria in the throat can change probability of acquiring a streptococcal infection.

2. THE ROLE OF STREPTOCOCCAL VIRULENCE FACTORS IN PATHOGENESIS
a) Streptococcal Inhibitor of Complement
SIC or the closely related protein, DRS, is expressed by four GAS M-types which are historically associated with APSGN. Our laboratory is investigating whether these proteins have a role in APSGN pathogenesis. This work is being conducted in collaboration with Dr Ketheesan's group at James Cook University.

b) Streptococcal biofilms
Group A streptococcus can grow in specialised mode of growth called a biofilm. This mode of growth may be more similar to bacterial growth during natural infection than traditional liquid cultures. In other species, biofilms are more resistant to antibiotic treatments, and also express different sets of virulence genes than when strains are grown in liquid culture. We are investigating whether GAS in this mode of growth are able to transfer DNA to other species, potentially increasing the capacity of recipient strains to cause disease. We are also investigating whether well characterised group A streptococcal virulence factors (i.e. M-protein, SIC and speB) are expressed differently during biofilm formation.

3. NOVEL VACCINE STRATEGIES TO COMBAT STREPTOCOCCAL INFECTION
We are developing new strategies that may be useful in the intranasal delivery of a GAS vaccine. Our current approach involves combining two GAS vaccine candidates, J14 and H12 into a single recombinant construct. J14 has been shown to be effective at inducing opsonic protective antibody responses, and H12 has been shown to stimulate oral mucosal responses. By combining these two constructs we are aiming to develop a construct which can be delivered intranasally, and evoke mucosal antibodies. This project is being undertaken in collaboration with Professor Michael Good and Dr Michael Batzloff of the Molecular Immunology Laboratory at QIMR.

4. RAPID DIAGNSOSIS OF STREPTOCOCCAL INFECTION
a) Rapid antigen based diagnostic for group B streptococcus Group B streptococcus (GBS) is a bacterial pathogen asymptomatically carried by mothers. During birth the bacteria may be transferred from the mother to the new born infant and initiate severe and possibly fatal infections such as pneumonia, septicaemia and meningitis. The current gold-standard diagnostic test for GBS takes at least 16 hours, and is therefore unfeasible to use during parturition. Our laboratory is developing rapid antigen based diagnostic test that will identify GBS is less than 1hour.

b) Multiplex assay for the identification of streptococci
GAS, GGS and GBS are closely related bacterial species. Using genes that are common to these three species but absent in other bacterial species, we are developing a rapid PCR based assay that will detect these three human pathogens, The addition of additional PCR primers that are specific for GAS, GGS and GBS, will provide an even greater level of specificity. Our goal is a rapid multiplex diagnostic assay equal to current gold standard culture diagnostics.

Staff

Labhead:	Associate Professor Kadaba Sriprakash
Research Officer:	Dr David McMillan
Research Assistants:	Mr Michael Binks Ms Josephine Shera Ms Meru Sheel (PT)
PhD Students:	Mr Mark Davies Ms Melina Georgousakis Ms Therese Vu

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Funding

We gratefully acknowledge support from the following organisations and funding bodies:

National Health and Medical Research Council
Cooperative Research Centre for Aborginal Health
Cooperative Research Centre for Vaccine Technology

Student Projects

The Bacterial Pathogenesis Laboratory has projects available for Honours and PhD students. We also encourage undergraduate students to apply for student Summer Vacations Scholarships to undertake short research projects in our lab. For more information visit the QIMR student website.

Collaborators

Professor Michael Good, Queensland Institute of Medical Research, Brisbane.
Professor Mark Walker, University of Wollongong, Wollongong.
Professor Singh Chhatwal, Gesellschaft fur Biotechnologische Forschung mbH, Braunschweig, Germany.
Professor Debra Bessen, Yale University, New Haven, USA.
Drs N Ketheesan and R Norton, James Cook University, Townsville.
Dr R Beiko, Institute of Molecular Biology, The University of Queensland, Brisbane.
Dr K N Brahmadathan, Christian Medical College, Vellore, Tamil Nadu, India
Professor MS Shaila, Indian Institute of Science, Bangalore, India

Recent Publications

Publications since 2002

McMillan, D.J., Geffers, R., Buer, J., Vlaminckx, B.J.M., Sriprakash, K.S. and Chhatwal, G.S. Variations in the distribution of genes encoding virulence and extracellular proteins in group A streptococcus are largely restricted to eleven genomic loci. Microb Infect. In press.

Mackay I.M., Harnett, G., Jeoffreys, N., Bastian, I., Sriprakash. K.S., Siebert, D., and Sloots, T.P. (2006) Detection and discrimination of herpes simplex viruses, Haemophilus ducreyi, Treponema pallidum, and Calymmatobacterium (Klebsiella) granulomatis from genital ulcers. Clin Infect Dis 42:1431-8.

Sanderson-Smith, M., Batzloff, M., Sriprakash, K.S., Dowton, M., Ranson, M., and Walker, M.J. (2006) Divergence in the plasminogen-binding group a streptococcal M protein family: functional conservation of binding site and potential role for immune selection of variants. J Biol Chem 281:3217-26.

McMillan D.J., Beiko R.G., Geffers R, Buer J, Schouls L.M., Vlaminckx B.J., Wannet, W.J., Sriprakash K.S., Chhatwal G.S. (2006). Genes for the majority of group a streptococcal virulence factors and extracellular surface proteins do not confer an increased propensity to cause invasive disease. Clin Infect Dis 2006 43:884-91

Binks, M.J., Fernie-King, B.A., Seilly, D.J., Lachmann, P.J., and Sriprakash, K.S. (2005) Attribution of the various inhibitory actions of the streptococcal inhibitor of complement (SIC) to regions within the molecule. J Biol Chem 280: 20120-20125.

Chhatwal, G.S. and McMillan D.J. (2005) Uncovering the Mystery of Group A Streptococcal Invasion. Trends Mol Medicine 4:152-155.

Davies, M.R., Tran, T.N., McMillan, D.J., Gardiner, D.L., Currie, B.J., and Sriprakash, K.S. (2005) Inter-species genetic movement may blur the epidemiology of streptococcal diseases in endemic regions. Microbes Infect 7: 1128-1138.

Dey, N., McMillan, D.J., Yarwood, P.J., Joshi, R.M., Kumar, R., Good, M.F., Sriprakash, K.S., and Vohra, H. (2005) High diversity of group A Streptococcal emm types in an Indian community: the need to tailor multivalent vaccines. Clin Infect Dis 40: 46-51.

McMillan, D.J., and Chhatwal, G. S. (2005). Prospects for a group A streptococcal vaccine. Current Opin Mol Therapeutics 7:11-16.

McMillan D.J. An evaluation of Streptavax. Investigational Drug Database 7: 186-90.

Vohra H., Dey, N., Gupta, S., Sharma, A.K., Kumar, R., McMillan, D. J., and Good, M.F. (2005). M protein conserved region antibodies opsonise multiple strains of Streptococcus pyogenes with sequence variations in C-repeats. Res Microbiol 156:575-82.

Batzloff, M.R., Sriprakash, K.S., and Good, M.F. (2004) Vaccine development for group A streptococcus infections and associated disease. Curr Drug Targets 5: 57-69.

Binks, M., and Sriprakash, K.S. (2004) Characterization of a complement-binding protein, DRS, from strains of Streptococcus pyogenes containing the emm12 and emm55 genes. Infect Immun 72: 3981-3986.

Edwards, M.L., Fagan, P.K., Currie, B.J., and Sriprakash, K.S. (2004) The fibronectin-binding capacity and host cell adherence of Streptococcus pyogenes strains are discordant with each other. Microbes Infect 6: 1156-1162.

Edwards, M.L., Fagan, P.K., Towers, R.J., Currie, B.J., and Sriprakash, K.S. (2004) Inhibition of Streptococcus pyogenes adherence to HaCaT cells by a peptide corresponding to the streptococcal fibronectin-binding protein, SfbI, is strain dependent. Microbes Infect 6: 926-928.

Good MF, SRIPRAKASH KS and Kemp DJ (2004) Vaccine Control strategies against Group A streptococcal infections: Expectations, hopes and possible impact. In Streptococcal Pharyngitis. Issues Infectious Diseases. Base., Karger, Eds: Pechere JC and Kaplan EL Vol 3, pp 202-214

McArthur, J., Medina, E., Mueller, A., Chin, J., Currie, B.J., Sriprakash, K.S., Talay, S.R., Chhatwal, G.S., and Walker, M.J. (2004) Intranasal vaccination with streptococcal fibronectin binding protein Sfb1 fails to prevent growth and dissemination of Streptococcus pyogenes in a murine skin infection model. Infect Immun 72: 7342-7345.

McArthur, J., Schulze, K., Chin, J., Currie, B.J., Sriprakash, K.S., Talay, S.R., Chhatwal, G.S., Guzman, C.A., and Walker, M.J. (2004) Immune responses of a liposome/ISCOM vaccine adjuvant against streptococcal fibronectin binding protein 1 (Sfb1) in mice. Indian J Med Res 119 Suppl: 115-120.

McGregor, K.F., Bilek, N., Bennett, A., Kalia, A., Beall, B., Carapetis, J.R., Currie, B.J., Sriprakash, K.S., Spratt, B.G., and Bessen, D.E. (2004) Group A streptococci from a remote community have novel multilocus genotypes but share emm types and housekeeping alleles with isolates from worldwide sources. J Infect Dis 189: 717-723.

McKay, F.C., McArthur, J.D., Sanderson-Smith, M.L., Gardam, S., Currie, B.J., Sriprakash, K.S., Fagan, P.K., Towers, R.J., Batzloff, M.R., Chhatwal, G.S., Ranson, M., and Walker, M.J. (2004) Plasminogen binding by group A streptococcal isolates from a region of hyperendemicity for streptococcal skin infection and a high incidence of invasive infection. Infect Immun 72: 364-370.

McMillan, D.J., Batzloff, M.R., Browning, C.L., Davies, M.R., Good, M.F., Sriprakash, K.S., Janulczyk, R., and Rasmussen, M. (2004) Identification and assessment of new vaccine candidates for group A streptococcal infections. Vaccine 22: 2783-2790.

McMillan, D.J., Davies, M.R., Browning, C.L., Good, M.F., and Sriprakash, K.S. (2004) Prospecting for new group A streptococcal vaccine candidates. Indian J Med Res 119 Suppl: 121-125. McMillan, D.J., Davies, M.R., Good, M.F., and Sriprakash, K.S. (2004) Immune response to superoxide dismutase in group A streptococcal infection. FEMS Immunol Med Microbiol 40: 249-256.

Ramachandran, V., McArthur, J.D., Behm, C.E., Gutzeit, C., Dowton, M., Fagan, P.K., Towers, R., Currie, B., Sriprakash, K.S., and Walker, M.J. (2004) Two distinct genotypes of prtF2, encoding a fibronectin binding protein, and evolution of the gene family in Streptococcus pyogenes. J Bacteriol 186: 7601-7609.

Towers, R.J., Gal, D., McMillan, D., Sriprakash, K.S., Currie, B.J., Walker, M.J., Chhatwal, G.S., and Fagan, P.K. (2004) Fibronectin-binding protein gene recombination and horizontal transfer between group A and G streptococci. J Clin Microbiol 42: 5357-5361.

Binks, M., McMillan, D., and Sriprakash, K.S. (2003) Genomic location and variation of the gene for CRS, a complement binding protein in the M57 strains of Streptococcus pyogenes. Infect Immun 71: 6701-6706.

Edwards, M.L., Fagan, P.K., Smith-Vaughan, H., Currie, B.J., and Sriprakash, K.S. (2003) Strains of Streptococcus pyogenes from severe invasive infections bind HEp2 and HaCaT cells more avidly than strains from uncomplicated infections. J Clin Microbiol 41: 3936-3938.

Towers, R.J., Fagan, P.K., Talay, S.R., Currie, B.J., Sriprakash, K.S., Walker, M.J., and Chhatwal, G.S. (2003) Evolution of sfbI encoding streptococcal fibronectin-binding protein I: horizontal genetic transfer and gene mosaic structure. J Clin Microbiol 41: 5398-5406.

Delvecchio, A., Currie, B.J., McArthur, J.D., Walker, M.J., and Sriprakash, K.S. (2002) Streptococcus pyogenes prtFII, but not sfbI, sfbII or fbp54, is represented more frequently among invasive-disease isolates of tropical Australia. Epidemiol Infect 128: 391-396.

DelVecchio, A., Maley, M., Currie, B.J., and Sriprakash, K.S. (2002) NAD-glycohydrolase production and speA and speC distribution in Group A streptococcus (GAS) isolates do not correlate with severe GAS diseases in the Australian population. J Clin Microbiol 40: 2642-2644.

Gillen, C.M., Towers, R.J., McMillan, D.J., Delvecchio, A., Sriprakash, K.S., Currie, B., Kreikemeyer, B., Chhatwal, G.S., and Walker, M.J. (2002) Immunological response mounted by Aboriginal Australians living in the Northern Territory of Australia against Streptococcus pyogenes serum opacity factor. Microbiology 148: 169-178.

Sriprakash, K.S., Hartas, J., and White, A. (2002) Antibodies to streptococcal inhibitor of complement function and M peptides in a post-streptococcal glomerulonephritis endemic region of Australia. J Med Microbiol 51: 589-594.