Year

2004

Degree Name

University of Wollongong. School of Biological Sciences

Department

School of Biological Sciences

Abstract

Group A streptococci (GAS) are one of the major bacterial pathogens affecting the health of Indigenous Australians. The Aboriginal population of the Northern Territory (NT) suffer some of the highest rates of serious GAS-associated disease in the world including acute rheumatic fever, acute post-streptococcal glomerulonephritis and severe invasive disease. Streptococcal infections are endemic most of which are benign conditions such as impetigo with up to 70% of school-aged Aboriginal children having GAS-infected skin sores.

As part of this study, data on 100 Northern Territory GAS isolates characterised in previous studies was collated and analysed to identify relationships between disease presentation, in vitro binding of human extracellular and plasma proteins, and the presence of genes encoding important streptococcal fibronectin- and plasminogenbinding proteins. This analysis showed that GAS present on the skin constitute the reservoir from which invasive disease isolates originate. Of the factors tested here only the presence of the fibronectin-binding protein FbaB was significantly linked to the incidence of invasive disease although the presence of this gene did not affect binding of fibronectin in vitro. On the other hand, the presence of genes encoding streptococcal fibronectin-binding protein I (SfbI) and serum opacity factor (SOF) was significantly associated with increased fibronectin binding. The presence of sof was linked to the presence of sfbl, despite these genes residing at remote loci, and the copresence of the two genes affected binding of fibronectin in an accumulative manner. The presence of the two variants of fibronectin-binding protein Protein F2 was also strongly linked to the presence of sof with sof-positive strains having fbaB and sol-negative strains harbouring fbaB.

By determining anti-SfbI and anti-SOF antibody titres in the serum and saliva of individuals living in remote Aboriginal communities, it was shown that natural infection in an endemic area results in significantly bigher antibody levels against these proteins. SfbI has been shown to be an effective vaccine antigen against both homologous and heterologous challenge. Using recombinant proteins expressed in this study, it was shown that the protective epitopes reside in the highly conserved carboxy-terminal fibronectin-binding repeat domain (FBRD).

Isolates of several streptococcal species including 71 group A, 6 group C and 5 group G streptococci were screened for the presence of sfbI and the genes were sequenced. Analysis of the DNA sequence data revealed that SfbI exhibits considerable variation in the amino-terminal aromatic-rich domain which has arisen through horizontal exchange of DNA both within and between GAS and GGS strains/species. Eleven recombinant SfbI proteins were cloned and expressed each representing a distinct sfbI sequence type (ST). The original SfbI protein was also truncated to remove its fibronectin-binding domains. The ability of the different SfbI variants to bind fibronectin was measured by surface plasmon resonance. This showed that the amino-terminal variation, and not the variable numbers of fibronectin-binding repeats, impacts on the amount of fibronectin bound by Sfbl. The truncated SfbI protein showed affinity for the amino-terminal 29 kDa fragment of fibronectin (Fn29), however, variability in the amino-terminus was also shown to affect binding of the 42 kDa fragment of fibronectin (Fn42). Probing of an SfbI peptide library revealed a novel, highly conserved Fn29-binding motif in the aromatic-rich domain.

Since variability in the aromatic-rich domain is due to horizontal transfer, the generation of new alleles is relatively rapid when compared to point mutations in a "trial-and-error" context. While no link: has been shown between the severity of streptococcal disease and the presence of sfbI, fibronectin binding by SfbI has been directly implicated in bacterial adherence and in the integrin-mediated uptake of GAS by human epithelial cells. It is likely that the introduction of penicillin in 1945 would have provided sufficient impetus for the selection of GAS strains with the ability to reside intracellularly. It is also likely that intraspecific recombination within sfbI has played a role in producing SfbI proteins with increased avidity for fibronectin. Horizontal gene transfer is also likely to have played a role in generating the mosaic structure observed in the fibronectin-collagen-T (FCT) region which has resulted in the tightly regulated expression of SfbI, SOF and Protein F2 in response to different environmental stimuli. Whether this increases the fitness of the strains by enhancing the ability to colonise or be internalised by human epithelial cells is a complex issue to resolve, however, it is our intention to use the rSfbI proteins purified in this study to elucidate on the ability of the different SfbI variants to adhere and be internalised by human tissue culture cells.

The high rates of streptococcal carriage seen in Indigenous people in remote NT communities provides an excellent opportunity forthe exchange of bacterial genetic material. Skin sores are frequently infected not only with multiple strains of GAS but also mixtures of GAS, GCS and GGS. The high degree of diversity reported in the NT streptococcal population is evidence that such transfer is frequent and ongoing.

Endemic streptococcal skin infection is likely to significantly increase the rates of intra/interspecific genetic exchange posing the significant risk of producing newly emergent, highly virulent streptococcal strains. The emm gene is also affected by horizontal transfer and recombination has resulted in many new emm STs in the NT. Even if the polyvalent M-protein-based vaccines currently on trial covered M types circulating in the NT, the high rate of recombination is likely to result in selection for new M proteins. Since the protective FBRD of StbI is not affected by horizontal transfer, an SfbI-based vaccine is more likely to elicit enduring immunity against StbIpositive GAS.

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