Regulation of M-like proteins SiMA and SiMB of Streptococcus iniae, and potential as cross-protective vaccines in Lates calcarifer

Aviles, Fabian (2012). Regulation of M-like proteins SiMA and SiMB of Streptococcus iniae, and potential as cross-protective vaccines in Lates calcarifer MPhil Thesis, School of Biological Sciences, The University of Queensland.

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Author Aviles, Fabian
Thesis Title Regulation of M-like proteins SiMA and SiMB of Streptococcus iniae, and potential as cross-protective vaccines in Lates calcarifer
Formatted title
Regulation of M-like proteins SiMA and SiMB of Streptococcus iniae, and potential as cross-protective vaccines in Lates calcarifer
School, Centre or Institute School of Biological Sciences
Institution The University of Queensland
Publication date 2012-03
Thesis type MPhil Thesis
Supervisor Andrew Barnes
Sandra Degnan
Total pages 96
Total colour pages 2
Total black and white pages 94
Language eng
Subjects 0707 Veterinary Sciences
0605 Microbiology
Formatted abstract
Streptococcus iniae is a fish pathogen of global significance causing invasive infections in a broad range of fresh and saltwater fish resulting in high economic losses in aquaculture, wild fish kills and occasional zoonotic infections. Vaccination against S. iniae is complicated by serotypic variation determined by capsular polysaccharide. M or M-like proteins are a major virulence factor in S. iniae, impeding phagocytosic clearance by binding host humoral factors. In S. iniae M-like proteins, SiMA and SiMB are encoded by the emm-like genes simA and simB. In contrast to other Streptococcal pathogens, M-like proteins are highly conserved in S. iniae with all virulent strains to date expressing the simA variant. This lack of diversity coupled with a critical role in virulence makes these proteins important targets for future vaccines, but they are poorly expressed in normal laboratory culture. The first aim of this study is to determine how SiMA is regulated in S. iniae in order to optimise culture conditions for increasing expression of simA and potentially improving vaccines that protect across serotypes.

The gene (mgx), encoding a putative multigene regulator MgX, was identified upstream of the simA gene, along with a possible MgX binding site in the -35 region of the simA promoters. Electrophoretic Mobility Shift Assay using recombinant MgX protein confirmed binding to the -35 promoter region of the simA gene. qRT-PCR was then employed to investigate expression of mgx and simA in S. iniae under differing culture conditions.

The highest level of simA expression was observed during exponential growth phase under iron limitation with a 20-fold increase in relative expression (normalised against gyrA) compared to growth in Todd-Hewitt broth. Expression of simA correlated closely with expression of mgX under the various culture conditions and growth phases analysed, supporting a role for MgX as a key regulator of simA expression in S. iniae.

Based on these results, a vaccination and challenge experiment was conducted in barramundi to determine whether recombinant SiMA is a) protective against Streptococcus iniae infection and b) cross-protective against an antigenically different capsular serotype. A challenge model was established resulting in 60% mortality in adjuvant-only controls. Formalin-killed bacterins prepared from the challenge strain resulted in 100% protection when given intraperitoneally in oil-adjuvant. Formalin killed bacterins prepared from a serotypically heterologous strain resulted in significantly reduced protection, even when culture conditions were manipulated to optimise SiMA expression. Recombinant SiMA protein was not protective against the challenge strain, in spite of eliciting specific antibody response in vaccinated fish.

In order to elucidate the role of specific antibody in protection, the challenge strain of S. iniae was incubated with serum derived from a cohort of vaccinated fish from each group in the challenge experiment, and the effect of the sera on phagocytosis and respiratory burst by head kidney leucocytes was analysed by Flow-cytometry. Interestingly, no increase in oxidative activity or phagocytosis was observed with the addition of antisera. Moreover, all sera and antisera elicited a significantly lower response compared to controls where bacteria were not incubated with serum. This lack of clear specific-antibody mediated opsonisation in spite of 100% protection against challenge with the homologous bacterin, suggests that other immune parameters are responsible for the observed protection of challenged fish. Depression of the HKL responses in the presence of serum may be indicative of binding of host serum components by the bacteria, thereby reducing the ability of leucocytes to detect them and respond.
Keyword Vaccine
M protein
Barramundi lates-calcarifer
Streptococcus iniae
Antibody response
Infectious disease

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Created: Tue, 25 Sep 2012, 22:56:15 EST by Mr Fabian Aviles on behalf of Scholarly Communication and Digitisation Service