Staphylococcus aureus evasion of host innate immunity

Abstract

Staphylococcus aureus is an opportunistic pathogen which can colonize the human nasal mucosae and the skin, as well as cause fatal diseases such as sepsis. S. aureus infections are challenging to treat because of antibiotic resistance, and there is currently no available vaccine, heightening the importance of expanding our understanding of virulence factors to develop new therapeutic approaches. S. aureus employs a diverse array of virulence factors to facilitate immune evasion and promote infection. In this study, we focus on investigating surface evasins, extracellular proteases and biofilm formation as major aspects of S. aureus pathogenesis.

The first objective was to assess the relative importance of evasins expressed on the bacterial surface. Since S. aureus express two essential surface evasins, Spa and Sbi, which complicate assessment due to non-specific binding to the Fc domain of IgG, we have generated a novel antibody-independent probe, biotinylated Sbi-IV, specifically targeting C3d domain. The Sbi-IV probe enables the assessment of C3 deposition on the bacterial surface via flow cytometry while mitigating non-specific binding issues. Through its application, we have identified that Spa and ClfA are relatively more important than other evasins considering complement evasion. In addition, preliminary data suggest that ClfB may exhibit complement evasion abilities similar to those observed for ClfA.

To enhance our comprehension of extracellular proteases and biofilm formation in clinical isolates, we employed k-mer-based genome-wide association study (GWAS) in chapters 4 and 5 to identify novel loci associated with specific phenotypes. To enable high-throughput quantification of extracellular protease activity, we optimized the casein plates assay to a casein hydrolysis assay (CHA) and phenotyped 134 clinical isolates from CC8. The phenotyping results have unveiled variability in both protease activity and biofilm formation. The protease activity of bacteraemia isolates is statistically significantly lower than that of isolates from SSTI and carriage. Meanwhile, the biofilm formation in bacteraemia isolates is lower than those from SSTI and carriage, although the difference is not statistically significant. The application of k-mer-based GWAS led to the identification of 31 novel loci associated with protease activity and 5 novel loci associated with biofilm formation. Disruption of these loci resulted in a discernible change in each respective phenotype.

To gain a better insight into the specific immune evasion mechanisms used by S. aureus we examined the pathogen’s interaction with the soluble complement inhibitor, C4b binding protein (C4BP). Previous studies indicated that S. aureus recruits C4BP using a 11 specific cell wall anchored surface protein and that bound C4BP limits complement deposition on the staphylococcal surface. Using flow cytometric based bacterial-protein binding assays we observed no interaction between S. aureus and C4BP. Moreover, we offer a precautionary warning that C4BP isolated from plasma can be co-purified with minute quantities of human IgG, which can distort binding analysis between S. aureus and human derived proteins. Combined our data indicates that recruitment of C4BP is not a complement evasion strategy employed by S. aureus.

Date of Award	26 Jun 2024
Original language	English
Awarding Institution	University of Bath
Supervisor	Maisem Laabei (Supervisor), Jean Van Den Elsen (Supervisor) & Vicky Hunt (Supervisor)