The importance of Staphylococcal superantigen-like proteins (SSLs) in Staphylococcus aureus pathogenicity; with a focus on SSL11

Abstract

Staphylococcus aureus (S.aureus) is a common human pathogen that resides asymptomatically in almost a third of the world’s population. This bacterium has a high socioeconomic impact and has the ability to cause a variety of diseases including life-threatening sepsis and staphylococcal toxic shock syndrome. A family of 14 virulence factors known as Staphylococcal Superantigen-Like proteins (SSLs) has been identified that have affinity for molecules found in the innate immune system and aid with immune evasion. Within this family, a protein called SSL11 is carbohydrate binding and is able to bind to sialyllactosamine (sLacNAc) with high affinity. In this way, SSL11 is able to evade immune surveillance as it interferes with immune recognition of sialyated receptors. SSL11 is always co-transcribed with ORF401, an adjacent gene that has yet to be investigated. A S.aureus strain was characterised in the Vernon Jansen Unit (VJU) at the University of Auckland, named JSNZ. This strain then had different ssl genes removed to provide knockout strains for laboratory use. JSNZ:: ssl11.orf401, JSNZ:: ssl11, JSNZ:: orf401, JSNZ:: ssl and wildtype JSNZ were used in this thesis. In whole blood killing assays survival of bacteria was shown to decrease with the removal of the ssl.orf401 genes in comparison to wildtype, while the addition of either SSL11 or ORF401 recombinant protein to the JSNZ:: ssl.orf401 returned survival back to wildtype levels. This trend was seen again when repeating this assay using JSNZ:: ssl and JSNZ:: orf401 strains instead of JSNZ:: ssl11.orf401 with recombinant protein. Following the whole blood killing assays, bacteria was injected into live, Galleria mellonella (G. mellonella) an insect (wax moth larvae) that is becoming more frequently used to screen potential proteins and immune target sites among other things. After injection, bacteria lacking the entire complement of ssl genes, or lacking the ssl11.orf401 genes showed decreased sickness compared to wildtype in G. mellonella models. Both intraperitoneal and subcutaneous murine models of infection were used to observe how the knockout strains differed to wildtype in an in vivo situation. Intraperitoneal studies showed high bacterial recovery in the kidneys, liver and spleen of mice infected with wildtype, with lower recoveries in mice infected with JSNZ:: ssl, and lower still in mice infected with JSNZ:: ssl11.orf401. Cellular data from subcutaneous studies showed a decrease in the number of neutrophils in both the spleen and lymph nodes of mice infected with JSNZ:: ssl compared to that of wildtype S. aureus. Decreases were also seen in the number of macrophages and CD8+/CD69+ T cells present in the JSNZ:: ssl treatment group compared to wildtype. The removal of the ssl genes or the ssl11.orf401 genes results in the reduced survival of S. aureus in nearly all assays and models of infection. However, the addition of either SSL11 or ORF401 is able to significantly increase survival back to wildtype levels compared to strains without ssl11.orf401 genes. This would appear to indicate a redundancy between the ssl11 and orf401 genes.