Prediction Of Virulence Genes In Staphylococcus Argenteus And Characterization Of Two Novel Superantigens

Staphylococcus argenteus and S.schweitzeri were proposed to be novel species closely related to S.aureus in 2015,which were theretofore considered as two genetically divergent lineages within S.aureus.The three species were redefined as the S.aureus complex(SAC).Most of S.argenteus strains were isolated from humans and could cause infections the same as S.aureus.S.schweitzeri has not been reported to be associated with human infections,in spite to that some isolates were from humans,but has been generally isolated from nonhuman primates and bats.The three species are very similar on phenotypes,and there is a lower level of divergence among them at genomic level than that among many other close related species.Because of its associations with human health,S.argenteus was the focus for the following aspects in this study.It is the first report of S.argenteus present in Eastern China.To determine whether S.argenteus is present in China,89 of 839(10.6%)“S.aureus” isolates obtained from Eastern China were found forming white colonies.To test the 89 isolates belonging to S.aureus or S.argenteus,the core genes based on a SAC pan-genome definition were screened,and S.aureus was found to have a 180 bp deletion in the gene sequence of a hypothetical nonribosomal peptide synthetase(NRPS),compared with S.argenteus and S.schweitzeri.Then,the 89 white isolates were tested by PCR targeting the indel region of the NRPS gene.The results demonstrated that the 89 strains were divided into three groups: six strains(6.8%)had PCR products,the size of which was in accordance with that of S.argenteus;75(84.3%)strains' PCR products were in accordance with S.aureus;and eight strains had no detectable PCR product.The six,75 and eight strains were further identified to be S.argenteus,S.aureus and other bacteria,respectively,using 16 S r RNA gene,rpo B gene and multilocus sequence typing(MLST).It is suggested that the PCR-based method targeting the NRPS gene can simultaneously identify and distinguish S.argenteus and S.aureus,together with observation on the colony color.Six S.argenteus isolates were found to be present in Eastern China.Systematical genomic analysis revealed that S.argenteus,harbors many virulence genes and had spread internationally.The genomes of five S.argenteus strains(selected from the six S.argenteus strains identified in this study)were sequenced,and the sequence data on representative genomes of S.aureus,S.argenteus and S.schweitzeri were downloaded from NCBI.A total of 51 SAC genomes were subjected to a pan-genome definition.The three species had divergent core genomes and rare interspecific recombination was observed among their core genes.However,some subtypes of staphylococcal cassette chromosome mec(SCCmec)elements and prophages were present in different species.Of the 111 tested virulence genes of S.aureus,85(76.6%)homologous genes were found in S.argenteus.There was no difference in virulence gene content between S.argenteus and S.aureus,but the sequence of most core virulence genes was divergent.Considering the cases of S.argenteus causing human infections and harboring the virulence genes mentioned above,S.argenteus should have a similar pathogenic potential as S.aureus.Furthermore,the geographic distribution of the widespread S.argenteus sequence type 2250(ST2250),showed ambiguous biogeographical structure among geographically isolated populations,demonstrating an international spread of this pathogen.S.argenteus ST2250 harbors two lineage-specific novel superantigens.Two genes were assigned to homologous gene families with known staphylococcal enterotoxin(SE)gene in the virulence gene prediction,but the related proteins were distinct from known SEs with low amino acid sequence similarities(<65.3%).Further analysis indicated that the two putative SE genes were located upstream of some v Sa? genetic islands of SAC,and the related proteins may represent two novel SEs,designated SEl26 and SEl27,respectively.In the S.aureus strains,m RNA encoding SEl26 and SEl27 were highly expressed in the early stationary phase of cultivation,whereas high levels of expression were found in S.argenteus and S.schweitzeri strains at the log phase.The recombinant SEl26 and SEl27 also exhibited superantigenic activity in human peripheral blood mononuclear cells(PBMCs)and mouse splenocytes.From these results,we identified SEl26 and SEl27 to be two novel SE-like toxins for their superantigenic activity and low identities(<10%)with known SEs,which was used to define different types of staphylococcal superantigens.This study suggested that S.argenteus was present in Eastern China,had strong pathogenetic potential at genomic level,and could produce novel superantigen toxins.The two-step procedure based on phenotype observation and NRPS-PCR is able to promote the studies on S.argenteus epidemiology,and the identification of SEl26 and SEl27 enriches the virulence gene reservoir of SAC.