Virulence Determinants Of Community-associated Methicillin-resistant Staphylococcus Aureus Genotype ST72

In the past decade, community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA), strains of MRSA that are able to infect otherwise healthy patients outside of hospital settings, have recently arisen in many locations around the globe. Different clones are found to dominate in different countries. In the United States, clone USA300(sequence type ST8) is predominant. This and many other CA-MRSA clones harbor the genes coding for Panton-Valentine leukocidin (PVL), a toxin causing lysis of human neutrophils. Therefore, PVL has often been discussed as a potential factor determining virulence of CA-MRSA strains. However, the role of this leukotoxin in CA-MRSA pathogenesis is still controversial. Several CA-MRSA clones have been found that do not harbor PVL genes, and further investigation using animal infection models indicated that extraordinary virulence of CA-MRSA is only in part, and incomparatively rare types of infections such as severe lung infection, due to PVLOne such PVL-negative strain is the ST72strain HL1, the main cause of CA-MRSA infections in Korea. The genome sequences of two key PVL-positive CA-MRSA strains (USA300, USA400) have been reported, but we lack information on the more recently found PVL-negative CA-MRSA strains. Here, we report the entire genome sequence of CA-MRSA strain HL1and analyze its gene content with a focus on virulence factors. Our results show that this strain does not have considerable differences in virulence factor content compared to other CA-MRSA strains (USA300, USA400), indicating that other toxins do not substitute for the lack of PVL in ST72. HL1harbors the vSaa, vSaP, and vSay genomic islands and the ΦSa3prophage. The vSaa, vSaβ and vSay genomic islands occur in most S. aureus strains and harbor a series of virulence factors. These islands and the OSa3prophage are also present in the USA300and USA400CA-MRSA strains. Although the overall genetic composition differs, there are no considerable differences in known virulence determinants. A genome-wide analysis on103virulence genes to determine whether virulence factors present in HL1are also present in USA300and USA400further confirmed that the overall composition of virulence genes in HL1, USA300, and USA400is almost identical.28surface proteins, identified by the sortase substrate LPXTG motif, did not show differences in composition between the three analyzed strains. This finding is in accordance with the notion that differential expression of widespread virulence determinants, rather than the acquisition of additional virulence factors on mobile genetic elements, such as PVL, is responsible for the increased virulence of CA-MRSA compared to hospital-associated MRS A.To further investigate the role of virulence determinants in the success of the epidemic CA-MRSA strain HL1, we tested HL1and HL1derivetives, in which psma operon, hla, agr system were deleted (HL1△psma, HL1△hla, HL1△agr), in vivo and vitro to compare their virulence. In addition, we include three HL1mutants in which certain unique genes from HL1genome as hypothetical virulence genes were deleted in our experiments, and used PVL-positive CA-MRSA strain LAC to compare. Abcesses caused by HL1△hla and HL1△agr had significantly less area than those caused by the HL1and other HL1derivetives, indicating hla and agr are more important than psma and other genes for staphylococcal abscess formation in the mouse skin infection model. Of note, mouse abscesses caused by HL1were similar in size to those caused by LAC strain. Furthermore, we analyzed other in vitro parameters with HL1and HL1derivetives, and the results showed that PSMa has apronounced impact on hemolysis and neutrophils lysis capabilities of HL1. In summary, our study indicates that expression of core genome-encoded toxins such as hla and psma, not acquisition of MGEs carrying PVL, play an important role in the pathogenesis of ST72HL1.