| Outline of Annual Research Achievements |
We screened Nebraska Transposon Mutant Library of USA300 and found that 8 mutants with disruption in 0750, 0553, 0980, 2320, 0321, 0657, 0942 and yjbI genes had reduced virulence in silkworm and mice infection models. By whole genome sequence analysis, we confirmed the disruption of each gene and absence of insertions at other positions. We did not find single nucleotide polymorphism in the mutants (except 0750::Tn, which had a single amino acid substitution in CshB (Arg133Gly). We introduced plasmids containing the intact genes into each mutant and determined their pathogenicity in silkworms. We found that the introduction of six genes (0750::Tn, 0553::Tn, 0980::Tn, 2320::Tn, 0321::Tn, and 0657::Tn) complemented the pathogenicity while introduction of 0942 in 0942::Tn resulted in loss of pathogenicity. We further found that introducing the yjbI in yjbI::Tn did not recover the pathogenicity, while the introduction of operon consisting of yjbI and yjbH genes, and only yjbH gene complemented the pathogenicity in both the yjbI::Tn and yjbH::Tn mutants. We performed RNA-seq analysis of yjbI::Tn and yjbH::Tn mutants and revealed that yjbI mutant has significantly decreased expression of yjbH gene (113 fold downregulated), suggesting that the observed phenotypes in yjbI may be as a result of the decreased expression of yjbH. We found that disruption of yjbI and yjbH genes significantly affected the expression of more than 200 genes which included decreased expression of several virulence factors related genes such as spA, sarS, aur, sspA, sspB, sspP, splA, sbi, splF, and lukE.
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| Current Status of Research Progress |
Current Status of Research Progress
1: Research has progressed more than it was originally planned.
Reason
As planned previously, we constructed the strains by introduction of plasmids containing the intact genes into the respective mutants and confirmed the roles of these genes in pathogenicity in silkworms. This is considered an important progress towards this research. The current status of the research is ahead of the plan, as we have already performed the in vitro RNA-seq analysis of yjbI::Tn and yjbH::Tn mutants and in vitro RNA-seq analysis of other strains is under progress.
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| Strategy for Future Research Activity |
In the next period, we plan to perform RNA-seq analysis of all the gene-disrupted mutants. The RNA-seq analysis will be performed first in vitro and then in vivo in mice according to a method established in our laboratory to selectively lyse host cells and enrich S. aureus cells. Gene expression patterns will be compared between wild-type and gene-disrupted mutants, and genes-complemented and overexpressed strains by RNA-seq analysis. Expression of selected candidate genes will then be analyzed by real time RT-PCR. This will help to identify the target genes possibly regulated by the identified genes, and to elucidate the mechanism of the genes in pathogenesis. In addition, exoprotein profiling will be performed by sodium dodecyl sulfate- polyacrylamide gel electrophoresis of the culture supernatants of the mutants, wild-type and genes-complemented strains in order to find the exoproteins regulated by the respective genes. Recombinant proteins will be expressed and purified for elucidating the mechanism of the respective proteins involved in virulence.
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