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United States Department of Agriculture

Agricultural Research Service

Research Project: MOLECULAR MECHANISMS OF PATHOGENIC BACTERIA INTERACTIONS WITH PLANT SURFACES AND ENVIRONMENTAL MATRICES

Location: Environmental Microbial & Food Safety Laboratory

Title: Genetic basis for the resistance of Staphylococcus aureus to peptidoglycan hydrolase by comparative transcriptome and whole genome sequence analysis

Author
item Yan, Xianghe
item Xie, Yanping
item Donovan, David
item Swift, Steven
item Li, Charles
item Gehring, Andrew
item Irwin, Peter
item He, Yiping

Submitted to: American Society for Microbiology
Publication Type: Abstract Only
Publication Acceptance Date: 5/2/2017
Publication Date: N/A
Citation: N/A

Interpretive Summary:

Technical Abstract: Background: Lysostaphin is a glycyl-glycine bacteriocin peptidoglycan hydrolase secreted by Staphylococcus simulans for degrading the peptidoglycan moieties in Staphylococcus aureus cell walls which result in cell lysis. There are known mechanisms of resistance to lysostaphin, e.g. serine in place of glycine residues introduced into the peptidoglycan pentaglycine cross bridge or a shortened cross bridge, but we are interested in identifying more generic resistance mechanisms to peptidoglycan hydrolases via repeated exposure of Staphylococcus aureus (Newman_2010; indicating our cultures of S. aureus Newman in year 2010) to sub-lethal concentrations of engineered triple-acting staphylolytic peptidoglycan hydrolases. The resultant mutant (1801_2010) we identified has significant resistance to lysostaphin relative to the wild type (>500 fold increase in minimum inhibitory concentration (MIC)), but does not appear to be due to either of the known resistance genes resulting in the phenotypes described above. Materials and Methods: In this study, both DNA- and RNA-seq libraries of the S. aureus Newman_2010 and 1801_2010 strains were constructed for genome-wide DNA sequence and RNA level transcriptome analysis. Reverse transcription qPCR was used to confirm the RNA-seq results. Computational analyses, including gene ontology and KEGG pathway enrichment analysis, were also employed. Pooled RNA-seq sequencing data were used to compare genome-wide SNP and InDel results between the mutant 1801_2010 and wild type. Results and Conclusion: Comparative transcriptome analysis and de novo short-read transcriptome assembly revealed that significant transcriptional changes in response to lysostaphin are associated with membrane, cell wall, and their related genes (e.g., amidase, peptidase, holin, and phospholipase D/transphosphatidylase, etc.). The RNA-seq based differential expression of genes was confirmed by reverse transcription qPCR. Moreover, these results are consistent with the observed structural changes at the DNA and RNA levels. These structural changes present in the genes encoding membrane/cell surface proteins and the perturbation in gene expression probably contribute to the increased resistance of S. aureus to lysostaphin. The findings of this work could provide insight into the design of new antimicrobial agents.

Last Modified: 10/17/2017
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