Effects of gram-negative bacterial outer membrane permeabilization on Serratia marcescens' gene expression

Abstract

Our laboratory has focused on understanding the effects of outer membrane permeabilizer compound 48/80 on the intrinsic resistance of gram-negative bacteria to hydrophobic antibacterial agents such as the biocide triclosan. Previously obtained RNAseq data were analyzed to identify gene expression changes potentiated by compound 48/80 in the opportunistic pathogen Serratia marcescens. These data indicated greatly upregulated expression of three genes, each involved in repair of outer membrane damage by other antimicrobial agents. The objective of the present study was to clarify the bacterial response to compound 48/80 treatment using RT-qPCR to follow gene expression, with the ultimate goal of establishing a proposed mechanism of action for compound 48/80-induced outer membrane permeability. Previous work indicated that S. marcescens is one of the few species of bacteria intrinsically resistant to triclosan, and that compound 48/80 induces transient sensitization to triclosan. The RNAseq analyses revealed a 50-fold increase in expression of slyB, phoP, and phoQ subsequent to compound 48/80 administration and qPCR primers were created in order to further investigate their regulation. The Bacterial and Viral Bioinformatics Resource Center (BV-BRC) analysis tools confirmed the upregulation of the aforementioned genes and provided Fragments Per Kilobase Million Mapped Reads (FPKM) to enable selection of an appropriate qPCR housekeeping gene, yfiR. RNA has been prepared from expression time-courses in preparation for the RTqPCR process, in which qPCR primers for slyB, phoQ, phoP and yfiR will be used to measure expression changes observed over time.