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

Our laboratory has focused on the effects of outer membrane permeabilizer compound 48/80 on the intrinsic resistance mechanisms of gram-negative bacteria to hydrophobic antibacterial agents such as the biocide triclosan. To study gene expression changes potentiated by compound 48/80 in Serratia marcescens, previously obtained RNAseq data were further analyzed. Original analysis of RNAseq data indicated that, among the genes upregulated, three are known to play roles in the outer membrane when damaged by other antimicrobial agents. The objective of the present study was to clarify the bacterial response to treatment, 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.

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. A more recent analysis of RNAseq data was obtained using the Pathosystems Resources Integration Center (Patric) analysis tool (https://www.patricbrc.org), as well as a newly re-annotated genome. These data allowed for fine tuning of the earlier analysis to provide more detailed information on the upregulated genes. The Patric analysis tool allowed for the in-depth observation of genomic expression levels.

Fragments per Million Mapped Reads allowed for selection of appropriate qPCR housekeeping genes. Patric analysis reconfirmed the upregulation of the aforementioned genes. RNAseq results were reanalyzed to prepare for a qPCR-based expression time-course and to identify appropriate housekeeping gene candidates. qPCR primers for slyB, phoQ, phoP, and housekeeping genes will be tested and calibrated to allow the observation of expression changes over time.