Post-transcriptional regulation of porin expression in Escherichia coli and its impacts on antibiotic resistance
Fellow: Sushovan Dam
Supervisor: Jean-Marie Pagès (Aix Marseille Université)
Co-supervisor: Dr. Muriel Masi (Aix Marseille Université)
The global emergence of multi drug resistant bacteria, particularly in Gram-negative species, is a growing threat to antibiotic therapy. In Gram-negative species, this can be attributed to altered porin expression and β-lactamase production.
Gene regulation by small non-coding RNAs (sRNAs) has been recognized as an important post-transcriptional regulatory mechanism for several years. In Gram-negative bacteria such as Escherichia coli and Enterobacter spp., these RNAs control stress response and translation of outer membrane proteins (OMPs) and therefore are key regulators of environmental stress. The two classical porins OmpF and OmpC in E. coli have been shown to be post-transcriptionally regulated by the MicF and MicC sRNAs, respectively.
In this work, we have investigated the expression profile of the MicC sRNA, its impact on porin expression and consequently its effect on antibiotic resistance. First, we used transcriptional lacZ fusions to screen through numerous stress conditions and genetic backgrounds. These data pointed to the putative role of sigma E (RpoE) and the DNA binding protein H-NS as well as several antimicrobials in the induction of MicC. Then, increased MicC levels were confirmed by Northern blot analysis and porin expression profiles were evaluated by Western blot analysis in carefully chosen stress conditions and mutants. Furthermore, we have performed RNA-seq experiments to decipher the global transcriptional impact of overexpressed and stress-induced MicC in E. coli MC4100 cells.
Previous studies have showed that many parallels can be drawn between the MicF and MicC sRNAs: primarily, both repress the expression of abundant porins by base pairing near the ribosome binding site, thereby blocking translation, and both are encoded opposite to another porin gene (i.e. the micF-ompC and micC-ompN regions). Here, we show that micC and the adjacent ompN gene share a common transcriptional regulation, reminiscent to what is observed for micF and the adjacent ompC gene. OmpN is an uncharacterized quiescent porin that could play a key role in the control of bacterial lifestyle and adaptation to stressful conditions, particularly to antibiotics. To test this hypothesis, we have raised antibodies against a specific OmpN peptide and analyzed the expression of OmpN in series of E. coli and E. aerogenes clinical isolates.
Fig 1: Genetic arrangement of OmpC and OmpN Fig 2: Identification of factors that controls micC/ompN expression by plasmidic fusions and and elucidating how this eventually impacts the Multi Drug Resistance phenotype
Altogether, our results highlight the pathways that suppress the major porin OmpC via the MicC sRNA, which ultimately impairs the antibiotic permeation and hence increases bacterial resistance.