Deciphering the mechanism of E. coli resistance to a membrane-targeting antimicrobial peptide through genomic and transcriptomic approaches
Jeanette Velásquez Guzmán: New Mexico Consortium
<div>Antimicrobial peptides (AMPs) are essential components of host innate immunity, representing the first line of defense in bacterial clearance. However, bacteria can develop resistance to AMPs. Using <em>Escherichia coli</em> strain BL21-Gold (DE3) as a model, we investigated the mechanism of bacterial resistance to AMPs. The strain was allowed to evolve resistance against an amphipathic 11 residue helical peptide (or P11). The minimal inhibitory concentration (MIC) of the resistant strain is 13-fold higher than that of the wildtype (or susceptible) strain. Genome sequencing of the resistant <em>E. coli</em> derivative revealed insertions and deletions in several genes. Through comparative genome analysis, we detected transposase insertions in genes involved in outer membrane (<em>asmA</em>) and lipopolysaccharide (<em>waaP</em>) biosynthesis. The <em>asmA</em> gene encodes assembly protein asmA, which is involved outer membrane assembly; whereas <em>waaP</em> encodes lipopolysaccharide core heptose (I) kinase, required for the heptose phosphorylation in lipopolysaccharide (LPS) core. We also detected a transposase insertion in the <em>Dihydrouridine synthase C</em> (<em>dusC</em>) gene, which encodes tRNA-dihydrouridine<sup> </sup>synthase. Knocking out these three genes resulted in 3-fold increase of the MIC compared to the control. Several mutations in genes that encode proteins are involved in interactions with phospholipids and membrane permeabilization of P11. Overall, our data suggest the collective action of genic and intergenic mutations contribute to resistance. Based on these observations, we designed a next-generation, 26 residue AMP (P26) that overcomes the bacterial resistance mechanism. Transcriptome profiling analysis is underway and should also be highly informative.</div>
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