Abstract
Clinical development of antibiotics with novel mechanisms of action to kill pathogenic bacteria is challenging, in part, due to the inevitable emergence of resistance. A phenomenon of potential clinical importance that is broadly overlooked in preclinical development is heteroresistance, an often-unstable phenotype in which subpopulations of bacterial cells show decreased antibiotic susceptibility relative to the dominant population. Here, we describe a new globomycin analog, G0790, with potent activity against the Escherichia coli type II signal peptidase LspA and uncover two novel resistance mechanisms to G0790 in the clinical uropathogenic E. coli strain CFT073. Building on the previous finding that complete deletion of Lpp, the major Gram-negative outer membrane lipoprotein, leads to globomycin resistance, we also find that an unexpectedly modest decrease in Lpp levels mediated by insertion-based disruption of regulatory elements is sufficient to confer G0790 resistance and increase sensitivity to serum killing. In addition, we describe a heteroresistance phenotype mediated by genomic amplifications of lspA that result in increased LspA levels sufficient to overcome inhibition by G0790 in culture. These genomic amplifications are highly unstable and are lost after as few as two subcultures in the absence of G0790, which places amplification-containing resistant strains at high risk of being misclassified as susceptible by routine antimicrobial susceptibility testing. In summary, our study uncovers two vastly different mechanisms of resistance to LspA inhibitors in E. coli and emphasizes the importance of considering the potential impact of unstable and heterogenous phenotypes when developing antibiotics for clinical use.IMPORTANCE Despite increasing evidence suggesting that antibiotic heteroresistance can lead to treatment failure, the significance of this phenomena in the clinic is not well understood, because many clinical antibiotic susceptibility testing approaches lack the resolution needed to reliably classify heteroresistant strains. Here we present G0790, a new globomycin analog and potent inhibitor of the Escherichia coli type II signal peptidase LspA. We demonstrate that in addition to previously known mechanisms of resistance to LspA inhibitors, unstable genomic amplifications containing lspA can lead to modest yet biologically significant increases in LspA protein levels that confer a heteroresistance phenotype.
Highlights
Clinical development of antibiotics with novel mechanisms of action to kill pathogenic bacteria is challenging, in part, due to the inevitable emergence of resistance
Our efforts to identify potent GBM analogs were driven by whole bacterial cell activity assays due to the low translatability of in vitro biochemical activity [34, 35]
Published data have demonstrated that resistance can occur via either deletion of lpp or removal of the C-terminal lysine that eliminates the peptidoglycan-linked Lpp form [31]
Summary
Clinical development of antibiotics with novel mechanisms of action to kill pathogenic bacteria is challenging, in part, due to the inevitable emergence of resistance. Many clinical laboratories use nonselective culture techniques to grow bacterial isolates from patient samples [13], and there are currently no established antimicrobial susceptibility tests to identify heteroresistance in the hospital setting For these reasons, heteroresistance phenotypes can escape detection and potentially pose a significant hurdle to the appropriate administration of antibiotics to patients. LspA inhibitors are proposed to cause bacterial cell death by leading to the accumulation of the peptidoglycan-linked form of Lpp in the inner membrane Consistent with this hypothesis, deletion of lpp leads to resistance to certain inhibitors of lipoprotein biosynthesis and transport [30,31,32,33]. Given the challenges associated with identifying new antibiotic leads that can efficiently penetrate the asymmetric Gram-negative bacterial cell envelope from initial hits identified in high-throughput screens [34, 35], we decided to use the natural
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