Unstable Mechanisms of Resistance to Inhibitors of Escherichia coli Lipoprotein Signal Peptidase

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.

Single-molecule nanopore sequencing reveals extreme target copy number heterogeneity in arylomycin-resistant mutants

“…1 C ). The MinION data add direct evidence to the growing recognition that clonal bacterial population can exhibit substantial heterogeneity with respect to CN ( 3 , 7 ) and are consistent with highly dynamic temporal expansion and/or contraction of the lepB repeat array ( Fig. 1 A ) during clonal evolution.…”

“…CN heterogeneity in bacteria is of special interest because it is a likely explanation for isolates that exhibit phenotypic heterogeneity with respect to antibiotic resistance, so-called heteroresistance ( 3 , 5 – 7 ). The underlying hypothesis is that variable gene dosages exist within such populations, something that has not been directly ascertained at the level of individual DNA molecules.…”

“…Gene amplification is a common mechanistic explanation for unstable resistance phenotypes, both in vitro and in clinical isolates ( 3 – 7 ). Amplification instability is driven by the stochasticities associated with the frequent formation and loss of the amplifications themselves, selection, and the costs that maintenance of amplified DNA impose on fitness ( 8 , 9 ).…”

Inhibition of Escherichia coli Lipoprotein Diacylglyceryl Transferase Is Insensitive to Resistance Caused by Deletion of Braun’s Lipoprotein

“…However, one could speculate that if G2824 binds to the conserved phosphatidylglycerol binding site in Lgt, mutations disrupting G2824 binding might be incompatible with bacterial viability. In fact, no on-target resistance mutations have ever been identified against the LspA inhibitor globomycin (55) or its improved analog, G0790 (54), which is believed to act as a substrate mimic and bind the highly conserved LspA active site (56). As recent publications have revealed significant insights into the potential mechanisms of diacylglyceryl on May 11, 2021 by guest http://jb.asm.org/…”

“…This is not unexpected and is detected in most published reports when using sucrose gradient centrifugation to examine membrane localization of Lpp in WT E. coli membranes ( 22 , 23 , 53 ), suggesting this is unlikely due to the use of lptD imp4213 -expressing cells. Interestingly, separation of IM and OM in globomycin-treated cells was more challenging, likely explained by the significant accumulation of membranes as detected by electron microscopy after inhibition of LspA ( 54 ). While we were unable to raise on-target resistant mutants to G2824, we cannot fully rule out that G2824 does not have additional targets in bacterial cells.…”