Two Small RNAs Conserved in Enterobacteriaceae Provide Intrinsic Resistance to Antibiotics Targeting the Cell Wall Biosynthesis Enzyme Glucosamine-6-Phosphate Synthase

Abstract

Formation of glucosamine-6-phosphate (GlcN6P) by enzyme GlcN6P synthase (GlmS) represents the first step in bacterial cell envelope synthesis. In Escherichia coli, expression of glmS is controlled by small RNAs (sRNAs) GlmY and GlmZ. GlmZ activates the glmS mRNA by base-pairing. When not required, GlmZ is bound by adapter protein RapZ and recruited to cleavage by RNase E inactivating the sRNA. The homologous sRNA GlmY activates glmS indirectly. When present at high levels, GlmY sequesters RapZ by an RNA mimicry mechanism suppressing cleavage of GlmZ. The interplay of both sRNAs is believed to adjust GlmS synthesis to the needs of the cell, i.e., to achieve GlcN6P homeostasis. Bacilysin (tetaine) and Nva-FMDP are dipeptide antibiotics that impair cell envelope synthesis by inhibition of enzyme GlmS through covalent modification. However, although taken up efficiently, these antibiotics are less active against E. coli for reasons unknown so far. Here we show that the GlmY/GlmZ circuit provides resistance. Inhibition of GlmS causes GlcN6P deprivation leading to activation of GlmY and GlmZ, which in turn trigger glmS overexpression in a dosage-dependent manner. Mutation of glmY or glmZ disables this response and renders the bacteria highly susceptible to GlmS inhibitors. Thus, E. coli compensates inhibition of GlmS by increasing its synthesis through the GlmY/GlmZ pathway. This mechanism is also operative in Salmonella indicating that it is conserved in Enterobacteriaceae possessing these sRNAs. As GlmY apparently responds to GlcN6P, co-application of a non-metabolizable GlcN6P analog may prevent activation of the sRNAs and thereby increase the bactericidal activity of GlmS inhibitors against wild-type bacteria. Initial experiments using glucosamine-6-sulfate support this possibility. Thus, GlcN6P analogs might be considered for co-application with GlmS inhibitors in combined therapy to treat infections caused by pathogenic Enterobacteriaceae.

Small RNA ‐binding protein RapZ mediates cell envelope precursor sensing and signaling in Escherichia coli

“…( Figs 5 and 6). In conclusion, the increase of GlmY* steady-state levels observed upon GlcN6P depletion in the current ( Fig 1A) and previous work (Reichenbach et al, 2008(Reichenbach et al, , 2009Khan et al, 2016) results from two distinct activities of RapZ: upregulation of glmY transcription through QseE/QseF and stabilization of GlmY* through its binding. We further identify a negative feedback loop that limits glmY expression under GlcN6P starvation.…”

“…Once replenished, GlcN6P releases RapZ from complexes with GlmY*, which is in turn rapidly degraded due to lack of protection (Figs and ). In conclusion, the increase of GlmY* steady‐state levels observed upon GlcN6P depletion in the current (Fig A) and previous work (Reichenbach et al , , ; Khan et al , ) results from two distinct activities of RapZ: upregulation of glmY transcription through QseE/QseF and stabilization of GlmY* through its binding.…”

“…To trigger GlcN6P depletion, we used Nva‐FMDP, a synthetic derivative of an antibiotic, which selectively inhibits GlmS enzymatic activity and causes exhaustion of intracellular GlcN6P (Chmara et al , ). We previously demonstrated that Nva‐FMDP upregulates glmS expression in a concentration‐dependent manner through activation of the GlmY/GlmZ system and the presence of an exogenous amino sugar overrides this effect (Khan et al , ). Cultures grown to exponential phase were split, and sub‐cultures were provided with a sub‐inhibitory concentration of Nva‐FMDP or H 2 O as mock control.…”

“…If not available, GlcN6P must be synthesized by GlmS (Milewski, ). GlmS is also target for antibiotics produced by other microorganisms and GlmY/GlmZ provide protection as they overcome inhibition by increasing GlmS amounts (Khan et al , ), a defense that could not be achieved by allosteric regulation of the enzyme. Hence, the need for GlcN6P synthesis may strongly vary during the bacterial life cycle and GlmS activity needs tight and instant control.…”

“…Sponging of protein or sRNA by decoy RNAs has emerged as a widespread principle in bacterial post-transcriptional regulation (Sonnleitner & Bläsi, 2014;Miyakoshi et al, 2015;Romeo & Babitzke, 2018). GlmY specifically accumulates and counters GlmZ decay, when GlcN6P levels decrease (Reichenbach et al, 2008;Khan et al, 2016). Accordingly, GlmS amounts increase and GlcN6P is replenished.…”