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

Muna A Khan,Boris Görke,Yvonne Göpel,Slawomir Milewski

doi:10.3389/fmicb.2016.00908

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.

Highlights

The continuous increase and spread of antibiotic resistance mechanisms among bacterial pathogens represents a major threat for public health
Our findings indicate that compounds suppressing GlmY and/or GlmZ would increase the efficacy of GlmS inhibitors when applied in combination
The β-galactosidase activities were determined. Both assays consistently detected a strong upregulation of glmS expression in the Nva-FMDP treated cells, which was suppressed by the addition of GlcN (Figures 2B, bottom panel and 4B, columns left)

Summary

Introduction

The continuous increase and spread of antibiotic resistance mechanisms among bacterial pathogens represents a major threat for public health. The initial steps in this pathway collectively referred to as hexosamine pathway, have been rarely considered as drug targets. The hexosamine pathway generates UDP– N-acetyl-glucosamine (UDP–GlcNAc) and is initiated by key enzyme glucosamine-6-phosphate (GlcN6P) synthase GlmS, which synthesizes GlcN6P from L-glutamine and fructose-6phosphate (Figure 1A; Milewski, 2002; Teplyakov et al, 2002). GlmS is essential unless exogenous amino sugars such as glucosamine (GlcN) are available in adequate amounts. GlcN can be taken up and converted to GlcN6P, thereby bypassing GlmS (Figure 1A) (Plumbridge, 2015). The GlcN concentration in human bodily fluids is too low to sustain growth of glmS mutants making GlmS essential for enteric bacteria colonizing the human host (Persiani et al, 2007; Kim et al, 2013; Bennett et al, 2016)

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Conclusion