The hydrolase LpqI primes mycobacterial peptidoglycan recycling

Abstract

Growth and division by most bacteria requires remodelling and cleavage of their cell wall. A byproduct of this process is the generation of free peptidoglycan (PG) fragments known as muropeptides, which are recycled in many model organisms. Bacteria and hosts can harness the unique nature of muropeptides as a signal for cell wall damage and infection, respectively. Despite this critical role for muropeptides, it has long been thought that pathogenic mycobacteria such as Mycobacterium tuberculosis do not recycle their PG. Herein we show that M. tuberculosis and Mycobacterium bovis BCG are able to recycle components of their PG. We demonstrate that the core mycobacterial gene lpqI, encodes an authentic NagZ β-N-acetylglucosaminidase and that it is essential for PG-derived amino sugar recycling via an unusual pathway. Together these data provide a critical first step in understanding how mycobacteria recycle their peptidoglycan.

Fig. 1

Overview of PG recycling. a The basic building block of PG is GlcNAc-MurNAc-pentapeptide. Enzymes produced by the bacterium or the host are able to cleave every major linkage in PG. b Known PG-recycling machinery is variable with respect to the localisation of NagZ and the subsequent conversion to GlcNAc-1P or UDP-GlcNAc/MurNAc. All known MurNAc recovery systems that sustain bacterial growth (as opposed to strictly recycling e.g. P. putida) terminate at MurQ in the cytoplasm

Fig. 2

LpqI is an authentic NagZ-type enzyme. a Reactions including 1 μM LpqI and the indicated chromogenic substrates at 1 mM were incubated at 37 °C and release of pNP or 4MU was followed by absorbance or fluorescence respectively. Cleaveage of 4MU-GlcNAc is indicated by blue triangles. b LpqI was incubated with increasing concentrations of 4MU-GlcNAc. The rate of 4MU release was plotted and the curve fit with the Michaelis–Menton equation using GraphPad Prism 7.0. (n = 3, error bars are ± SD). c Thin-layer chromatograph of reaction products showing that LpqI is able to release GlcNAc from soluble muropeptides derived from M. smegmatis mc²155 PG. d The active site of LpqI is highly conserved as evidenced by the similar positioning of key binding residues observed in the GlcNAc, 1,6-anhydroMurNAc complex with NagZ_Pa (PDB: 5G3R) the main chains of which have been aligned with an RMSD of 2.3 Å using LSQ KAB

Fig. 3

M. tuberculosis and M. bovis BCG are able to recycle MurNAc. a M. bovis BCG WT was inoculated at a starting OD₆₀₀ of 0.1 in Sauton’s minimal media containing glycerol (blue diamonds), MurNAc (orange triangles), or no carbon (grey circles) and growth was monitored daily by taking OD₆₀₀ readings at the indicated time points (n = 3; error bars are ± SD). b M. tuberculosis H37Rv was washed and then serially diluted into fresh carbon-free minimal media. 10 μL of each dilution was spotted onto Sauton’s agar containing the indicated carbon sources at 5 mM. c Growth of M. bovis BCG on 5 mM MurNAc, GlcNAc, l-lactate, d-lactate, and glycerol was evaluated in aerated (blue) or static 5% CO₂ (orange) cultures using a resazurin assay (n = 3; error bars are ± SD). d Mid-exponential M. bovis BCG was grown in minimal media with 5 mM glycerol including 1 mM 4MU-d-lactate (blue circles) with constant agitation. At the indicated times, the 4MU fluorescence of the samples was determined in a BMG Polarstar plate reader (n = 3; error bars are ± SD) and compared with controls without cells (orange squares) or without 4MU-d-lactate (grey triangles)

Fig. 4

M. bovis BCG is able to recycle PG. a M. bovis BCG WT, ∆lpqI, ∆lpqI::lpqI and ∆lpqI::Empty Vector were incubated with 1 mM 4MU-GlcNAc in minimal media. After 3 days the fluorescence of the cultures were measured (n = 3; error bars are ± SD). b M. bovis BCG WT (grey diamonds) and ∆lpqI (red triangles) were simultaneously evaluated for release of cell wall peptides and growth (n = 3; error bars are ± SD). c M. bovis BCG WT (dark grey bars), ∆lpqI (red bars), and ∆lpqI::lpqI (light grey bars) were evaluated for their growth using MurNAc (5 mM), GlcNAc-MurNAc (2 mM), soluble PG from M. smegmatis mc²155 wild-type or ∆namH (5 and 2.5 mM, respectively), and soluble PG, which had been pre-digested wth LpqI (striped bars) as sole carbon sources using a resazurin assay (n = 3; *** = p < 0.001; ** = p < 0.005). Statistical significance determined using a two-tailed t-test in GraphPad Prism 7

Fig. 5

Loss of LpqI leads to lysozyme and antibiotic resistance. a–e M. bovis BCG WT (blue), ∆lpqI (red), and ∆lpqI::lpqI (grey) were incubated with increasing concentrations of lysozyme or antibiotics at a starting OD₆₀₀ of 0.1. After 7 days incubation total growth was assessed using a resazurin assay, where total fluorescence correlates with respiration and growth (n = 3; error bars are ± SD; Amox./Clav—amoxicillin plus clavulanic acid; Chlor.—chloramphenicol). f M. bovis BCG WT, ∆lpqI and ∆lpqI::lpqI and ∆lpqI::EV were incubated with EtBr and the rate of EtBr uptake was monitored as an increase in fluorescence. No significant differences were found in pairwise t tests across all strains (n = 3; error bars are ± SD). Statistical significance determined using a two-tailed t test

Fig. 6

Peptidoglycan recovery pathway in pathogenic mycobacteria. Based on our observations we can propose the following model for PG recycling and recovery in mycobacteria. Cleavage of the cell wall by endogenous autolysins or host-derived lysozyme generates muropeptides. Some of this material undergoes limited release to stimulate the host immune system. The remainder are subsequently degraded by amidases and other peptidases. LpqI then cleaves GlcNAc-MurNAc, which is followed by d-lactyl-ether cleavage. Lactate can then be used by the cell under aerobic conditions and GlcNAc (or its derivatives) are most likely released. Perturbation of this system by deleting LpqI leads to increased resistance to anti-mycobacterial agents