The RipA and RipB Peptidoglycan Endopeptidases Are Individually Nonessential to Mycobacterium smegmatis

Martinelli, Daniel; Pavelka, Martin S.

doi:10.1128/jb.00059-16

Cited by 22 publications

(23 citation statements)

References 40 publications

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“…RipA and RipB are not essential in M. tuberculosis. A recent study in M. smegmatis demonstrated that ripA is not essential, provided that ripB, coding for another D,Lendopeptidase and located downstream of ripA within the same operon, is expressed (28). To examine whether ripA and ripB are essential in M. tuberculosis, we first generated a merodiploid strain by expressing the ripA and ripB genes from an integrated streptomycin resistance-conferring plasmid in the attL5 locus (WT::ripAB) ( Fig.…”

Section: Resultsmentioning

confidence: 99%

Peptidoglycan Hydrolases RipA and Ami1 Are Critical for Replication and Persistence of Mycobacterium tuberculosis in the Host

Healy

Gouzy

Ehrt

2020

mBio

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Synthesis and cleavage of the cell wall polymer peptidoglycan (PG) are carefully orchestrated processes and are essential for the growth and survival of bacteria. Yet, the function and importance of many enzymes that act on PG in Mycobacterium tuberculosis remain to be elucidated. We demonstrate that the activity of the N-acetylmuramyl-L-alanine amidase Ami1 is dispensable for cell division in M. tuberculosis in vitro yet contributes to the bacterium's ability to persist during chronic infection in mice. Furthermore, the D,L-endopeptidase RipA, a predicted essential enzyme, is dispensable for the viability of M. tuberculosis but required for efficient cell division in vitro and in vivo. Depletion of RipA sensitizes M. tuberculosis to rifampin and to cell envelope-targeting antibiotics. Ami1 helps sustain residual cell division in cells lacking RipA, but the partial redundancy provided by Ami1 is not sufficient during infection, as depletion of RipA prevents M. tuberculosis from replicating in macrophages and leads to dramatic killing of the bacteria in mice. Notably, RipA is essential for persistence of M. tuberculosis in mice, suggesting that cell division is required during chronic mouse infection. Despite the multiplicity of enzymes acting on PG with redundant functions, we have identified two PG hydrolases that are important for M. tuberculosis to replicate and persist in the host. IMPORTANCE Tuberculosis (TB) is a major global heath burden, with 1.6 million people succumbing to the disease every year. The search for new drugs to improve the current chemotherapeutic regimen is crucial to reducing this global health burden. The cell wall polymer peptidoglycan (PG) has emerged as a very successful drug target in bacterial pathogens, as many currently used antibiotics target the synthesis of this macromolecule. However, the multitude of genes encoding PGsynthesizing and PG-modifying enzymes with apparent redundant functions has hindered the identification of novel drug targets in PG synthesis in Mycobacterium tuberculosis. Here, we demonstrate that two PG-cleaving enzymes are important for virulence of M. tuberculosis. In particular, the D,L-endopeptidase RipA represents a potentially attractive drug target, as its depletion results in the clearance of M. tuberculosis from the host and renders the bacteria hypersusceptible to rifampin, a frontline TB drug, and to several cell wall-targeting antibiotics.

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Section: Resultsmentioning

confidence: 99%

Peptidoglycan Hydrolases RipA and Ami1 Are Critical for Replication and Persistence of Mycobacterium tuberculosis in the Host

Healy

Gouzy

Ehrt

2020

mBio

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“…P-values have been computed using the Ranksum test adjusted for multiple testing. Martinelli & Pavelka, 2016). We further refer to this strain as RipAB knockdown (KD).…”

Section: Catalytically Inactive Marp-s343a Leads To the Formation Of mentioning

confidence: 99%

“…Here, we identified RipA-a peptidoglycan hydrolase (Hett et al, 2008;Martinelli & Pavelka, 2016)-as a MarP substrate. MarP cleaves RipA and this processing is essential for RipA's activity.…”

Section: Introductionmentioning

confidence: 99%

Mycobacterium tuberculosis protease MarP activates a peptidoglycan hydrolase during acid stress

et al. 2017

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(Mtb) can persist in the human host in a latent state for decades, in part because it has the ability to withstand numerous stresses imposed by host immunity. Prior studies have established the essentiality of the periplasmic protease MarP for Mtb to survive in acidified phagosomes and establish and maintain infection in mice. However, the proteolytic substrates of MarP that mediate these phenotypes were unknown. Here, we used biochemical methods coupled with supravital chemical probes that facilitate imaging of nascent peptidoglycan to demonstrate that during acid stress MarP cleaves the peptidoglycan hydrolase RipA, a process required for RipA's activation. Failure of RipA processing in MarP-deficient cells leads to cell elongation and chain formation, a hallmark of progeny cell separation arrest. Our results suggest that sustaining peptidoglycan hydrolysis, a process required for cell elongation, separation of progeny cells, and cell wall homeostasis in growing cells, may also be essential for Mtb's survival in acidic conditions.

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“…9 In M. tuberculosis, Rv1477 and Rv1478 are individually non-essential and the knock out phenotype leads to lysozyme sensitivity (decreased cell wall stability) and increased susceptibility to various antibiotics. 10 Both proteins encoded in the operon harbor a NlpC/P60 domain 11…”

Section: Introductionmentioning

confidence: 99%

The structure of the N‐terminal module of the cell wall hydrolase RipA and its role in regulating catalytic activity

et al. 2018

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RipA plays a vital role during cell division of Mycobacterium tuberculosis by degrading the cell wall peptidoglycan at the septum, allowing daughter cell separation. The peptidoglycan degrading activity relies on the NlpC/P60 domain, and as it is potentially harmful when deregulated, spatial and temporal control is necessary in this process. The N-terminal domain of RipA has been proposed to play an inhibitory role blocking the C-terminal NlpC/P60 domain. Accessibility of the active site cysteine residue is however not limited by the presence of the N-terminal domain, but by the lid-module of the inter-domain linker, which is situated in the peptide binding groove of the crystal structures of the catalytic domain. The 2.2 Å resolution structure of the N-terminal domain, determined by Se-SAD phasing, reveals an all-α-fold with 2 long α-helices, and shows similarity to bacterial periplasmic protein domains with scaffold-building role. Size exclusion chromatography and SAXS experiments are consistent with dimer formation of this domain in solution. The SAXS data from the periplasmic two-domain RipA construct suggest a rigid baton-like structure of the N-terminal module, with the catalytic domain connected by a 24 residue long flexible linker. This flexible linker allows for a catalytic zone, which is part of the spatiotemporal control of peptidoglycan degradation.

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The RipA and RipB Peptidoglycan Endopeptidases Are Individually Nonessential to Mycobacterium smegmatis

Cited by 22 publications

References 40 publications

Peptidoglycan Hydrolases RipA and Ami1 Are Critical for Replication and Persistence of Mycobacterium tuberculosis in the Host

Peptidoglycan Hydrolases RipA and Ami1 Are Critical for Replication and Persistence of Mycobacterium tuberculosis in the Host

Mycobacterium tuberculosis protease MarP activates a peptidoglycan hydrolase during acid stress

The structure of the N‐terminal module of the cell wall hydrolase RipA and its role in regulating catalytic activity

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