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

Abstract

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.

Bacterial Cell Wall Quality Control during Environmental Stress

“…(ii) Intracellular pathogens carry genes that encode pH specialist cell wall enzymes. pH-sensitive cell wall enzymes have also been identified in Salmonella enterica serotype Typhimurium and Mycobacterium tuberculosis (62)(63)(64)(65). Although not environmental generalists like E. coli, these pathogens experience pH stress in host intracellular compartments, wherein they persist and replicate.…”

“… M. tuberculosis genes encode two semiredundant dl -endopeptidases, RipA and RipB. While production of either enzyme is sufficient to support normal growth of M. tuberculosis in rich medium at neutral pH, RipA becomes conditionally essential for growth in acidic medium and in chronic infections ( 64 , 65 ). RipA abundance is regulated through MarP, a periplasmic serine protease required to maintain intracellular pH homeostasis ( 66 ).…”

Structural Modeling of Cell Wall Peptidase CwpFM (EntFM) Reveals Distinct Intrinsically Disordered Extensions Specific to Pathogenic Bacillus cereus Strains

“…Additionally, they are implicated in bacterial adhesion and invasiveness [4], persistence in the host [5] and in the initiation step of biofilm formation [6], therefore contributing to bacterial pathogenicity. Moreover, the resulting cell wall fragments are recycled as signaling molecules to trigger bacterial communication, immune response or antibiotics resistance [5,7]. Finally, they also prime the insertion of supramolecular structures like secretion, flagella or pili systems [8].…”

Handling the Hurdles on the Way to Anti-tuberculosis Drug Development

“…Mycobacterial genes that are shown to be essential under in vitro growth conditions are usually expected to be fundamental for the in vivo infection context also. However, context-dependent roles have been shown, for instance, for Ami1 (N-acetylmuramyl-Lalanine amidase; Rv3717) and RipA (D,L-endopeptidase; Rv1477) enzymes, which are involved in synthesis and cleavage of mycobacterial peptidoglycan polymer (Healy et al, 2020). Ami1 was shown to be dispensable for cell division of M. tuberculosis in different growth contexts in vitro (e.g., macrophage infection, nutrient starvation, and nitric oxide exposure), but it was critical for persistence in the lungs of infected mice (Healy et al, 2020).…”