The transpeptidase PbpA and noncanonical transglycosylase RodA of Mycobacterium tuberculosis play important roles in regulating bacterial cell lengths

Abstract

The cell wall of () is a complex structure that protects the pathogen in hostile environments. Peptidoglycan (PG), which helps determine the morphology of the cell envelope, undergoes substantial remodeling under stress. This meshwork of linear chains of sugars, cross-linked through attached peptides, is generated through the sequential action of enzymes termed transglycosylases and transpeptidases. The genome encodes two classical transglycosylases and four transpeptidases, the functions of which are not fully elucidated. Here, we present work on the yet uncharacterized transpeptidase PbpA and a nonclassical transglycosylase RodA. We elucidate their roles in regulating growth and survival of pathogenic mycobacteria. We find that RodA and PbpA are required for regulating cell length, but do not affect mycobacterial growth. Biochemical analyses show PbpA to be a classical transpeptidase, whereas RodA is identified to be a member of an emerging class of noncanonical transglycosylases. Phosphorylation of RodA at Thr-463 modulates its biological function. In a guinea pig infection model, RodA and PbpA are found to be required for both bacterial survival and formation of granuloma structures, thus underscoring the importance of these proteins in mediating mycobacterial virulence in the host. Our results emphasize the fact that whereas redundant enzymes probably compensate for the absence of RodA or PbpA during growth, the two proteins play critical roles for the survival of the pathogen inside its host.

Peptidoglycan precursor synthesis along the sidewall of pole-growing mycobacteria

“…We next sought to address whether these molecules could be used to build the peptidoglycan polymer. Transglycosylases from both the PBP (penicillin-binding proteins) and SEDS (shape, elongation, division, and sporulation) families stitch peptidoglycan precursors into the existing meshwork ( Figure 3—figure supplement 1 , [ Boutte et al, 2016 ; Hayashi et al, 2016 ; Zhao et al, 2017 ; Cho et al, 2016 ; Meeske et al, 2016 ; Leclercq et al, 2017 ; Arora et al, 2018 ]). If peptidoglycan precursors are polymerized along the lateral surface of the mycobacterial cell, at least a subset of these periplasmic enzymes must be present at the sidewall to assemble the biopolymer.…”

Peptidoglycan precursor synthesis along the sidewall of pole-growing mycobacteria

“…We next sought to address whether these molecules could be used to build the peptidoglycan polymer. Transglycosylases from both the PBP (penicillin-binding proteins) and SEDS (shape, elongation, division, and sporulation) families stitch peptidoglycan precursors into the existing meshwork ( Figure 3—figure supplement 1 , (14, 32, 51, 55–58)). If peptidoglycan precursors are polymerized along the lateral surface of the mycobacterial cell, at least a subset of these periplasmic enzymes must be present at the sidewall to assemble the biopolymer.…”

Delineating FtsQ-mediated regulation of cell division in Mycobacterium tuberculosis

“…5Ј and 3Ј genomic flank sequences of sepI-VA Msm (ϳ800 bp each) were amplified, and allelic exchange substrate (AES) was generated as described earlier (85,86). Linearization of AES, electroelution, and mutant generation and confirmation were performed as described previously (87).…”