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

Divya Arora,Yogesh Chawla,Basanti Malakar,Archana Singh,Vinay Kumar Nandicoori

doi:10.1074/jbc.m117.811190

Abstract

The cell wall of Mycobacterium tuberculosis (Mtb) 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 Mtb 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 in vitro growth and in vivo 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 in vitro growth, the two proteins play critical roles for the survival of the pathogen inside its host.

Highlights

The cell wall of Mycobacterium tuberculosis (Mtb) is a complex structure that protects the pathogen in hostile environments
We find that RodA and PbpA are required for regulating cell length, but do not affect mycobacterial growth
Lipid II anchored to the intracellular membrane is flipped into the periplasmic space by flippase followed by transglycosylation, wherein the sugar moieties are linked to the existing chain through glycosidic bonds, via transglycosylases

Summary

Results

To delineate the role of RodA and PbpA on mycobacterial cell morphology regulation, we sought to determine the impact. Whereas the WT and deletion mutants grown in 7H9 medium showed no significant difference in cell wall ultrastructure (Fig. 5, a and c), we observed conspicuous changes in the cell wall architecture of Mtb⌬p and Mtb⌬rp in Sauton’s medium as compared with the Mtb and Mtb⌬r strains (Fig. 5, b and d). In persisters analysis, wherein Msm WT and mutant cultures were exposed to 10 ␮g/ml isoniazid, both Msm⌬r and Msm⌬p mutants showed a 10-fold decline in cfu (Fig. 5f) Taken together, these data suggest that both RodA and PbpA may play a role in combating survival under different stress conditions. Disease progression as assessed by the gross evaluation of lungs and spleen (data not shown) and lung bacillary load 4 weeks postinfection revealed marginal differences between infection by Mtb versus the mutant strains, suggesting that the absence of rodA and pbpA had no impact on mycobacterial survival in the mice model as host (Fig. 9a). Based on compromised survival studies and lower histopathological scores, obtained in the case of all of the deletion mutants (Fig 10), we suggest that RodA and PbpA play crucial roles in imparting mycobacterial virulence in the host

Discussion

Experimental procedures

Ethics statement