Abstract
Penicillin binding proteins (PBPs) are the target of numerous antimicrobial agents that disrupt bacterial cell wall synthesis. In mycobacteria, cell elongation occurs through insertion of nascent cell wall material in the sub-polar region, a process largely driven by High Molecular Weight PBPs. In contrast, the function of DD-carboxypeptidases (DD-CPases), which are Low Molecular Weight Class 1C PBPs, in mycobacteria remains poorly understood. Mycobacterium smegmatis encodes four putative DD-CPase homologues, which display homology to counterparts in Escherichia coli. Herein, we demonstrate that these are expressed in varying abundance during growth. Deletion of MSMEG_1661, MSMEG_2433 or MSMEG_2432, individually resulted in no defects in growth, cell morphology, drug susceptibility or spatial incorporation of new peptidoglycan. In contrast, deletion of MSMEG_6113 (dacB) was only possible in a merodiploid strain expressing the homologous M. tuberculosis operon encoding Rv3627c (dacB), Rv3626c, Rv3625c (mesJ) and Rv3624c (hpt), suggestive of essentiality. To investigate the role of this operon in mycobacterial growth, we depleted gene expression using anhydrotetracycline-responsive repressors and noted reduced bipolar peptidoglycan synthesis. These data point to a possible role for this four gene operon, which is highly conserved across all mycobacterial species, in regulating spatial localization of peptidoglycan synthesis.
Highlights
Penicillin binding proteins (PBPs) are a group of evolutionary related bacterial proteins that have formed the basis of successful antibiotic chemotherapy for decades due to their vulnerability to β-lactam antibiotics
This notion is supported by several studies that demonstrated physiological roles for low molecular weight (LMW) PBPs: (i) Over-expression of Rv2911 from Mycobacterium tuberculosis, in the non-pathogenic saprophyte, Mycobacterium smegmatis, resulted in reduced growth, altered colony morphology, defective sliding motility and decreased biofilm formation[25]; (ii) deletion of dacB2 from M. tuberculosis inhibited growth in minimal media under acidic conditions, and resulted in enhanced survival in THP-1 human macrophage-like cells[25]; (iii) the β-lactam antibiotic, meropenem, directly inhibited DacB2 through covalent linkage with the enzyme which resulted in polar swelling, eventually leading to cell lysis[26] and (iv) expression of an M. smegmatis LMW PBP with DD-CPase and beta-lactamase activity, MSMEG_2433, corrected morphological defects in a septuple PBP mutant of E. coli[27]
Protein sequences corresponding to E. coli LMW PBP homologues were used in a BLAST analysis to identify mycobacterial homologues
Summary
Penicillin binding proteins (PBPs) are a group of evolutionary related bacterial proteins that have formed the basis of successful antibiotic chemotherapy for decades due to their vulnerability to β-lactam antibiotics. This notion is supported by several studies that demonstrated physiological roles for LMW PBPs: (i) Over-expression of Rv2911 (dacB2) from Mycobacterium tuberculosis, in the non-pathogenic saprophyte, Mycobacterium smegmatis, resulted in reduced growth, altered colony morphology, defective sliding motility and decreased biofilm formation[25]; (ii) deletion of dacB2 from M. tuberculosis inhibited growth in minimal media under acidic conditions, and resulted in enhanced survival in THP-1 human macrophage-like cells[25]; (iii) the β-lactam antibiotic, meropenem, directly inhibited DacB2 through covalent linkage with the enzyme which resulted in polar swelling, eventually leading to cell lysis[26] and (iv) expression of an M. smegmatis LMW PBP with DD-CPase and beta-lactamase activity, MSMEG_2433, corrected morphological defects in a septuple PBP mutant of E. coli[27] These studies suggest that mycobacterial DD-CPases function to the E. coli homologues in regulating cross-linking and maintaining cell shape. We attempt to expand the current understanding of DD-CPase function in mycobacterial PG synthesis by constructing and characterizing mutant and knockdown strains defective for putative DD-CPase-encoding genes in M. smegmatis
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