Characterization of M23- domain activators of peptidoglycan degrading amidases in mycobacterium tuberculosis

Abstract

Mycobacterium tuberculosis, the causative agent of Tuberculosis (TB), continues to claim a substantial number of human lives globally, a situation that has continued for centuries. South Africa is one of the designated high burdened countries where the high number of people with HIV are most vulnerable to TB infection and disease. Although TB is curable with 6 months of chemotherapy, this outcome can be thwarted by the emergence of drug resistance. Multiple and extensive drug resistant TB are harder to treat, take longer to cure using drugs associated with severe side-effects. New, faster-curing drugs are urgently needed against targets in M. tuberculosis that are not targeted by current drugs. The broader objective of this research was to explore the peptidoglycan core of the M. tuberculosis cell wall for novel drug targets. Since mycobacterial growth depends on the synthesis, degradation and maintenance of the cell wall, enzymes involved in these processes are promising drug targets. Various enzymes required for remodelling of the cell wall, at the stem-peptide of the peptidoglycan core, have been shown to be necessary for mycobacterial growth, division and virulence. Peptidoglycan M23 endopeptidases are necessary for growth and virulence in numerous bacteria but have not been identified in M. tuberculosis. This research focused on identifying M23 endopeptidase-encoding genes in M. tuberculosis and to characterise a potential role for these genes in M. tuberculosis growth. Three putative endopeptidase domain containing genes, encoded at Rv0950c, Rv2891 and Rv3786c were identified. Rv0950c is the most conserved among the three homologues, with a predicted, hydrolytic active-site and was therefore the focus of further study. A deletion mutant strain of M. tuberculosis lacking Rv0950c was generated and studied. Loss of Rv0950c caused cell shortening and modulated incorporation of mono peptide and di-peptide fluorescent peptidoglycan probes at the side-wall of cells during axenic growth and following cell wall damage. These phenotypes were reversed upon genetic complementation and suggest a role for Rv0950c in cell length maintenance and peptidoglycan stem-peptide formation, likely for maturation of peptidoglycan at the M. tuberculosis side wall. Transcriptomic analyses further demonstrated that Rv0950c is expressed at exponential growth under axenic conditions, and further upregulated under nutrient-limited stress. A Rv2891 deletion mutant strain was also generated and studied. The loss of Rv2891 did not alter cell morphology but patterns of fluorescent mono-peptide and Vancomycin labelling point to a role for Rv2891 in nascent, 4-3 cross-link remodelling, which needs to be confirmed by genetic complementation. The work therefore presents foundational characterisation of novel M23 endopeptidases in M. tuberculosis.