Browsing by Author "Asmal, Rukaya"

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    Characterization of putative DD-carboxypeptidase-encoding genes in Mycobacterium smegmatis
    (Nature Research, 2019-03) Ealand, Christopher S.; Asmal, Rukaya; Mashigo, Lethabo; Campbell, Lisa; Kana, Bavesh D.
    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 anhydrotetracyclineresponsive 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.
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    Identification and cellular localization of DD-carboxypeptidase-interacting proteins in mycobacterium smegmatis
    (2015-09-17) Asmal, Rukaya
    Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis (TB), is responsible for approximately 1.5 million deaths worldwide; a pandemic which is further exacerbated by the emergence of multi- and extensively drug resistant strains. The extensive morbidity and mortality associated with TB has brought about an urgent need for new drugs that promise to shorten treatment duration and reduce the daily pill burden. In this context, the mycobacterial cell wall, particularly the peptidoglycan (PG) layer, is a promising source of new drug targets due to the extensive number of bacterial-specific enzymes that are responsible for biosynthesis of this macromolecular component in bacterial cells. PG biosynthesis and cross-linking involves a number of membrane-bound as well as cytoplasmic enzymes, including penicillin-binding proteins (PBPs), which are the targets for beta-lactam antibiotics. In this study we further elucidate the role of DD-Carboxypeptidases (DD-CPases), a group of low molecular mass PBPs that play a crucial role in the biosynthesis and remodelling of PG in a variety of organisms. These enzymes cleave the terminal D-Alanine residue in PG and are postulated to be involved in preventing the formation of inappropriate cross-links. To identify possible interacting partners for two of the five putative DD-CPases (DacB and MSMEG_2433) in Mycobacterium smegmatis, a full-coverage, representative genomic library was constructed as fusions with the Gal4 DNA binding domain in a yeast two-hybrid vector. The genes encoding dacB and MSMEG_2433 were cloned as fusions with the Gal4 activation domain and these clones were used to screen the library for putative interacting partners. This analysis identified fifteen possible interacting partners in total for these two DD-CPases. DacB interacting proteins included a tetracycline regulator, an acyltransferase, as well as a subunit of the FoF1 ATP synthase. Putative interacting proteins identified for MSMEG_2433 included the same tetracycline regulator that was identified for DacB, a helicase, and PBP1A/1B (PonA2). PonA2 is of particular interest as its identification points to a role for DacB in PG cross-linking. For cellular localization studies, C-terminal fusions with the mCherry; Venus and rsEGFP fluorescent proteins were created for DacB and MSMEG_2433. Fluorescence microscopy revealed weak or diffused signal from the mCherry and Venus fusion proteins, in contrast, the rsEGFP fusion yielded specific, robust signal and was used for further analysis. Localization studies revealed that both DacB and MSMEG_2433 localized to the cell pole either in a mono-polar or bi-polar fashion similar to that observed with other cell division proteins such as Wag31 and ParB. Furthermore, MSMEG_2433 also localized to the septum in some cases. Collectively, the data from this study suggests that DD-CPases may interact with other PG hydrolysing enzymes to exert their function in mycobacteria. Moreover, localization confirms an important role for these enzymes in cell growth and expansion, which occurs at cell poles in mycobacteria.