Browsing by Author "Campbell, Lisa"

Now showing 1 - 2 of 2

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.
Recombinant production and activity assessment of growth stimulatory proteins in the resuscitation of mycobacterium tuberculosis
(2020) Campbell, Lisa
Mycobacterium tuberculosis (Mtb) is able to enter a non-culturable, dormant-like, state under stressful conditions to evade eradication. When growth permissive conditions resume, Mtb can exit this non-culturable state and resume active replication. Non-culturable populations are of great interest in clinical settings as they can contribute to misdiagnoses and inaccurate treatment monitoring. Resuscitation promoting factors (Rpfs) have been implicated as stimulatory growth factors for resuscitation from the non-culturable state, together with their interacting partner, Rpf-interacting protein A (RipA). In this study the resuscitation capabilities of Resuscitation promoting factor B (RpfB) and RipA against Mtb were evaluated. The catalytic regions of RipA and RpfB were expressed and purified and their muralytic activity was assesed using four protein activity assays. The resuscitation capabilities of RipA and RpfB in Mycobacterial growth indicator tube (MGIT) assays was evaluated using carbon starvation-derived non-culturable Mtb from the laboratory strain (H37Rv). It was observed that RipA and RpfB supplementation was able to stimulate the resuscitation of non-culturable, carbon-starved H37Rv, significantly decreasing its time-topositivity (TTP) in MGIT assays compared to un-supplemented MGIT media. Upon investigating clinical strains of Mtb, no significant reduction in TTP of non-culturable, carbon-starved Beijing and LAM isolates was reported. Supplementation of RipA and RpfB in MGIT assays to improve the detection of clinical isolates of Mtb and enhance their TTP was evaluated using a South African cohort of 30 sputum specimens. Overall RipA and RpfB supplementation yielded no significant improvements to MGIT TTP, regardless of initial colony forming units, patient gender, immunology or diagnostic data; however, 20% of individual specimens did benefit from the supplementation, yielding improved TTP when compared to un-supplemented MGITs. No significant adverse effects were observed during supplementation and given the positive yield in some specimens, it is suggested that the addition of RipA and RpfB could improve the detection of Mtb in clinical specimens.