Browsing by Author "Ismail, Zaahida Sheik"

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    Characterization of DD-carboxypeptidase function in mycobacteria: genetic knockout and recombinant protein production
    (2016-10-12) Ismail, Zaahida Sheik
    Tuberculosis (TB), a disease caused by the pathogenic bacterium Mycobacterium tuberculosis (Mtb), is responsible for killing over one million people each year with an alarming number categorized as multidrug resistant (MDR) or extensively drug resistant (XDR) infections. These high numbers, coupled with other factors such as the ability of Mtb to adapt to its host, its synergistic relationship with Human Immunodeficiency Virus (HIV) and the protracted treatment regimen required to treat TB has resulted in the urgent need for new TB drugs. In this regard, the peptidoglycan (PG) layer of the mycobacterial cell wall, which requires an array of enzymes for synthesis of this mesh-like polymer, have been of particular interest. The PG consists of sugars cross-linked by stem peptides and is synthesized, cross-linked and remodeled by carefully regulated enzymes such as penicillin binding proteins (PBPs) which perform the final crosslinking step in PG biosynthesis. This study focuses on a specific group of low molecular weight PBPs, namely the DD-carboxypeptidases (DD-CPases), which are responsible for regulating the amount of cross-links found in the PG by cleaving the terminal D-Alanine (D-Ala) from the stem peptide of nascent PG units. To date, these proteins have remained largely uncharacterized in mycobacteria. To investigate the functions of these proteins in Mycobacterium smegmatis, two double knockout mutants lacking different combinations of the DD-CPase-encoding genes (MSMEG_1661, MSMEG_2432 and/or MSMEG_2433) were created using two-step allelic exchange and assessed using a range of phenotypic analyses. In addition recombinant protein production of these DD-CPases as well as MSMEG_6113 was attempted. We were unable to create a double knockout mutant lacking both MSMEG_2432 and MSMEG_2433 from an existing single mutant strain, suggesting that the operonic structure of these two genes may require a different approach. We were able to generate two double knockout mutants, ΔMSMEG_1661 ΔMSMEG_2432 and ΔMSMEG_2433 ΔMSMEG_1661, lacking two DDCPases. Southern blot and gene expression analyses confirmed loss of the respective genes from M. smegmatis. The ΔMSMEG_1661 ΔMSMEG_2432 mutant and ΔMSMEG_2433 ΔMSMEG_1661 mutant displayed no alterations in colony morphology, biofilm formation, sliding motility and sodium dodecyl sulphate (SDS) sensitivity. However, loss of two DD-CPases resulted in increased sensitivity of M. smegmatis to vancomycin and a range of β-lactams antibiotics. Analysis of cellular morphology using transmission electron microscopy (TEM) demonstrated that the septum was fully formed in all strains but in some cases was not degraded during daughter cell separation. Scanning electron microscopy (SEM) and spatial localization of new PG units using BODIPY-labeled vancomycin revealed that late division processes were hampered in these double knockout mutants with new PG inserted across the cell as well as cells with multiple unresolved septa. To create recombinant derivatives of MSMEG_6113, MSMEG_1661, MSMEG_2432 and MSMEG_2433, proteins were His-tagged and purified using affinity chromatography. However, under all conditions tested soluble protein could not be acquired. Collectively, these data provide the first evidence that the DD-CPases of M. smegmatis may play a direct role in the late cell division process that lead to daughter cell separation in mycobacteria.
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    Novel hinge insertions in South African HIV-1 subtype C protease: implications for biochemical and biophysical properties
    (2022) Ismail, Zaahida Sheik
    Although treatable, human-immunodeficiency virus infections remain distressingly high in sub-Saharan Africa, particularly due to the prevalence of the subtype C variant in this location. While advances have been made in tackling drug resistance globally, subtype C infections are majorly understudied specifically with regards to the effect of mutations in this subtype on drug resistance to currently available inhibitors. The HIV protease is responsible for the production of new infectious viral progenies. Cleavage of the Gag and Gag-Pol polyprotein precursors by the protease produces mature viral particles that can infect new host cells. The indispensable role of the HIV protease in the lifecycle of the virus makes it a major drug target for combatting this disease. Mutations in the protease accumulate both within and distal to the active site, often contributing to drug resistance. More recently, insertions within the hinge region of the protease have been identified. The effect of these hinge region insertions on the HIV protease is not fully understood. The study was based on a clinical isolate containing two insertions, Asn and Leu, at position 38 in the hinge region as well as four mutations: K20R, E35D, R57K and V82I (L38↑N↑L +4 ). The aim of this study was to determine the direct effect of these hinge region insertions on the characteristics of the protease. As such, a variant subtype C protease containing only the double insertion of Asn and Leu was created (L38↑N↑L -4 ). The variant L38↑N↑L -4 protease was successfully overexpressed and purified using a newly adapted ion-exchange chromatography purification protocol. Circular dichroism and size exclusion-high performance liquid chromatography experiments showed that the secondary and oligomeric structures of the variant protease was similar to the wild-type (WT). Active site titrations indicated that the purification of L38↑N↑L -4 resulted in a decrease of actively prepared sample in comparison to the WT. The specific activity and turnover number of L38↑N↑L -4 was significantly reduced in comparison to the WT while the catalytic efficiency was increased. Induced-fit docking studies showed that the WT, L38↑N↑L -4 and L38↑N↑L +4 do bind the protease inhibitors atazanavir (ATV), darunavir (DRV), ritonavir (RTV) and saquinavir (SQV) albeit with different affinities. In vitro displacement titration experiments confirmed that L38↑N↑L -4 does bind these inhibitors. The binding affinity of L38↑N↑L -4 to the protease inhibitors ATV, RTV and SQV was significantly reduced with changes to the overall binding energetics of these vi reactions. Darunavir did not display any change in binding affinity to L38↑N↑L -4 ; however, the overall energetics of this reaction was altered in comparison to the WT. In addition, the stability of these L38↑N↑L -4 complexes were significantly diminished implicating these insertions in drug binding interactions. Differential scanning calorimetry results showed that the transition temperature of the apo L38↑N↑L -4 protease was 5 °C higher than the apo WT protease indicating the role of these insertions in the stability of the protease. Molecular dynamic simulations confirmed the increased structural stability of the apo mutated proteases which exhibited more flexible hinge regions than the WT. Additionally, both hinge region mutants, L38↑N↑L -4 and L38↑N↑L +4 , exhibited a mainly closed conformation thus explaining the decreased catalytic properties of the protease. Furthermore, the presence of inhibitors altered the dynamics, conformations, and flexibility of the three proteases. The inhibitor bound mutated proteases, L38↑N↑L -4 and L38↑N↑L +4 , displayed more flexible flap and hinge regions in comparison to the WT. Except for SQV, the presence of inhibitors shifted the flap conformers of the mutated proteases from closed to semiopen as well. Further analysis indicated changes in the number of hydrophobic interactions, hydrogen bonding and water bridge formations between the proteases and the inhibitors. Altogether, these results implicate the hinge region insertions in drug binding affinities and interactions with protease inhibitors as well as structural stability, dynamics, and flexibility of the protease both in the absence and presence of protease inhibitors.