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Elucidation of the roles of the carbonic anhydrase enzymes, CanA & CanB, in the physiology of Mycobacterium smegmatis

- Jackson, Gabriella Teresa

Authors: Jackson, Gabriella Teresa
Date: 2024-04-04
Subjects: Uncatalogued
Language: English
Type: Academic theses , Master's theses , text
Identifier: http://hdl.handle.net/10962/435299 , vital:73145
Description: The bacterial pathogen Mycobacterium tuberculosis (Mtb) is the causative agent of tuberculosis (TB) and one of the leading infectious causes of death globally. The success of Mtb as a pathogen depends on its ability to detect and respond to a variety of physical and chemical stresses it encounters during infection of its human host. These environmental stresses include shifts in temperature, oxygen concentration, osmolarity and nutrient availability. Mtb is, in addition, exposed to changes in pH and CO2 concentration in the intracellular and extracellular environments it inhabits, which the bacterium has to adapt to in order to ensure its growth, survival and/or persistence during infection. Carbonic anhydrases (CAs) are a widely distributed family of enzymes that catalyse the reversible hydration of carbon dioxide (CO2) to bicarbonate (HCO3−) in the reaction: CO2 + H2O ⇄ HCO3− + H+. In microbes, CA activity is important for the activity of enzymes involved in carbon fixation as well as for maintaining pH homeostasis. Mtb is known to express three CAs, encoded by the Rv3588c, Rv1284 and Rv3273 genes (canA, canB and canC, respectively). The role(s) of these CA enzymes in the physiology of Mtb and other mycobacterial species, such as Mycobacterium smegmatis (Msm), has not been elucidated to date. To gain insights into the function of the CanA and CanB enzymes in mycobacterial species, we generated both canA and canB knockdown (KD) and knockout (KO) mutants in the fast-growing mycobacterial species, Msm, and analysed their growth phenotypes under several growth conditions where CA activity is known to be required. Notably, Msm lacks the CanC homologue, which makes it an ideal surrogate to focus on CanA and CanB. The Msm canA KD mutant was found to display a growth defect following anhydrotetracycline (ATc)-mediated gene silencing at atmospheric (low) CO2 concentrations [~0.035% CO2 (v/v)]. The growth defect could be rescued by incubating cells at physiological (high) CO2 concentrations [~5% CO2 (v/v)] or by supplementing the growth media with either HCO3− or the metabolic end-products of certain HCO3−-dependent-carboxylase enzymes at low CO2 concentrations. The ability of these compounds to rescue the growth of the canA KD mutants was, however, dependent on the extent of ATc-mediated gene silencing, suggesting that the canA gene is required for Msm growth at both low and high CO2 concentrations. This was confirmed by our findings that canA could only be genetically inactivated when a second copy of the gene was provided on the chromosome in trans, regardless of the CO2 concentration used. In contrast to our observations for canA, no differences in the growth phenotypes of the Msm wild type (WT) and canB KD or knockout (KO) mutant strains were observed following silencing or inactivation of the canB gene at either low or high CO2 concentrations or different pH values. These observations suggest that, in contrast to canA, the canB gene is dispensable for the growth of Msm under standard laboratory growth conditions. The canB KO mutant strain, nevertheless, displayed a slight decrease in its ability to form biofilms when compared to the WT strain, which could be restored by genetic complementation. CanB activity may, therefore, be required to promote bacterial growth and/or survival under biofilm conditions where CO2 diffusion into cells is limited, a phenomenon that has recently been observed in other microbes. Further studies are required to confirm the role of CanB in biofilm formation and to determine how the different CA enzymes cooperate to promote the growth and survival of mycobacterial species in the various environments they are known to inhabit. , Thesis (MSc) -- Faculty of Science, Biochemistry and Microbiology, 2024
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Date Issued: 2024-04-04

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Biochemical and genetic analysis of the Mycobacterium smegmatis CnoX Chaperedoxin

- Watkins, Ariana Heloise Jo

Authors: Watkins, Ariana Heloise Jo
Date: 2023-03-29
Subjects: Uncatalogued
Language: English
Type: Academic theses , Master's theses , text
Identifier: http://hdl.handle.net/10962/422403 , vital:71939
Description: Mycobacterium (M.) tuberculosis (Mtb) encounters numerous physical and chemical stresses associated with host immunity during infection. These include exposure to reactive oxygen, chlorine and nitrogen species, low pH, hypoxia, nutrient starvation, and metal toxicity. Cellular proteins are particularly susceptible to damage by these stresses, and the ability to prevent their irreversible damage is consequently crucial for bacterial growth and survival. Mtb employs a network of proteins that includes chaperones, disaggregases, and proteases to maintain the integrity of its proteome. The chaperedoxin, CnoX, is a recently identified stress-inducible chaperone that combines redox and holdase activities to prevent the over-oxidation and aggregation of proteins in E. coli and other proteobacterial species. In this study, we identified orthologs of the E. coli CnoX (EcCnoX) in Mtb and M. smegmatis (Msm). Bioinformatics analysis of the Mtb and Msm CnoX orthologs (MtCnoX and MsCnoX, respectively) revealed that they possess similar domains, domain architectures and predicted tertiary structures as previously characterised CnoX enzymes, i.e. an N-terminal thioredoxin (Trx) domain fused to a C-terminal TPR-motif containing domain. The EcCnoX, MsCnoX, and MtCnoX enzymes were expressed as recombinant, His-tagged proteins in E. coli and purified to near homogeneity. Biochemical analysis of the recombinant CnoX enzymes revealed that the MsCnoX and MtCnoX both lack thiol-disulphide oxidoreductase (thioredoxin) activity, as evidenced by their inability to catalyse the reduction of the disulphide bonds of insulin in vitro. Both mycobacterial CnoX enzymes displayed activity as chaperones (holdases) during thermal aggregation assays of the model substrate, malate dehydrogenase (MDH). In contrast to previously reported findings for EcCnoX, the holdase activity of the mycobacterial CnoX enzymes was constitutive and did not require exposure to hypochlorous acid (HOCl) for activation. To establish the physiological role of CnoX in Msm, cnoX knockdown (KD) and knockout (KO) mutants were generated using CRISPRi-mediated gene silencing or homologous recombination, respectively. Consistent with previous findings, CnoX activity was not essential for the growth of Msm under conventional growth conditions. Reducing or eliminating CnoX activity in the Msm KD or KO mutants, respectively, did not confer increased sensitivity to HOCl as has been observed for an E. coli cnoX mutant. Reduced CnoX activity in Msm did, however, confer sensitivity to the superoxide generator, plumbagin, and front-line antitubercular drugs rifampicin and isoniazid. The combination of biochemical and physiological data presented suggests that MsCnoX may function as a holdase for substrates following proteotoxic damage induced by certain types of oxidants, a line of investigation that will be pursued in future studies. , Thesis (MSc) -- Faculty of Science, Biochemistry and Microbiology, 2023
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Date Issued: 2023-03-29

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