Dissertation Submitted to the Faculty of Health Sciences,
University of the Witwatersrand, Johannesburg, in the
fulfillment of the requirement for the degree of Master of
Science in Medicine by Research.
Johannesburg, 2017 / Electron transport and respiration in Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis (TB) occurs through the use of the aa3 type cytochrome c oxidase (CcO) under normoxic conditions or cytochrome bd oxidase (CbdO) under microaerophillia. Using these oxidases, Mtb couples substrate level oxidation to generation of metabolic energy in the form of adenosine triphosphate (ATP) under aerobic conditions. The presence of the CbdO is expected to allow for growth and survival of Mtb in the oxygen restricted environment of the human TB granuloma, thus rendering it an important enzyme for further study. CbdO is comprised of two structural subunits, CydA (subunit I) and CydB (subunit II), which are encoded by the cydAB operon. Both subunits span the cytoplasmic membrane to form part of the mycobacterial electron transport chain. Notably, two other genes that are transcribed separately from the cydAB operon, the cydDC operon, have been proposed to be required for the synthesis of a functional CbdO. Based on amino acid sequence and structural predictions, the cydDC encode heterodimeric members of the ATP Binding Cassette ABC-type transporters. In other organisms, the cydDC functions to transport reducing agents to the periplasm, thus contributing to periplasmic redox homeostasis. In this study we aimed to analyze the function of the cydDC genes in Mycobacterium smegmatis. Through bioinformatics analyses, it was demonstrated that the CydDC subunits retain conserved residues associated with the ABC domain and adopt a three-dimensional fold that is similar to their counterparts in Escherichia coli. However, the published sequence of M. smegmatis suggests that cydC is a pseudogene, which was inconsistent with the demonstrated evidence of a functional CbdO in this organism. In this study, using standard DNA sequencing, it was demonstrated that the CBTBR laboratory strain of M. smegmatis does not harbor a cydC pseudogene but rather has a functional cydC gene. Next, we interrogated the function of the M. smegmatis and Mtb cydDC genes by heterologous expression in an E. coli cydD mutant. Heterologous expression of the Mtb cydDC genes restored CbdO biogenesis in the E. coli mutant. Using various microbiological approaches, we demonstrated that the mycobacterial cydDC was able to revert the stationary phase exit defect, high temperature sensitivity and increased oxidative stress susceptibility defects of the E. coli cydD mutant.
Collectively, these data provided strong evidence that the mycobacterial cydDC genes encode a functional transporter that contributes to periplasmic redox homeostasis. Following this, we generated a double deletion mutant of the cydDC operon in M smegmatis. We confirmed the genetic integrity of the ΔcydDC strain by Southern Blot analysis and proved by absorption difference spectroscopy that this strain is defective in the ability to synthesize a functional CbdO, as measured by the lack of a heme d peak in membrane preparations from the mutant. In addition, the ΔcydDC mutant displayed increased sensitivity to oxidative stress and a reduced ability to exit stationary phase, phenotypic defects that were consistent with the lack of CbdO. In summary, this study provides the first evidence that loss of the M. smegmatis cydDC genes affects CbdO biogenesis. These data also confirm that the CydDC ABC-type transporter most likely transports reducing equivalents that allow for maturation of CbdO in the periplasm. Collectively, our observations advance the understanding of the mycobacterial electron transport chain and provide new evidence to assist in the development of CbdO as a TB drug target. / MT2017
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:wits/oai:wiredspace.wits.ac.za:10539/23320 |
| Date | January 2017 |
| Creators | Moeketsi, Moseki Raymond |
| Source Sets | South African National ETD Portal |
| Language | English |
| Detected Language | English |
| Type | Thesis |
| Format | application/pdf |
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