Browsing by Author "Bartizal, Tom Jack"

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    Advancing the understanding of the PE/PPE protein families through PPE_MPTR protein expression and analysis of pe/ppe single nucleotide variants in differentially drug sensitive Mycobacterium tuberculosis.
    (Stellenbosch : Stellenbosch University, 2022-11) Bartizal, Tom Jack; Sampson, Samantha; Kriel, Nastassja; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Biomedical Sciences. Molecular Biology and Human Genetics.
    ENGLISH ABSTRACT: Mycobacterium tuberculosis is one of the leading causes of infection and death by a single microorganism. The M. tuberculosis genome contains two prominent gene families, encoding proteins known as the PE (proline-glutamate) and PPE (proline-proline-glutamate) families. Several of these are essential for M. tuberculosis pathogenesis, however, the majority of the PE/PPE proteins, especially those within the PPE_MPTR subfamily are understudied. This is apparent regarding the sub-cellular localisation of PPE_MPTR proteins which may interact at the host-pathogen interface. Some functional aspects have also been overlooked, such as the potential for pe/ppe variants to contribute to drug resistance in M. tuberculosis. These genes are often overlooked due to their polymorphic nature, as well as the challenges associated with short-read sequencing technologies to reliably assemble their highly repetitive and GC-rich sequences. For the first part of our study, we aimed to identify the sub-cellular localisation of select PPE_MPTR proteins (PPE_MPTR10, -24, -40, 42, -53 and -62) by expressing them as fluorescently tagged proteins in Mycobacterium smegmatis. We successfully amplified all six genes from M. tuberculosis H37Rv DNA, and successfully cloned ppe_mptr10 into the pCG expression vector. Unfortunately, no protein expression was detected and we were unable to determine the localisation of PPE_MPTR10 within M. smegmatis. For the second aim of our study, we made use of a newly developed analytical pipeline to screen whole-genome sequencing (WGS) data and identify high-confidence pe/ppe singlenucleotide variants (SNVs) associated with drug resistance profiles. Nine SNVs were predicted to be unique to either drug susceptible, multi- or extensively-drug resistant (DS, MDR or XDR) classes of M. tuberculosis. We aimed to verify these findings by PCR and Sanger sequencing of targeted SNVs in an independent set of clinical M. tuberculosis isolates. The SNVs were determined to be true variants identified by the pipeline present in clinical isolates but absent from the reference M. tuberculosis H37Rv. None of the target variants were found to be unique to the drug resistance class for which they were originally predicted. The successful identification of pe/ppe variants by genomic analysis demonstrates the potential to screen these repetitive GC-rich regions for genetic variants. Future studies will be required to establish the role of PPE_MPTR proteins in M. tuberculosis pathogenesis.