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Title: Understanding molecular mechanisms of Mycobacterium tuberculosis persistence
Author: Ndhlovu, Victor Sengelwayo
ISNI:       0000 0004 7656 8276
Awarding Body: University of Liverpool
Current Institution: University of Liverpool
Date of Award: 2019
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Background: Although Mtb strains have been shown to exhibit nucleotide level similarity of >99%, such similarity is rarely replicated in the phenotype. This phenotypic heterogeneity has been demonstrated by a subpopulation of cells called 'persisters'. Persisters constitute a minority of the organisms found in clinical specimens but statistical modelling studies suggest that they predominate after the first few days of treatment. The rate of their elimination is likely the principal determinant of the length of therapy and likelihood of relapse. Understanding the complete biology of persistence is among the most important scientific barriers to shortening TB treatment. Methods: A total of 64 bacteriologically confirmed Mtb clinical isolates from Malawian patients recruited for a cohort study of determinants of treatment outcome were genotyped using LSP-PCR to determine the molecular epidemiology of TB in Blantyre, Malawi and to evaluate the possible impact of lineage on outcome. Eighteen of the 64 isolates including two 'persister' strains defined by lipid body staining and clinical relapse were further subjected to whole genome sequencing using Single Molecule Real Time (SMRT) sequencing in order to assess differences in the methylome among these strains. Using bioinformatics analysis DNA methylation patterns were determined and factors leading to loss/ absence of methylation could then be elucidated. Results: The 64 isolates genotyped clustered into 4 main Mtb lineages (1-4) with lineage 4 predominating. There was differential occurrence of methylation across the strains. Methylation was found to occur within three different confidently defined sequence motifs (CTGGAG, CACGCAG and GATN4RTAC) across all the isolates influenced by three different methyltransferases in a lineage specific manner. Three different methylation disrupting mutations within methyltransferase genes E270A in mamA, P360L in hsdM the recently characterized S253L mutation in mamB were confirmed. For the first time, total loss of methylation in MamB was reported due to the S253L mutation. No compelling correlation between DNA methylation pattern and persistence during treatment was observed, at the level of genic or intergenic regions or within specific operons of interest previously associated with persistence in vitro. Conclusions: Four main Mtb lineages occur in Blantyre Malawi and DNA methylation in these isolates were lineage specific. Specific mutations are responsible for disrupting methylation within various Mtb strains and methylation disrupting mutations could be reliably used to assign Mtb lineages for some lineages. Methylation patterns did not differ significantly between persister and non-persister isolates, though current limitations in direct extraction of DNA from clinical samples and numbers of paired isolates from representative longitudinal studies suggest that further work is needed to definitively answer this question.
Supervisor: Davies, Geraint ; Caws, Maxine ; Mandala, Wilson Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral