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Investigation of the role of gene deletions in the virulence of an outbreak strain of Mycobacterium tuberculosis

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posted on 24.09.2018, 07:52 authored by Ahmed Saleh R. Al-Obaidi
In 2001, a large tuberculosis outbreak took place at Crown Hills School, Leicester, UK. This outbreak had unusual features including a high number of infected individuals with rapid development of symptoms. Genotypic investigation of the causative strain, Mycobacterium tuberculosis CH, identified unique deletions of five loci compared with the reference strain M. tuberculosis H37Rv genome. Among the five deleted loci, a locus involving two adjacent genes named rv1995 and rv1996 was found. There was an evolutionary clue that these two genes were the last deletions within the genome of CH. Phenotypically, CH grew less rapidly, was less resistant to low pH and H2O2, but stimulated more production of IL-10 and IL-6 from monocyte derived macrophages than H37Rv. The aim of this project was to determine the contribution of the deletion of rv1995 and universal stress protein encoding gene, rv1996 to the phenotype and hypervirulence of the CH strain. The present study involved construction of shuttle expression vectors for rv1995 and rv1996. Both genes were amplified from the H37Rv genome, ligated successfully with the digested pSMT3 plasmid, and then cloned giving two expression vectors, pAAO1 and pAAO3 for rv1995 and rv1996 respectively. Both vectors were sent to Imperial College and inserted into CH to enable expression of rv1995 and rv1996, and investigated. Thereafter, knocking out of the target genes from candidate strains and phenotypic characterisation were planned. Suicide vectors for rv1995 and rv1996 were developed using insertional mutagenesis strategy and named as pANO1 and pANO2, respectively. Using PCR and bioinformatics analysis, it was shown that rv1995 and rv1996 are present in M. bovis BCG and absent from M. smegmatis. Thus, the constructed suicide vectors were transformed into the BCG and H37Rv strains to knock out the sequences of target genes from their genomes by homologous recombination. H37Rv recombinants were stored, whereas BCG recombinants were used as a model for investigation. The mutant successfully created was for rv1996 and named as M. bovis BCG rv1996::hygR. Phenotypic in vitro experiments showed that there was no significant difference between the growth rates of the wildtype BCG and its mutant. Both strains showed similar levels of resistance to various concentrations of NaNO2 and to H2O2. However, the mutant strain showed less acid resistance than the BCG wildtype strain. It was expected that deletion of rv1996 from CH would be the one responsible for microbiological attenuation of CH compared to H37Rv based on the prediction of the universal stress protein function in other bacteria. However, it can be concluded from this work that rv1996 had a minor or no role on exposure of M. tuberculosis to H2O2 and NO stress under the experimental conditions followed in this work. Only a difference on exposure to acid stress was found on disruption of the gene in BCG. This was compatible with the results provided from the Imperial College team on complementation of CH by pAAO3 carrying rv1996, whereas complementation by pAAO1 carrying rv1995 induced no change in any of the investigated phenotypic characters. This is the first study to implicate rv1996 in a role on exposure of mycobacteria to acid stress. It could be also hypothesised that deletion of rv1996 as a dosR regulated gene might be associated with failure of latency development, therefore, increasing the likelihood of active disease and propensity of CH to cause the outbreak. Lastly, the substantial role of rv1995/96 locus deletion as a phylogenetic marker of the CH strain is evident. More work is needed to confirm these findings and to identify other factors that might be related to the CH phenotype.

History

Supervisor(s)

Oggioni, Marco Rinaldo; Ketley, Julian

Date of award

30/08/2018

Author affiliation

Department of Infection, Immunity and Inflammation

Awarding institution

University of Leicester

Qualification level

Doctoral

Qualification name

PhD

Language

en

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