DNA metabolism in mycobacteria

PhD - ScienceSpecialised mechanisms have evolved in pathogenic bacteria to enable adaptation to hostile and fluctuating host environments. Inducible mutagenesis, in particular, has been implicated in the emergence of antibiotic- and stress-resistant mutants. This thesis examined mycobacterial DNA metabolism with specific emphasis on the roles of multiple Y-family polymerases in the evolution of inter-strain variation and drug resistance in M. tuberculosis. The contribution of the nrdZ-encoded class II ribonucleotide reductase (RNR) to the maintenance of dNTP pools for replication and repair under hypoxic conditions was also explored. In addition, the co-factor requirement of NrdZ prompted an investigation into the biosynthesis and transport of adenosylcobalamin (AdoCbl) in M. tuberculosis. The data suggest that the mycobacterial Y-polymerases are tightly regulated and restricted to specialised damage-free repair or replication restart. Disruptions in individual M. smegmatis mc2155 DinB (pol IV) homologues resulted in novel antibiotic-resistance polymorphisms that were suggestive of non-redundant function. In contrast, abrogation of all error-prone polymerase activity failed to impair long-term competitive survival of mc2155 in vitro. Similarly, heterologous overexpression of M. tuberculosis pol IV homologues did not increase spontaneous mutation rates in wild-type mc2155, or complement damage hypersensitivity. However, treatment of M. smegmatis with gyrase inhibitors confirmed the differential induction of pol IV homologues in response to replication stalling and demonstrated elevated rates of spontaneous mutagenesis as a result of GyrB inhibition. The class II RNR does not appear to play a significant role in mycobacterial pathogenesis. Specifically, NrdZ was unable to substitute for the class I RNR under aerobic conditions in vitro, and a M. tuberculosis ÄnrdZ deletion mutant was not impaired in its ability to adapt to hypoxia in vitro. Similarly, infection of immunocompetent mice suggested that nrdZ is not required for the survival or virulence of M. tuberculosis in vivo. Disruptions in genes required for AdoCbl and methionine biosynthesis revealed that complex regulatory functions govern mycobacterial methionine and AdoCbl homeostasis. Loss of early (cobK) or late (cobU) stage AdoCbl biosynthetic enzymes had no effect on the growth of M. tuberculosis H37Rv in vitro. In contrast, deletion of the B12-independent methionine synthase (metE) resulted in impaired growth on solid media that could be rescued by vitamin B12 but not Lmethionine supplementation, simultaneously demonstrating the ability of M. tuberculosis to transport exogenous vitamin B12. Significantly, double ÄcobU/ÄmetE and ÄcobK/ÄmetE deletion mutants in which all predicted methionine synthase activity was eliminated, were not impaired for growth in liquid minimal media, suggesting that M. tuberculosis H37Rv possesses alternative mechanisms for methionine generation. Finally, the attenuated virulence of the ÄcobU and ÄmetE deletion mutants in vivo in immunocompetent mice indicated the relevance of AdoCbl biosynthesis to mycobacterial pathogenesis

Translating genomics research into control of tuberculosis: lessons learned and future prospects

Genomics research has enabled crucial insights into the adaptive evolution of Mycobacterium tuberculosis as an obligate human pathogen. Here, we highlight major recent advances and evaluate the potential for genomics approaches to inform tuberculosis control efforts in high-burden settings

Effective Visualization of Tuberculosis Three-Drug Assays: A Design Study

The rise in multidrug-resistant tuberculosis means that new drugs and new drug combinations are needed to address the problems associated with current treatments. Drug screening facilities aim to identify new high quality drug compounds, or novel drug combinations, for treatment of tuberculosis. The experimental drug assay procedure produces multivariate data that is difficult to analyse and onerous to process in order to determine which drug combinations should be pursued for further development. In this design study, we have developed a visualization tool to assist with analysis and processing of this multidimensional data. The tool was developed with an iterative user-centred design process, beginning with a low fidelity paper mock-up through to the deployment of a fully functional, computer-based prototype that expert users judge to be both usable and effective

Minimal-medication approaches to treating schizophrenia

UK guidelines for treating people diagnosed with schizo phrenia currently emphasise the primacy of antipsychotic medication, with or without psycho-socially based interventions as circumstances dictate. We now see increasing calls, most notably from mental health service users, for the provision of ‘whole-person-based’, minimal-medication approaches to treating people with this diagnosis. This article is intended to locate the development of such approaches within the history of modern and pre-modern psychiatry and, in doing so, summarise the available evidence base that underpins their efficacy

Novel approach to estimate tuberculosis transmission in primary care clinics in sub-Saharan Africa: protocol of a prospective study.

INTRODUCTION Tuberculosis (TB) transmission is difficult to measure, and its drivers are not well understood. The effectiveness of infection control measures at healthcare clinics and the most appropriate intervention strategies to interrupt transmission are unclear. We propose a novel approach using clinical, environmental and position-tracking data to study the risk of TB transmission at primary care clinics in TB and HIV high burden settings in sub-Saharan Africa. METHODS AND ANALYSIS We describe a novel and rapid study design to assess risk factors for airborne TB transmission at primary care clinics in high-burden settings. The study protocol combines a range of different measurements. We will collect anonymous data on the number of patients, waiting times and patient movements using video sensors. Also, we will collect acoustic sound recordings to determine the frequency and intensity of coughing. Environmental data will include indoor carbon dioxide levels (CO2 in parts per million) and relative humidity. We will also extract routinely collected clinical data from the clinic records. The number of Mycobacterium tuberculosis particles in the air will be ascertained from dried filter units using highly sensitive digital droplet PCR. We will calculate rebreathed air volume based on people density and CO2 levels and develop a mathematical model to estimate the risk of TB transmission. The mathematical model can then be used to estimate the effect of possible interventions such as separating patient flows or improving ventilation in reducing transmission. The feasibility of our approach was recently demonstrated in a pilot study in a primary care clinic in Cape Town, South Africa. ETHICS AND DISSEMINATION The study was approved by the University of Cape Town (HREC/REF no. 228/2019), the City of Cape Town (ID-8139) and the Ethics Committee of the Canton Bern (2019-02131), Switzerland. The results will be disseminated in international peer-reviewed journals

Isolation and characterization of new phenolic siderophores with antimicrobial properties from Pseudomonas sp. UIAU-6B

We thank Russell Gray, Subha Arjuna, and Shan Wang of the Marine Biodiscovery Centre, Aberdeen for assisting in NMR, mass spectrometry and Marfey analyses, respectively. We also appreciate the support of Dr. Soji Fakoya and Blessing Akinboyo of Ondo State University of Science and Technology, Okitipupa, Nigeria, for their support in the collection of sediments. This work was supported by funding from TETFund, Nigeria for a University Senate Based research.Peer reviewedPublisher PD

Identification of Antimycobacterial Natural Products from a Library of Marine Invertebrate Extracts

Tuberculosis (TB) remains a public health crisis, requiring the urgent identification of new anti-mycobacterial drugs. We screened several organic and aqueous marine invertebrate extracts for their in vitro inhibitory activity against the causative organism, Mycobacterium tuberculosis. Here, we report the results obtained for 54 marine invertebrate extracts. The chemical components of two of the extracts were dereplicated, using 1H NMR and HR-LCMS with GNPS molecular networking, and these extracts were further subjected to an activity-guided isolation process to purify the bioactive components. Hyrtios reticulatus yielded heteronemin 1 and Jaspis splendens was found to produce the bengamide class of compounds, of which bengamides P 2 and Q 3 were isolated, while a new derivative, bengamide S 5, was putatively identified and its structure predicted, based on the similarity of its MS/MS fragmentation pattern to those of other bengamides. The isolated bioactive metabolites and semi-pure fractions exhibited M. tuberculosis growth inhibitory activity, in the rang

The Influence of HIV on the Evolution of Mycobacterium tuberculosis.

HIV significantly affects the immunological environment during tuberculosis coinfection, and therefore may influence the selective landscape upon which M. tuberculosis evolves. To test this hypothesis whole genome sequences were determined for 169 South African M. tuberculosis strains from HIV-1 coinfected and uninfected individuals and analyzed using two Bayesian codon-model based selection analysis approaches: FUBAR which was used to detect persistent positive and negative selection (selection respectively favoring and disfavoring nonsynonymous substitutions); and MEDS which was used to detect episodic directional selection specifically favoring nonsynonymous substitutions within HIV-1 infected individuals. Among the 25,251 polymorphic codon sites analyzed, FUBAR revealed that 189-fold more were detectably evolving under persistent negative selection than were evolving under persistent positive selection. Three specific codon sites within the genes celA2b, katG, and cyp138 were identified by MEDS as displaying significant evidence of evolving under directional selection influenced by HIV-1 coinfection. All three genes encode proteins that may indirectly interact with human proteins that, in turn, interact functionally with HIV proteins. Unexpectedly, epitope encoding regions were enriched for sites displaying weak evidence of directional selection influenced by HIV-1. Although the low degree of genetic diversity observed in our M. tuberculosis data set means that these results should be interpreted carefully, the effects of HIV-1 on epitope evolution in M. tuberculosis may have implications for the design of M. tuberculosis vaccines that are intended for use in populations with high HIV-1 infection rates

Synthesis and biological evaluation of bis-N2, N2′-(4-hydroxycoumarin-3-yl) ethylidene]-2, 3-dihydroxysuccinodihydrazides

A series of N2,N2′-bis[4-hydroxycoumarin-3-yl)ethylidene]-2,3-dihydroxysuccino-hydrazides, containing 4-hydroxycoumarin, hydrazine and tartaric acid moieties, have been prepared and examined for possible biological activity. Several of these compounds exhibit promising HIV-1 integrase inhibition (IC50 = 3.5 μM), and anti-T. brucei (32% viability) and anti-mycobacterial (Visual MIC90 = 15.63 μM) activity

Correction for Kipkorir et al., "De Novo Cobalamin Biosynthesis, Transport, and Assimilation and Cobalamin-Mediated Regulation of Methionine Biosynthesis in Mycobacterium smegmatis"

Volume 203, no. 7, e00620-20, 2021, https://doi.org/10.1128/JB.00620-20. Page 1: This article was published on 8 March 2021 with Stephanie S. Dawes missing from the byline. The byline was updated in the version posted on 21 January 2022. The following corrections were also made in that version. Page 1, footnote box: The following footnote was added. “§Present address: Stephanie S. Dawes, Laboratory of Structural Biology, School of Biological Sciences and Maurice Wilkins Centre for Molecular Biodiscovery, University of Auckland, Auckland, New Zealand.” Page 14, Acknowledgments, line 3: “and Stephanie Dawes for providing the DmetE cobK::hyg mutant” was deleted. Page 14, Acknowledgments, lines 1 and 2 from bottom: “(to V.M. and D.F.W.)” was changed to “(to S.S.D., V.M., and D.F.W.).