1,721,035 research outputs found
CRISPRI-based high-throughput functional genomic approaches for use in mycobacteria
No full textIn the 20 years since the pioneering publication of the genome of Mycobacterium tuberculosis, significant efforts have been made to complete functional annotation of the genome. However, these efforts have generally been performed on a single-gene basis, ensuring slow progress and leaving large portions of the genome unannotated. High-throughput approaches to understanding the functional genome, such as transposon-insertion sequencing, have been developed and applied to mycobacteria in a variety of conditions; however, they have several limitations, particularly in their ability to study genes essential for viability. The recent optimisation of inducible CRISPR-interference for mycobacteria offers the potential to expand the high-throughput functional genomic toolkit. This thesis utilises CRISPR-interference for the development and validation of two high-throughput functional genomic approaches in the model mycobacterium M. smegmatis. The first approach combines large-scale pooled oligonucleotide synthesis and nextgeneration sequencing, and is termed CRISPRi-Seq. A pooled library of 11 367 mutants, targeting 2 385 M. smegmatis genes with M. tuberculosis homologues, was constructed and used to infer gene essentialities which were compared with corresponding predictions from transposon-insertion sequencing data. This process validated the CRISPRi-Seq technique and identified practical considerations for its future use. The second approach utilises data derived from CRISPRi-Seq to create an arrayed library of 263 individual M. smegmatis inducible CRISPRi mutants targeting essential genes. This library is applied to a quantitative imaging pipeline to produce detailed data-driven profiles of the morphological impact of essential gene suppression. These morphological profiles are used to statistically predict genetic function, as well as antimicrobial mechanism-of-action. The two novel approaches developed in this work represent valuable technical advances and produce large datasets of functional genomic data which are available interactively online. Taken individually, or in combination, these methodologies can be utilised to increase fundamental understanding of mycobacteria, including the pathogenic M. tuberculosi
Structure/function analyses indicate novel roles for mycobacterial DnaQ homologs in genome maintenance
No full textMycobacterial DNA metabolism is of increasing interest as both an underexplored source of new targets for anti-tuberculosis (TB) drug development and for its potential role in the emergence of drug-resistant Mycobacterium tuberculosis strains. However, the redundancy implied by the sizable complement of DNA replication and repair pathways complicates investigations of gene function. There are, moreover, multiple examples in mycobacteria of apparent fusion – or hybrid – proteins in which N-and C-terminal domains appear to provide discrete functions. Both challenges apply to the mycobacterial DnaQ homologs – comprising separate DnaQ and DnaQ-UvrC hybrid proteins – which, by analogy to model organisms such as E. coli, have traditionally been assumed to fulfil proofreading roles in DNA replication owing to the presence of conserved exonuclease domains. Phylogenetic analysis of DnaQ-like proteins revealed a unique domain composition specific to the Mycobacterium genus comprising a conserved BRCA1 C Terminus (BRCT) domain in DnaQ. Owing to the presence of the BRCT domain and based on the phenotypes observed in domain-targeted mutants, it appeared that the activity of DnaQ protein (M. tuberculosis Rv3711c; M. smegmatis MSMEG_6275) might be linked to the mycobacterial gyrases that are responsible for DNA negative supercoiling following replication. The phylogenetic analysis also revealed highly conserved nucleotide excision repair (NER) proteins among bacteria; however, some species like Actinobacteria, possess both a canonical UvrC along with a DnaQ-UvrC protein. The mycobacterial DnaQ-UvrC (M. tuberculosis Rv2191; M. smegmatis MSMEG_4259) N- terminal was shown to be structurally very similar to that of DnaQ and its C-terminal to that of UvrC, giving the ability to bind either the β-clamp or the components of the UvrABC system. Opposing phenotypes between DnaQ-UvrC (M. tuberculosis Rv2191; M. smegmatis MSMEG_4259) and uvrC deletion imply a DNA-damage specific NER in mycobacteria. This was further confirmed using a combination of gene knockout, site-directed mutants and CRISPRi of NER genes, namely uvrB and uvrC, in DNA damaging conditions. However, further work is required to elucidate the precise functions of DnaQ and DnaQ-UvrC, and their contribution to genome dynamics in a family of organisms that includes major human and animal pathogens
Visualising the Mycobacterial Mutasome
Get PDFAn SOS-inducible DNA repair system has been linked to transient hyper-mutation and the development of drug resistance in Mycobacterium tuberculosis. Previous work has established that this “mycobacterial mutasome” comprises the specialist DNA polymerase, DnaE2, and accessory factors of unknown function, ImuA′ and ImuB. However, the molecular interactions and sub-cellular recruitment dynamics enabling mutasome function remain poorly understood. Here, a panel of fluorescent strains of M. smegmatis was developed to investigate expression and subcellular localization of ImuA′ and ImuB in live mycobacteria exposed to genotoxic agents. Using fluorescence microscopy, it was observed that, during prolonged genotoxic stress, single M. smegmatis cells exhibited an elongated cell phenotype and apparent aneuploidy – potentially providing an environment for recombination between differentially mutated chromosomes. Furthermore, ImuB was seen to associate with the dnaNencoded β clamp in discrete foci during mutagenic DNA repair. In contrast, ImuA′ did not exhibit similar localization and instead appeared to diffuse throughout the bacillus. A mutant ImuB protein deficient in the β clamp-binding motif failed to colocalize with the β clamp, reinforcing the inferred essentiality of the ImuB-β clamp protein-protein interaction for mutasome recruitment and induced mutagenesis. Additionally, exposure of M. smegmatis to griselimycin, a novel β clamp-targeting natural product antibiotic, prevented ImuB-β clamp co-localization during SOS induced mutagenesis, an observation confirmed by superresolution, threedimensional interferometric photo-activated light microscopy. These results establish the capacity of griselimycin to inhibit DNA replication as well as prevent DNA damage-induced mutagenesis by disrupting mutasome assembly and activity. Notably, this differentiates griselimycin from other inhibitors of DNA metabolic function which carry the often-unavoidable liability of accelerating drug-resistance by inducing mutagenic DNA repair. In turn, it suggests the potential application of griselimycin as an anti-evolution agent in novel therapeutic regimens designed to protect existing tuberculosis drugs
Riboswitch regulation of methionine metabolism and vitamin B12 uptake in mycobacteria – implications for drug susceptibility and pathogenesis
Get PDFAlterations in the genetic capacity for cobamide biosynthesis have been identified as potentially critical in the evolution of Mycobacterium tuberculosis from a putative environmental ancestor. Moreover, recent studies have implicated cobamide biosynthesis pathway genes in the adaptation of the bacillus to intracellular pathogenesis. Although mycobacteria retain essential biochemical reactions that require cobamides, the specific role of these co-factors during tuberculosis (TB) disease remains unresolved. This thesis aimed to examine the production, uptake, and utilization of cobamides in mycobacteria using M. smegmatis as a model. To this end, the genetic capacity for de novo production and uptake of cobamide in host-associated and environmental mycobacteria was assessed, followed by direct validation in M. smegmatis. A combination of genetics, gene expression analysis, live-cell time-lapse microscopy and targeted metabolite and protein analysis via mass spectrometry (MS) was then employed to investigate cobamide riboswitch-dependent regulation of methionine biosynthesis in M. smegmatis. Results indicated that, in wild-type M. smegmatis, de novo cobamide biosynthesis ensured constitutive repression of metE, the gene encoding the mycobacterial cobalamin-independent methionine synthase. Owing to this repression, metH, a gene encoding the cobalamin-dependent methionine synthase, was found to be conditionally essential for bacillary replication in vitro. Drug susceptibility testing to investigate the link between cobamides and the intrinsic resistance to anti-folate antibiotics confirmed novel mycobacterial vulnerabilities in cobamide-related methionine metabolism, indicating that the outcomes of cobamidedependent regulation may have relevance to mycobacterial pathogenesis and drug discovery. In contrast to M. tuberculosis, which was previously shown to transport exogenous CNCbl readily, M. smegmatis poorly assimilated exogenous co-factor despite the presence of multiple putative cobamide transporters. However, uptake was enhanced in a mutant requiring CNCbl for growth. Elucidating the factors which regulate cobamide biosynthesis and co-factor utilization in M. smegmatis, an environmental mycobacterium, might provide a lens through which to consider the differential regulation and utilization of cobamides in M. tuberculosis, an obligate pathogen with a limited host range
Catching a glimpse: the visualization of Mycobacterium tuberculosis from TB patient bioaerosols
Get PDFTransmission between hosts is crucial for the success and survival of the obligate human pathogen and aetiological agent of tuberculosis (TB), Mycobacterium tuberculosis (Mtb). Despite this, little is known about how and when Mtb is aerosolized nor the key metabolic and morphological determinants driving successful transmission. To address these knowledge gaps, my doctoral research sought to develop a microscopic method for the detection of aerosolized Mtb following liquidcapture within the respiratory aerosol sampling chamber (RASC). This was achieved through the combination of the mycobacterial cell wall probe, 4-N,Ndimethylamino-1,8-naphthalimide-trehalose (DMN-tre), with the arraying of bioaerosol samples on bespoke nanowell devices amenable to fluorescence microscopy. With this method, a median of 14 live Mtb bacilli (range 0-36) were detected in 90% of confirmed TB patients following 60 minutes of bioaerosol sampling. Three distinct DMN-tre staining patterns were identified among aerosolized Mtb, strongly suggestive of metabolic heterogeneity. Moreover, a low proportion of patients produced Mtb in small clumps. These observations highlight the advantages of using microscopy over conventional culture- or molecular-based techniques for probing the metabolic and morphological characteristics of aerosolized Mtb. Applying this method in a second study, we sought to understand how and when Mtb is aerosolized. To this end, we aimed to compare the aerosolization of Mtb and total particulate matter from patients with TB during three respiratory manoeuvres: tidal breathing (TiBr), forced vital capacity (FVC), and cough. Although total particle counts were 4.8-fold greater in cough samples than either TiBr or FVC, all three manoeuvres returned similar rates of positivity for Mtb. No correlation was observed between total particle production and Mtb count. Instead, for total Mtb counts, the variability between individuals was greater than the variability between sampling manoeuvres. Finally, when modelled using 24-hour breath and cough frequencies, our data indicate that TiBr might contribute more than 90% of the daily aerosolized Mtb among symptomatic TB patients. Assuming the number of viable Mtb organisms detected provides a proxy measure of patient infectiousness, this method suggests that TiBr is a significant contributor to TB transmission. In developing a novel platform for the detection of aerosolized Mtb, this work has suggested the need to re-examine old assumptions about Mtb transmission
Whole-genome transposon mutagenesis to elucidate the genetic requirements for vitamin B12 biosynthesis and assimilation in mycobacteria
Get PDFComparative genomic analyses have identified an altered capacity for cobalamin biosynthesis as a critical step in the evolution of the pathogenic Mycobacterium tuberculosis Complex strains from a common environmental ancestor. However, resolving the full gene complement involved in the complex, multi-step pathway for de novo cobalamin biosynthesis, assimilation, and salvage in different mycobacterial species is challenging. A genome-scale approach was adopted to yield detailed genetic maps of de novo cobalamin biosynthesis in M. smegmatis, a non-pathogenic saprophyte. To this end, a combination of whole-genome transposon (Tn) mutagenesis and next generation sequencing (TnSeq) was applied in M. smegmatis ΔmetE, a gene-deletion mutant in which the cobalamin-independent methionine synthase is inactivated, rendering the cobalamin-dependent isoform, MetH, essential for viability. Following growth of the metE mutant in rich medium, genomic DNA was extracted, amplified by PCR, and subjected to high-throughput sequencing to quantify all Tn junctions. Thereafter, the library was cultivated in defined minimal medium to enable identification of all conditionally essential genes – including those required for de novo cobalamin biosynthesis. A ∆metE library comprising 400,000 individual Tn insertion mutants (cfu/ml) was generated. Of the predicted 6,716 genes in the M. smegmatis genome, 213 genes were identified as essential for growth on rich agar while 356, 301, and 337 genes were identified as essential in unsupplemented, cyanocobalamin (CNCbl; vitamin B12)-supplemented and cobalt-supplemented Sauton's minimal medium, respectively. A total of 424 genes were identified as essential across all conditions tested with only 10, 13 and 24 genes (ES plus GD) uniquely required for growth in unsupplemented, CNCbl-supplemented and cobalt supplemented Sauton's minimal medium, respectively. On average, predicted cobalamin pathway genes were underrepresented in number of Tn insertions and read counts, indicating the likely essentiality of these genes during growth of the metE mutant in minimal medium. Notably, elucidation of cobalamin biosynthetic and assimilatory genes required the analysis of libraries exposed to CNCbl-unsupplemented minimal media for extended durations, probably reflecting the need to exhaust the organism's capacity for co-factor storage and recycling. Utilizing targeted silencing of individual genes by CRISPR interference, candidate cobalamin biosynthesis genes were validated, providing functional evidence of their essentiality for metE survival in minimal medium, in turn supporting the validity of the cobalamin biosynthetic pathway constructed from the TnSeq results. In addition, the results add further evidence in support of the functionality of the cobalamin riboswitch upstream of metE. This is an important observation as it suggests the potential to apply an analogous approach in M. tuberculosis, a major human pathogen whose ability to synthesize cobalamins remains unresolved. Moreover, elucidating the genetic requirements for optimal growth under specific conditions can inform our basic understanding of mycobacterial physiology and pathogenicity, identifying potential vulnerabilities for novel anti-tuberculosis therapeutics
Structural analysis of induced mutagenesis A’ protein from mycobacterium tuberculosis and of a thermophillic GH9 cellulase
Get PDFMasters of ScienceThe three-dimensional structures of proteins are important in understanding their function and interaction with ligands and other proteins. In this work, the structures of two proteins, ImuA’ from mycobacterium tuberculosis and GH9 C1 cellulase from a metagenomic library, were analysed using structural biological and modelling techniques. The gene encoding ImuA’ was amplified by two-step PCR, cloned, and expressed in E. coli. The recombinant ImuA’ produced was found to be largely insoluble. The insoluble protein was successfully solubilized in 8M urea but refolding the protein to its native structure was unsuccessful. By homology modelling, a 3D model of ImuA’ was obtained from a partly homologous protein RecA. In comparison to RecA, ImuA’ appears to lack some loop amino acids critical for DNA binding. Hence ImuA’ is postulated to not bind DNA. The second protein, GH9 C1 cellulase, was produced in E. coli. The protein was purified by chromatographic techniques and crystallized in a precipitant to protein ratio of 1:2 by hanging and sitting drop crystallization methods. The reservoir solution was made up of 15-30% (w/v) PEG 3350, 200 mM salt and 100 mM Tris-HCL pH 7.5-8.5. The protein crystals only diffracted x-rays to 4 å resolution which could not be used to obtain a crystal structure of the protein. The diffraction data, however, showed the crystal to be monoclinic with space group P2. Homology modelling revealed GH9 C1 cellulase to be a two domain protein with a smaller N-terminal Ig-like domain and a larger catalytic domain.The catalytic domain retains two ca2+ binding sites, which potentially stabilize the active site conformation and increase thermostability of the protein. Overall GH9 C1 cellulase is structurally similar to other GH9 cellulases, suggesting that its catalytic mechanism may be conserved
Developing methods to prioritize in vitro drug combinations against Mycobacterium tuberculosis: fusidic acid as potential combination partner with known antitubercular agents
Get PDFThe tuberculosis (TB) epidemic remains a major threat to public health globally, and is exacerbated by the escalating number of multi-drug resistant cases. These factors have highlighted the urgent need for new effective therapies or different approaches to augment the efficacy of current anti-TB drugs. Synergistic drug combinations present a feasible strategy towards expanding TB treatment options. Despite reported successes with combination screening, as well as the current reliance on combination therapy for TB, this approach remains largely underexplored. Evidence suggests that utilizing synergistic combinations might enable existing clinically-approved drugs to be readily re-purposed for TB treatment, including against multi-(MDR) and extensively- (XDR) drug resistant strains for current therapies are often ineffective. This thesis focused on the development and application of improved methods to identify and advance novel drug combinations for TB therapy. There were two key aspects to this work: firstly, exploring mechanisms of synergy between fusidic acid (FSA), a natural product antibiotic, and current anti-TB agents and, secondly, characterizing antibiotic action by delineating bacteriostatic and bactericidal compounds
Going Beyond Counting First Authors in Author Co-citation Analysis
Get PDFThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
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