Roles of the Methylcitrate and Methylmalonyl-COA Pathways in Mycobacterial Metabolism and Pathogenesis

Mycobacterium tuberculosis has been a human pathogen for the history of mankind, but we are only now beginning to understand how it is able to survive and persist indefinitely in the host. Understanding carbon metabolism of the pathogen during infection is key, not only as a source of potential drug targets, but also for elucidating the environment in vivo, so that drugs can be tested under relevant conditions. Studies have revealed that, during infection, M. tuberculosis relies on gluconeogenic carbon sources rather than sugars. Fatty acids, cholesterol, and amino acids have all been demonstrated as usable carbon sources in vitro and can all generate propionyl-CoA. The methylcitrate cycle, which, in M. tuberculosis, uses a bifunctional isocitrate lyase/methylisocitrate lyase (ICL/MCL), is one of the two routes for metabolism of propionyl-CoA. A mutant strain of M. tuberculosis lacking the ICL/MCL was rapidly cleared from the lungs of infected mice. However, the upstream enzymes of this pathway have been demonstrated to be dispensable for infection and survival in the mouse model. The methylmalonyl- CoA route of propionyl-CoA utilization can be activated in vitro by addition of the vitamin B12 cofactor of the methylmalonyl-CoA mutase. This route may buffer the loss of the methylcitrate cycle in vivo, depending on B12 availability or production in the host. The work here examines the relative use of the methylcitrate cycle and methylmalonyl-CoA pathways in M. tuberculosis and in the related, nonpathogenic species, M. smegmatis, using genetic mutants of either or both of the metabolic routes. It is shown here that, as for M. tuberculosis, M. smegmatis preferentially uses the methylcitrate cycle for growth on propionate. In the absence of the methylcitrate cycle M. smegmatis, in contrast to M. tuberculosis, can eventually endogenously activate the methylmalonyl-CoA pathway in vitro, presumably through B12 synthesis. In mutants of both species, lacking both pathways, the use of other carbon sources in the media is inhibited in the presence of propionate. This dominant inhibition implies the accumulation of toxic metabolites derived from the inability to metabolize propionate, as has been suggested by previous studies. To detect propionate-derived intermediates, metabolite analysis by targeted liquid chromatography-tandem mass spectrometry (LC-MS/MS) was used in this study. Accumulation of these metabolites under propionate exposure was identified in a strain of M. smegmatis impaired in both metabolic routes, but not in the wild-type. These studies also revealed similar accumulation under glucose growth, where the mutant strain displayed a slight growth defect, and also under no-carbon conditions, where the mutant demonstrated a survival defect compared to wild-type. These findings suggest a role of the propionate pathways for endogenously derived propionyl-CoA as well as during starvationinduced amino acid and/or fatty acid mobilization. The M. tuberculosis mutant strains generated here were tested in the mouse infection model. The methylmalonyl-CoA mutase was found to be individually dispensable for growth in vivo. However, a strain with the additional deletion of the methylcitrate cycle was attenuated during the early stage of infection and caused less tissue pathology, even after the bacterial burden reached wild-type levels. While propionate metabolism may not be required per se for in vivo growth, the suggested accumulation of toxic intermediates, demonstrated here in M. smegmatis, may indicate a required role for ICL/MCL in M. tuberculosis for detoxification of propionyl-CoA in vivo

The characterisation of polymicrobial bacterial communities in the lower respiratory tract of individuals with chronic pulmonary disease

Microbial diversity encompasses the whole of the Earth’s biosphere and is incredibly vast. The microbial diversity of three disparate micro-environments using two culture-independent techniques (denaturing gradient gel electrophoresis (DGGE) and 454-pyrosequencing) were revealed. Five commercially available DNA polymerase (pol) enzymes were assessed in determining the bacterial community generated in sandy soil. The V3 region of the 16S rRNA gene was targeted for amplification by polymerase chain reaction (PCR). Using a PCR-DGGE approach, different DNA pols exhibited differences in the DGGE profiles produced. Both high-fidelity DNA pols Ex Taq™ Hot Start (HS) and Platinum® Pfx detected greater microbial diversity present within sandy soil than the other DNA polymerase enzymes. We employed Ex Taq™ HS to characterise the microbial communities present in two chronic respiratory tract diseases, non-cystic fibrosis bronchiectasis (nCFBR) and chronic obstructive pulmonary disease (COPD). Seventy individuals expectorated sputum, and using 16S and 28S rRNA PCR-DGGE polymicrobial communities were revealed. From the 70 patients investigated, 20 presented with symptoms consistent with an exacerbation, the remainder being clinically stable. Demographic and culture data were used in constrained ordination analyses to identify any significant associations between these data and changes in the sputum microbiota. The data presented indicates that bacterial lung communities in adult nCFBR patients have distinct differences between exacerbating and clinically stable episodes. Persistent colonisation by Pseudomonas aeruginosa is significantly associated with reduced lung function, and is negatively correlated with Haemophilus influenzae carriage. Bacterial communities seem to be predominantly assembled by stochastic processes. Fungal taxa present were scarce. Stable COPD populations have been previously investigated using culture-dependent techniques. Eleven clinically stable COPD patients had a bronchoalveolar lavage (BAL) fluid taken from the right lower lobe. Both 16S and 28S rRNA PCR-DGGE was performed on all clinical samples from extracted DNA. Co-migration of bands was then compared to a 16S and 28S standard ladder consisting of pure cultivars. Additionally, execution of 454-pyrosequencing and interrogation of the V3-V5 region of 16S rRNA genes resulted in 1799 unique OTUs being identified. Dominant bacterial genera identified were Streptococcus, Arthrobacter, and Staphylococcus respectively. Bacterial taxa identified were then subjected to multivariate statistical analysis to identify relationships between the microbial communities and patient phenotypes. Metagenomic analysis demonstrated that heterogeneous bacterial populations exist in all eleven individuals. This preliminary study shows that the lungs of COPD sufferers are colonised with multiple species of bacteria and demonstrate that a complex microbial community is present. Furthermore, bacterial phylotypes resolved to class-level indicated three potential drivers of community structure within the COPD lung microbiome: lung function, moderate and severe COPD progression, and smoking status in cohort. The identification of a greater number of bacterial taxa was also apparent in culture-negative patients using both PCR-DGGE and 454-pyrosequencing approaches

Computational approaches to find transcriptomic and epigenomic signatures of latent TB in HIV patients

Abstract: HIV infection promotes the progression of latent infection of Mtb to the active disease with the primary challenge of diagnosis being the development of efficient and sensitive methods to detect latent TB in HIV infected individuals. Previous studies have identified transcriptional signatures for active TB along with signatures predicting the risk of active TB disease in latent TB infected individuals or those with other diseases. Existing studies have also identified characteristic genes for active TB in HIV infected patients. However, no studies have identified predictive transcriptional signatures that discriminate latent TB from active TB disease in HIV positive persons as well epigenetic mechanisms associated with latent TB/HIV coinfection. The aim of this study was to develop a computational pipeline using statistical modelling and machine learning (ML) methods to identify a transcriptomic signature associated with latent TB in HIV positive patients and to identify candidate epigenetic modifications for future studies. A novel pipeline, that leverages statistical differential expression analyses (OPLS-DA) and supervised ML and feature selection methods, was applied to an existing transcriptomic dataset (NCBI GEO repository accession number GSE37250) and the outcome of the two methodologies were integrated to define a gene signature characterising the progression of latent to active TB in HIV infected patients. Enrichment analysis was performed on the transcriptomic panel of genes to predict candidate epigenetic marks in latent TB/HIV coinfection. An 11-gene minimal signature was identified of which the expression levels discriminate between latent TB and active TB in HIV positive patients. A broader analysis of DEGs identified by the ML and OPLS-DA revealed enrichment of pathways related to T- and B-cell receptor signalling, metabolic processes, insulin signalling, endocrine resistance and ATP-binding. Candidate epigenetic alterations associated with latent TB in the HIV positive cohort were identified using transcription factor (TF), histone modification (HM) and miRNA enrichment analyses. This novel integrative approach to identify a discriminative latent TB gene signature provided new insights into the response mechanism of HIV co-infection with Mtb, and pathways that merit further investigation was identified. The genes of interest identified may provide novel diagnostic and therapeutic targets for latent TB in patients who are HIV positive.M.Sc. (Biochemistry

Characterization of mycobacteria SPP. and antimycobacterial activities of plant derived compounds from Anacardiaceae family

The treatment of tuberculosis (TB) is currently a challenge due to multi- and extensively drug resistant strains of Mycobacterium tuberculosis. Mycobacterium bovis and M. tuberculosis cause clinically indistinguishable tuberculosis in humans. Both M. bovis and M. tuberculosis have been isolated from humans and animals. Plant species contain antimicrobial compounds that may lead to new anti-TB drugs. To conduct in vitro antimycobacterial assays, it is important to include current clinical isolates as new strains of bacteria might be circulating under the ongoing climate change environment. The overall goal and objectives of this study were to isolate and characterize mycobacteria species from South Africa, to test some selected plant species of the Anacardiaceae family for antimycobacterial activity using some of the newly isolated and reference strains of mycobacteria followed by cytotoxicity evaluation of the most active plant species, and finally the isolation and characterization of at least one compound from the most active and least toxic plant. This study led to the discovery of a new isolate of Mycobacterium Avium Complex species from black wildebeest. Other non-tuberculous mycobacteria and M. bovis isolates were identified from other animal species. Five out of 15 plant species screened showed good activity against Mycobacterium species. Five antimycobacterial compounds were isolated from Searsia undulata, the most active plant species. Two out of the five compounds were identified, and one compound appears to be novel, but both compounds have been isolated for the first time from Searsia undulata. An incidental finding was the potential anticancer property of extracts of Searsia undulata. Recommended future activities include isolation and identification of more active compounds from Searsia undulata which were visible in bioautography analysis, as well as synergy evaluation of antimycobacterial activities of the different compounds with current anti-tubercular drugs.Environmental SciencesPh. D. (Environmental Science

Cystic Fibrosis

Cystic Fibrosis - Heterogeneity and Personalized Treatment provides the latest research and clinical evidence for clinicians, scientists and researchers involved in the care of patients with cystic fibrosis (CF). This book outlines the burden of the CF microbiome, utilisation of CF registries to impact future care, the sequelae of hepatobiliary complication, the use of upcoming technologies to provide patient-centred care, and provides an overview of cystic fibrosis transmembrane regulator (CFTR) modulators. Looking after patients with CF is highly rewarding, allowing those of us to combine our dedication and problem-solving skills to create a personalized approach. This book is invaluable for those involved in the care of CF patients

2021 Student Symposium Research and Creative Activity Book of Abstracts

The UMaine Student Symposium (UMSS) is an annual event that celebrates undergraduate and graduate student research and creative work. Students from a variety of disciplines present their achievements with video presentations. It’s the ideal occasion for the community to see how UMaine students’ work impacts locally – and beyond. The 2021 Student Symposium Research and Creative Activity Book of Abstracts includes a complete list of student presenters as well as abstracts related to their works

A study of the diagnosis, treatment and epidemiology of Mycobacterium abscessus in patients with cystic fibrosis

Members of the Mycobacterium abscessus complex (MABSC) are a highly antibiotic-resistant complex of organisms within the genus Mycobacterium, increasingly acknowledged as a significant cause of lung infection in patients with cystic fibrosis (CF)and associated with poor clinical outcomes. Current methods of isolation of MABSC are hindered by the fact that they grow at a slower rate in culture than other microorganisms with many patient samples having to be discarded due to the overgrowth of more rapidly growing species. Decontamination of samples has shown to have an adverse effect upon the viability of MABSC, therefore improvements in the isolation of MABSC are urgently required in order to offer the possibility of a more rapid and accurate diagnosis. A novel medium (RGM) was developed for the isolation of MABSC. Commercially available pre-poured media were compared with RGM and challenged with isolates of rapidly growing mycobacteria and other species. In addition, in a multi-centre study sputum samples collected from patients with CF were inoculated onto RGM medium, BCSA and standard automated liquid culture method and assessed for growth. RGM demonstrated superior sensitivity over currently used methods without any requirement for decontamination and could easily be incorporated into any laboratory alongside routine culture for other CF pathogens. Chromogenic and fluorogenic substrates were investigated for the possibility of differentiating between subspecies within the MABSC complex. However, the results established that these would not provide any additional benefit to RGM. Possible environmental sources were explored in order to establish how patients with CF were acquiring MABSC. Although person-to-person transmission has been suggested, there are very few reports to substantiate this at present and many questions remain unanswered. In this study, MABSC was not isolated from any of the environments screened. Finally, a selection of antimicrobials were investigated against MABSC with the purpose of ascertaining susceptibility and whether any may be used for a more successful treatment outcome. There were no clinically applicable results therefore further work is required in this area. To conclude, RGM is a novel culture medium, which can be embedded alongside routine culture for other CF pathogens without any requirement for decontamination. This means that all respiratory samples submitted from patients with CF can be conveniently cultured for NTM, considerably improving the service offered to clinicians and patients. Furthermore, it is likely that formal AFB culture methods could be replaced by use of such a medium, potentially enabling substantial savings in terms of materials and labour time

2020 Student Symposium Research and Creative Activity Book of Abstracts

The UMaine Student Symposium (UMSS) is an annual event that celebrates undergraduate and graduate student research and creative work. Students from a variety of disciplines present their achievements with video presentations. It’s the ideal occasion for the community to see how UMaine students’ work impacts locally – and beyond. The 2020 Student Symposium Research and Creative Activity Book of Abstracts includes a complete list of student presenters as well as abstracts related to their works