List of Titles

Analyzing Microsporidia sp. MB from sequence to biology: comprehensive exploration of the genome, protein structures, and functions through extensive bioinformatics analysis

- Ang'ang'o, Lilian Mbaisi

Authors: Ang'ang'o, Lilian Mbaisi
Date: 2024-10-11
Subjects: Microsporidia , Whole genome sequencing , Proteins Structure , Symbiont , Malaria Prevention , Vector control
Language: English
Type: Academic theses , Doctoral theses , text
Identifier: http://hdl.handle.net/10962/466480 , vital:76734 , DOI https://doi.org/10.21504/10962/466480
Description: Microsporidia are spore-forming intracellular organisms classified as the earliest divergent group within the Fungi kingdom. Microsporidia have been found widely affecting different hosts, including both vertebrates and invertebrates. The pathogenicity of microsporidia depends on their species and the host species they infect. Due to their obligate intracellular nature, microsporidia have extensively evolved. This is illustrated by their highly variable genome sizes and gene content. Being minimalist eukaryotes, the microsporidia genome is often associated with extreme gene reduction and compaction. However, these interesting microorganisms retain particular genes that help them acquire specific host nutrients, thereby relying heavily on their host for survival and proliferation. The mode of sexual reproduction of microsporidia has not been well-studied. Harnessing microsporidia in the laboratory is often a challenge, however, the advances in computational tools have made it cheaper and quicker to accurately predict and annotate these organisms to understand their mechanism of infection. Understanding the protein structure and function of these unique organisms is the baseline for providing insights into their biology and survival in their respective hosts. Microsporidia genomes contain a large proportion of hypothetical proteins of which their functions are not described. Vittaforma corneae ATCC 50505 was used as a model to highlight the functions and structure of these otherwise unknown proteins. A systematic annotation pipeline employing exhaustive computational tools was devised to carefully annotate the hypothetical proteins of V. corneae, aiming to characterize their structure and function.The genome of the novel microsporidian, Microsporidia sp. MB, a Plasmodium-transmission-blocking symbiont isolated from Anopheles mosquitoes in Sub-Saharan Africa, was sequenced, assembled, and annotated. The genome was found to contain over 2000 putative genes spanning its 5.9 Mb size and contained minimal repeats. Comparative phylogenomic analysis of Microsporidia sp. MB grouped this symbiont within the Enterocytozoonida (clade IV) microsporidia, clustering with its closest relative – V. corneae. Using robust computational techniques, prediction and characterization of the putative proteins of Microsporidia sp. MB was conducted. The decay of several proteins in the glycolytic pathway is one unique characteristic associated with microsporidia. The proteins retained or lost often vary across the microsporidian taxon. This study highlights the retention of most of the proteins involved in the glycolytic pathway in Microsporidia sp. MB. The available genome dataset of Microsporidia sp. MB was further used to infer its mode of sexual reproduction. The symbiont appears to have several meiotic-related gene orthologs, suggesting that it is capable of sexual reproduction. These findings describe the basic biology of Microsporidia sp. MB and provide a basis for future Next-Generation Sequencing, RNA sequencing experiments ultimately informing the application of this microorganism as a biological malaria control tool. , Thesis (PhD) -- Faculty of Science, Biochemistry, Microbiology and Bioinformatics, 2024
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Date Issued: 2024-10-11

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Structural dynamic investigation of the mutation-induced resistance mechanisms of Mycobacterium tuberculosis DNA-directed RNA polymerase against Rifampicin

- Monama, Mokgerwa Zacharia

Authors: Monama, Mokgerwa Zacharia
Date: 2024-10-11
Subjects: Uncatalogued
Language: English
Type: Academic theses , Doctoral theses , text
Identifier: http://hdl.handle.net/10962/466849 , vital:76792 , DOI https://doi.org/10.21504/10962/466849
Description: Emerging resistant strains of Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis (TB) disease, continue to plague mankind and reduce the efficacies of antitubercular therapies that have been an effective defence against TB for decades. More specifically, mutations located in the β subunit of the multisubunit Mtb RNA replicative machinery, RNA polymerase (RNAP), have been well established as the reason behind resistance to the first-line antitubercular drug rifampicin (RIF), which has resulted in therapeutic failure in several clinical cases. Additionally, elusive details pertaining to the underlying mechanisms associated with RIF resistance due to the presence of Mtb-RNAP-β mutations, have resulted in setbacks in the development of novel and effective drugs that might be able to curb the ongoing threat. Hence, in this investigation, we attempted to resolve the involved Mtb-RNAP structural events at the molecular level to discern potentially important details regarding the nine clinically relevant Mtb-RNAP-β missense mutations under investigation. Hence, for the first time, we conducted an in-silico RIF resistance investigation using the Mtb-RNAP complex. To accomplish the set-out task, we first employed the use of more traditional post-MD analytical approaches such as root mean square deviation, root mean square fluctuation, radius of gyration, center of mass distance analyses, hydrogen bond occupancies, and binding free energy calculations, to conduct a global analysis of the mutated Mtb-RNAP proteins referencing RIF efficacy. Our findings revealed that the mutations may have a perturbation effect resulting in the disruption of essential structural dynamics attributed to the protein’s catalytic functions. This was for instance observed for the βfork loop 2 domain, the β’zinc-binding domain, the β’ trigger loop domain, and the β’jaw domain, which happen to be in line with previously reported experiments detailing changes in RNAP processivity. Complementarily, some of the mutations more specifically perturbed the RIF binding pocket (RIF-BP) which observably led to the reorientation of RIF from the native or active orientation needed to obstruct the processive addition of nucleoside triphosphates to the growing RNA transcript. The mutation-induced repositioning from the active RIF orientation was also reflected through the loss of essential interactions between RIF and the RIF-BP along with the loss of binding affinities captured for a majority of the mutant proteins. In conjunction with traditional analytical approaches, we further employed computational alanine scanning, weighted contact map analyses, and dynamic residue network (DRN) analyses, a novel approach that delineates residue-residue communication pathways through several metrics, to further elucidate how a set of clinically relevant mutations affect Mtb-RNAP function. With that, we were able to observe several key changes in residue importance and interactions that may be instrumental in bringing about RIF resistance and the compensatory conformational changes we observed among the mt systems through global analysis. Furthermore, we identified persistent hubs that may be particularly important in maintaining transcriptional activities in the presence and absence of the investigated mutations and RIF that could serve as potential resistance markers for future therapeutic investigations. We believe these findings will significantly aid future efforts in the discovery of new treatment options with the potential to overcome antitubercular resistance. , Thesis (PhD) -- Faculty of Science, Biochemistry, Microbiology and Bioinformatics, 2024
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Date Issued: 2024-10-11

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