Integrated Genomic, Transcriptomic and Phenotypic Analysis of Mycobacterium Tuberculosis to Identify Potential Therapeutic Targets and Biomarkers
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Date
2025-05-10
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Addis Ababa University
Abstract
Introduction: Mycobacterium tuberculosis (Mtb), the pathogen responsible for tuberculosis (TB), remains a major global health challenge, particularly due to increasing drug resistance. Sub-lineage 4.2.2.2 is the most prevalent and frequently isolated strain among the various Mtb lineages and sub-lineages in treatment refractory Ethiopian TB patients, making treatment a daunting task. Beyond, the well-characterized mutation, the mechanisms involved in driving drug resistance appear to be more complex. The study findings provided valuable opportunities for identifying potential targets for developing new drugs or vaccines or biomarkers, supporting efforts to combat
TB effectively.
Objectives: To investigate the growth, drug susceptibility, gene expression, and molecular mechanisms of diverse Mtb lineages, with a focus on Ethiopian sub-lineage 4.2.2.2, to enhance understanding of TB pathogenesis and drug resistance, inform treatment strategies, and identify potential therapeutic targets.
Methods: Drug susceptibility was assessed through whole-genome analysis and phenotypic testing using the BACTEC MGIT™ 960 system. RNA sequencing analysis was performed by isolating RNA from thirty-six Mtb strains during the mid-logarithmic growth phases. Quality control and taxonomic identification of reads were performed, followed by alignment and quantification using Hisat2 and feature Counts. Differential gene expression (DGE) was analyzed with DESeq2 in R, involving normalization, dispersion estimation, and filtering. DEGs were identified using a log2(fold change) > 0.85 as a threshold.
Results: A discrepancy was observed between the phenotypic resistance profiles and the predictions based on whole-genome data, with the latter indicating a wider range of resistance. For instance, the missense mutations in rpoB (p.Ser450Leu) and katG (p.Ser315Thr) were detected, but there was no corresponding change in phenotypic drug sensitivity to rifampicin and isoniazid, respectively. RNA sequencing revealed reduced expression of six genes (Rv0096, Rv2780, Rv3136, Rv3136A, Rv3137, and Rv3230c) among drug-resistant Mtb isolates, although direct link to known resistance mechanisms is lacking. In Mtb sub-lineage 4.2.2.2 clinical isolates, seven DEGs with unique SNPs were identified, six upregulated (Rv0331, Rv0720, Rv1993c, Rv2030c, Rv2034, and Rv3129) and one downregulated (Rv0997a), previously associated with virulence, lipid metabolism, stress response, and metal transport; however, functional validation in this context is necessary. Sub-lineage 4.2.2.2 exhibited enhanced DosR regulon gene expression compared to other lineages, which may contribute to its adaptability in Ethiopia; however, this
association requires further validation. Sub-lineages 4.1.2.1 and 4.2.2.2 displayed higher maximum growth concentrations (Cmax), indicating superior growth efficiency and adaptability, possibly enhancing pathogenicity and resistance. While all strains were phenotypically susceptible, minimum inhibitory concentration (MIC) values varied, with sub-lineages 4.1.2.1 and 4.2.2.2 matching WHO’s critical thresholds, except for rifampicin. Lineage 3 showed increased drug sensitivity, requiring lower concentrations of rifampicin, isoniazid, and streptomycin.
Conclusions: The identified differentially expressed genes and their associated networks could be useful in unraveling the complexities of Mtb drug resistance and in understanding the impact that drug resistance conferring mutations have on the physiology of drug-resistant Mtb. Moreover, potential targets for future therapeutic development have been presented. These results emphasize the need to account for lineage-specific variations in Mtb isolates to optimize treatment regimens and enhance tuberculosis control strategies, especially in regions with genetically diverse Mtb populations such as Ethiopia.
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Keywords
Mycobacterium tuberculosis, lineage, drug-resistance, variation, gene expression, Ethiopia, optimized treatment