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Description
Tuberculosis (TB) is an airborne infectious disease caused by Mycobacterium tuberculosis (Mtb). With early diagnosis and treatment, most cases of TB may be cured. Yet, it remains the leading cause of human deaths due to an infectious disease. The emergence of drug-resistant TB poses a major challenge to its treatment and control. Pyrazinamide (PZA) is a drug that shortens the treatment period for TB. An association between the pncA gene and PZA-resistance in Mtb has been proposed by previous studies. However, an estimated 17% of TB cases that are resistant to PZA do not carry mutations in pncA. This suggests the existence of alternate genes that have yet to be associated with PZA resistance. The goal of this project was to identify alternate genes in the Mtb genome that may be involved with clinical resistance to PZA. Discovery of novel PZA resistance genes may help to further elucidate the mechanisms through which PZA acts against Mtb and may offer new biomarkers for rapid and accurate molecular diagnostic tests for PZA drug resistance. An additional objective of this project was to establish country-specific mutation patterns in the pncA gene of PZA-resistant samples analyzed. For this project, publicly-available whole genome sequencing (WGS) Mtb accession numbers were identified from literature review and their respective genomes were downloaded from the National Center for Biotechnology Information (NCBI) site for analysis. The WGS analysis pipeline involved trimming with seqtk, mapping with Burrows-Wheeler Aligner (BWA), variant calling with SAMtools, and annotation and processing with BCFtools and snpEff. The annotated variants were compared using Microsoft Excel to find variants that were unique to PZA-resistant specimens with no mutations in their pncA gene (i.e., unexplained PZA-resistant specimens). WGS analysis and comparisons found a large number of variants that were unique to unexplained PZA-resistant cases. A total of 1,254 unique nonsynonymous variants were found in this group, representing 868 loci and 369 of the identified genes in the reference Mtb genome. Analysis also identified 161 variants affecting the pncA gene in other PZA-resistant specimens. These pncA variants were traced to 14 countries. This study found that a large number of the nonsynonymous variants that were unique to the unexplained PZA-resistant genomes affected proteins related to Mtb pathogenicity (PE-PGRS), membrane secretion and macrophage function (PPE), lipid virulence factor transport (polyketide synthase), and efflux pumps and membrane transport/energetics (membrane/transmembrane proteins). Nonsynonymous variants affecting fatty acid biosynthesis (Fas1) were also found, which agrees with studies found in the literature. Finally, the data compiled through this study identified country-specific mutation patterns in pncA that span Africa, Asia, Europe, and South America.
