Tuberculosis is an extremely lethal human ailment that has probably been there for thousands of years, ranking as the greatest cause of mortality among all diseases. The number of MDR-TB diagnoses is increasing, even though there are currently significantly fewer drug-susceptible TB cases reported globally, highlighting the need for advancement in this area (Xi et al., 2022; Chitale et al., 2022). In this study, the drug resistance was the highest to rifampicin in isolates, followed by isoniazid, ethambutol, streptomycin, fluoroquinolones, ethionamide, and aminoglycosides. 65 isolates were confirmed to be MDR-TB, and among them, one isolate was processed for genome sequencing and analysis to investigate the genetic physiology and association with the reference strain.
RAST analysis and NCBI genome BLAST analysis revealed that strain MNPK1 is closely related to the Beijing type strain with only 1% differences. While there are variations in the frequencies of the Beijing-type strain, it remains uncertain whether these differences in Beijing frequencies are due to selective isolation, increased prevalence, or geographical factors (Stucki et al., 2016). In general, it is known the high virulence and mobility of Beijing strains articulate concerns for public health surveillance about their presence in Pakistan (Yar et al., 2018; Ibrahim et al., 2018). Various comparative genome analysis was conducted while evaluating the precision of the genomes obtained in this study. The arrangement of coding sequences in circular genomes was likewise not significantly different from that of reference strains. The obtained genomes are credible because of the great similarity in genomic regions and composition. Moreover, the MNPK1, MNPK, and SWLPK strains showed Indo-Oceanic and Asian lineages by RAST based on its virulent, drug-resistant, and stress-associated gene characteristics, suggesting epidemiological links among the strains (Xi et al., 2022; Chitale et al., 2022).
Recent years have witnessed significant progress in comprehending the molecular underpinnings of mycobacterial pathogenicity, virulence, and persistence, primarily driven by advances in genomics technologies (Stucki et al., 2016). Notably, strains M. tuberculosis MNPK1, SWLPK, and MNPK are found to contain numerous genes associated with virulence. These genes include components of the type VII secretion system (T7SS), enzymes related to various lipid pathways, cell surface proteins, as well as regulators and proteins involved in signal transduction systems. In most of the cases, M. tuberculosis's pathogenesis relies on the secretion of critical virulence factors, such as the 6 kDa early secreted antigenic target ESAT-6 (EsxA) and its partner, the 10 kDa culture filtrate protein CFP-10 (EsxB), via the ESX-1 secretion system. It's worth noting that ESX-1 serves as the prototype for the recently recognized type VII secretion systems present in various actinobacteria (Samten et al., 2009). The PE/PPE proteins of M. tuberculosis exhibit common N-terminal motifs featuring Pro-Glu (PE) or Pro-Pro-Glu (PPE) residues, along with a notably diverse C-terminus. A substantial portion of these proteins are secreted through type VII secretion systems and are primarily found in pathogenic M. tuberculosis, as reported by Simeone et al. Additionally, it's worth noting that many of the virulence genes present in M. tuberculosis strains are shared with non-pathogenic mycobacteria, underscoring their conservation (Malhotra et al., 2017).
These findings imply that pathogenic mycobacterial species such as strain MNPK1, MNPK and SWLPK have adapted their genomes to the intracellular environment with minimal acquisition of exclusive virulence genes, transitioning from a free-living lifestyle. M. tuberculosis strains MNPK1, MNPK, and SWLPK carry a repertoire of drug-resistant genes conferring resistance to critical drugs like isoniazid, rifampicin, capreomycin, ethambutol, ethionamide, and ofloxacin. The presence of these drug resistance genes not only establishes their link with the Beijing family genotype of M. tuberculosis but also holds significance for the development of drug resistance gene-based markers tailored for strains of M. tuberculosis found in Pakistan (Khan et al., 2022).
HSPs, a highly maintained class of proteins found in all cellular compartments of M. tuberculosis strain MNPK1 and membranes, act as chaperones. HSPs are also crucial in defending cells from stimuli that might cause stress or harm (Canova et al., 2009; Yadav et al., 2014). Eight heat shock genes were identified in M. tuberculosis strain MNPK1, MNPK, and SWLPK that were conserved in all three strains. In bacterial cytosol, GroEL1 plays a crucial role in folding proteins and the prevention of protein aggregates. Mycobacteria are unique among bacteria because their genomes encode more than one copy of GroEL (hsp60) (Canova et al., 2009; Yadav et al., 2014). serB2 was among the genes with 1% sequence variation from the M. tuberculosis strain H37Rv. hsp70 (dnaK) plays a major role in folding catalysts and cellular networking of molecular chaperones. dnaJ and grpE cochaperone regulate the stream of unfolded proteins into and out of the substrate-binding domain of dnaK by controlling the nucleotide-bound state of dnaK(Harrison 2003; Khalid et al., 2016). Overall identification of all key HSPs in strains MNPK1, MNPK, and SWLPK reveal that, like the H37Rv reference strain, these strains may have higher drug resistance features and virulence.
Moreover, the differential gene expression was noted with all three growth conditions compared to the corresponding control; it was obvious that dnaK, GrpE, GroEL1, GroEL2, SerB2, DnaJ2, DnaJ1 and hspX of the genes were upregulated under sputum and ASM growth conditions. While dnaK, GrpE, dnaJ1, dnaJ2, and hspX genes were upregulated under 7H9 culture conditions. The DnaK operon in Mycobacterium consists of the dnaK, grpE, dnaJ1, and hspR genes. These genes have a common promoter that allows for their coordinated expression; hspR functions as a repressor to negatively regulate the other genes in the operon(Tyedmers et al., 2016; Geluk et al., 2007). Furthermore, without stress, the DnaK operon plays a non-redundant function necessary for proper cell development. Our differential gene expression study not only confirmed the findings of prior research showing that genes in the dnaK operon are crucial for the bacilli's viability and continued infection but also revealed the niche adaptation. While the expressions of GroEL1, GroEL2, and SerB2, were downregulated under 7H9. GroEL1 is the primary chaperonin protein in M. tuberculosis. The groEL1 gene encodes it and is highly conserved among mycobacterial species (Cao et al., 2014). GroEL1 assists in the folding and maturation of a wide range of client proteins, including enzymes, structural proteins, and regulatory factors. So overall up-regulation of both GroEL1 (Nachappa et al., 2022; Bandyopadhyay et al., 2012) and GroEL2 seems to be important HSPs in M. tuberculosis, contributing to the bacterium's adaptation, survival, and virulence under stressful conditions.