Mycobacterium Tuberculosis Genes Research Articles

Evaluation of a real-time PCR assay performance to detect Mycobacterium tuberculosis, rifampicin, and isoniazid resistance in sputum specimens: a multicenter study in two major cities of Indonesia.

Tuberculosis (TB) is one of the major global health issues due to its high mortality rate, especially in low- and middle-income countries. One of the key success points of the TB eradication program is early TB diagnosis, which requires rapid and accurate diagnostic testing. This study aimed to evaluate the performance of a newly developed RT-PCR kit (Indigen MTB/DR-TB RT-PCR) in a routine TB clinical setting. A multi-fluorescence RT-PCR assay was designed and developed to detect regions within IS6110, rpoB, katG, and inhA of the Mycobacterium tuberculosis (MTB) genes. Sputum specimens were obtained from suspected TB patients who visited TB healthcare facilities in two major cities of Indonesia from September 2022 to May 2023. Specimens were assessed using Indigen MTB/DR-TB RT-PCR, acid-fast bacillus (AFB) smear microscopy, MTB culture, and drug susceptibility testing (DST) methods. Fisher's exact test (χ2) was used to analyze the Indigen performance relative to culture methods. The performance of Indigen MTB/DR-TB RT-PCR to detect MTB was assessed using 610 sputum specimens obtained from suspected patients. The overall sensitivity and specificity were 94.12% (95% CI: 90.86-96.48%) and 98.32% (95% CI: 96.20-99.46%), respectively. When the analysis was performed on AFB smear-negative TB subjects (386 subjects), a lower sensitivity level was found at 78.57% (95% CI: 68.26-86.78%), while the specificity level remained similar at 98.34% (95% CI: 96.18-99.46%). The overall performance of Indigen MTB/DR-TB RT-PCR to detect MTB showed substantial agreement with the MTB culture method (kappa value 0.93). In comparison to DST, the sensitivity and specificity levels of Indigen to detect RIF resistance or INH resistance were 78.2% (95% CI: 61.8-90.2%) and 82.8% (95% CI: 64.2-94.2%), respectively, while the specificity level for both groups was at 100% (95% CI, 87.7-100%). Indigen MTB/DR-TB RT-PCR demonstrated reliable performance for TB molecular diagnostic testing and can be implemented in routine TB diagnostic settings.

An HLA-E-targeted TCR bispecific molecule redirects T cell immunity against Mycobacterium tuberculosis

Peptides presented by HLA-E, a molecule with very limited polymorphism, represent attractive targets for T cell receptor (TCR)-based immunotherapies to circumvent the limitations imposed by the high polymorphism of classical HLA genes in the human population. Here, we describe a TCR-based bispecific molecule that potently and selectively binds HLA-E in complex with a peptide encoded by the inhA gene of Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis in humans. We reveal the biophysical and structural bases underpinning the potency and specificity of this molecule and demonstrate its ability to redirect polyclonal T cells to target HLA-E-expressing cells transduced with mycobacterial inhA as well as primary cells infected with virulent Mtb. Additionally, we demonstrate elimination of Mtb-infected cells and reduction of intracellular Mtb growth. Our study suggests an approach to enhance host T cell immunity against Mtb and provides proof of principle for an innovative TCR-based therapeutic strategy overcoming HLA polymorphism and therefore applicable to a broader patient population.

Identification of positively selected genes in Mycobacterium tuberculosis from southern Xinjiang Uygur autonomous region of China.

Tuberculosis (TB), mainly caused by Mycobacterium tuberculosis (Mtb), remains a serious public health problem. Increasing evidence supports that selective evolution is an important force affecting genomic determinants of Mtb phenotypes. It is necessary to further understand the Mtb selective evolution and identify the positively selected genes that probably drive the phenotype of Mtb. This study mainly focused on the positive selection of 807 Mtb strains from Southern Xinjiang of China using whole genome sequencing (WGS). PAML software was used for identifying the genes and sites under positive selection in 807 Mtb strains. Lineage 2 (62.70%) strains were the dominant strains in this area, followed by lineage 3 (19.45%) and lineage 4 (17.84%) strains. There were 239 codons in 47 genes under positive selection, and the genes were majorly associated with the functions of transcription, defense mechanisms, and cell wall/membrane/envelope biogenesis. There were 28 codons (43 mutations) in eight genes (gyrA, rpoB, rpoC, katG, pncA, embB, gid, and cut1) under positive selection in multi-drug resistance (MDR) strains but not in drug-susceptible (DS) strains, in which 27 mutations were drug-resistant loci, 9 mutations were non-drug-resistant loci but were in drug-resistant genes, 2 mutations were compensatory mutations, and 5 mutations were in unknown drug-resistant gene of cut1. There was a codon in Rv0336 under positive selection in L3 strains but not in L2 and L4 strains. The epitopes of T and B cells were both hyper-conserved, particularly in the T-cell epitopes. This study revealed the ongoing selective evolution of Mtb. We found some special genes and sites under positive selection which may contribute to the advantage of MDR and L3 strains. It is necessary to further study these mutations to understand their impact on phenotypes for providing more useful information to develop new TB interventions.

Genetically encoded transcriptional plasticity underlies stress adaptation in Mycobacterium tuberculosis

Transcriptional regulation is a critical adaptive mechanism that allows bacteria to respond to changing environments, yet the concept of transcriptional plasticity (TP) – the variability of gene expression in response to environmental changes – remains largely unexplored. In this study, we investigate the genome-wide TP profiles of Mycobacterium tuberculosis (Mtb) genes by analyzing 894 RNA sequencing samples derived from 73 different environmental conditions. Our data reveal that Mtb genes exhibit significant TP variation that correlates with gene function and gene essentiality. We also find that critical genetic features, such as gene length, GC content, and operon size independently impose constraints on TP, beyond trans-regulation. By extending our analysis to include two other Mycobacterium species -- M. smegmatis and M. abscessus -- we demonstrate a striking conservation of the TP landscape. This study provides a comprehensive understanding of the TP exhibited by mycobacteria genes, shedding light on this significant, yet understudied, genetic feature encoded in bacterial genomes.

Mechanistic insights into the conformational changes and alterations in residual communications due to the mutations in the pncA Gene of Mycobacterium tuberculosis: A computational perspective for effective therapeutic solutions

Due to its emerging resistance to first-line anti-TB medications, tuberculosis (TB) is one of the most contagious illness in the world. According to reports, the effectiveness of treating TB is severely impacted by drug resistance, notably resistance caused by mutations in the pncA gene-encoded pyrazinamidase (PZase) to the front-line drug pyrazinamide (PZA). The present study focused on investigating the resistance mechanism caused by the mutations D12N, T47A, and H137R to better understand the structural and molecular events responsible for the resistance acquired by the pncA gene of Mycobacterium tuberculosis (MTB) at the structural level. Bioinformatics analysis predicted that all three mutations were deleterious and located near the active centre of the pncA, affecting its functional activity. Furthermore, molecular dynamics simulation (MDS) results established that mutations significantly reduced the structural stability and caused the rearrangement of FE2+ in the active centre of pncA. Moreover, essential dynamics analysis, including principal component analysis (PCA) and free energy landscape (FEL), concluded variations in the protein motion and decreased conformational space in the mutants. Additionally, the mutations potentially impacted the network topologies and altered the residual communications in the network. The complex simulation study results established the significant movement of the flap region from the active centre of mutant complexes, further supporting the flap region’s significance in developing resistance to the PZA drug. This study advances our knowledge of the primary cause of the mechanism of PZA resistance and the structural dynamics of pncA mutants, which will help us to design new and potent chemical scaffolds to treat drug-resistant TB (DR-TB).

Uncovering the roles of Mycobacterium tuberculosis melH in redox and bioenergetic homeostasis: implications for antitubercular therapy.

This study unveils the pivotal role played by the melH gene in Mycobacterium tuberculosis and in Mycobacterium marinum in combatting the detrimental impact of oxidative conditions during infection. This investigation revealed notable alterations in the level of cytokinin-associated aldehyde, para-hydroxybenzaldehyde, as well as the redox buffer ergothioneine, upon deletion of melH. Moreover, changes in crucial cofactors responsible for electron transfer highlighted melH's crucial function in maintaining a delicate equilibrium of redox and bioenergetic processes. MelH prefers epoxide small substrates with a phenyl substituted substrate. These findings collectively emphasize the potential of melH as an attractive target for the development of novel antitubercular therapies that sensitize mycobacteria to host stress, offering new avenues for combating tuberculosis.

Prokaryotic Expression and Bioinformatic Analysis of Rv3432c From Mycobacterium tuberculosis

To express the protein enconded by the Rv3432c gene of Mycobacterium tuberculosis (M.tb) in vitro by prokaryotic expression, to analyze the structure of the Rv3432c protein by using bioinformatics software, and to explore for new drug targets against M.tb. The Rv3432c gene was amplified by PCR using the genomic DNA of the inactivated M.tb strain H37Rv as the template and a recombinant plasmid was constructed with the expression vector pET-28a. The expression products were analyzed by SDS-PAGE and purified using affinity chromatography. The biological properties of Rv3432c were analyzed with Protparam, the Pfam online tool, SOMPA, Protscale, TMHMM Signalp 6.0, NetPhos3.1, SUMOsp 2.0, and SWISS-MODEL. pET-28a-Rv3432c recombinant plasmid sequencing results were fully consistent with those of the target gene. SDS-PAGE analysis showed that the fusion protein existed in the form of a soluble protein with a relative molecular mass of about 55×103, which matched the expected size. ProtParam analysis showed that the Rv3432c protein was hydrophilic (showing a GRAVY value of －0.079). Rv3432c was a protein with no transmembrane structural domains or signal peptide. The secondary structure of Rv3432c mainly consisted of random coils (39.78%) and α-helix (39.57%) and was relatively loosely structured. We successfully constructed a prokaryotic expression plasmid of the Rv3432c protein and analyzed its structure using bioinformatics, laying the foundation for further research on the role of Rv3432c in the pathogenesis and progression of tuberculosis as well as the identification of new drug targets against M.tb.

A protocol for cloning, expression, and purification of Lysine exporter (LysE) gene of Mycobacterium tuberculosis

Background Tuberculosis (TB) is among the deadliest diseases and a significant cause of illnessacross the globe. Several studies on mycobacterial proteins, such as proteases and transporters that are essential for survival and pathogenesis have aimed to develop an efficient anti-tubercular agent. In mycobacterium, lysine exporter (LysE) is an amino acid transporter and a probable target for an anti-tubercular agent as it is responsible for bacterial growth inhibition and is also absent in the widely used Bacillus Calmette-Guérin (BCG) vaccine. Methods Some studies have purified LysE using different protocols. This study describes a protocol for purifying different constructs of LysE, focusing on its hydrophobic region using immobilized metal affinity chromatography (IMAC) after expressing LysE gene in a bacterial expression system. pET vector (pET28a) is used as an expression vector. Amplified LysE gene is ligated with the pET28a vector, and the resultant plasmid is then transformed into E. coli cells. The vector has a histidine tag that makes the purification process convenient. After IMAC, the samples will be subjected to size-exclusion chromatography for further purification. Results Cloning and amplification findings will be analyzed using 1% agarose gel, and protein expression and purification outcomes will be examined using sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Domain-specific constructs of LysE can be further analyzed as an anti-tubercular agent. Conclusions Despite being a potential anti-tubercular target, research is quite limited on this protein. Therefore, we aim to purify LysE protein for further analysis. Similar protocols have already been implemented to purify several other bacterial proteins with >95% purity.

Mutation Characteristics of inhA and katG Genes in Isoniazid-Resistant Mycobacterium Tuberculosis Patients in Xinjiang

Objective: To analyze the mutation characteristics of inhA and katG genes in isoniazid-resistant Mycobacterium tuberculosis in Xinjiang. Methods: The katG and inhA in 148 strains of isoniazid-resistant Mycobacterium tuberculosis were amplified through fluorescence quantitative PCR, and the amplified products were sequenced and compared. Results:The inhA gene mutation rate of 148 strains of isoniazid-resistant mycobacterium tuberculosis was 13.51% (20/148), among which the inhA gene mutation rate among patients of Han, Uygur, and Kazakh ethnicity were 15.87%, 13.21%, and 17.65%, respectively. There was no significant difference in the inhA mutation rate among nationalities (c2 = 2.897, P > 0.05). The mutation rate of the katG gene was 84.46% (125/148), among which the mutation rates of patients of Han, Uyghur, and Kazak ethnicities were 82.54%, 84.91%, and 76.47%, respectively. The Hui and other ethnic groups were all affected by the katG gene mutation. There was no significant difference in the mutation rate of the katG gene among different ethnicities (c2 = 3.772, P > 0.05). The mutation rates of the inhA gene in southern Xinjiang, northern Xinjiang, and other provinces were 18.60%, 9.28%, and 37.50%, respectively. The mutation rates of the inhA gene in different regions were statistically different (c2 = 6.381, P < 0.05). There was no significant difference in the inhA mutation rate between patients from southern and northern Xinjiang (c2 = 2.214, P > 0.05) and between southern Xinjiang and other provinces (c2 =1.424, P > 0.05). However, the mutation rate of the inhA gene in patients from other provinces was higher than that in northern Xinjiang (c2 = 5.539, P < 0.05). The mutation rates of the katG gene in southern Xinjiang, northern Xinjiang, and other provinces were 81.40%, 87.63%, and 62.50%, respectively. There was no significant difference in the mutation rates of the katG gene among different regions (c2 = 3.989, P > 0.05). Conclusion: katG gene mutation was predominant in isoniazid-resistant tuberculosis patients in Xinjiang Uygur Autonomous Region, and inhA and katG gene mutation were no different among different ethnic groups.

The comprehensive computational study on PE11 gene of Mycobacterium tuberculosis involved in virulence

The comprehensive computational study on PE11 gene of Mycobacterium tuberculosis involved in virulence

The Effect of a Mutation in the vapC46 Gene of Mycobacterium tuberculosis on the Functional Activity of the VapC46 Toxin

The Effect of a Mutation in the vapC46 Gene of Mycobacterium tuberculosis on the Functional Activity of the VapC46 Toxin

Gene recoding by synonymous mutations creates promiscuous intragenic transcription initiation in mycobacteria.

Mycobacterium tuberculosis (Mtb) is the causative agent of tuberculosis, one of the deadliest infectious diseases worldwide. Previous studies have established that synonymous recoding to introduce rare codon pairings can attenuate viral pathogens. We hypothesized that non-optimal codon pairing could be an effective strategy for attenuating gene expression to create a live vaccine for Mtb. We instead discovered that these synonymous changes enabled the transcription of functional mRNA that initiated in the middle of the open reading frame and from which many smaller protein products were expressed. To our knowledge, this is one of the first reports that synonymous recoding of a gene in any organism can create or induce intragenic transcription start sites.

Structural dynamics insights into the M306L, M306V, and D1024N mutations in Mycobacterium tuberculosis inducing resistance to ethambutol.

Resistance to anti-tuberculosis drugs, especially ethambutol (EMB), has been widely reported worldwide. EMB resistance is caused by mutations in the embB gene, which encodes the arabinosyl transferase enzyme. This study aimed to detect mutations in the embB gene of Mycobacterium tuberculosis from Papua and to evaluate their impact on the effectiveness of EMB. We analyzed 20 samples of M. tuberculosis culture that had undergone whole-genome sequencing, of which 19 samples were of sufficient quality for further bioinformatics analysis. Mutation analysis was performed using TBProfiler, which identified M306L, M306V, D1024N, and E378A mutations. In sample TB035, the M306L mutation was present along with E378A. The binding affinity of EMB to arabinosyl transferase was calculated using AutoDock Vina. The molecular docking results revealed that all mutants demonstrated an increased binding affinity to EMB compared to the native protein (-0.948 kcal/mol). The presence of the M306L mutation, when coexisting with E378A, resulted in a slight increase in binding affinity compared to the M306L mutation alone. The molecular dynamics simulation results indicated that the M306L, M306L + E378A, M306V, and E378A mutants decreased protein stability. Conversely, the D1024N mutant exhibited stability comparable to the native protein. In conclusion, this study suggests that the M306L, M306L + E378A, M306V, and E378A mutations may contribute to EMB resistance, while the D1024N mutation may be consistent with continued susceptibility to EMB.

Spontaneous mutational patterns and novel mutations for bedaquiline and clofazimine resistance in Mycobacterium tuberculosis.

The 2022 World Health Organization guidelines recommend use of two core anti-tuberculosis (TB) drugs, bedaquiline (BDQ) and clofazimine (CFZ), for treatment of drug-resistant (DR)-TB. However, several mutated Mycobacterium tuberculosis (MTB) genes, conferring BDQ and CFZ resistance, have been reported that predominantly arose from sporadic mutations that have not been comprehensively characterized. Herein, MTB clinical isolates collected from drug-susceptible (DS)-, multidrug-resistant (MDR)-, and extensively drug-resistant (XDR)-TB patients were cultured in vitro with BDQ or CFZ to generate progeny strains with resistance to these drugs. Progeny strains exposed to CFZ exhibited increased CFZ minimum inhibitory concentrations (MICs) that exceeded MIC increases of BDQ-exposed progeny strains. Notably, mmpR and pepQ mutations accounted for 83% and 17% of BDQ-induced spontaneous gene mutations, respectively, and 86% and 14% of CFZ-induced spontaneous gene mutations, respectively. Analyses of predicted mutation-induced changes in amino acid sequences and structures of MmpR and PepQ mutants revealed several point mutations affected sequence conversation and functionality as an underlying mechanism for observed acquired BDQ/CFZ resistance. Moreover, our results revealed differences in patterns of BDQ- and CFZ-induced acquired spontaneous mutations that may enhance our understanding of MTB BDQ/CFZ-resistance mechanisms. IMPORTANCE This study of MTB drug resistance mechanisms revealed patterns of spontaneous MTB mutations associated with acquired BDQ and CFZ resistance that arose after clinical MTB isolates were cultured in vitro with BDQ or CFZ. Results of protein sequence and structural analyses provided insights into potential mechanisms underlying associations between MTB gene mutations and DR phenotypes. Taken together, these results revealed differences in acquired BDQ and CFZ resistance mechanisms as a new perspective that may enhance our understanding of BDQ/CFZ resistance mechanisms and facilitate the development of new methods for detecting MTB drug resistance genes.

A protocol for cloning, expression, and purification of Lysine exporter (LysE) gene of Mycobacterium tuberculosis.

Tuberculosis (TB) is among the deadliest diseases and a significant cause of illnessacross the globe. Several studies on mycobacterial proteins, such as proteases and transporters that are essential for survival and pathogenesis have aimed to develop an efficient anti-tubercular agent. In mycobacterium, lysine exporter (LysE) is an amino acid transporter and a probable target for an anti-tubercular agent as it is responsible for bacterial growth inhibition and is also absent in the widely used Bacillus Calmette-Guérin (BCG) vaccine. Some studies have purified LysE using different protocols. This study describes a protocol for purifying different constructs of LysE, focusing on its hydrophobic region using immobilized metal affinity chromatography (IMAC) after expressing LysE gene in a bacterial expression system. pET vector (pET28a) is used as an expression vector. Amplified LysE gene is ligated with the pET28a vector, and the resultant plasmid is then transformed into E. coli cells. The vector has a histidine tag that makes the purification process convenient. After IMAC, the samples will be subjected to size-exclusion chromatography for further purification. Cloning and amplification findings will be analyzed using 1% agarose gel, and protein expression and purification outcomes will be examined using sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Domain-specific constructs of LysE can be further analyzed as an anti-tubercular agent. Despite being a potential anti-tubercular target, research is quite limited on this protein. Therefore, we aim to purify LysE protein for further analysis. Similar protocols have already been implemented to purify several other bacterial proteins with >95% purity.

Real-time recombinase-aided amplification assay for rapid amplification of the IS1081 gene of Mycobacterium tuberculosis.

Mycobacterium tuberculosis (MTB), the etiological agent of tuberculosis (TB), is the leading cause of death due to a single infectious agent worldwide. Rapid and accurate diagnosis of MTB is critical for controlling TB especially in resource-limited countries, since any diagnosis delay increases the chances of transmission. Here, a real-time recombinase-aided amplification (RAA) assay targeting conserved positions in IS1081 gene of MTB, is successfully established to detect MTB. The intact workflow was completed within 30 min at 42 °C with no cross-reactivity observed for non-tuberculous mycobacteria and other clinical bacteria, and the detection limit for recombinant plasmid of MTB IS1081 was 163 copies/reaction at 95% probability, which was approximately 1.5-fold increase in analytical sensitivity for the detection of MTB, compared to conventional quantitative real-time PCR (qPCR; 244 copies/reaction). Furthermore, the result of clinical performance evaluation revealed an increased sensitivity of RAA assay relative to qPCR was majorly noted in the specimens with low bacteria loads. Our results demonstrate that the developed real-time RAA assay is a convenient, sensitive, and low-cost diagnostic tool for the rapid detection of MTB.

A CRISPR-Cas12a-based platform for ultrasensitive rapid highly specific detection of Mycobacterium tuberculosis in clinical application.

Tuberculosis, caused by Mycobacterium tuberculosis (MTB), is the second leading cause of death after COVID-19 pandemic. Here, we coupled multiple cross displacement amplification (MCDA) technique with CRISPR-Cas12a-based biosensing system to design a novel detection platform for tuberculosis diagnosis, termed MTB-MCDA-CRISPR. MTB-MCDA-CRISPR pre-amplified the specific sdaA gene of MTB by MCDA, and the MCDA results were then decoded by CRISPR-Cas12a-based detection, resulting in simple visual fluorescent signal readouts. A set of standard MCDA primers, an engineered CP1 primer, a quenched fluorescent ssDNA reporter, and a gRNA were designed targeting the sdaA gene of MTB. The optimal temperature for MCDA pre-amplification is 67°C. The whole experiment process can be completed within one hour, including sputum rapid genomic DNA extraction (15 minutes), MCDA reaction (40 minutes), and CRISPR-Cas12a-gRNA biosensing process (5 minutes). The limit of detection (LoD) of the MTB-MCDA-CRISPR assay is 40 fg per reaction. The MTB-MCDA-CRISPR assay does not cross reaction with non-tuberculosis mycobacterium (NTM) strains and other species, validating its specificity. The clinical performance of MTB-MCDA-CRISPR assay was higher than that of the sputum smear microscopy test and comparable to that of Xpert method. In summary, the MTB-MCDA-CRISPR assay is a promising and effective tool for tuberculosis infection diagnosis, surveillance and prevention, especially for point-of-care (POC) test and field deployment in source-limited regions.

Mycobacterium tuberculosis Gene Expression Associated With Fluoroquinolone Resistance and Efflux Pump Inhibition.

We evaluated the relationship between response to efflux pump inhibition in fluoroquinolone-resistant Mycobacterium tuberculosis (Mtb) isolates and differences in gene expression and expression quantitative trait loci (eQTL). We determined ofloxacin minimum inhibitory concentration (MIC) for ofloxacin-resistant and -susceptible Mtb isolates without and with the efflux pump inhibitor verapamil. We performed RNA sequencing (RNA-seq), whole genome sequencing (WGS), and eQTL analysis, focusing on efflux pump, transport, and secretion-associated genes. Of 42 ofloxacin-resistant Mtb isolates, 27 had adequate WGS coverage and acceptable RNA-seq quality. Of these 27, 7 had >2-fold reduction in ofloxacin MIC with verapamil; 6 had 2-fold reduction, and 14 had <2-fold reduction. Five genes (including Rv0191) had significantly increased expression in the MIC fold change >2 compared to <2 groups. Among regulated genes, 31 eQTLs (without ofloxacin) and 35 eQTLs (with ofloxacin) had significant allele frequency differences between MIC fold change >2 and <2 groups. Of these, Rv1410c, Rv2459, and Rv3756c (without ofloxacin) and Rv0191 and Rv3756c (with ofloxacin) have previously been associated with antituberculosis drug resistance. In this first reported eQTL analysis in Mtb, Rv0191 had increased gene expression and significance in eQTL analysis, making it a candidate for functional evaluation of efflux-mediated fluoroquinolone resistance in Mtb.

Assessment of Haematological Parameters in Drug-Resistant TB

Mycobacterium tuberculosis causes tuberculosis (TB), the world's deadliest infectious illness. In addition to the lungs, bone marrow is also impacted by tuberculosis. Significant haematological abnormalities can be found in TB patients. This means that these haematological markers can be used to assess a patient's diagnosis, prognosis, and treatment outcome. The goals of this study are (a) to assess the hematological characteristics of TB patients, and (b) to examine the impact of rifampin (RIF) resistance on the prevalence of mutations in the whole rpoB gene of Mycobacterium tuberculosis. Fifty people participated in the study after being chosen through a systematic random sample process. From each participant, about 4 milliliters of blood were taken from a vein and estimated for the haematological parameters like Hb, WBC, Lymphocyes, neutrophils, Erythrocyte sedimentation rate and platelet count. Fifty tuberculosis isolates had their whole rpoB genes sequenced so that we could analyze the positions of the codons and the frequency with which they occurred. When compared to healthy controls, the values of hemoglobin and other blood indices were significantly lower or abnormal (p&lt;0.05). ESR values were alarmingly increased in the subjects along with platelets (P value &lt; 0.05). All of our 25 isolates exhibited four types of mutations at four RRDR positions (codons 510, 516, 522 and 526). We found codons 526 showed a high level (92%) of RIF resistance or mutations when compared to other positions. An easy and affordable way to forecast the progression of the illness and keep track of complications in underdeveloped nations is to test the haematological parameters of tuberculosis patients. RIF resistance is linked to certain mutations in the rpoB gene that may impact how RpoB and RIF interact. These results may be used to create new antibiotics and create cutting-edge diagnostic tools for TB medication resistance.

A protocol for cloning, expression, and purification of Lysine exporter (LysE) gene of Mycobacterium tuberculosis

Background: Tuberculosis (TB) is among the deadliest diseases and a significant cause of illness across the globe. Several studies on mycobacterial proteins, such as proteases and transporters that are essential for survival and pathogenesis have aimed to develop an efficient anti-tubercular agent. In mycobacterium, lysine exporter (LysE) is an amino acid transporter and a probable target for an anti-tubercular agent as it is responsible for bacterial growth inhibition and is also absent in the widely used Bacillus Calmette-Guérin (BCG) vaccine. Methods: Some studies have purified LysE using different protocols. This study describes a protocol for purifying different constructs of LysE, focusing on its hydrophobic region using immobilized metal affinity chromatography (IMAC) after expressing LysE gene in a bacterial expression system. pET vector (pET28a) is used as an expression vector. Amplied LysE gene is ligated with the pET28a vector, and the resultant plasmid is then transformed into E. coli cells. The vector has a histidine tag that makes the purification process convenient. After IMAC, the samples will be subjected to size-exclusion chromatography for further purification. Results: Cloning and amplification findings will be analyzed using 1% agarose gel, and protein expression and purification outcomes will be examined using sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Domain-specific constructs of LysE can be further analyzed as an anti-tubercular agent. Conclusions: Despite being a potential anti-tubercular target, research is quite limited on this protein. Therefore, we aim to purify LysE protein for further analysis. Similar protocols have already been implemented to purify several other bacterial proteins with &gt;95% purity.