An overview of next generation sequencing strategies and genomics tools used for tuberculosis research.

Tuberculosis (TB) is a grave public health concern and is considered the foremost contributor to human mortality resulting from infectious disease. Due to the stringent clonality and extremely restricted genomic diversity, conventional methods prove inefficient for in-depth exploration of minor genomic variations and the evolutionary dynamics operating in Mycobacterium tuberculosis (M.tb) populations. Until now, the majority of reviews have primarily focused on delineating the application of whole-genome sequencing (WGS) in predicting antibiotic resistant genes, surveillance of drug resistance strains, and M.tb lineage classifications. Despite the growing use of next generation sequencing (NGS) and WGS analysis in TB research, there are limited studies that provide a comprehensive summary of there role in studying macroevolution, minor genetic variations, assessing mixed TB infections, and tracking transmission networks at an individual level. This highlights the need for systematic effort to fully explore the potential of WGS and its associated tools in advancing our understanding of TB epidemiology and disease transmission. We delve into the recent bioinformatics pipelines and NGS strategies that leverage various genetic features and simultaneous exploration of host-pathogen protein expression profile to decipher the genetic heterogeneity and host-pathogen interaction dynamics of the M.tb infections. This review highlights the potential benefits and limitations of NGS and bioinformatics tools and discusses their role in TB detection and epidemiology. Overall, this review could be a valuable resource for researchers and clinicians interested in NGS-based approaches in TB research.

MiR-23a-5p modulates mycobacterial survival and autophagy during mycobacterium tuberculosis infection through TLR2/MyD88/NF-κB pathway by targeting TLR2.

Autophagy plays a pivotal role in activating the antimicrobial host defense against Mycobacterium tuberculosis (M.tb.). The emerging roles of microRNAs (miRNAs) in regulating immune responses have attracted increasing attention in recent years. Appreciating the potential of host-directed therapies designed to control autophagy during mycobacterial infection, we focused on the influence of miR-23a-5p on the activation of macrophage autophagy during M.tb. infection in bone marrow-derived macrophages (BMDMs) and murine RAW264.7 cells. Here, we demonstrated that M.tb.-infection of macrophages lead to markedly enhanced expression of miR-23a-5p in a time- and dose-dependent manner. Furthermore, forced expression of miR-23a-5p accelerated the survival rate of intracellular mycobacteria, while transfection with miR-23a-5p inhibitors attenuated mycobacterial survival. More importantly, overexpression of miR-23a-5p dramatically prevented M.tb.-induced activation of autophagy in macrophages, whereas inhibitors of miR-23a-5p remarkably accelerated M.tb.-induced autophagy. Mechanistically, miR-23a-5p is able to modulate TLR2/MyD88/NF-κB signaling activity by targeting TLR2 in RAW264.7 cells in response to M.tb.-infection. Collectively, these findings demonstrated that miR-23a-5p modulated the innate host defense by promoting mycobacteria survival and inhibiting the activation of autophagy against M.tb. through TLR2/MyD88/NF-κB pathway by targeting TLR2, which may provide a promising therapeutic target for tuberculosis.

TLR-4/miRNA-32-5p/FSTL1 signaling regulates mycobacterial survival and inflammatory responses in Mycobacterium tuberculosis-infected macrophages.

Macrophages play a pivotal role in host immune response against mycobacterial infection, which is tightly modulated by multiple factors, including microRNAs. The purpose of the present study was to investigate the biological function and potential mechanism of miR-32-5p in human macrophages during Mycobacterium tuberculosis (M.tb) infection. The results demonstrated that miR-32-5p was robustly enhanced in THP-1 and U937 cells in response to M.tb infection. TLR-4 signaling was required for upregulation of miR-32-5p induced by M.tb infection. Additionally, the introduction of miR-32-5p strongly increased the survival rate of intracellular mycobacteria, whereas inhibition of miR-32-5p suppressed intracellular growth of mycobacteria during M.tb challenged. Furthermore, forced expression of miR-32-5p dramatically attenuated the accumulation of inflammatory cytokines IL-1ß, IL-6 and TNF-α induced by M.tb infection. Conversely, downregulated expression of miR-32-5p led to enhancement in these inflammatory cytokines. More importantly, our study explored that Follistatin-like protein 1 (FSTL1) was a direct and functional target of miR-32-5p. qRT-PCR and western blot analysis further validated that miR-32-5p negatively regulated the expression of FSTL1. Mechanistically, re-expression of FSTL1 attenuated the ability of miR-32-5p to promote mycobacterial survival. Meanwhile, miR-32-5p-mediated inhibition of the inflammatory cytokine production were completely reversed by overexpression of FSTL1. Collectively, our findings demonstrated a novel role of TLR-4/miRNA-32-5p/FSTL1 in the modulation of host defense against mycobacterial infection, which may provide a better understanding of the pathogenesis of tuberculosis and useful information for developing potential therapeutic interventions against the disease.

A novel enzyme-linked ligand-sorbent assay (ELLSA) to screening pulmonary tuberculosis: a retrospective cross-sectional study.

BACKGROUND: Little knowledge of antigen existence in the pulmonary tuberculosis (PTB) patient serum impeded its development in antigen detection technology, despite its considerable potential. METHODS: Human ligand proteins and their adsorbent Mycobacterium tuberculosis (M.tb) proteins in the serum of PTB patients were identified using human protein chip (HuProt™) and LC-MS/MS, successively. The monoclonal antibody of ligand proteins, C5orf24, and polyclonal antibody of 9 M.tb proteins were prepared on mice and rabbits which were used to develop a novel enzyme-linked ligand-sorbent assay (ELLSA). The 412 volunteers were divided into the PTB group (n = 250) and the healthy control (n = 162). The PTB group was further divided into ATB (n = 131), LTBI (n = 18), Clinical diagnosis (n = 18), and Suspected (n = 73). All samples were tested by ELLSA to evaluate the diagnostic performance of ELLSA in PTB patients. RESULTS: Nine ligand proteins specific to PTB patients were identified on chips, with Chromosome 5 Open Reading Frame 24 (C5orf24) and kinocilin (KNCN) showing significantly higher signals. Proteomic analysis of the C5orf24-and KNCN-adsorbent protein complexes revealed 10 and 10 of the M.tb proteins, respectively. According to the composition reference of standard, the ELLSA based on C5orf24 ligand demonstrated a higher sensitivity of 69.6% and specificity of 90.18% in ATB patients and had a sensitivity of 64.22% in bacterial negative pulmonary tuberculosis, whereas the sensitivity of MGIT 960 and Xpert M.tb/RIF were 0%, respectively. CONCLUSIONS: M.tb proteins in serum can be enriched by ligand proteins and detected by ELLSA which proved to have excellent diagnostic performance for PTB.

HBHA induces IL-10 from CD4+ T cells in patients with active tuberculosis but IFN-γ and IL-17 from individuals with Mycobacterium tuberculosis infection.

Background: To effectively control tuberculosis (TB), it is crucial to distinguish between active TB disease and latent TB infection (LTBI) to provide appropriate treatment. However, no such tests are currently available. Immune responses associated with active TB and LTBI are dynamic and exhibit distinct patterns. Comparing these differences is crucial for developing new diagnostic methods and understanding the etiology of TB. This study aimed to investigate the relationship between pro- and anti-inflammatory CD4+ cytokine production following stimulation with two types of latency-associated Mycobacterium tuberculosis (M.tb) antigens to allow differentiation between active TB and LTBI. Methods: Cryopreserved PBMCs from patients with active TB disease or LTBI were stimulated overnight with replication-related antigen [ESAT-6/CFP-10 (E/C)] or two latency-associated antigens [heparin-binding hemagglutinin (HBHA) and alpha-crystallin-like protein (Acr)]. Responses were analyzed using multiparameter flow cytometry: active TB disease (n=15), LTBI (n=15) and ELISA: active TB disease (n=26) or LTBI (n=27). Results: CD4+ central memory T cells (Tcm) specific to E/C and CD4+ effector memory T cells specific to Acr and HBHA were higher in LTBI than in TB patients. IFN-γ+Tcm and IL-17+ Tem cells was higher in the LTBI group (p= 0.012 and p=0.029 respectively), but IL-10+ Tcm was higher in the active TB group (p= 0.029) following HBHA stimulation. Additionally, following stimulation with HBHA, IL-10 production from CD4+ T cells was significantly elevated in patients with active TB compared to those with LTBI (p= 0.0038), while CD4+ T cell production of IL-17 and IFN-γ was significantly elevated in LTBI compared to active TB (p= 0.0076, p< 0.0001, respectively). HBHA also induced more CCR6+IL-17+CD4Tcells and IL-17+FoxP3+CD25+CD4Tcells in LTBI than in TB patients (P=0.026 and P=0.04, respectively). HBHA also induced higher levels of IFN-γ+IL-10+CD4+ T cells in patients with active TB (Pp=0.03) and higher levels of IFN-γ+IL-17+ CD4+ T cells in those with LTBI (p=0.04). HBHA-specific cytokine production measured using ELISA showed higher levels of IFN-γ in participants with LTBI (P=0.004) and higher levels of IL-10 in those with active TB (P=0.04). Conclusion: Stimulation with HBHA and measurement of CD4+ T cell production of IFN-γ, IL-10, and IL-17 could potentially differentiate active TB from LTBI. The characteristics of cytokine-expressing cells induced by HBHA also differed between participants with active TB and LTBI.

Mycobacterium tuberculosis and host interactions in the manifestation of tuberculosis.

The final step of epigenetic processes is changing the gene expression in a new microenvironment in the body, such as neuroendocrine changes, active infections, oncogenes, or chemical agents. The case of tuberculosis (TB) is an outcome of Mycobacterium tuberculosis (M.tb) and host interaction in the manifestation of active and latent TB or clearance. This comprehensive review explains and interprets the epigenetics findings regarding gene expressions on the host-pathogen interactions in the development and progression of tuberculosis. This review introduces novel insights into the complicated host-pathogen interactions, discusses the challengeable results, and shows the gaps in the clear understanding of M.tb behavior. Focusing on the biological phenomena of host-pathogen interactions, the epigenetic changes, and their outcomes provides a promising future for developing effective TB immunotherapies when converting gene expression toward appropriate host immune responses gradually becomes attainable. Overall, this review may shed light on the dark sides of TB pathogenesis as a life-threatening disease. Therefore, it may support effective planning and implementation of epigenetics approaches for introducing proper therapies or effective vaccines.

Spermine enhances the activity of anti-tuberculosis drugs.

IMPORTANCE: This is the first study that attempted to demonstrate the mechanisms of reactive oxygen species (ROS) generation by spermine (Spm) in Mycobacterium tuberculosis (M.tb). Furthermore, this is the first study to demonstrate that it is able to enhance the activity of currently available and World Health Organization (WHO)-approved tuberculosis (TB) drugs. Spermine can easily be obtained since it is already found in our diet. Moreover, as opposed to conventional antibiotics, it is less toxic to humans since it is found in millimolar concentrations in the body. Finally, with the difficulty of curing TB with conventional antibiotics, this study suggests that less toxic molecules, such as Spm, could in a long-term perspective be incorporated in a TB regimen to boost the treatment.

Ligand-based pharmacophore modelling, virtual screening and docking studies to identify potential compounds against FtsZ of Mycobacterium tuberculosis.

BACKGROUND AND INTRODUCTION: Tuberculosis (TB) is caused by Mycobacterium tuberculosis (M.tb) which is the most common cause of death from bacterial illness. Millions of victims of TB infections have been recorded including 20,800 deaths amongst HIV positive individuals. Hence, there is a rising need for new and active compounds against M. tb protein targets especially as there is a persistent resistance to the current drug treatment regime. AIM: This study identifies new potential compounds against the M. tb target protein ftsZ via pharmacophore modelling, QSAR analysis and docking studies. METHOD: Inhibitors with known PIC50 were used as a training set and the pharmacophore features (1 aromatic center, 2 hydrophobic, 2 hydrogen bond acceptors and 1 hydrogen bond donor) were validated against four test set compounds. The identified hits were subjected to rigorous ADMET properties and docked using PyRx. DS visualizer was used in binding interactions study. Stability was measured based on the total number of interactions and preference given to the number of hydrogen bond interactions. RESULTS: Based on the number of interactions, hydrogen bonds, extensive virtual screening and ADMET filtration, 40 compounds have been identified as potential inhibitors of ftsZ with only 3 considered to be the best leads. SIGNIFICANCE OF RESEARCH: The identified compounds have potential of being drug candidate against Mycobacterium tuberculosis and may possess a novel mechanistic route in inhibiting the resistant strains.

Gene Expression Study of Host and Mycobacterium tuberculosis Interactions in the Manifestation of Acute Tuberculosis.

Mycobacterium tuberculosis (M.tb) could induce type IV hypersensitivity. The chemotaxis of the leukocytes toward the site of infection and producing matrix metalloproteinases (MMPs) are key factors in the immune pathogenesis of tuberculosis (TB). Mononuclear cells were isolated from bronchoalveolar lavage (BAL) specimens, and the target from genomic DNA was used for qPCR TB diagnosis and cDNA for specific RT-qPCR gene expression. The subjects were then classified into TB+ and TB- groups, and the expression levels of CFP-10, ESAT-6, CCR1, CCR12 and MMP3,9 were evaluated. The mean level of CCR1 expression in TB+ and TB- patients' BAL was 1.71 ± 0.78 and 0.5 ± 0.22, respectively, which was statistically different (p = 0.01). The CCR2 level, in TB+ (2.07 ± 1.4), was higher than in TB- patients (1.42 ± 0.89, p = 0.01). The MMP9 expression in TB+ was 2.56 ± 0.68, also higher than in TB- patients (1.13 ± 0.35), while MMP3 was lower in TB+ (0.22 ± 0.09) than in TB- (0.64 ± 0.230, p = 0.05). The CCR2/CCR1 and MMP3/MMP9 balance in TB+ were reduced, compared to the TB-. The CFP-10 and ESAT-6 were highly expressed in TB+ patients. The CFP-10 expression had a strong negative correlation with albumin (r = - 0.93, p = 0.001), and a negative correlation with neutrophil (r = - 0.444, p = 0.1 with 90% CI). The MMP-9 expression showed a positive correlation with WBC count (r = 0.61, p = 0.02), in TB+, and had a negative correlation with BMI (r = 0.59, p = 0.02) in TB-. The M.tb CFP-10 might be implicated in lowering CCR2 and MMP3 expression in favour of M.tb dissemination. Moreover, the balance of CCR2/CCR1 and MMP3/MMP9 can be used as prognostic factors in the severity of TB.

Vaccines against Tuberculosis: Where Are We Now?

Tuberculosis (TB) is among the top 10 leading causes of death in low-income countries. Statistically, TB kills more than 30,000 people each week and leads to more deaths than any other infectious disease, such as acquired immunodeficiency syndrome (AIDS) and malaria. TB treatment is largely dependent on BCG vaccination and impacted by the inefficacy of drugs, absence of advanced vaccines, misdiagnosis improper treatment, and social stigma. The BCG vaccine provides partial effectiveness in demographically distinct populations and the prevalence of multidrug-resistant (MDR) and extensively drug-resistant (XDR) TB incidences demands the design of novel TB vaccines. Various strategies have been employed to design vaccines against TB, such as: (a) The protein subunit vaccine; (b) The viral vector vaccine; (c) The inactivation of whole-cell vaccine, using related mycobacteria, (d) Recombinant BCG (rBCG) expressing Mycobacterium tuberculosis (M.tb) protein or some non-essential gene deleted BCG. There are, approximately, 19 vaccine candidates in different phases of clinical trials. In this article, we review the development of TB vaccines, their status and potential in the treatment of TB. Heterologous immune responses generated by advanced vaccines will contribute to long-lasting immunity and might protect us from both drug-sensitive and drug-resistant TB. Therefore, advanced vaccine candidates need to be identified and developed to boost the human immune system against TB.

Atosiban and Rutin exhibit anti-mycobacterial activity - An integrated computational and biophysical insight toward drug repurposing strategy against Mycobacterium tuberculosis targeting its essential enzyme HemD.

With the advancements of high throughput computational screening procedures, drug repurposing became the privileged framework for drug discovery. The structure-based drug discovery is the widely used method of drug repurposing, consisting of computational screening of compounds and testing them in-vitro. This current method of repurposing leaves room for mechanistic insights into how these screened hits interact with and influence their targets. We addressed this gap in the current study by integrating highly sensitive biophysical methods into existing computational repurposing methods. We also corroborated our computational and biophysical findings on H37Rv for the anti-mycobacterial action of selected drugs in-vitro and ex-vivo conditions. Atosiban and Rutin were screened as highly interacting hits against HemD through multi-stage docking and were cross-validated in biophysical studies. The affinity of these drugs (K ~ 106 M-1) was quantified using fluorescence quenching studies. Differential Scanning Fluorimetry (DSF) and urea-based chemical denaturation studies revealed a destabilizing effect of these drugs on target which was further validated using MD simulations. Conformational rearrangements of secondary structures were established using CD spectra and intrinsic fluorescence. Furthermore, Atosiban and Rutin inhibited M.tb growth in-vitro and ex-vivo while remaining non-toxic to mice peritoneal macrophages.

Effects of Mycobacterium tuberculosis lineages and regions of difference (RD) virulence gene variation on tuberculosis recurrence.

Background: China ranks second in the incidence of tuberculosis (TB), and the virulence and infectivity of Mycobacterium tuberculosis (M.tb) in different lineages are different. The variation of virulence genes in the M.tb regions of difference (RD) may be the reason for differences in pathogenicity. Studying the relationship between virulence gene mutations in the RD region of clinical strains of M.tb and TB relapse can provide basic data for the study of TB prevention and control. Methods: A total of 155 M.tb clinical strains were collected in Kashgar Prefecture. Whole-genome sequencing (WGS) was conducted, and mutations in virulence genes in the M.tb RD region were analyzed. The maximum likelihood method was implemented using IQ-TREE software. Logistic regression was used to analyze the relationship between lineage, RD region virulence gene variation, and patient relapse. Results: The 155 strains of M.tb in Kashgar Prefecture belong to 3 M.tb lineages: L2 (45.80%), L3 (32.90%), and L4 (21.30%). In relapsed patients, L2 (70.83%, 17/24) was significantly higher than the other lineages (29.17%, 7/24; P<0.05). Relapse was significantly correlated with L2 [odds ratio (OR) =3.505; 95% confidence interval (CI): 1.341-9.158; P=0.011]. In the virulence genes of the RD region, g.4357804 (T→G, OR =4.278; 95% CI: 1.594-11.481; P=0.004), g.4359653 (C→T, OR =3.356; 95% CI: 1.303-8.644; P=0.012), and g.2627618 (C→A, OR =2.676; 95% CI: 1.101-6.502; P=0.030) mutations were significantly associated with patient relapse. The mutation frequencies of g.4357804, g.4359653, and g.2627618 in L2 were significantly higher than those in the non-L2 group (P<0.05). Conclusions: Patients infected with L2 are more prone to relapse, and RD region virulence gene variation is an important factor for the strong pathogenicity and easy relapse after infection associated with L2.

Tuberculosis: The success tale of less explored dormant Mycobacterium tuberculosis.

Mycobacterium tuberculosis (M.tb) is an intracellular pathogen that predominantly affects the alveolar macrophages in the respiratory tract. Upon infection, the activation of TLR2 and TLR4- mediated signaling pathways leads to lysosomal degradation of the bacteria. However, bacterium counteracts the host immune cells and utilizes them as a cellular niche for its survival. One distinctive mechanism of M.tb to limit the host stress responses such as hypoxia and nutrient starvation is induction of dormancy. As the environmental conditions become favorable, the bacteria resuscitate, resulting in a relapse of clinical symptoms. Different bacterial proteins play a critical role in maintaining the state of dormancy and resuscitation, namely, DevR (DosS), Hrp1, DATIN and RpfA-D, RipA, etc., respectively. Existing knowledge regarding the key proteins associated with dormancy and resuscitation can be employed to develop novel therapies. In this review we aim to highlight the current knowledge of bacterial progression from dormancy to resuscitation and the gaps in understanding the transition from dormant to active state. We have also focused on elucidating a few therapeutic strategies employed to prevent M.tb resuscitation.

EST12 regulates Myc expression and enhances anti-mycobacterial inflammatory response via RACK1-JNK-AP1-Myc immune pathway.

c-Myc (Myc) is a well-known transcription factor that regulates many essential cellular processes. Myc has been implicated in regulating anti-mycobacterial responses. However, its precise mechanism in modulating mycobacterial immunity remains elusive. Here, we found that a secreted Rv1579c (early secreted target with molecular weight 12 kDa, named EST12) protein, encoded by virulent Mycobacterium tuberculosis (M.tb) H37Rv region of deletion (RD)3, induces early expression and late degradation of Myc protein. Interestingly, EST12-induced Myc was further processed by K48 ubiquitin proteasome degradation in E3 ubiquitin ligase FBW7 dependent manner. EST12 protein activates JNK-AP1-Myc signaling pathway, promotes Myc binding to the promoters of IL-6, TNF-α and iNOS, then induces the expression of pro-inflammatory cytokines (IL-6 and TNF-α)/inducible nitric oxide synthase (iNOS)/nitric oxide (NO) to increase mycobacterial clearance in a RACK1 dependent manner, and these effects are impaired by both Myc and JNK inhibitors. Macrophages infected with EST12-deficiency strain (H37RvΔEST12) displayed less production of iNOS, IL-6 and TNF-α. In conclusion, EST12 regulates Myc expression and enhances anti-mycobacterial inflammatory response via RACK1-JNK-AP1-Myc immune pathway. Our finding provides new insights into M.tb-induced immunity through Myc.

Characterization of active/binding site residues of peptidyl-tRNA hydrolase using biophysical and computational studies.

All mRNAs cannot be translated into full-length proteins due to ribosome-stalling that leads to release of peptidyl-tRNA which can be lethal for bacterial survival. The enzyme peptidyl-tRNA hydrolase (PtH) hydrolyses the ester bond between nascent peptide and tRNA of peptidyl-tRNA and rescues the cells from toxicity. PtH is an essential enzyme in bacteria and inhibiting this crucial enzyme can serve to combat bacterial diseases. But due to lack of understanding about the catalytic mechanism of PtH, its inhibitors have not been developed. In this work, we have carried out the binding studies of M. tuberculosis and E. coli PtH with the peptidyl-tRNA analogue (puromycin) using ITC, FTIR, CD experiments followed by docking and MD simulations to identify the potential active site residues that would help to design PtH inhibitors. Binding studies of puromycin with both PtH by ITC experiments demonstrate similar thermodynamic parameters and three fold difference in their KD. CD and FTIR studies detected changes in secondary structure composition of PtH in the presence of puromycin with different degree of perturbation. Though interactions with puromycin are conserved in both proteins, modelling studies revealed that water mediated interactions in M. tb-PtH resulting in higher affinity to puromycin.

Immunogenicty and immunoprotective effect of Mycobacterium tuberculosis Ag85B-Fc2a DNA vaccine / 中国生物制品学杂志

@#Objective To evaluate the immunogenicity and protective effect of Mycobacterium tuberculosis(M.tb) Ag85B-Fc2a DNA vaccine in mice,so as to provide experimental basis for the development of the vaccine.Methods The recombinant plasmid pcD-Ag85B-Fc2a was identified by double digestion and sequencing,and then transfected into CHO-K1 cells.The expression of fusion protein Ag85B-Fc2a was detected by Western blot.Vector pcDNA3.1(+) and recombinant plasmid pcDAg85B-Fc2a were injected into female C57BL/6J mice through thigh muscle respectively(control group and immunization group),with 10 mice in each group,and booster immunization was carried out two weeks after the initial immunization.At14 d,28 d and 42 d after the initial immunization,serum samples was separated,and the titers of IgG antibody in the serum were detected by indirect ELISA.At 42 d after the initial immunization,the spleen of mice was taken aseptically and made into single cell suspension,and the proportions of CD4~+and CD8~+T cells were detected by flow cytometry.The remaining mice were injected with M.tb H37Ra through the tail vein 42 d after the initial immunization at a dose of 10~6 CFU/mouse.After 28 d of challenge,the lung and spleen of mice were collected aseptically.The number of bacteria in the left lung and spleen was measured by plate method,and the bacteria in the right lung was detected by auramine O fluorescence staining.Results Double digestion and sequencing results showed that the recombinant plasmid pcD-Ag85B-Fc2a was constructed correctly.After transfection into CHO-K1 cells,the fusion protein Ag85B-Fc2a with a relative molecular mass of about 70 000was detected.The Ag85B-specific IgG antibody titer in serum of mice in the immunization group was 1:3 200,1:12 160,and 1:12 800 at 14 d,28 d,and 42 d after the initial immunization,respectively,but no antibody titer was detected in the serum samples of control group.At 42 d after the initial immunization,the percentages of CD4~+ T cells in mouse spleen of control group and immunization group were(23.61±0.64)% and(26.92±0.80)%,and the percentages of CD8~+T cells were(14.12±0.87)% and(18.78±0.94)%,respectively,with significant differences(t=3.23 and 3.64,respectively,each P <0.05).After infection with M.tb H37Ra for 28 d,the numbers of bacteria were(4.73±0.13) and(3.81±0.14)CFU in the left lung,and(5.02±0.19) and(4.30±0.13) C.FU in the spleen of control group and immunization group,respectively,with significant differences(t=4.65 and 3.12,respectively,each P <0.01).The bacteria loading in the right lung was consistent with that in the left lung.Conclusion Ag85B-Fc2a DNA vaccine can induce specific humoral and cellular immune effects in mice,and can produce good protective effect against M.tb H37Ra infection.

Comparison of QFT-Plus and QFT-GIT tests for diagnosis of M. tuberculosis infection in immunocompetent Korean subjects.

BACKGROUND: QFT-Plus is a recently developed next-generation QuantiFERON-TB Gold In-Tube (QFT-GIT) test. Unlike the QFT-GIT test, it includes a TB2 antigen tube with peptides that may stimulate CD8+ T cells. This study evaluated the diagnostic performance of QFT-Plus and compared it with that of QFT-GIT. METHODS: QFT-Plus and QFT-GIT tests were performed in 33 patients with active tuberculosis (TB) and 57 healthy controls including subjects with latent TB infection (LTBI). Positivity and negativity of IFN-γ responses were compared between tests, and total concordance of the outcome was analyzed. RESULTS: Positive and negative outcomes of QFT-Plus and QFT-GIT tests showed substantial agreement (91.1%, kappa=0.8). The sensitivity and the specificity of QFT-Plus (93.9% sensitivity, 92.6% specificity) were similar with those of QFT-GIT (93.9% sensitivity, 100% specificity). Of eight discordant results, five (5.56%) and three (3.3%) were positive in QFT-GIT alone and QFT-plus alone, respectively. Reactivity in the TB2 tube contributes to the difference between QFT-GIT and QFT-Plus. Median IFN-γ production in TB2 (10.0 IU/mL in TB, 3.850 IU/mL in LTBI, P=0.001) is significantly higher in the TB group than the LTBI group. The QFT-Plus did not clearly discriminate between active TB and latent TB, although it showed significantly lower IFN-γ concentrations compared with the QFT-GIT in individuals with LTBI (3.850 vs. 7.205 IU/mL). CONCLUSIONS: Similar accuracy of detecting Mycobacterium tuberculosis infection was observed for QFT-Plus and QFT-GIT tests in immunocompetent patients and healthy controls, including those with LTBI. Improved efficacy for identifying M. tuberculosis infection was not found with the QFT-Plus, but further studies in a larger population may confirm the clinical significance of positive response in the TB2 tube of QFT-Plus.

Inhibition mechanism of L,D-transpeptidase 5 in presence of the ß-lactams using ONIOM method.

Tuberculosis (TB) is one of the world's deadliest diseases resulting from infection by the bacterium, Mycobacterium tuberculosis (M.tb). The L,D-transpeptidase enzymes catalyze the synthesis of 3 → 3 transpeptide linkages which are predominant in the peptidoglycan of the M.tb cell wall. Carbapenems is class of ß-lactams that inactivate L,D-transpeptidases by acylation, although differences in antibiotic side chains modulate drug binding and acylation rates. Herein, we used a two-layered our Own N-layer integrated Molecular Mechanics ONIOM method to investigate the catalytic mechanism of L,D-transpeptidase 5 (LdtMt5) by ß-lactam derivatives. LdtMt5 complexes with six ß-lactams, ZINC03788344 (1), ZINC02462884 (2), ZINC03791246 (3), ZINC03808351 (4), ZINC03784242 (5) and ZINC02475683 (6) were simulated. The QM region (high-level) comprises the ß-lactam, one water molecule and the Cys360 catalytic residue, while the rest of the LdtMt5 residues were treated with AMBER force field. The activation energies (ΔG#) were calculated with B3LYP, M06-2X and ωB97X density functionals with 6-311++G(2d, 2p) basis set. The ΔG# for the acylation of LdtMt5 by the selected ß-lactams were obtained as 13.67, 20.90, 22.88, 24.29, 27.86 and 28.26 kcal mol-1respectively. Several of the compounds showed an improved ΔG# when compared to the previously calculated energies for imipenem and meropenem for the acylation step for LdtMt5. This model provides further validation of the catalytic inhibition mechanism of LDTs with atomistic detail.

Identification of potent L,D-transpeptidase 5 inhibitors for Mycobacterium tuberculosis as potential anti-TB leads: virtual screening and molecular dynamics simulations.

Virtual screening is a useful in silico approach to identify potential leads against various targets. It is known that carbapenems (doripenem and faropenem) do not show any reasonable inhibitory activities against L,D-transpeptidase 5 (LdtMt5) and also an adduct of meropenem exhibited slow acylation. Since these drugs are active against L,D-transpeptidase 2 (LdtMt2), understanding the differences between these two enzymes is essential. In this study, a ligand-based virtual screening of 12,766 compounds followed by molecular dynamics (MD) simulations was applied to identify potential leads against LdtMt5. To further validate the obtained virtual screening ranking for LdtMt5, we screened the same libraries of compounds against LdtMt2 which had more experimetal and calculated binding energies reported. The observed consistency between the binding affinities of LdtMt2 validates the obtained virtual screening binding scores for LdtMt5. We subjected 37 compounds with docking scores ranging from - 7.2 to - 9.9 kcal mol-1 obtained from virtual screening for further MD analysis. A set of compounds (n = 12) from four antibiotic classes with ≤ - 30 kcal mol-1 molecular mechanics/generalized born surface area (MM-GBSA) binding free energies (ΔGbind) was characterized. A final set of that, all ß-lactams (n = 4), was considered. The outcome of this study provides insight into the design of potential novel leads for LdtMt5. Graphical abstract.

Application of metabolomics to drug discovery and understanding the mechanisms of action of medicinal plants with anti-tuberculosis activity.

Human tuberculosis (TB) is amongst the oldest and deadliest human bacterial diseases that pose major health, social and economic burden at a global level. Current regimens for TB treatment are lengthy, expensive and ineffective to emerging drug resistant strains. Thus, there is an urgent need for identification and development of novel TB drugs and drug regimens with comprehensive and specific mechanisms of action. Many medicinal plants are traditionally used for TB treatment. While some of their phytochemical composition has been elucidated, their mechanisms of action are not well understood. Insufficient knowledge on Mycobacterium tuberculosis (M.tb) biology and the complex nature of its infection limit the effectiveness of current screening-based methods used for TB drug discovery. Nonetheless, application of metabolomics tools within the 'omics' approaches, could provide an alternative method of elucidating the mechanism of action of medicinal plants. Metabolomics aims at high throughput detection, quantification and identification of metabolites in biological samples. Changes in the concentration of specific metabolites in a biological sample indicate changes in the metabolic pathways. In this paper review and discuss novel methods that involve application of metabolomics to drug discovery and the understanding of mechanisms of action of medicinal plants with anti-TB activity. Current knowledge on TB infection, anti-TB drugs and mechanisms of action are also included. We further highlight metabolism of M. tuberculosis and the potential drug targets, as well as current approaches in the development of anti-TB drugs.