Anti-tuberculosis lead molecules from natural products targeting <Emphasis Type="Italic">Mycobacterium tuberculosis</Emphasis> ClpC1

Tuberculosis (TB) is a serious and potentially fatal disease caused by Mycobacterium tuberculosis (M. tb). The occurrence of multidrug-resistant (MDR) and extensively drug-resistant (XDR) M. tb is a significant public health concern because most of the anti-TB drugs that have been in use for over 40 years are no longer effective for the treatment of these infections. Recently, new anti-TB lead compounds such as cyclomarin A, lassomycin, and ecumicin, which are cyclic peptides from actinomycetes, have shown potent anti-TB activity against MDR and XDR M. tb as well as drug-susceptible M. tb in vitro. The target molecule of these antibiotics is ClpC1, a protein that is essential for the growth of M. tb. In this review, we introduce the three anti-TB lead compounds as potential anti-TB therapeutic agents targeting ClpC1 and compare them with the existing anti-TB drugs approved by the US Food and Drug Administration.     

In silico discovery of potential drug molecules to improve the treatment of isoniazid-resistant Mycobacterium tuberculosis

The emergence of multidrug-resistant Mycobacterium tuberculosis (M.tb) has become one of the major hurdles in the treatment of tuberculosis (TB). Drug-resistant M.tb has evolved with various strategies to avoid killing by the anti-tubercular drugs. Thus, there is a rising need to develop effective anti-TB drugs to improve the treatment of these strains. Traditional drug design approach has earned little success due to time and the cost involved in the process of development of anti-infective drugs. Numerous reports have demonstrated that several mutations in the drug target sites cause emergence of drug-resistant M.tb strains. In this study, we performed computational mutational analysis of M.tb inhA, fabD, and ahpC genes, which are the primary targets for first-line isoniazid (INH) drug. In silico virtual drug screening was performed to identify the potent drugs from a ChEMBL compound library to improve the treatment of INH-resistant M.tb. Further, these compounds were analyzed for their binding efficiency against active drug binding cavity of M.tb wild-type and mutant InhA, FabD and AhpC proteins. The drug efficacy of predicted lead compounds was verified by molecular docking using M.tb wild-type and mutant InhA, FabD and AhpC protein template models. Different in silico and pharmacophore analysis predicted three potent lead compounds with better drug-like properties against both M.tb wild-type and mutant InhA, FabD, and AhpC proteins as compared to INH drug, and thus may be considered as effective drugs for the treatment of INH-resistant M.tb strains. We hypothesize that this work may accelerate drug discovery process for the treatment of drug-resistant TB.

Communicated by Ramaswamy H. Sarma     

Genetics-directed drug discovery for combating Mycobacterium tuberculosis infection

Mycobacterium tuberculosis (Mtb), the pathogen of tuberculosis (TB), is one of the most infectious bacteria in the world. The traditional strategy to combat TB involves targeting the pathogen directly; however, the rapid evolution of drug resistance lessens the efficiency of this anti-TB method. Therefore, in recent years, some researchers have turned to an alternative anti-TB strategy, which hinders Mtb infection through targeting host genes. In this work, using a theoretical genetic analysis, we identified 170 Mtb infection-associated genes from human genetic variations related to Mtb infection. Then, the agents targeting these genes were identified to have high potential as anti-TB drugs. In particular, the agents that can target multiple Mtb infection-associated genes are more druggable than the single-target counterparts. These potential anti-TB agents were further screened by gene expression data derived from connectivity map. As a result, some agents were revealed to have high interest for experimental evaluation. This study not only has important implications for anti-TB drug discovery, but also provides inspirations for streamlining the pipeline of modern drug discovery.     

Inhibitor binding studies of Mycobacterium tuberculosis MraY (Rv2156c): Insights from molecular modeling,docking, and simulation studies

Abstract

Tuberculosis (TB) is a contagious disease caused by Mycobacterium tuberculosis (M.tb) or tubercule bacillus, and H37Rv is the most studied clinical strain. The recent development of resistance to existing drugs is a global health-care challenge to control and cure TB. Hence, there is a critical need to discover new drug targets in M.tb. The members of peptidoglycan biosynthesis pathway are attractive target proteins for antibacterial drug development. We have performed in silico analysis of M.tb MraY (Rv2156c) integral membrane protein and constructed the three-dimensional (3D) structure model of M.tb MraY based on homology modeling method. The validated model was complexed with antibiotic muraymycin D2 (MD2) and was used to generate structure-based pharmacophore model (e-pharmacophore). High-throughput virtual screening (HTVS) of Asinex database and molecular docking of hits was performed to identify the potential inhibitors based on their mode of interactions with the key residues involved in M.tb MraY–MD2 binding. The validation of these molecules was performed using molecular dynamics (MD) simulations for two best identified hit molecules complexed with M.tb MraY in the lipid bilayer, dipalmitoylphosphatidyl-choline (DPPC) membrane. The results indicated the stability of the complexes formed and retained non-bonding interactions similar to MD2. These findings may help in the design of new inhibitors to M.tb MraY involved in peptidoglycan biosynthesis.     

Lineage specific histories of Mycobacterium tuberculosis dispersal in Africa and Eurasia

Mycobacterium tuberculosis (M.tb) is a globally distributed, obligate pathogen of humans that can be divided into seven clearly defined lineages. An emerging consensus places the origin and global dispersal of M.tb within the past 6,000 years: identifying how the ancestral clone of M.tb spread and differentiated within this timeframe is important for identifying the ecological drivers of the current pandemic. We used Bayesian phylogeographic inference to reconstruct the migratory history of M.tb in Africa and Eurasia and to investigate lineage specific patterns of spread from a geographically diverse sample of 552 M.tb genomes. Applying evolutionary rates inferred with ancient M.tb genome calibration, we estimated the timing of major events in the migratory history of the pathogen. Inferred timings contextualize M.tb dispersal within historical phenomena that altered patterns of connectivity throughout Africa and Eurasia: trans‐Indian Ocean trade in spices and other goods, the Silk Road and its predecessors, the expansion of the Roman Empire, and the European Age of Exploration. We found that Eastern Africa and Southeast Asia have been critical in the dispersal of M.tb. Our results further reveal that M.tb populations have grown through range expansion, as well as in situ, and delineate the independent evolutionary trajectories of bacterial subpopulations underlying the current pandemic.     

Recent insights into Mycobacterium tuberculosis through proteomics and implications for the clinic

Introduction: This review aimed at providing an update on the application of proteomics-based approaches to gain recent insights of Mycobacterium tuberculosis (M.tb) and its relevance to clinic. Proteomics and bioinformatics approaches helped in the identification and characterization of novel proteins. Studying M.tb, causative agent of tuberculosis (TB), at the proteomic level can contribute to the identification of proteins which can be considered as potential targets for developed drugs and can help us in better understanding the pathogen physiology.

Areas covered: In this review we have presented a comprehensive literature pertaining to role of proteomics in understanding M.tb. We have also focused on how the development and advancement in technology in the field of proteomics has augmented the research and played a pivotal role in answering many unexplored questions. Lastly, the application of proteomics to clinic has also been discussed.

Expert commentary: We envisage that proteomics has gained remarkable momentum over the years. Proteomics can play an important role in the discovery of biomarkers for TB and other diseases. Also, it can aid in development of effective vaccines and simple, rapid and cost-effective test for the diagnosis of TB which is crucial for the management and control of the disease.     

A review of medicinal plant of Middle East and North Africa (MENA) region as source in tuberculosis drug discovery

Tuberculosis (TB) is a disease that affects one-third of the world’s population. Although currently available TB drugs have many side effects, such as nausea, headache and gastrointestinal discomfort, no new anti-TB drugs have been produced in the past 30 years. Therefore, the discovery of a new anti-TB agent with minimal or no side effects is urgently needed. Many previous works have reported the effects of medicinal plants against Mycobacterium tuberculosis (MTB). However, none have focused on medicinal plants from the Middle Eastern and North African (MENA) region. This review highlights the effects of medicinal plants from the MENA region on TB. Medicinal plants from the MENA region have been successfully used as traditional medicine and first aid against TB related problems. A total of 184 plants species representing 73 families were studied. Amongst these species, 93 species contained more active compounds with strong anti-MTB activity (crude extracts and/or bioactive compounds with activities of 0–100 µg/ml). The extract of Inula helenium, Khaya senegalensis, Premna odorata and Rosmarinus officinalis presented the strongest anti-MTB activity. In addition, Boswellia papyrifera (Del) Hochst olibanum, Eucalyptus camaldulensis Dehnh leaves (river red gum), Nigella sativa (black cumin) seeds and genus Cymbopogon exhibited anti-TB activity. The most potent bioactive compounds included alantolactone, octyl acetate, 1,8-cineole, thymoquinone, piperitone, α- verbenol, citral b and α-pinene. These compounds affect the permeability of microbial plasma membranes, thus kill the mycobacterium spp. As a conclusion, plant species collected from the MENA region are potential sources of novel drugs against TB.     

Genome-Wide Expression Profiling Identifies Type 1 Interferon Response Pathways in Active Tuberculosis

Tuberculosis (TB), caused by Mycobacterium tuberculosis (M.tb), remains the leading cause of mortality from a single infectious agent. Each year around 9 million individuals newly develop active TB disease, and over 2 billion individuals are latently infected with M.tb worldwide, thus being at risk of developing TB reactivation disease later in life. The underlying mechanisms and pathways of protection against TB in humans, as well as the dynamics of the host response to M.tb infection, are incompletely understood. We carried out whole-genome expression profiling on a cohort of TB patients longitudinally sampled along 3 time-points: during active infection, during treatment, and after completion of curative treatment. We identified molecular signatures involving the upregulation of type-1 interferon (α/β) mediated signaling and chronic inflammation during active TB disease in an Indonesian population, in line with results from two recent studies in ethnically and epidemiologically different populations in Europe and South Africa. Expression profiles were captured in neutrophil-depleted blood samples, indicating a major contribution of lymphocytes and myeloid cells. Expression of type-1 interferon (α/β) genes mediated was also upregulated in the lungs of M.tb infected mice and in infected human macrophages. In patients, the regulated gene expression-signature normalized during treatment, including the type-1 interferon mediated signaling and a concurrent opposite regulation of interferon-gamma. Further analysis revealed IL15RA, UBE2L6 and GBP4 as molecules involved in the type-I interferon response in all three experimental models. Our data is highly suggestive that the innate immune type-I interferon signaling cascade could be used as a quantitative tool for monitoring active TB disease, and provide evidence that components of the patient’s blood gene expression signature bear similarities to the pulmonary and macrophage response to mycobacterial infection.     

Emergence and evolution of drug-resistant Mycobacterium tuberculosis in eastern China: A six-year prospective study

Understanding the emergence and evolution of drug resistance can inform public health intervention to combat tuberculosis (TB). In this prospective molecular epidemiological surveillance study from 2015 to 2021 in eastern China, we prospectively collected whole-genome sequencing and epidemiological data on TB patients. We dissect the ordering of drug resistance mutation acquisition for nine commonly used anti-TB drugs, and we found that the katG S315T mutation first appeared around 1959, followed by rpoB S450L (1969), rpsL L43A (1972), embB M306V (1978), rrs 1401 (1981), fabG1 (1982), pncA (1985) and folC (1988) mutations. GyrA gene mutations appeared after the year of 2000. We observed that the first expansion of Mycobacterium tuberculosis (M.tb) resistance population among eastern China appeared after the introduction of isoniazid, streptomycin and para-amino salicylic acid, and the second expansion after the ethambutol, rifampicin, pyrazinamide, ethionamide and aminoglycosides. We speculate these two expansions are linked with population shift historically. By geospatial analysis, we found drug-resistant isolates migrated within eastern China. With epidemiological data of clonal strains, we observed some strains can evolve continuously in individuals and transmit readily in a population. In conclusion, this study mirrored the emergence and evolution of drug-resistant M.tb in eastern China were linked to the sequence and timing of introduction of anti-TB drugs, and multiple factors may contribute to the resistant population enlarged. To resolve the epidemic of drug-resistant TB, it requires applying anti-TB drugs carefully and/or identifying resistant patients timely to prevent them from developing high-level resistance and transmitting to others.     

MiR‐140 modulates the inflammatory responses of Mycobacterium tuberculosis‐infected macrophages by targeting TRAF6

This study aimed to examine miR‐140 expression in clinical samples from tuberculosis (TB) patients and to explore the molecular mechanisms of miR‐140 in host‐bacterial interactions during Mycobacterium tuberculosis (M tb) infections. The miR‐140 expression and relevant mRNA expression were detected by quantitative real‐time PCR (qRT‐PCR); the protein expression levels were analysed by ELISA and western blot; M tb survival was measured by colony formation unit assay; potential interactions between miR‐140 and the 3′ untranslated region (UTR) of tumour necrosis factor receptor‐associated factor 6 (TRAF6) was confirmed by luciferase reporter assay. MiR‐140 was up‐regulated in the human peripheral blood mononuclear cells (PBMCs) from TB patients and in THP‐1 and U937 cells with M tb infection. Overexpression of miR‐140 promoted M tb survival; on the other hand, miR‐140 knockdown attenuated M tb survival. The pro‐inflammatory cytokines including interleukin 6, tumour necrosis‐α, interleukin‐1β and interferon‐γ were enhanced by M tb infection in THP‐1 and U937 cells. MiR‐140 overexpression reduced these pro‐inflammatory cytokines levels in THP‐1 and U937 cells with M tb infection; while knockdown of miR‐140 exerted the opposite actions. TRAF6 was identified to be a downstream target of miR‐140 and was negatively modulated by miR‐140. TRAF6 overexpression increased the pro‐inflammatory cytokines levels and partially restored the suppressive effects of miR‐140 overexpression on pro‐inflammatory cytokines levels in THP‐1 and U937 cells with M tb infection. In conclusion, our results implied that miR‐140 promoted M tb survival and reduced the pro‐inflammatory cytokines levels in macrophages with M tb infection partially via modulating TRAF6 expression.     

Interferon-Gamma Assay in Combination with Tuberculin Skin Test Are Insufficient for the Diagnosis of Culture-Negative Pulmonary Tuberculosis

Objective

Early diagnosis of infectious cases and treatment of tuberculosis (TB) are important strategies for reducing the incidence of this disease. Unfortunately, traditional TB diagnostic methods are time-consuming and often unreliable. This study compared the accuracy and reliability of the tuberculin skin test (TST) and interferon (IFN)-γ-based assay (IGRA) for the diagnosis of active pulmonary TB Polish cases that could or could not be confirmed by M. tuberculosis (M.tb) culture.

Methods

In total, 126 adult patients with clinically active TB or non-mycobacterial, community-acquired lung diseases (NMLD) hospitalised at the Regional Specialised Hospital of Tuberculosis, Lung Diseases and Rehabilitation in Tuszyn, Poland were enrolled in the present study. Sensitivity, specificity, positive predicted value (PPV), negative predicted value (NPV), and analytic accuracy (Acc) of TST and IGRA testing for the diagnosis of culture-positive and culture-negative TB patients were calculated. The quantities of IFN-γ produced in the response to M.tb specific antigens (TB Ag – Nil) in the cultures of blood from patients with active TB and NMLD patients were also analysed.

Results

The IGRA sensitivity in culture-positive and culture-negative TB patients was similar, measuring 65.1% and 55.6%, respectively. The sensitivity of TST did not differ from the parameters designated for IGRA, measuring 55.8% in culture-positive and 64.9% in culture-negative TB. The sensitivity of TST and IGRA was age-dependent and decreased significantly with the age of the patients. No differences in the frequency or intensity of M.tb-stimulated IFN-γ production, as assessed by IGRA testing between culture-positive and culture-negative TB were noticed. Significantly lower concentrations of IFN-γ were observed in patients with advanced TB forms compared with those with mild or moderate TB pathologies.

Conclusions

Our results do not show that a combination of IGRA and TST might be a step forward in the diagnosis of culture-negative TB cases. However, M. tuberculosis-stimulated IFN-γ levels might help to assess the extent of pulmonary TB lesions.     

Structure-based in-silico rational design of a selective peptide inhibitor for thymidine monophosphate kinase of mycobacterium tuberculosis

Tuberculosis still remains one of the most deadly infectious diseases. The emergence of drug resistant strains has fuelled the quest for novel drugs and drug targets for its successful treatment. Thymidine monophosphate kinase (TMPK) lies at the point where the salvage and de novo synthetic pathways meet in nucleotide synthesis. TMPK in M.tb has emerged as an attractive drug target since blocking it will affect both the pathways involved in the thymidine triphosphate synthesis. Moreover, the unique differences at the active site of TMPK enzyme in M.tb and humans can be exploited for the development of ideal drug candidates. Based on a detailed evaluation of known inhibitors and available three-dimensional structures of TMPK, several peptidic inhibitors were designed. In silico docking and selectivity analysis of these inhibitors with TMPK from M.tb and human was carried out to examine their differential binding at the active site. The designed tripeptide, Trp-Pro-Asp, was found to be most selective for M.tb. The ADMET analysis of this peptide indicated that it is likely to be a drug candidate. The tripeptide so designed is a suitable lead molecule for the development of novel TMPK inhibitors as anti-tubercular drugs.     

Theoretical basis for reducing time-lines to the determination of positive Mycobacterium tuberculosis cultures using thymidylate kinase (TMK) assays

Background  

In vitro culture of pathogens on growth media forms a "pillar" for both infectious disease diagnosis and drug sensitivity profiling. Conventional cultures of Mycobacterium tuberculosis (M.tb) on Lowenstein Jensen (LJ) medium, however, take over two months to yield observable growth, thereby delaying diagnosis and appropriate intervention. Since DNA duplication during interphase precedes microbial division, "para-DNA synthesis assays" could be used to predict impending microbial growth. Mycobacterial thymidylate kinase (TMKmyc) is a phosphotransferase critical for the synthesis of the thymidine triphosphate precursor necessary for M.tb DNA synthesis. Assays based on high-affinity detection of secretory TMKmyc levels in culture using specific antibodies are considered. The aim of this study was to define algorithms for predicting positive TB cultures using antibody-based assays of TMKmyc levels in vitro.     

IL-18 and related function proteins associated with tuberculosis severity and screening for active TB among patients with non-mycobacterial community-acquired pneumonia (CAP)

BackgroundDifferentiation of active pulmonary tuberculosis (TB) from non-mycobacterial community-acquired pneumonia (CAP) still remains a diagnostic challenge.ObjectiveThe study aimed to quantify the IL-18, IFN-γ, IL-18BP, IL-37, and IP-10 levels in serum and Mycobacterium tuberculosis (M.tb) antigens-stimulated blood cultures from TB or CAP patients and explore if the proteins can be a useful basis for discriminating these diseases.MethodsIn total, 124 Polish adults, including mild/moderate (M/MTB) or advanced (ATB) TB patients, and CAP patients, were enrolled in the study. The concentrations of IL-18, IL-18BP, IFN-γ, IL-37, and IP-10 in sera and M.tb-stimulated cultures were measured by ELISA.ResultsThe most specific and sensitive serum proteins discriminating TB from CAP were IP-10 and IL-18BP; however, IP-10 had the highest AUC in the ROC curve for the diagnosis. Serum IP-10 and IL-18BP levels increased significantly in M/MTB or ATB groups. The IL-18BP elevation in ATB group was accompanied by an increase in IL-18. No single protein measured in M.tb-stimulated cultures differed TB from CAP patients.ConclusionsThe combined analysis of serum IL-18BP and IP-10 might be considered as an auxiliary tool in the differentiation of TB from CAP.     

Nanobead-based interventions for the treatment and prevention of tuberculosis

Tuberculosis (TB), caused by Mycobacterium tuberculosis, is one of the most devastating bacterial diseases to affect humans. M. tuberculosis is a robust pathogen that has evolved the capacity to survive and grow inside macrophage phagosomes. A cocktail of antibiotics has long been successfully used against M. tuberculosis but is becoming less effective owing to the emergence of multidrug resistance. The only available preventive vaccine, using Mycobacterium bovis bacille Calmette-Guérin, is considered to be ineffective against adult pulmonary TB, the most prevalent form of the disease. Here, we review the potential use of biodegradable nanoparticle-based anti-TB drug delivery systems that have been shown to be more effective against M. tuberculosis in animal models than conventional antibiotic treatment regimens. This technology also has substantial potential for vaccination and other therapeutic strategies against TB and other infectious diseases.     

Mycobacterium tuberculosis EspB binds phospholipids and mediates EsxA‐independent virulence

The type‐VII ESX‐1 secretion apparatus, encoded by the esx‐1 genetic locus, is essential for the export of EsxA and EsxB, two major virulence factors of Mycobacterium tuberculosis. ESX‐1 also requires the products of the unlinked espACD operon for optimal function and these proteins are considered integral parts of the secretion apparatus. Here we show that the espACD operon is not necessary for the secretion of EspB, another ESX‐1 substrate, and this unimpeded secretion of EspB is associated with significant residual virulence. Upon further investigation, we found that purified EspB can facilitate M. tb virulence even in the absence of EsxA and EsxB, and may do so by binding the bioactive phospholipids phosphatidic acid and phosphatidylserine, both of which are potent bioactive molecules with prominent roles in eukaryotic cell signalling. Our findings provide new insights into the impact of the espACD operon on the ESX‐1 apparatus and reveal a distinct virulence function for EspB with novel implications in M. tb‐host interactions.     

Role of Tissue Factor in Mycobacterium tuberculosis-Induced Inflammation and Disease Pathogenesis

Tuberculosis (TB) is a chronic lung infectious disease characterized by severe inflammation and lung granulomatous lesion formation. Clinical manifestations of TB include hypercoagulable states and thrombotic complications. We previously showed that Mycobacterium tuberculosis (M.tb) infection induces tissue factor (TF) expression in macrophages in vitro. TF plays a key role in coagulation and inflammation. In the present study, we investigated the role of TF in M.tb-induced inflammatory responses, mycobacterial growth in the lung and dissemination to other organs. Wild-type C57BL/6 and transgenic mice expressing human TF, either very low levels (low TF) or near to the level of wild-type (HTF), in place of murine TF were infected with M.tb via aerosol exposure. Levels of TF expression, proinflammatory cytokines and thrombin-antithrombin complexes were measured post M.tb infection and mycobacterial burden in the tissue homogenates were evaluated. Our results showed that M.tb infection did not increase the overall TF expression in lungs. However, macrophages in the granulomatous lung lesions in all M.tb-infected mice, including low TF mice, showed increased levels of TF expression. Conspicuous fibrin deposition in the granuloma was detected in wild-type and HTF mice but not in low TF mice. M.tb infection significantly increased expression levels of cytokines IFN-γ, TNF-α, IL-6 and IL-1ß in lung tissues. However, no significant differences were found in proinflammatory cytokines among the three experimental groups. Mycobacterial burden in lungs and dissemination into spleen and liver were essentially similar in all three genotypes. Our data indicate, in contrast to that observed in acute bacterial infections, that TF-mediated coagulation and/or signaling does not appear to contribute to the host-defense in experimental tuberculosis.     

The Role of Selection in Shaping Diversity of Natural M. tuberculosis Populations

Mycobacterium tuberculosis (M.tb), the cause of tuberculosis (TB), is estimated to infect a new host every second. While analyses of genetic data from natural populations of M.tb have emphasized the role of genetic drift in shaping patterns of diversity, the influence of natural selection on this successful pathogen is less well understood. We investigated the effects of natural selection on patterns of diversity in 63 globally extant genomes of M.tb and related pathogenic mycobacteria. We found evidence of strong purifying selection, with an estimated genome-wide selection coefficient equal to −9.5×10⁻⁴ (95% CI −1.1×10⁻³ to −6.8×10⁻⁴); this is several orders of magnitude higher than recent estimates for eukaryotic and prokaryotic organisms. We also identified different patterns of variation across categories of gene function. Genes involved in transport and metabolism of inorganic ions exhibited very low levels of non-synonymous polymorphism, equivalent to categories under strong purifying selection (essential and translation-associated genes). The highest levels of non-synonymous variation were seen in a group of transporter genes, likely due to either diversifying selection or local selective sweeps. In addition to selection, we identified other important influences on M.tb genetic diversity, such as a 25-fold expansion of global M.tb populations coincident with explosive growth in human populations (estimated timing 1684 C.E., 95% CI 1620–1713 C.E.). These results emphasize the parallel demographic histories of this obligate pathogen and its human host, and suggest that the dominant effect of selection on M.tb is removal of novel variants, with exceptions in an interesting group of genes involved in transportation and defense. We speculate that the hostile environment within a host imposes strict demands on M.tb physiology, and thus a substantial fitness cost for most new mutations. In this respect, obligate bacterial pathogens may differ from other host-associated microbes such as symbionts.     

DNA vaccine with discontinuous T‐cell epitope insertions into HSP65 scaffold as a potential means to improve immunogenicity of multi‐epitope Mycobacterium tuberculosis vaccine

DNA‐based vaccine is a promising candidate for immunization and induction of a T‐cell‐focused protective immune response against infectious pathogens such as Mycobacterium tuberculosis (M. tb). To induce multi‐functional T response against multi‐TB antigens, a multi‐epitope DNA vaccine and a ‘protein backbone grafting’ design method is adopted to graft five discontinuous T‐cell epitopes into HSP65 scaffold protein of M. tb for enhancement of epitope processing and immune presentation. A DNA plasmid with five T‐cell epitopes derived from ESAT‐6, Ag85B, MTB10.4, PPE25 and PE19 proteins of H37Rv strain of M. tb genetically inserted into HSP65 backbone was constructed and designated as pPES. After confirmation of its in vitro expression efficiency, pPES DNA was i.m. injected into C57BL/6 mice with four doses of 50 µg DNA followed by mycobacterial challenge 4 weeks after the final immunization. It was found that pPES DNA injection maintained the ability of HSP65 backbone to induce specific serum IgG. ELISPOT assay demonstrated that pPES epitope‐scaffold construct was significantly more potent to induce IFN‐γ⁺ T response to five T‐cell epitope proteins than other DNA constructs (with epitopes alone or with epitope series connected to HSP65), especially in multi‐functional‐CD4⁺ T response. It also enhanced granzyme B⁺ CTL and IL‐2⁺ CD8⁺ T response. Furthermore, significantly improved protection against Mycobacterium bovis BCG challenge was achieved by pPES injection compared to other DNA constructs. Taken together, HSP65 scaffold grafting strategy for multi‐epitope DNA vaccine represents a successful example of rational protein backbone engineering design and could prove useful in TB vaccine design.