Virulence Factor Of Mycobacterium Tuberculosis Research Articles

In-silico predictive binding affinity and ADMET of novel n-[5-(2-fluorophenyl)-1, 3, 4-oxadiazole-carboxamide derivatives towards prot

Abstract: The present study experimentally investigated the nitro-substituted heteroaromatic carboxamidesas effective against PKnGof Mycobacterium tuberculosis. Here we designed 15 heteroaromatic carboxamides[A1-B5]. The activities of the compounds can be explained in terms of docking results and biological activity by ADMET prediction. Protein Kinase G is a thioredoxin-fold containing eukaryoticlike serine/threonine Protein Kinase is a virulence factor in Mycobacterium tuberculosis. In present study the compounds A7, A2, A5& A6 are the candidates that shows the good results in Molecular Docking and these are tested against the reference compound that is stand Anti-TB agent Isoniazid.

TB impairs DNA repair.

A Mycobacterium tuberculosis virulence factor promotes foam cell formation by inhibiting DNA repair.

Review Article: Virulence Factors of Mycobacterium Tuberculosis

Mycobacterium tuberculosis (MTB) causes active TB infections that result in pulmonary tuberculosis (PTB), relapse even after treatment, and latent TB. Tuberculosis is a bacterium airborne pulmonary infectious disease. Extra pulmonary tuberculosis (EPTB) results from an illness which is too severe with Mycobacterium tuberculosis entering into the circulatory system. A really bad situation with further multi-drug TB. In the nation, pulmonary TB is spreading as well as reemerging. Recent findings of an increase in cases in the area pose a mortality burden and infection spread risk. The group of bacteria genetically organisms known as the Mycobacterium tuberculosis complex (MTBC) are accountable for human as well as animal tuberculosis. Among the primary reasons of mortality or morbidity worldwide continues to remain this sickness even now. The mycobacteria infiltrate the host via breathing that is phagocytated by macrophage as they reach the respiratory tract. It may cause the bacteria responsible to be quickly destroyed or cause an aggressive TB disease. Precisely a result of its human immunological reaction, multiple distinct virulence indicators have emerged among MTBC subgroups. The purpose of this research is to discuss the bacterial genes or enzymes that are to be crucial to determining the pathogenicity of MTBC strains through in vivo infections paradigm. As a way to eradicate various illnesses as well as get closer to a future without infections such as tuber emerging medicines or therapies must take into account the virulence aspects of MTBC.

Virulence Factors of Mycobacterium tuberculosis as Modulators of Cell Death Mechanisms.

Mycobacterium tuberculosis (Mtb) modulates diverse cell death pathways to escape the host immune responses and favor its dissemination, a complex process of interest in pathogenesis-related studies. The main virulence factors of Mtb that alter cell death pathways are classified according to their origin as either non-protein (for instance, lipomannan) or protein (such as the PE family and ESX secretion system). The 38 kDa lipoprotein, ESAT-6 (early antigen-secreted protein 6 kDa), and another secreted protein, tuberculosis necrotizing toxin (TNT), induces necroptosis, thereby allowing mycobacteria to survive inside the cell. The inhibition of pyroptosis by blocking inflammasome activation by Zmp1 and PknF is another pathway that aids the intracellular replication of Mtb. Autophagy inhibition is another mechanism that allows Mtb to escape the immune response. The enhanced intracellular survival (Eis) protein, other proteins, such as ESX-1, SecA2, SapM, PE6, and certain microRNAs, also facilitate Mtb host immune escape process. In summary, Mtb affects the microenvironment of cell death to avoid an effective immune response and facilitate its spread. A thorough study of these pathways would help identify therapeutic targets to prevent the survival of mycobacteria in the host.

Multi-omics-based characterization of the influences of Mycobacterium tuberculosis virulence factors EsxB and PPE68 on host cells.

Mycobacterium tuberculosis, the ancient master of causing tuberculosis, is one of the most successful pathogens capable of persistently colonizing host lungs. The EsxB (CFP-10) of ESX-1 system and PPE68 of the PPE family contribute to the virulence of M. tuberculosis. However, the virulence potential and pathogenetic characteristics of these two proteins during M. tuberculosis infection remain unclear. In this study, two prokaryotic expressionplasmids for EsxB or PPE68 of M. tuberculosis were constructed and the recombinant proteins His-EsxB or His-PPE68 were purified. The proteome and transcriptome of MH-S cells treated with His-EsxB or His-PPE68 were explored, followed by validating the expression of the identified differentially expressed genes (DEGs) using quantitative PCR. A total of 159/439 specific proteins or 633/1117 DEGs were obtained between control and His-EsxB or His-PPE68 treated groups in the MH-S proteomes and transcriptomes. Additionally, 37/60 signal pathways were predicted in the His-EsxB or His-PPE68 treated groups and "Cytokine-cytokine receptor interaction" was the most represented pathway. Furthermore, the expression of the DEGs (IL-1β, IL-6, and TNF-α) was significantly upregulated, suggesting that these DEGs contributed to the host response during EsxB or PPE68 treatment. These findings provide detailed information on developing an effective intervention strategy to control M. tuberculosis infection.

Mycobacterial protein PE_PGRS30 induces macrophage apoptosis through prohibitin 2 mitochondrial function interference.

PE_PGRS30 belongs to the PE_PGRS protein family and is characterized by a conserved Pro-Glu (PE) domain and a typically polymorphic GC-rich sequence (PGRS) domain. PE_PGRS30 is a virulence factor of Mycobacterium tuberculosis that induces macrophage cell death. We found that RAW264.7 cells and murine alveolar macrophages underwent apoptosis in response to PE_PGRS30. The host protein prohibitin 2 (PHB2) was identified as a target molecule. PE_PGRS30 and PHB2 interact via the PGRS domain and mitochondrial targeting sequence, respectively. PHB2 overexpression reduced macrophage apoptosis in response to PE_PGRS30. PE_PGRS30 co-localized with PHB2, not in mitochondria, but in lysosomes. The maintenance of mitochondrial structure by PHB2 was impaired in response to the PGRS domain. These results indicated that PE_PGRS30 reduces PHB2 in mitochondria, resulting in mitochondrial dysfunction and cellular apoptosis.

Culture filtrate protein 32 as a potential target to attenuate the heterogeneous antibody response against Mycobacterium tuberculosis Antigens in different endemic settings.

We previously reported the development of an enzyme-linked immunosorbent assay for the detection of the immunoglobulin G (IgG) response to Mycobacterium tuberculosis virulence factor - culture filtrate protein 32 (CFP32). The assay achieved high performance in comparing healthy Bacillus Calmette-Guerin-vaccinated controls with active tuberculosis (TB) patients from the Tunisian population. Herein, we aimed to assess the anti-CFP32 IgG response in suspected or confirmed active pulmonary TB individuals in different endemic settings. Serum samples were obtained from 224 donors from African and Latin American countries with variable levels of TB endemicity and different ethnical origins. Receiver operating characteristic curve was used to evaluate the performance of the serological assay. The area under the curve was 0.70. The use of a cutoff level of 0.65 gave 67% and 68% sensitivity and specificity, respectively, regardless of ethnicity and endemicity. Except for the suspected Latin American group, overall multiple comparisons of medians pointed out the stability of the anti-CFP32 IgG response across the different endemic settings. Therefore, endemicity and ethnicity seem not to affect anti-CFP32 IgG response, mainly for detecting confirmed active TB individuals. These findings suggest that the inclusion of CFP32 epitopes in multi-antigen TB assay could attenuate serological differences related to heterogeneous endemicity and ethnicity. For this purpose, we further identified B-cell epitopes belonging to CFP32 by an in silico analysis.

Diagnosis of paediatric tuberculosis by optically detecting two virulence factors on extracellular vesicles in blood samples

Sensitive and specific blood-based assays for the detection of pulmonary and extrapulmonary tuberculosis would reduce mortality associated with missed diagnoses, particularly in children. Here we report a nanoparticle-enhanced immunoassay read by dark-field microscopy that detects two Mycobacterium tuberculosis virulence factors (the glycolipid lipoarabinomannan and its carrier protein) on the surface of circulating extracellular vesicles. In a cohort study of 147 hospitalized and severely immunosuppressed children living with HIV, the assay detected 58 of the 78 (74%) cases of paediatric tuberculosis, 48 of the 66 (73%) cases that were missed by microbiological assays, and 8 out of 10 (80%) cases undiagnosed during the study. It also distinguished tuberculosis from latent-tuberculosis infections in non-human primates. We adapted the assay to make it portable and operable by a smartphone. With further development, the assay may facilitate the detection of tuberculosis at the point of care, particularly in resource-limited settings.

Mycobacterium tuberculosis latency-associated antigen Rv1733c SLP improves the accuracy of differential diagnosis of active tuberculosis and latent tuberculosis infection.

Background:Differential diagnosis of active tuberculosis (ATB) and latent tuberculosis infection (LTBI) has been a challenge for clinicians in high TB burden countries. The purpose of this study was to improve the accuracy of differential diagnosis of ATB and LTBI by using fluorescent immunospot (FluoroSpot) assay to detect specific Th1 cell immune responses. The novel mycobacterium tuberculosis (MTB) latency-associated antigens Rv1733c and synthetic long peptides derived from Rv1733c (Rv1733c SLP) were used based on virulence factors early secreting antigen target-6 (ESAT-6) and culture filtrate protein-10 (CFP-10).Methods:Fifty-seven ATB cases, including 20 pathogen-confirmed ATB and 37 clinically diagnosed ATB, and 36 LTBI cases, were enrolled between January and December 2017. FluoroSpot assay was used to detect the interferon γ (IFN-γ) and interleukin 2 (IL-2) secreted by the specific T cells after being stimulated with MTB virulence factors ESAT-6 and CFP-10, MTB latency-associated antigens Rv1733c and Rv1733c SLP. The receiver operating characteristic (ROC) curve was used to define the best cutoff value of latency-associated antigens in the use of differentiating ATB and LTBI. The sensitivity, specificity, predictive value, and likelihood ratio of ESAT-6 and CFP-10-FluoroSpot combined with latency-associated antigen in the differential diagnosis of ATB and LTBI were also calculated.Results:Following the stimulation with Rv1733c and Rv1733c SLP, the frequency of single IL-2-secreting T cells stimulated by Rv1733c SLP had the largest area under the ROC curve, which was 0.766. With a cutoff value of 1 (spot-forming cells [SFCs]/2.5 × 105 peripheral blood mononuclear cells) for frequency, the sensitivity and specificity of distinguishing ATB from LTBI were 72.2% and 73.7%, respectively. ESAT-6 and CFP-10-FluoroSpot detected the frequency and proportion of single IFN-γ-secreting T cells; the sensitivity and specificity of distinguishing ATB from LTBI were 82.5% and 66.7%, respectively. Combined with the frequency of single IL-2-secreting T cells stimulated by Rv1733c SLP on the basis of ESAT-6 and CFP-10-FluoroSpot, the sensitivity and specificity increased to 84.2% and 83.3%, respectively.Conclusion:Rv1733c SLP, combined with ESAT-6 and CFP-10, might be used as a candidate antigen for T cell-based tuberculosis diagnostic tests to differentiate ATB from LTBI.

Determination of variable region sequences from hybridoma immunoglobulins that target Mycobacterium tuberculosis virulence factors.

Mycobacterium tuberculosis (Mtb) infects one-quarter of the world's population. Mtb and HIV coinfections enhance the comorbidity of tuberculosis (TB) and AIDS, accounting for one-third of all AIDS-associated mortalities. Humoral antibody to Mtb correlates with TB susceptibility, and engineering of Mtb antibodies may lead to new diagnostics and therapeutics. The characterization and validation of functional immunoglobulin (Ig) variable chain (IgV) sequences provide a necessary first step towards developing therapeutic antibodies against pathogens. The virulence-associated Mtb antigens SodA (Superoxide Dismutase), KatG (Catalase), PhoS1/PstS1 (regulatory factor), and GroES (heat shock protein) are potential therapeutic targets but lacked IgV sequence characterization. Putative IgV sequences were identified from the mRNA of hybridomas targeting these antigens and isotype-switched into a common immunoglobulin fragment crystallizable region (Fc region) backbone, subclass IgG2aκ. Antibodies were validated by demonstrating recombinant Ig assembly and secretion, followed by the determination of antigen-binding specificity using ELISA and immunoblot assay.

Serum-Based Diagnosis of Pediatric Tuberculosis by Assay of Mycobacterium tuberculosis Factors: a Retrospective Cohort Study.

Diagnosis of pediatric tuberculosis (TB) is often complicated by its nonspecific symptoms, paucibacillary nature, and the need for invasive specimen collection techniques. However, a recently reported assay that detects Mycobacterium tuberculosis virulence factors in serum can diagnose various TB manifestations, including paucibacillary TB cases, in adults with good sensitivity and specificity. The current study examined the ability of this M. tuberculosis biomarker assay to diagnose pediatric TB using archived cryopreserved serum samples drawn from children ≤18 years of age who were screened for suspected TB as part of a prospective population-based active surveillance study. In this analysis, any detectable level of either of the M. tuberculosis virulence factors CFP-10 and ESAT-6 was considered direct evidence of TB. Serum samples from 105 children evaluated for TB (55 TB cases and 50 close contacts without TB) were analyzed. The results of this analysis yielded sensitivity of 85.5% (95% confidence interval [CI], 73.3 to 93.5). Similar diagnostic sensitivities were observed for culture-positive (87.5%; 95% CI, 67.6 to 97.3) and culture-negative (83.9%; 95% CI, 66.3 to 94.5) TB cases and for culture negative pulmonary (77.8%; 95% CI, 40.0 to 97.2) and extrapulmonary (86.4%; 95% CI, 65.1 to 97.1) TB cases. These results suggest that serum biomarker analysis holds significant promise for rapid and sensitive diagnosis of pediatric TB cases, including extrapulmonary or paucibacillary TB cases. The ability to use frozen samples for this analysis should also permit assays to be performed at central sites, without a requirement for strict timelines for sample analysis.

PE_PGRS33, an Important Virulence Factor of Mycobacterium tuberculosis and Potential Target of Host Humoral Immune Response.

PE_PGRS proteins are surface antigens of Mycobacterium tuberculosis (Mtb) and a few other pathogenic mycobacteria. The PE_PGRS33 protein is among the most studied PE_PGRSs. It is known that the PE domain of PE_PGRS33 is required for the protein translocation through the mycobacterial cell wall, where the PGRS domain remains available for interaction with host receptors. Interaction with Toll like receptor 2 (TLR2) promotes secretion of inflammatory chemokines and cytokines, which are key in the immunopathogenesis of tuberculosis (TB). In this review, we briefly address some key challenges in the development of a TB vaccine and attempt to provide a rationale for the development of new vaccines aimed at fostering a humoral response against Mtb. Using PE_PGRS33 as a model for a surface-exposed antigen, we exploit the availability of current structural data using homology modeling to gather insights on the PGRS domain features. Our study suggests that the PGRS domain of PE_PGRS33 exposes four PGII sandwiches on the outer surface, which, we propose, are directly involved through their loops in the interactions with the host receptors and, as such, are promising targets for a vaccination strategy aimed at inducing a humoral response.

Priming mycobacterial ESX-secreted protein B to form a channel-like structure.

ESX-1 is a major virulence factor of Mycobacterium tuberculosis, a secretion machinery directly involved in the survival of the microorganism from the immune system defence. It disrupts the phagosome membrane of the host cell through a contact-dependent mechanism. Recently, the structure of the inner-membrane core complex of the homologous ESX-3 and ESX-5 was resolved; however, the elements involved in the secretion through the outer membrane or those acting on the host cell membrane are unknown. Protein substrates might form this missing element. Here, we describe the oligomerisation process of the ESX-1 substrate EspB, which occurs upon cleavage of its C-terminal region and is favoured by an acidic environment. Cryo-electron microscopy data shows that quaternary structure of EspB is conserved across slow growing species, but not in the fast growing M. smegmatis. EspB assembles into a channel with dimensions and characteristics suitable for the transit of ESX-1 substrates, as shown by the presence of another EspB trapped within. Our results provide insight into the structure and assembly of EspB, and suggests a possible function as a structural element of ESX-1.

Rv3091, An Extracellular Patatin-Like Phospholipase in Mycobacterium tuberculosis, Prolongs Intracellular Survival of Recombinant Mycolicibacterium smegmatis by Mediating Phagosomal Escape.

Patatin-like phospholipases (PLPs) are important virulence factors of many pathogens. However, there are no prevailing studies regarding PLPs as a virulence factor of Mycobacterium tuberculosis (Mtb). Analysis of Rv3091, a putative protein of Mtb, shows that it belongs to the PLPs family. Here, we cloned and expressed the rv3091 gene in Mycobacterium smegmatis and, subsequently, conducted protein purification and characterization. We show that it possesses phospholipase A1, phospholipase A2, and lipase activity. We confirm the putative active site residues, namely, Ser214 and Asp407, using site directed mutagenesis. The Rv3091 is an extracellular protein that alters the colony morphology of M. smegmatis. The presence of Rv3091 enhances the intracellular survival capability of M. smegmatis in murine peritoneal macrophages. Additionally, it promotes M. smegmatis phagosomal escape from macrophages. Moreover, Rv3091 significantly increased the survival of M. smegmatis and aggravated lesions in C57BL/6 J murine lungs in vivo. Taken together, our results indicate that Rv3091 as an extracellular PLP that is critical to the pathogenicity of mycobacterium as it allows mycobacterium to utilize phospholipids for its growth and provides resistance to phagosome killing, resulting in its enhanced intracellular survival.

The ESX-1 Virulence Factors Downregulate miR-147-3p in Mycobacterium marinum-Infected Macrophages.

As important virulence factors of Mycobacterium tuberculosis, EsxA and EsxB not only play a role in phagosome rupture and M. tuberculosis cytosolic translocation but also function as modulators of host immune responses by modulating numerous microRNAs (miRNAs). Recently, we have found that mycobacterial infection downregulated miR-148a-3p (now termed miR-148) in macrophages in an ESX-1-dependent manner. The upregulation of miR-148 reduced mycobacterial intracellular survival. Here, we investigated miR-147-3p (now termed miR-147), a negative regulator of inflammatory cytokines (e.g., interleukin-6 [IL-6] and IL-10), in mycobacterial infection. We infected murine RAW264.7 macrophages with Mycobacterium marinum, a surrogate model organism for M. tuberculosis, and found that the esxBA-knockout strain (M. marinum ΔesxBA) upregulated miR-147 to a level that was significantly higher than that induced by the M. marinum wild-type (WT) strain or by the M. marinum ΔesxBA complemented strain, M. marinum ΔesxBA/pesxBA, suggesting that the ESX-1 system (potentially EsxBA and/or other codependently secreted factors) is the negative regulator of miR-147. miR-147 was also downregulated by directly incubating the macrophages with the purified recombinant EsxA or EsxB protein or the EsxBA heterodimer, which further confirms the role of the EsxBA proteins in the downregulation of miR-147. The upregulation of miR-147 inhibited the production of IL-6 and IL-10 and significantly reduced M. marinum intracellular survival. Interestingly, inhibitors of either miR-147 or miR-148 reciprocally compromised the effects of the mimics of their counterparts on M. marinum intracellular survival. This suggests that miR-147 and miR-148 share converged downstream pathways in response to mycobacterial infection, which was supported by data indicating that miR-147 upregulation inhibits the Toll-like receptor 4/NF-κB pathway.

Classification and functional analyses of putative virulence factors of Mycobacterium tuberculosis: A combined sequence and structure based study

The emergence of the drug-resistant mechanisms in Mycobacterium tuberculosis poses the biggest challenges to the current therapeutic measures, which necessitates the identification of new drug targets. The Hypothetical Proteins (HPs), a class of functionally uncharacterized proteins, may provide a new class of undiscovered therapeutic targets. The genome of M. tuberculosis contains 1000 HPs with their sequences were analyzed using a variety of bioinformatics tools and the functional annotations were performed. The functions of 662 HPs were successfully predicted and further classified 483 HPs as enzymes, 141 HPs were predicted to be involved in the diverse cellular mechanisms and 38 HPs may function as transporters and carriers proteins. Furthermore, 28 HPs were predicted to be virulent in nature. Amongst them, the HP P95201, HP P9WM79, HP I6WZ30, HP I6 × 9T8, HP P9WKP3, and HP P9WK89 showed the highest virulence scores. Therefore, these proteins were subjected to extensive structure analyses and dynamics of their conformations were investigated using the principles of molecular dynamics simulations, each for a 150 ns time scale. This study provides a deeper understanding of the undiscovered drug targets and the generated outputs will facilitate the process of drug design and discovery against the infection of M. tuberculosis.

Nα-Acetylation of the virulence factor EsxA is required for mycobacterial cytosolic translocation and virulence

The Mycobacterium tuberculosis virulence factor EsxA and its chaperone EsxB are secreted as a heterodimer (EsxA:B) and are crucial for mycobacterial escape from phagosomes and cytosolic translocation. Current findings support the idea that for EsxA to interact with host membranes, EsxA must dissociate from EsxB at low pH. However, the molecular mechanism by which the EsxA:B heterodimer separates is not clear. In the present study, using liposome-leakage and cytotoxicity assays, LC-MS/MS-based proteomics, and CCF-4 FRET analysis, we obtained evidence that the Nα-acetylation of the Thr-2 residue on EsxA, a post-translational modification that is present in mycobacteria but absent in Escherichia coli, is required for the EsxA:B separation. Substitutions at Thr-2 that precluded Nα-acetylation inhibited the heterodimer separation and hence prevented EsxA from interacting with the host membrane, resulting in attenuated mycobacterial cytosolic translocation and virulence. Molecular dynamics simulations revealed that at low pH, the Nα-acetylated Thr-2 makes direct and frequent "bind-and-release" contacts with EsxB, which generates a force that pulls EsxB away from EsxA. In summary, our findings provide evidence that the Nα-acetylation at Thr-2 of EsxA facilitates dissociation of the EsxA:B heterodimer required for EsxA membrane permeabilization and mycobacterial cytosolic translocation and virulence.

Early secreted antigenic target 6-kDa from Mycobacterium tuberculosis enhanced the protective innate immunity of macrophages partially via HIF1α

Early secreted antigenic target 6-kDa protein (ESAT6) is an essential virulence factor of Mycobacterium tuberculosis (MTb). However, ESAT6 helped fighting MTb infection according to vaccine studies. It's unclear whether ESAT6 confers protection via enhancing the innate immunity of macrophages, which are the first-line defense against MTb. We profiled the global transcriptional changes and characterized the innate immunity of THP-1 macrophages treated with ESAT6. We found ESAT6 promoted the phagocytosis ability, enhanced reactive oxygen species (ROS) generation and accelerated glucose metabolism in macrophages. Meanwhile, ESAT6 induced a distinctive phenotype of macrophages with a concurrence of pro-inflammatory and anti-inflammatory cytokines and chemokines. ESAT6 increased the expression of HIF1α mRNA and protein. Interfering HIF1α with siRNA defected the capacity of phagocytosis and ROS generation as well as glucose metabolism. Thus, ESAT6 enhanced the protective innate immunity of macrophages partially via HIF1α. This study provided clues for developing therapies against tuberculosis by targeting ESAT6.

Novel thiazolidinedione-hydroxamates as inhibitors of Mycobacterium tuberculosis virulence factor Zmp1

Zinc metalloprotease 1 (Zmp1) is an extracellular enzyme, which has been found essential for the intracellular survival and pathogenesis of Mycobacterium tuberculosis. In this work, we designed and synthesized a series of novel thiazolidinedione-hydroxamates and evaluated in silico their drug-likeness behavior. Then, their inhibitory properties towards a recombinant Zmp1 from Mycobacterium tuberculosis were analyzed by MALDI-TOF MS. Nine of the tested compounds were found to inhibit the enzymatic reaction more effectively than the generic metalloprotease inhibitor phosphoramidon. Furthermore, the synthesized thiazolidinedione-hydroxamate hybrids were evaluated for their in vitro antimycobacterial activity and acute cytotoxicity using whole-cell assays. Results showed that none of the hybrids exhibited acute cytotoxicity against RAW264.7 macrophages. Whereas extracellular antimycobacterial activity was limited, RAW264.7 macrophage infection results showed that a majority of the hybrids inhibited the intracellular growth of Mycobacterium tuberculosis at a concentration of 100 and 10 μM. The thiazolidinedione-hydroxamate compound 2n was considered to be the best candidate of the evaluated library.

Mimicking Halimane Synthases: Monitoring a Cascade of Cyclizations and Rearrangements from Epoxypolyprenes.

We have developed and rationalized a biomimetic transformation mimicking halimane synthases based on a Lewis acid-catalyzed cascade of cyclizations and rearrangements of epoxypolyprenes. Two rings, three stereogenic centers, and a new double bond were generated in a single chemical operation. Based on this cascade transformation, we achieved a unified strategy toward the stereoselective total syntheses of halimene-type terpenoids and analogues as a proof-of-concept study. This method has been applied to the rapid synthesis of diterpene isotuberculosinol, a virulence factor of Mycobacterium tuberculosis as a representative example.