Tuberculosis (TB) still threatens human life despite the availability of childhood vaccination and modern treatment regimens due to the emergence of tuberculosis with extended resistance profiles. The mouse active TB model remains the gold standard for evaluating anti-TB drugs and vaccines. However, in vivo experiments raise ethical concerns and are time-consuming. We established an Akaluc-based bioluminescence platform to enable rapid drug screening in culture media and THP‑1 cells. Codon-optimized Akaluc expression in mycobacteria was accomplished by assortment of vectors and promoters. Bioluminescence kinetics were evaluated in culture media and THP‑1 cells with or without drug treatment, and optimized by adjusting time points and substrate concentrations. TokeOni at a concentration of 10 nM/100 µL produced the highest bioluminescence compared to other tested concentrations and substrates. Among the tested promoter-plasmid constructs, the Ag85B promoter in pMV261 generated the strongest bioluminescence in Mycobacterium smegmatis and Mycobacterium bovis. Bioluminescence fluctuated with bacterial growth, peaking during the log phase and gradually declining during the stationary phase. A positive correlation was observed between bioluminescence and CFU reduction in vitro upon treatment with sensitive drugs.

Objective: This study aimed to evaluate the pooled sequencing of mixed samples with different minimum inhibitory concentrations (MIC) of bedaquiline (BDQ) and clarithromycin to identify genes associated with drug resistance and explore those that could predict cross-resistance to both drugs. Additionally, it aimed to preliminarily investigate the association between single-nucleotide polymorphisms (SNPs) from pooled samples and the progression of drug resistance.Material and Methods: Mycolicibacterium smegmatis wild type (WT) and hup B (MDP1) knock-out strains were obtained from the Bacteriology Laboratory, Graduate School of Medical and Dental Science, Niigata University. Drug-resistant strains were generated through stepwise passaging on drug-containing agar plates, followed by MIC determination. Whole-genome sequencing was performed using the MinION Nanopore platform, with pooled samples categorized based on MIC levels. Bioinformatics analysis, including SNP identification and annotation, was conducted using burrows wheeler aligner, Samtools, genome analysis toolkit, and SnpEff pipelines. Molecular docking and molecular dynamics simulations were employed for in silico validation of SNP-drug interactions. SNP frequency was calculated, and statistical analysis was performed using a paired t-test in Prism® software.Results: High-impact SNPs that changed the amino acid were found in both pooled samples, one in pooled sample A (atpA p.Glu429Gln) and 5 in pooled sample B (MSMEG_0639, MSMEG_1867, MSMEG_4765, and MSMEG_6302).Conclusion: Pooled samples of BDQ and clarithromycin-resistant M. smegmatis strains exhibited mutations in the genes associated with the efflux family and the adenosine triphosphate synthase protein. A pooled sample sequencing method can be used to identify variant sites and predict genes associated with cross-resistance.

The World Health Organization (WHO) recommends whole genome sequencing (WGS) to support the detection of drug-resistant tuberculosis and the use of the new regimen BPaLM (Bedaquiline-Pretomanid-Linezolid-Moxifloxacin) to treat drug-resistant tuberculosis (TB). Genomic profiles of Mycobacterium tuberculosis (MTB) are needed to understand the regimen’s future impact comprehensively. In this study, we performed WGS to Mycobacterium tuberculosis isolates from rifampicin resistant tuberculosis patients at Dr. Soetomo Hospital. A total of 15 samples consisting of 14 clinical isolate samples (8 BPaLM and 6 Individualized treated patients) and one laboratory H37Rv reference strain were sequenced using the Illumina MiSeq platform and analyzed using Galaxy, TBProfiler, Mykrobe, and ResFinder. Lineage analysis revealed that L2.2 was the predominant strain (48

Mycobacterium intracellulare is a major etiological agent of the recently expanding Mycobacterium avium–intracellulare complex pulmonary disease (MAC-PD). Therapeutic regimens that include a combination of macrolides and antituberculous drugs have been implemented with limited success. To identify novel targets for drug development that accommodate the genomic diversity of M. avium–intracellulare, we subjected eight clinical MAC-PD isolates and the type strain ATCC13950 to genome-wide profiling to comprehensively identify universally essential functions by transposon sequencing (TnSeq). Among these strains, we identified 131 shared essential or growth-defect-associated genes by TnSeq. Unlike the type strain, the clinical strains showed increased requirements for genes involved in gluconeogenesis and the type VII secretion system under standard growth conditions, the same genes required for hypoxic pellicle-type biofilm formation in ATCC13950. Consistent with the central role of hypoxia in the evolution of M. intracellulare, the clinical MAC-PD strains showed more rapid adaptation to hypoxic growth than the type strain. Importantly, the increased requirements of hypoxic fitness genes were confirmed in a mouse lung infection model. These findings confirm the concordant genetic requirements under hypoxic conditions in vitro and hypoxia-related conditions in vivo and highlight the importance of using clinical strains and host-relevant growth conditions to identify high-value targets for drug development.

Tuberculosis (TB) is one of the leading causes of death due to infectious disease. The sole established vaccine against TB is the Mycobacterium bovis Bacillus Calmette–Guerin (BCG) vaccine. However, owing to the lack of durable immunity with the BCG vaccine and its risk of infection, safer vaccines that can also be used as boosters are needed. Here, we examined whether membrane vesicles (MVs) from BCG (BCG-MVs) isolated from BCG statically cultured in nutrient-restricted Sauton’s medium (s-MVs) and from BCG planktonically cultured in nutrient-rich medium commonly used in the laboratory (p-MVs) could be used as novel TB vaccines. MVs are extracellular vesicles produced by various bacteria, including mycobacteria. Differences in the culture conditions affected the morphology, contents, immunostimulatory activity and immunogenicity of BCG-MVs. s-MVs presented greater immunostimulatory activity than p-MVs via the induction of TLR2 signaling. Mouse immunization experiments revealed that s-MVs, but not p-MVs, induced mycobacterial humoral and mucosal immunity, especially when administered in combination with adjuvants. In a BCG challenge experiment using BCG Tokyo type I carrying pMV361-Km, subcutaneous vaccination with s-MVs reduced the bacterial burden in the mouse lung to a level similar to that after intradermal vaccination with live BCG. Furthermore, the administration of s-MVs induced a significant lipopolysaccharide-induced proinflammatory response in macrophages in vitro. These results indicate that BCG-MVs obtained from static culture in Sauton’s medium induce not only humoral immunity against mycobacteria but also trained immunity, which can allow the clearance of infectious agents other than mycobacteria. Together, these findings highlight the immunological properties of BCG-MVs and the availability of acellular TB vaccines that confer broad protection against various infectious diseases.

BACKGROUND:Nontuberculous mycobacterial disease has emerged worldwide over the past 20 years. However, there are currently few reports on the established technique for constructing knockout mutants of nontuberculous mycobacteria. Therefore, gene recombination techniques for nontuberculous mycobacteria require further research. RESULTS:We constructed vector pPR23LHR that harbors the ribosomal protein S12 gene (rpsL+) as a dominant negative selection marker and the hygromycin (Hyg) and lacZ cassettes as positive selection markers. We constructed knockout mutants of proteasomal genes, which we found to be required for hypoxic pellicle formation in Mycobacterium intracellulare by functional genomic analysis. The knockout mutants showed impaired hypoxic pellicle formation, consistent with previous data using epoxomicin, a proteasomal inhibitor. CONCLUSIONS:Our findings demonstrate that rpsL+ is an efficient dominant negative selection marker for gene recombination in nontuberculous mycobacteria. Our temperature-sensitive rpsL+ method for the construction of knockout mutants will facilitate functional assays to validate the virulence factors of nontuberculous mycobacteria and the pathogenesis of nontuberculous mycobacterial disease.

Mycobacterium intracellulare is a major etiological agent of the recently expanding Mycobacterium avium–intracellulare complex pulmonary disease (MAC-PD). Therapeutic regimens that include a combination of macrolides and antituberculous drugs have been implemented with limited success. To identify novel targets for drug development that accommodate the genomic diversity of M. avium–intracellulare , we subjected eight clinical MAC-PD isolates and the type strain ATCC13950 to genome-wide profiling to comprehensively identify universally essential functions. Among these strains, we identified 131 shared essential or growth-defect-associated genes. Unlike the type strain, the clinical strains showed an increased requirement for genes involved in gluconeogenesis and the type VII secretion system under standard growth conditions, the same genes required for hypoxic pellicle-type biofilm formation in ATCC13950. Consistent with the central role of hypoxia in the evolution of M. intracellulare , the clinical MAC-PD strains showed more rapid adaptation to hypoxic growth than the type strain. Importantly, the increased essentiality of hypoxic fitness genes was confirmed in a mouse lung infection model. These findings confirm the concordant gene essentiality under hypoxic conditions in vitro and hypoxia-related conditions in vivo , and highlight the importance of using clinical strains and host-relevant growth conditions to identify high-value targets for drug development.

IntroductionThere is no useful method to discriminate between latent tuberculosis infection (LTBI) and active pulmonary tuberculosis (PTB). This study aimed to investigate the potential of cytokine profiles to discriminate between LTBI and active PTB using whole-blood stimulation with Mycobacterium tuberculosis (MTB) antigens, including latency-associated antigens.Materials and methodsPatients with active PTB, household contacts of active PTB patients and community exposure subjects were recruited in Manila, the Philippines. Peripheral blood was collected from the participants and used for whole-blood stimulation (WBS) with either the early secretory antigenic target and the 10-kDa culture filtrate protein (ESAT-6/CFP-10), Rv3879c or latency-associated MTB antigens, including mycobacterial DNA-binding protein 1 (MDP-1), α-crystallin (Acr) and heparin-binding hemagglutinin (HBHA). Multiple cytokine concentrations were analyzed using the Bio-Plex™ multiplex cytokine assay.ResultsA total of 78 participants consisting of 15 active PTB patients, 48 household contacts and 15 community exposure subjects were eligible. The MDP-1-specific IFN-γ level in the active PTB group was significantly lower than that in the household contact group (p < 0.001) and the community exposure group (p < 0.001). The Acr-specific TNF-α and IL-10 levels in the active PTB group were significantly higher than those in the household contact (TNF-α; p = 0.001, IL-10; p = 0.001) and community exposure (TNF-α; p < 0.001, IL-10; p = 0.01) groups. However, there was no significant difference in the ESAT-6/CFP-10-specific IFN-γ levels among the groups.ConclusionThe patterns of cytokine profiles induced by latency-associated MTB antigens using WBS have the potential to discriminate between LTBI and active PTB. In particular, combinations of IFN-γ and MDP-1, TNF-α and Acr, and IL-10 and Acr are promising. This study provides the first demonstration of the utility of MDP-1-specific cytokine responses in WBS.

Mycobacteria are the major human pathogens with the capacity to become dormant persisters. Mycobacterial DNA-binding protein 1 (MDP1), an abundant histone-like protein in dormant mycobacteria, induces dormancy phenotypes, e.g. chromosome compaction and growth suppression. For these functions, the polycationic intrinsically disordered region (IDR) is essential. However, the disordered property of IDR stands in the way of clarifying the molecular mechanism. Here we clarified the molecular and structural mechanism of DNA compaction by MDP1. Using high-speed atomic force microscopy, we observed that monomeric MDP1 bundles two adjacent DNA duplexes side-by-side via IDR. Combined with coarse-grained molecular dynamics simulation, we revealed the novel dynamic DNA cross-linking model of MDP1 in which a stretched IDR cross-links two DNA duplexes like double-sided tape. IDR is able to hijack HU function, resulting in the induction of strong mycobacterial growth arrest. This IDR-mediated reversible DNA cross-linking is a reasonable model for MDP1 suppression of the genomic function in the resuscitable non-replicating dormant mycobacteria.

Tuberculosis remains a large health threat, despite the availability of the tuberculosis vaccine, BCG. As BCG efficacy gradually decreases from adolescence, BCG-Prime and antigen-booster may be an efficient strategy to confer vaccine efficacy. Mycobacterial DNA-binding protein 1 (MDP1, namely Rv2986c, hupB or HU) is a major Mycobacterium tuberculosis protein that induces vaccine-efficacy by co-administration with CpG DNA. To produce MDP1 for booster-vaccine use, we have created recombinant MDP1 produced in both Escherichia coli (eMDP1) and Mycolicibacterium smegmatis (mMDP1), an avirulent rapid-growing mycobacteria. We tested their immunogenicity by checking interferon (IFN)-gamma production by stimulated peripheral blood cells derived from BCG-vaccinated individuals. Similar to native M. tuberculosis MDP1, we observed that most lysin resides in the C-terminal half of mMDP1 are highly methylated. In contrast, eMDP1 had less post-translational modifications and IFN-gamma stimulation. mMDP1 stimulated the highest amount of IFN-gamma production among the examined native M. tuberculosis proteins including immunodominant MPT32 and Antigen 85 complex. MDP1-mediated IFN-gamma production was more strongly enhanced when combined with a new type of CpG DNA G9.1 than any other tested CpG DNAs. Taken together, these results suggest that the combination of mMDP1 and G9.1 possess high potential use for human booster vaccine against tuberculosis.

Vaccination is an important factor in public health. The recombinant bacillus Calmette Guerin (rBCG) vaccine, which expresses foreign antigens, is expected to be a superior vaccine against infectious diseases. Here, we report a new recombination platform in which the BCG Tokyo strain is transformed with nucleotide sequences encoding foreign protein fused with the MPB70 immunogenic protein precursor. By RNA-sequencing, mpb70 was found to be the most transcribed among all known genes of BCG Tokyo. Small oligopeptide, namely, polyhistidine tag, was able to be expressed in and secreted from rBCG through a process in which polyhistidine tag fused with intact MPB70 were transcribed by an mpb70 promoter. This methodology was applied to develop an rBCG expressing the receptor binding domain (RBD) of severe acute respiratory syndrome coronavirus 2. Immunoblotting images and mass spectrometry data showed that RBD was also secreted from rBCG. Sera from mice vaccinated with the rBCG showed a tendency of weak neutralizing capacity. The secretion was retained even after a freeze-drying process. The freeze-dried rBCG was administered to and recovered from mice. Recovered rBCG kept secreting RBD. Collectively, our recombination platform offers stable secretion of foreign antigens and can be applied to the development of practical rBCGs.

Accurate point-of-care testing (POCT) is critical for managing tuberculosis (TB). However, current antibody-based diagnosis shows low specificity and sensitivity. To find proper antigen candidates for TB diagnosis by antibodies, we assessed IgGs responsiveness to Mycobacterium tuberculosis proteins in pulmonary TB (PTB) patients. We employed major secreted proteins, such as Rv1860, Ag85C, PstS1, Rv2878c, Ag85B, and Rv1926c that were directly purified from M. tuberculosis . In the first screening, we found that IgG levels were significantly elevated in PTB patients only against Rv1860, PstS1, and Ag85B among tested antigens. However, recombinant PstS1 and Ag85B from Escherichia coli (E. coli) couldn’t distinguish PTB patients and healthy controls (HC). Recombinant Rv1860 was not checked due to its little expression. Then, the 59 confirmed PTB patients from Soetomo General Academic Hospital, Surabaya, Indonesia, and 102 HC were tested to Rv1860 and Ag85B only due to the low yield of the PstS1 from M. tuberculosis . The ROC analysis using native Ag85B and Rv1860 showed an acceptable area under curve for diagnosis, which is 0.812 (95% CI 0.734–0.890, p < 0.0001) and 0.821 (95% CI 0.752–0.890, p < 0.0001). This study indicates that taking consideration of native protein structure is key in developing TB’s POCT by antibody-based diagnosis.

The basic, intrinsically disordered regions of eukaryotic histones and their bacterial counterparts are presumed to act as signaling hubs to regulate the compaction of chromosomes or nucleoids and various DNA processes such as gene expression, recombination, and DNA replication. Posttranslational modifications (PTMs) on these regions are pivotal in regulating chromosomal or nucleoid compaction and DNA processes. However, the low sequence complexity and the presence of short lysine-rich repeats in the regions have hindered the accurate determination of types and locations of PTMs using conventional proteomic procedures. We described a limited proteolysis protocol using trypsin to analyze PTMs on mycobacterial DNA-binding protein 1 (MDP1), a nucleoid-associated protein in mycobacterial species that possesses an extended, lysine-rich, intrinsically disordered region in its C -terminal domain. This limited proteolysis approach successfully revealed significant methylation on many lysine residues in the C-terminal domain of MDP1 purified from Mycobacterium tuberculosis, which was lacking in the corresponding region of recombinant MDP1 expressed in Escherichia coli.

Survival of the live attenuated Bacillus Calmette-Guérin (BCG) vaccine amidst harsh host environments is key for BCG effectiveness as it allows continuous immune response induction and protection against tuberculosis. Mycobacterial DNA binding protein 1 (MDP1), a nucleoid associated protein, is essential in BCG. However, there is limited knowledge on the extent of MDP1 gene regulation and how this influences BCG survival. Here, we demonstrate that MDP1 conditional knockdown (cKD) BCG grows slower than vector control in vitro , and dies faster upon exposure to antibiotics (bedaquiline) and oxidative stress (H 2 O 2 and menadione). MDP1-cKD BCG also exhibited low infectivity and survival in THP-1 macrophages and mice indicating possible susceptibility to host mediated stress. Consequently, low in vivo survival resulted in reduced cytokine (IFN-gamma and TNF-alpha) production by splenocytes. Temporal transcriptome profiling showed more upregulated (81–240) than downregulated (5–175) genes in response to MDP1 suppression. Pathway analysis showed suppression of biosynthetic pathways that coincide with low in vitro growth. Notable was the deferential expression of genes involved in stress response ( sigI ), maintenance of DNA integrity ( mutT1 ), REDOX balance ( WhiB3 ), and host interactions ( PE/PE_PGRS ). Thus, this study shows MDP1’s importance in BCG survival and highlights MDP1-dependent gene regulation suggesting its role in growth and stress adaptation.

BACKGROUND:Mycobacterium intracellulare is a major etiological agent of Mycobacterium avium-intracellulare pulmonary disease (MAC-PD). However, the characteristics of the virulence of M. intracellulare and the in vivo chemotherapeutic efficacy remain unclear. In this study, we examined the virulence of nine M. intracellulare strains with different clinical phenotypes and genotypes in C57BL/6 mice. RESULTS:We classified three types of virulence phenotypes (high, intermediate, and low) based on the kinetics of the bacterial load, histological lung inflammation, and neutrophilic infiltration. High virulence strains showed more severe neutrophilic infiltration in the lungs than intermediate and low virulence strains, with 6.27-fold and 11.0-fold differences of the average percentage of neutrophils in bronchoalveolar lavage fluid, respectively. In particular, the high virulence strain M.i.198 showed the highest mortality in mice, which corresponded to the rapid progression of clinical disease. In mice infected with the drug-sensitive high virulence strain M019, clarithromycin-containing chemotherapy showed the highest efficacy. Monotherapy with rifampicin exacerbated lung inflammation with increased lymphocytic and neutrophilic infiltration into the lungs. CONCLUSIONS:The virulence phenotypes of clinical strains of M. intracellulare were diverse, with high virulence strains being associated with neutrophilic infiltration and disease progression in infected mice. These high virulence strains were proposed as a useful subject for in vivo chemotherapeutic experiments.

Abstract Mycobacteria are a continuous threat to human health. They include various species, such as Mycobacterium tuberculosis ( M. tuberculosis ), which is an intracellular parasite of mammals, and the most virulent and non-tuberculous mycobacteria (NTM), namely, M. avium , which are environmental bacteria causing intractable NTM diseases. An infection model of transparent zebrafish and fish-infectious M. marinum was established to better understand the in vivo behavior of mycobacteria under the pressure of host immune responses. However, the fish model does not fully replicate mammalian immunity. Here, we demonstrate that a clear, unobstructed brain/body imaging cocktail and computational analysis (CUBIC)-based infection (CUBIC-infection) analysis enables comprehensive mycobacterial profiling of the whole lung. We assessed the in vivo kinetics of mycobacterial infection along with fluorescent protein-expressing recombinant mycobacteria. We detected mycobacterium at a single bacterial level and counted bacterial numbers, which was comparable to the colony-forming units of organ homogenates. CUBIC-infection analysis distinguished in vivo spatiotemporal behavior of M. tuberculosis , M. tuberculosis variant Bacillus Calmette-Guerin, and M. avium in mice. Furthermore, it monitored spatiotemporal information on the therapeutic efficacies of anti-tuberculosis drugs and an anti-lymphangiogenesis agent. Our data suggest that CUBIC-infection analysis is a powerful tool for understanding mycobacterial infections in mammals and developing therapeutic agents.

Tuberculosis (TB) is fatal in elephants, hence protecting elephants from TB is key not only in the conservation of this endangered animal, but also to prevent TB transmission from elephants to humans. Most human TB cases arise from long-term asymptomatic infections. Significant diagnostic challenges remain in the detection of both infection and disease development from latency in elephants due to their huge bodies. In this study, we assessed cryopreserved sera collected for over 16 years, from the first Japanese treatment case of elephant TB. Semi-quantification of IgG levels to 11 proteins showed high detection levels of 3 proteins, namely ESAT6/CFP10, MPB83 and Ag85B. The level of IgG specific to these 3 antigens was measured longitudinally, revealing high and stable ESAT6/CFP10 IgG levels regardless of onset or treatment. Ag85B-specifc IgG levels were largely responsive to onset or treatment, while those of MPB83 showed intermediate responses. These results suggest that ESAT6/CFP10 is immunodominant in both asymptomatic and symptomatic phases, making it useful in the detection of infection. On the other hand, Ag85B has the potential to be a marker for the prediction of disease onset and in the evaluation of treatment effectiveness in elephants.

Pathogenic intracellular mycobacteria, such as Mycobacterium tuberculosis and Mycobacterium avium, which cause lung diseases, can grow in macrophages. Extracellular mycobacteria have been reported in the lungs, blood, and sputum of patients, indicating the involvement of these pathogens in disease progression. Erythrocytes are involved in the symptoms associated with pulmonary mycobacterial diseases, such as bloody sputum and hemoptysis; however, little attention has been paid to the role of erythrocytes in mycobacterial diseases. Herein, we found that Mycobacterium avium subsp. hominissuis (MAH) and Mycobacterium intracellulare colocalized with erythrocytes at the sites of lung infection, inside capillaries and necrotic areas of granulomas, using histopathological examinations. Electron microscopy showed that MAH adhered and entered human erythrocytes when they were cocultured in vitro. MAH adhered to erythrocytes through complement receptor 1 and cell-surface sialo-glycoproteins. Importantly, MAH grew vigorously without causing any pronounced damage to erythrocytes. This erythrocyte-mediated enhancement of MAH growth occurred extracellularly depending on its direct attachment to erythrocytes. In contrast, MAH failed to multiply inside erythrocytes. Similarly, erythrocytes augmented the growth of other pathogenic mycobacteria, such as M. intracellulare and M. tuberculosis. THP-1 cell-derived human macrophages preferentially phagocytosed erythrocytes that were attached to mycobacteria (compared to bacteria alone), suggesting that erythrocyte-attached mycobacteria are an efficient infectious source for macrophages. Our findings provide new insights into the pathogenesis of mycobacterial diseases and offer an alternative and useful strategy for treating mycobacterial disease. IMPORTANCE Pathogenic mycobacteria, such as Mycobacterium tuberculosis, Mycobacterium avium subsp. hominissuis (MAH), and Mycobacterium intracellulare, cause pulmonary infections as intracellular parasites of lung macrophages and epithelial cells. Here, using histopathological examinations we found that MAH and M. intracellulare colocalized with erythrocytes in lung infection sites. Subsequent studies demonstrated that direct interaction with erythrocytes enhances the extracellular proliferation of mycobacteria based on the following results: 1. MAH adhered and invaded human erythrocytes upon coculture in vitro; 2. MAH adhered to erythrocytes through complement receptor 1 and cell-surface sialo-glycoproteins; 3. MAH rapidly proliferated when directly attached to erythrocytes but not within them; 4. other mycobacteria, such as M. intracellulare and M. tuberculosis, also proliferated in the same way as MAH. The finding that pathogenic mycobacteria grow extracellularly in an erythrocyte-dependent manner is of considerable clinical importance for understanding disease progression and latent infection.

Tuberculosis remains a public health crisis and a health security threat. There is an urgent need to develop new antituberculosis drugs with novel modes of action to cure drug-resistant tuberculosis and shorten the chemotherapy period by sterilizing tissues infected with dormant bacteria. Lysocin E is an antibiotic that showed antibacterial activity against Staphylococcus aureus by binding to its menaquinone (commonly known as vitamin K2). Unlike S. aureus, menaquinone is essential in both growing and dormant Mycobacterium tuberculosis. This study aims to evaluate the antituberculosis activities of lysocin E and decipher its mode of action. We show that lysocin E has high in vitro activity against both drug-susceptible and drug-resistant Mycobacterium tuberculosis var. tuberculosis and dormant mycobacteria. Lysocin E is likely bound to menaquinone, causing M. tuberculosis membrane disruption, inhibition of oxygen consumption, and ATP synthesis. Thus, we have concluded that the high antituberculosis activity of lysocin E is attributable to its synergistic effects of membrane disruption and respiratory inhibition. The efficacy of lysocin E against intracellular M. tuberculosis in macrophages was lower than its potent activity against M. tuberculosis in culture medium, probably due to its low ability to penetrate cells, but its efficacy in mice was still superior to that of streptomycin. Our findings indicate that lysocin E is a promising lead compound for the development of a new tuberculosis drug that cures drug-resistant and latent tuberculosis in a shorter period.