The cell wall of mycobacteria plays a key role in interactions with the environment. Its ability to act as a selective filter is crucial to bacterial survival. Proteins in the cell wall enable this function by mediating the import and export of diverse metabolites, from ions to lipids to proteins. Identifying cell wall proteins is an important step in assigning function, especially as many mycobacterial proteins lack functionally characterized homologues. Current methods for protein localization have inherent limitations that reduce accuracy. Here we showed that although chemical labeling of live cells did not exclusively label surface proteins, protein tagging by the engineered peroxidase APEX2 within live Mycobacterium tuberculosis accurately identified the cytosolic and cell wall proteomes. Our data indicate that substrates of the virulence-associated Type VII ESX secretion system are exposed to the periplasm, providing insight into the currently unknown mechanism by which these proteins cross the mycobacterial cell envelope.

The development of tuberculosis (TB) vaccines has been hindered by the complex nature of Mycobacterium tuberculosis (M.tb) and the absence of clearly defined immune markers of protection. While Bacillus Calmette-Guerin (BCG) is currently the only licensed TB vaccine, its effectiveness diminishes in adulthood. In our previous research, we identified that boosting BCG with an intranasally administered chimpanzee adenovirus expressing the PPE15 antigen of M.tb (ChAdOx1.PPE15) improved its protection. To enhance the vaccine\'s efficacy, we combined PPE15 with the other three members of the Esx-5a secretion system and Ag85A into a multi-antigen construct (5Ag). Leveraging the mucosal administration safety of ChAdOx1, we targeted the site of M.tb infection to induce localized mucosal responses, while employing modified vaccinia virus (MVA) to boost systemic immune responses. The combination of these antigens resulted in enhanced BCG protection in both the lungs and spleens of vaccinated mice. These findings provide support for advancing ChAdOx1.5Ag and MVA.5Ag to the next stages of vaccine development.

Mycobacteria, such as the pathogen M. tuberculosis, utilize up to five paralogous type VII secretion systems to transport proteins across their cell envelope. Since these proteins associate in pairs that depend on each other for transport to a different extent, the secretion pathway to the bacterial surface remained challenging to address. Structural characterization of the inner-membrane embedded secretion machineries along with recent advances on the substrates\' co-dependencies for transport allow for the first time more detailed and testable models for secretion.

The cell wall of mycobacteria plays a key role in interactions with the environment and its ability to act as a selective filter is crucial to bacterial survival. Proteins in the cell wall enable this function by mediating the import and export of diverse metabolites from ions to lipids to proteins. Accurately identifying cell wall proteins is an important step in assigning function, especially as many mycobacterial proteins lack functionally characterized homologues. Current methods for protein localization have inherent limitations that reduce accuracy. Here we showed that protein tagging by the engineered peroxidase APEX2 within live Mycobacterium tuberculosis enabled the accurate identification of the cytosolic and cell wall proteomes. Our data indicate that substrates of the virulence-associated Type VII ESX secretion system are exposed to the Mtb periplasm, providing insight into the currently unknown mechanism by which these proteins cross the mycobacterial cell envelope.

Mycobacterium tuberculosis (MTB) causes one of the ancient diseases, Tuberculosis, affects people around the globe and its severity can be understood by its classification as a second infectious disease after COVID-19 and the 13th leading cause of death according to a WHO report. Despite having advanced diagnostic approaches and therapeutic strategies, unfortunately, TB is still spreading across the population due to the emergence of drug-resistance MTB and Latent TB infection (LTBI). We are seeking for effective approaches to overcome these hindrances and efficient treatment for this perilous disease. Therefore, there is an urgent need to develop drugs based on operative targeting of the bacterial system that could result in both efficient treatment and lesser emergence of MDR-TB. One such promising target could be the secretory systems and especially the Type 7 secretory system (T7SS-ESX) of Mycobacterium tuberculosis, which is crucial for the secretion of effector proteins as well as in establishing host-pathogen interactions of the tubercle bacilli. The five paralogous ESX systems (ESX-1 to EXS-5) have been observed by in silico genome analysis of MTB, among which ESX-1 and ESX-5 are substantial for virulence and mediating host cellular inflammasome. The bacterium growth and virulence can be modulated by targeting the T7SS. In the present review, we demonstrate the current status of therapeutics against MTB and focus on the function and cruciality of T7SS along with other secretory systems as a promising therapeutic target against Tuberculosis.

Non-tuberculous mycobacteria (NTM) are a large group of micro-organisms comprising more than 200 individual species. Most NTM are saprophytic organisms and are found mainly in terrestrial and aquatic environments. In recent years, NTM have been increasingly associated with infections in both immunocompetent and immunocompromised individuals, prompting significant efforts to understand the diverse pathogenic and signalling traits of these emerging pathogens. Since the discovery of Type VII secretion systems (T7SS), there have been significant developments regarding the role of these complex systems in mycobacteria. These specialised systems, also known as Early Antigenic Secretion (ESX) systems, are employed to secrete proteins across the inner membrane. They also play an essential role in virulence, nutrient uptake and conjugation. Our understanding of T7SS in mycobacteria has significantly benefited over the last few years, from the resolution of ESX-3 structure in Mycobacterium smegmatis, to ESX-5 structures in Mycobacterium xenopi and Mycobacterium tuberculosis. In addition, ESX-4, considered until recently as a non-functional system in both pathogenic and non-pathogenic mycobacteria, has been proposed to play an important role in the virulence of Mycobacterium abscessus; an increasingly recognized opportunistic NTM causing severe lung diseases. These major findings have led to important new insights into the functional mechanisms of these biological systems, their implication in virulence, nutrient acquisitions and cell wall shaping, and will be discussed in this review.

UNASSIGNED: Mycobacterium tuberculosis is the causative organism of tuberculosis, which is the most lethal disease after cancer in the current decade. The development of multidrug and broadly drug-resistant strains is making the problem of tuberculosis more and more critical. In the last 40 years, only one molecule has been added to the treatment regimen. Generally, drug design and development programs target proteins whose function is known to be essential to the bacterial cell. M. tuberculosis possesses specialized protein export systems like the SecA2 export pathway and ESX pathways.
UNASSIGNED: In the present communication, rational development of an antimycobacterial agent\'s targeting protein export system was carried out by integrating pocket modeling and virtual analysis.
UNASSIGNED: The 23 identified potential lead compounds were synthesized, characterized by physicochemical and spectroscopic methods like infrared and nuclear magnetic resonance spectroscopy, and further screened for antimycobacterial activity using isoniazid as standard. All the designed compounds showed profound antimycobacterial activity.
UNASSIGNED: We found that Q30, M9, M26, U8, and R26 molecules had significant desirable biological activity and specific interactions with Sec of mycobacteria. Further optimization of these leads is necessary for the development of potential antimycobacterial drug candidates with fewer side effects.

Mycobacteria use type VII secretion systems (T7SSs) to translocate a wide range of proteins across their diderm cell envelope. These systems, also called ESX systems, are crucial for the viability and/or virulence of mycobacterial pathogens, including Mycobacterium tuberculosis and the fish pathogen Mycobacterium marinum. We have previously shown that the M. tuberculosis ESX-5 system is unable to fully complement secretion in an M. marinum esx-5 mutant, suggesting species specificity in secretion. In this study, we elaborated on this observation and established that the membrane ATPase EccC5 , possessing four (putative) nucleotide-binding domains (NBDs), is responsible for this. By creating M. marinum-M. tuberculosis EccC5 chimeras, we observed both in M. marinum and in M. tuberculosis that secretion specificity of PE_PGRS proteins depends on the presence of the cognate linker 2 domain of EccC5 . This region connects NBD1 and NBD2 of EccC5 and is responsible for keeping NBD1 in an inhibited state. Notably, the ESX-5 substrate EsxN, predicted to bind to NBD3 on EccC5 , showed a distinct secretion profile. These results indicate that linker 2 is involved in species-specific substrate recognition and might therefore be an additional substrate recognition site of EccC5 .

The mycosin protease (MycP) is widely conserved in type VII secretion (T7S) systems throughout Actinobacteria. Within the T7S systems of mycobacteria, also known as the ESX systems, MycP is essential for secretion, which is probably linked to its stabilizing effect on the ESX membrane complex. However, it is unknown how this is mediated, as MycP is not a stable component of this complex. In this study, we set out to create a chimeric fusion protein of EccB5 and MycP5, based on a chimeric gene of eccB and mycP in the T7S locus of Bifidobacterium dentium. We show that this fusion protein is functional and capable of complementing ESX-5 secretion in both an eccB5 and a mycP5 knockout in Mycobacterium marinum. To study the ESX complex containing this fusion protein in more detail, we replaced the original eccB5 and mycP5 of the Mycobacterium xenopi esx-5 locus, reconstituted in Mycobacterium smegmatis, with the chimeric gene. The EccB5-MycP5 fusion construct also restored ESX-5 secretion under these double knockout conditions. Subsequent protein pulldowns on the central complex component EccC5 showed that under these conditions, the EccB5-MycP5 fusion was specifically copurified and a stable component of the ESX-5 complex. Based on our results, we can conclude that MycP5 carries out its essential function in secretion in close proximity to EccB5, indicating that EccB5 is the direct interaction partner of MycP5.

The ESX (or Type VII) secretion systems are protein export systems in mycobacteria and many Gram-positive bacteria that mediate a broad range of functions including virulence, conjugation, and metabolic regulation. These systems translocate folded dimers of WXG100-superfamily protein substrates across the cytoplasmic membrane. We report the cryo-electron microscopy structure of an ESX-3 system, purified using an epitope tag inserted with recombineering into the chromosome of the model organism Mycobacterium smegmatis. The structure reveals a stacked architecture that extends above and below the inner membrane of the bacterium. The ESX-3 protomer complex is assembled from a single copy of the EccB3, EccC3, and EccE3 and two copies of the EccD3 protein. In the structure, the protomers form a stable dimer that is consistent with assembly into a larger oligomer. The ESX-3 structure provides a framework for further study of these important bacterial transporters.