ESX-1 Secretion Research Articles

The regulatory functions of ESX-1 substrates, EspE and EspF, are separable from secretion.

Pathogenic mycobacteria are a significant global health burden. The ESX-1 secretion system is essential for mycobacterial pathogenesis. The secretion of ESX-1 substrates is required for phagosomal lysis, which allows the bacteria to enter the macrophage cytoplasm, induce a Type I IFN response, and spread to new host cells. EspE and EspF are dual-functioning ESX-1 substrates. Inside the mycobacterial cell, they regulate transcription of ESX-1-associated genes. Following secretion, EspE and EspF are essential for lytic activity. The link between EspE/F secretion and regulatory function has not been investigated. We investigated the relationship between EspE and EspF using molecular genetics in Mycobacterium marinum, a non-tuberculous mycobacterial species that serves as an established model for ESX-1 secretion and function in Mycobacterium tuberculosis. Our data support that EspE and EspF, which require each other for secretion, directly interact. The disruption of the predicted protein-protein interaction abrogates hemolytic activity and secretion but does not impact their gene regulatory activities in the mycobacterial cell. In addition, we predict a direct protein-protein interaction between the EsxA/EsxB heterodimer and EspF. Our data support that the EspF/EsxA interaction is also required for hemolytic activity and EspE secretion. Our study sheds light on the intricate molecular mechanisms governing the interactions between ESX-1 substrates, regulatory function, and ESX-1 secretion, moving the field forward.IMPORTANCETuberculosis (TB), caused by Mycobacterium tuberculosis, is a historical and pervasive disease responsible for millions of deaths annually. The rise of antibiotic and treatment-resistant TB, as well as the rise of infection by non-tuberculous mycobacterial species, calls for a better understanding of pathogenic mycobacteria. The ESX-1 secreted substrates, EspE and EspF, are required for mycobacterial virulence and may be responsible for phagosomal lysis. This study focuses on the mechanism of EspE and EspF secretion from the mycobacterial cell.

Deep mutational scanning of EccD3 reveals the molecular basis of its essentiality in the mycobacterium ESX secretion system.

Tuberculosis remains the deadliest infectious disease in the world and requires novel therapeutic targets. The ESX-3 secretion system, which is essential for iron and zinc homeostasis and thus M. tuberculosis survival, is a promising target. In this study, we perform a deep mutational scan on the ESX-3 core protein EccD3 in the model organism M. smegmatis. We systematically investigated the functional roles of 145 residues across the soluble ubiquitin-like domain, the conformationally distinct flexible linker, and selected transmembrane helices of EccD3. Our data combined with structural comparisons to ESX-5 complexes support a model where EccD3 stabilizes the complex, with the hinge motif within the linker being particularly sensitive to disruption. Our study is the first deep mutational scan in mycobacteria, which could help guide drug development toward novel treatment of tuberculosis. This study underscores the importance of context-specific mutational analyses for discovering essential protein interactions within mycobacterial systems.

Marine sponge microbe provides insights into evolution and virulence of the tubercle bacillus.

Reconstructing the evolutionary origins of Mycobacterium tuberculosis, the causative agent of human tuberculosis, has helped identify bacterial factors that have led to the tubercle bacillus becoming such a formidable human pathogen. Here we report the discovery and detailed characterization of an exceedingly slow growing mycobacterium that is closely related to M. tuberculosis for which we have proposed the species name Mycobacterium spongiae sp. nov., (strain ID: FSD4b-SM). The bacterium was isolated from a marine sponge, taken from the waters of the Great Barrier Reef in Queensland, Australia. Comparative genomics revealed that, after the opportunistic human pathogen Mycobacterium decipiens, M. spongiae is the most closely related species to the M. tuberculosis complex reported to date, with 80% shared average nucleotide identity and extensive conservation of key M. tuberculosis virulence factors, including intact ESX secretion systems and associated effectors. Proteomic and lipidomic analyses showed that these conserved systems are functional in FSD4b-SM, but that it also produces cell wall lipids not previously reported in mycobacteria. We investigated the virulence potential of FSD4b-SM in mice and found that, while the bacteria persist in lungs for 56 days after intranasal infection, no overt pathology was detected. The similarities with M. tuberculosis, together with its lack of virulence, motivated us to investigate the potential of FSD4b-SM as a vaccine strain and as a genetic donor of the ESX-1 genetic locus to improve BCG immunogenicity. However, neither of these approaches resulted in superior protection against M. tuberculosis challenge compared to BCG vaccination alone. The discovery of M. spongiae adds to our understanding of the emergence of the M. tuberculosis complex and it will be another useful resource to refine our understanding of the factors that shaped the evolution and pathogenesis of M. tuberculosis.

The SapM phosphatase can arrest phagosome maturation in an ESX-1 independent manner in Mycobacterium tuberculosis and BCG.

Mycobacterium tuberculosis (Mtb) is an intracellular pathogen that survives and grows in macrophages. A mechanism used by Mtb to achieve intracellular survival is to secrete effector molecules that arrest the normal process of phagosome maturation. Through phagosome maturation arrest (PMA), Mtb remains in an early phagosome and avoids delivery to degradative phagolysosomes. One PMA effector of Mtb is the secreted SapM phosphatase. Because the host target of SapM, phosphatidylinositol-3-phosphate (PI3P), is located on the cytosolic face of the phagosome, SapM needs to not only be released by the mycobacteria but also travel out of the phagosome to carry out its function. To date, the only mechanism known for Mtb molecules to leave the phagosome is phagosome permeabilization by the ESX-1 secretion system. To understand this step of SapM function in PMA, we generated identical in-frame sapM mutants in both the attenuated Mycobacterium bovis bacille Calmette-Guérin (BCG) vaccine strain, which lacks the ESX-1 system, and Mtb. Characterization of these mutants demonstrated that SapM is required for PMA in BCG and Mtb. Further, by establishing a role for SapM in PMA in BCG, and subsequently in a Mtb mutant lacking the ESX-1 system, we demonstrated that the role of SapM does not require ESX-1. We further determined that ESX-2 or ESX-4 is also not required for SapM to function in PMA. These results indicate that SapM is a secreted effector of PMA in both BCG and Mtb, and that it can function independent of the known mechanism for Mtb molecules to leave the phagosome.

ESAT-6 undergoes self-association at phagosomal pH and an ESAT-6-specific nanobody restricts M. tuberculosis growth in macrophages

Mycobacterium tuberculosis (Mtb) is known to survive within macrophages by compromising the integrity of the phagosomal compartment in which it resides. This activity primarily relies on the ESX-1 secretion system, predominantly involving the protein duo ESAT-6 and CFP-10. CFP-10 likely acts as a chaperone, while ESAT-6 likely disrupts phagosomal membrane stability via a largely unknown mechanism. we employ a series of biochemical analyses, protein modeling techniques, and a novel ESAT-6-specific nanobody to gain insight into the ESAT-6’s mode of action. First, we measure the binding kinetics of the tight 1:1 complex formed by ESAT-6 and CFP-10 at neutral pH. Subsequently, we demonstrate a rapid self-association of ESAT-6 into large complexes under acidic conditions, leading to the identification of a stable tetrameric ESAT-6 species. Using molecular dynamics simulations, we pinpoint the most probable interaction interface. Furthermore, we show that cytoplasmic expression of an anti-ESAT-6 nanobody blocks Mtb replication, thereby underlining the pivotal role of ESAT-6 in intracellular survival. Together, these data suggest that ESAT-6 acts by a pH-dependent mechanism to establish two-way communication between the cytoplasm and the Mtb-containing phagosome.

ESAT-6 undergoes self-association at phagosomal pH and an ESAT-6-specific nanobody restricts M. tuberculosis growth in macrophages.

Mycobacterium tuberculosis (Mtb) is known to survive within macrophages by compromising the integrity of the phagosomal compartment in which it resides. This activity primarily relies on the ESX-1 secretion system, predominantly involving the protein duo ESAT-6 and CFP-10. CFP-10 likely acts as a chaperone, while ESAT-6 likely disrupts phagosomal membrane stability via a largely unknown mechanism. we employ a series of biochemical analyses, protein modeling techniques, and a novel ESAT-6-specific nanobody to gain insight into the ESAT-6's mode of action. First, we measure the binding kinetics of the tight 1:1 complex formed by ESAT-6 and CFP-10 at neutral pH. Subsequently, we demonstrate a rapid self-association of ESAT-6 into large complexes under acidic conditions, leading to the identification of a stable tetrameric ESAT-6 species. Using molecular dynamics simulations, we pinpoint the most probable interaction interface. Furthermore, we show that cytoplasmic expression of an anti-ESAT-6 nanobody blocks Mtb replication, thereby underlining the pivotal role of ESAT-6 in intracellular survival. Together, these data suggest that ESAT-6 acts by a pH-dependent mechanism to establish two-way communication between the cytoplasm and the Mtb-containing phagosome.

New insights into the domain of unknown function (DUF) of EccC5, the pivotal ATPase providing the secretion driving force to the ESX-5 secretion system.

Type VII secretion (T7S) systems, also referred to as ESAT-6 secretion (ESX) systems, are molecular machines that have gained great attention due to their implications in cell homeostasis and in host-pathogen interactions in mycobacteria. The latter include important human pathogens such as Mycobacterium tuberculosis (Mtb), the etiological cause of human tuberculosis, which constitutes a pandemic accounting for more than one million deaths every year. The ESX-5 system is exclusively found in slow-growing pathogenic mycobacteria, where it mediates the secretion of a large family of virulence factors: the PE and PPE proteins. The secretion driving force is provided by EccC5, a multidomain ATPase that operates using four globular cytosolic domains: an N-terminal domain of unknown function (EccC5DUF) and three FtsK/SpoIIIE ATPase domains. Recent structural and functional studies of ESX-3 and ESX-5 systems have revealed EccCDUF to be an ATPase-like fold domain with potential ATPase activity, the functionality of which is essential for secretion. Here, the crystal structure of the MtbEccC5DUF domain is reported at 2.05 Å resolution, which reveals a nucleotide-free structure with degenerated cis-acting and trans-acting elements involved in ATP binding and hydrolysis. This crystallographic study, together with a biophysical assessment of the interaction of MtbEccC5DUF with ATP/Mg2+, supports the absence of ATPase activity proposed for this domain. It is shown that this degeneration is also present in DUF domains from other ESX and ESX-like systems, which are likely to exhibit poor or null ATPase activity. Moreover, based on an in silico model of the N-terminal region of MtbEccC5DUF, it is hypothesized that MtbEccC5DUF is a degenerated ATPase domain that may have retained the ability to hexamerize. These observations draw attention to DUF domains as structural elements with potential implications in the opening and closure of the membrane pore during the secretion process via their involvement in inter-protomer interactions.

Genomic and phenotypic characterization of Mycobacterium tuberculosis' closest-related non-tuberculous mycobacteria.

Four species of non-tuberculous mycobacteria (NTM) rated as biosafety level 1 or 2 (BSL-1/BSL-2) organisms and showing higher genomic similarity with Mycobacterium tuberculosis (Mtb) than previous comparator species Mycobacterium kansasii and Mycobacterium marinum were subjected to genomic and phenotypic characterization. These species named Mycobacterium decipiens, Mycobacterium lacus, Mycobacterium riyadhense, and Mycobacterium shinjukuense might represent "missing links" between low-virulent mycobacterial opportunists and the highly virulent obligate pathogen Mtb. We confirmed that M. decipiens is the closest NTM species to Mtb currently known and found that it has an optimal growth temperature of 32°C-35°C and not 37°C. M. decipiens showed resistance to rifampicin, isoniazid, and ethambutol, whereas M. lacus and M. riyadhense showed resistance to isoniazid and ethambutol. M. shinjukuense was sensitive to all three first-line TB drugs, and all four species were sensitive to bedaquiline, a third-generation anti-TB drug. Our results suggest these four NTM may be useful models for the identification and study of new anti-TB molecules, facilitated by their culture under non-BSL-3 conditions as compared to Mtb. M. riyadhense was the most virulent of the four species in cellular and mouse infection models. M. decipiens also multiplied in THP-1 cells at 35°C but was growth impaired at 37°C. Genomic comparisons showed that the espACD locus, essential for the secretion of ESX-1 proteins in Mtb, was present only in M. decipiens, which was able to secrete ESAT-6 and CFP-10, whereas secretion of these antigens varied in the other species, making the four species interesting examples for studying ESX-1 secretion mechanisms.IMPORTANCEIn this work, we investigated recently identified opportunistic mycobacterial pathogens that are genomically more closely related to Mycobacterium tuberculosis (Mtb) than previously used comparator species Mycobacterium kansasii and Mycobacterium marinum. We confirmed that Mycobacterium decipiens is the currently closest known species to the tubercle bacilli, represented by Mycobacterium canettii and Mtb strains. Surprisingly, the reference strain of Mycobacterium riyadhense (DSM 45176), which was purchased as a biosafety level 1 (BSL-1)-rated organism, was the most virulent of the four species in the tested cellular and mouse infection models, suggesting that a BSL-2 rating might be more appropriate for this strain than the current BSL-1 rating. Our work establishes the four NTM species as interesting study models to obtain new insights into the evolutionary mechanisms and phenotypic particularities of mycobacterial pathogens that likely have also impacted the evolution of the key pathogen Mtb.

Mycobacterium tuberculosis resides in lysosome-poor monocyte-derived lung cells during chronic infection.

Mycobacterium tuberculosis (Mtb) infects lung myeloid cells, but the specific Mtb-permissive cells and host mechanisms supporting Mtb persistence during chronic infection are incompletely characterized. We report that after the development of T cell responses, CD11clo monocyte-derived cells harbor more live Mtb than alveolar macrophages (AM), neutrophils, and CD11chi monocyte-derived cells. Transcriptomic and functional studies revealed that the lysosome pathway is underexpressed in this highly permissive subset, characterized by less lysosome content, acidification, and proteolytic activity than AM, along with less nuclear TFEB, a regulator of lysosome biogenesis. Mtb infection does not drive lysosome deficiency in CD11clo monocyte-derived cells but promotes recruitment of monocytes that develop into permissive lung cells, mediated by the Mtb ESX-1 secretion system. The c-Abl tyrosine kinase inhibitor nilotinib activates TFEB and enhances lysosome functions of macrophages in vitro and in vivo, improving control of Mtb infection. Our results suggest that Mtb exploits lysosome-poor lung cells for persistence and targeting lysosome biogenesis is a potential host-directed therapy for tuberculosis.

The antagonistic transcription factors, EspM and EspN, regulate the ESX-1 secretion system in M. marinum.

Bacterial pathogens use protein secretion systems to transport virulence factors and regulate gene expression. Among pathogenic mycobacteria, including Mycobacterium tuberculosis and Mycobacterium marinum, the ESAT-6 system 1 (ESX-1) secretion is crucial for host interaction. Secretion of protein substrates by the ESX-1 secretion system disrupts phagosomes, allowing mycobacteria cytoplasmic access during macrophage infections. Deletion or mutation of the ESX-1 system attenuates mycobacterial pathogens. Pathogenic mycobacteria respond to the presence or absence of the ESX-1 system in the cytoplasmic membrane by altering transcription. Under laboratory conditions, the EspM repressor and WhiB6 activator control transcription of specific ESX-1-responsive genes, including the ESX-1 substrate genes. However, deleting the espM or whiB6 gene does not phenocopy the deletion of the ESX-1 substrate genes during macrophage infection by M. marinum. In this study, we identified EspN, a critical transcription factor whose activity is masked by the EspM repressor under laboratory conditions. In the absence of EspM, EspN activates transcription of whiB6 and ESX-1 genes during both laboratory growth and macrophage infection. EspN is also independently required for M. marinum growth within and cytolysis of macrophages, similar to the ESX-1 genes, and for disease burden in a zebrafish larval model of infection. These findings suggest that EspN and EspM coordinate to counterbalance the regulation of the ESX-1 system and support mycobacterial pathogenesis.IMPORTANCEPathogenic mycobacteria, which are responsible for tuberculosis and other long-term diseases, use the ESX-1 system to transport proteins that control the host response to infection and promote bacterial survival. In this study, we identify an undescribed transcription factor that controls the expression of ESX-1 genes and is required for both macrophage and animal infection. However, this transcription factor is not the primary regulator of ESX-1 genes under standard laboratory conditions. These findings identify a critical transcription factor that likely controls expression of a major virulence pathway during infection, but whose effect is not detectable with standard laboratory strains and growth conditions.

Discovery of dual-active ethionamide boosters inhibiting the Mycobacterium tuberculosis ESX-1 secretion system

Drug-resistant Mycobacterium tuberculosis (Mtb) remains a major public health concern requiring complementary approaches to standard anti-tuberculous regimens. Anti-virulence molecules or compounds that enhance the activity of antimicrobial prodrugs are promising alternatives to conventional antibiotics. Exploiting host cell-based drug discovery, we identified an oxadiazole compound (S3) that blocks the ESX-1 secretion system, a major virulence factor of Mtb. S3-treated mycobacteria showed impaired intracellular growth and a reduced ability to lyse macrophages. RNA sequencing experiments of drug-exposed bacteria revealed strong upregulation of a distinct set of genes including ethA, encoding a monooxygenase activating the anti-tuberculous prodrug ethionamide. Accordingly, we found a strong ethionamide boosting effect in S3-treated Mtb. Extensive structure-activity relationship experiments revealed that anti-virulence and ethionamide-boosting activity can be uncoupled by chemical modification of the primary hit molecule. To conclude, this series of dual-active oxadiazole compounds targets Mtb via two distinct mechanisms of action.

ER-dependent membrane repair of mycobacteria-induced vacuole damage.

Tuberculosis still remains a global burden and is one of the top infectious diseases from a single pathogen. Mycobacterium tuberculosis, the causative agent, has perfected many ways to replicate and persist within its host. While mycobacteria induce vacuole damage to evade the toxic environment and eventually escape into the cytosol, the host recruits repair machineries to restore the MCV membrane. However, how lipids are delivered for membrane repair is poorly understood. Using advanced fluorescence imaging and volumetric correlative approaches, we demonstrate that this involves the recruitment of the endoplasmic reticulum (ER)-Golgi lipid transfer protein OSBP8 in the Dictyostelium discoideum/Mycobacterium marinum system. Strikingly, depletion of OSBP8 affects lysosomal function accelerating mycobacterial growth. This indicates that an ER-dependent repair pathway constitutes a host defense mechanism against intracellular pathogens such as M. tuberculosis.

From accurate genome sequence to biotechnological application: The thermophile Mycolicibacterium hassiacum as experimental model.

Mycobacteria constitute a large group of microorganisms belonging to the phylum Actinobacteria encompassing some of the most relevant pathogenic bacteria and many saprophytic isolates that share a unique and complex cell envelope. Also unique to this group is the extensive capability to use and synthesize sterols, a class of molecules that include active signalling compounds of pharmaceutical use. However, few mycobacterial species and strains have been established as laboratory models to date, Mycolicibacterium smegmatis mc2 155 being the most common one. In this work, we focus on the use of a thermophilic mycobacterium, Mycolicibacterium hassiacum, which grows optimally above 50°C, as an emerging experimental model valid to extend our general knowledge of mycobacterial biology as well as for application purposes. To that end, accurate genomic sequences are key for gene mining, the study of pathogenicity or lack thereof and the potential for gene transfer. The combination of long- and short-massive sequencing technologies is strictly necessary to remove biases caused by errors specific to long-reads technology. By doing so in M. hassiacum, we obtained from the curated genome clues regarding the genetic manipulation potential of this microorganism from the presence of insertion sequences, CRISPR-Cas, type VII ESX secretion systems, as well as lack of plasmids. Finally, as a proof of concept of the applicability of M. hassiacum as a laboratory and industrial model, we used this high-quality genome of M. hassiacum to successfully knockout a gene involved in the use of phytosterols as source of carbon and energy, using an improved gene cassette for thermostable selection and a transformation protocol at high temperature.

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.

Mycobacterium tuberculosis Evasion of Guanylate Binding Protein-Mediated Host Defense in Mice Requires the ESX1 Secretion System.

Cell-intrinsic immune mechanisms control intracellular pathogens that infect eukaryotes. The intracellular pathogen Mycobacterium tuberculosis (Mtb) evolved to withstand cell-autonomous immunity to cause persistent infections and disease. A potent inducer of cell-autonomous immunity is the lymphocyte-derived cytokine IFNγ. While the production of IFNγ by T cells is essential to protect against Mtb, it is not capable of fully eradicating Mtb infection. This suggests that Mtb evades a subset of IFNγ-mediated antimicrobial responses, yet what mechanisms Mtb resists remains unclear. The IFNγ-inducible Guanylate binding proteins (GBPs) are key host defense proteins able to control infections with intracellular pathogens. GBPs were previously shown to directly restrict Mycobacterium bovis BCG yet their role during Mtb infection has remained unknown. Here, we examine the importance of a cluster of five GBPs on mouse chromosome 3 in controlling Mycobacterial infection. While M. bovis BCG is directly restricted by GBPs, we find that the GBPs on chromosome 3 do not contribute to the control of Mtb replication or the associated host response to infection. The differential effects of GBPs during Mtb versus M. bovis BCG infection is at least partially explained by the absence of the ESX1 secretion system from M. bovis BCG, since Mtb mutants lacking the ESX1 secretion system become similarly susceptible to GBP-mediated immune defense. Therefore, this specific genetic interaction between the murine host and Mycobacteria reveals a novel function for the ESX1 virulence system in the evasion of GBP-mediated immunity.

Structure-function analysis of the dual functioning mycobacterial virulence factors EspE and EspF.

Mycobacterium tuberculosis is the world's second leading infectious disease killer, with approximately 1.5 million people succumbing to the disease annually. Protein export by the ESX-1 secretion system is essential for the virulence of pathogenic mycobacteria, including M. tuberculosis and nontuberculous mycobacteria (NTMs) such as Mycobacterium marinum. ESX-1 mediates rupture of the phagosomal membrane, allowing mycobacteria to gain access to the cytoplasm of host macrophages. Phagosomal lysis is required for host cell death and mycobacterial cell-to-cell spread. Our previous work demonstrated that the ESX-1 substrates EspE and EspF are required for the membranolytic activity of M. marinum. Our lab recently showed that EspE and EspF are the last set of substrates secreted by the ESX-1 system and require each other for secretion. Moreover, EspE and EspF regulate gene expression in the mycobacterial cytoplasm. Using a targeted genetic approach, we are conducting a structure-function analysis to determine if protein interaction is required for the hemolytic and regulatory activities of EspE and EspF. We show that the disruption of a predicted salt bridge between EspE and EspF abrogates hemolytic activity and EspE secretion. In addition, the protein-protein interface between EspF and EsxA is necessary for hemolytic activity. Disruption of this region leads to a substantial decrease in EspE secretion. This, combined with EsxB secretion data, shows that EspE-EspF interaction is required for secretion, but may not be required for gene regulation. These data provide new insight into the unique ESX-1 substrates EspE and EspF in gene regulation, protein secretion, and virulence.

Dysregulation of Mycobacterium marinum ESX-5 Secretion by Novel 1,2,4-oxadiazoles.

The ESX-5 secretion system is essential for the viability and virulence of slow-growing pathogenic mycobacterial species. In this study, we identified a 1,2,4-oxadiazole derivative as a putative effector of the ESX-5 secretion system. We confirmed that this 1,2,4-oxadiazole and several newly synthesized derivatives inhibited the ESX-5-dependent secretion of active lipase LipY by Mycobacterium marinum (M. marinum). Despite reduced lipase activity, we did not observe a defect in LipY secretion itself. Moreover, we found that several other ESX-5 substrates, especially the high molecular-weight PE_PGRS MMAR_5294, were even more abundantly secreted by M. marinum treated with several 1,2,4-oxadiazoles. Analysis of M. marinum grown in the presence of different oxadiazole derivatives revealed that the secretion of LipY and the induction of PE_PGRS secretion were, in fact, two independent phenotypes, as we were able to identify structural features in the compounds that specifically induced only one of these phenotypes. Whereas the three most potent 1,2,4-oxadiazoles displayed only a mild effect on the growth of M. marinum or M. tuberculosis in culture, these compounds significantly reduced bacterial burden in M. marinum-infected zebrafish models. In conclusion, we report a 1,2,4-oxadiazole scaffold that dysregulates ESX-5 protein secretion.

Functional Analysis of EspM, an ESX-1-Associated Transcription Factor in Mycobacterium marinum.

Pathogenic mycobacteria use the ESX-1 secretion system to escape the macrophage phagosome and survive infection. We demonstrated that the ESX-1 system is regulated by feedback control in Mycobacterium marinum, a nontuberculous pathogen and model for the human pathogen Mycobacterium tuberculosis. In the presence of a functional ESX-1 system, the WhiB6 transcription factor upregulates expression of ESX-1 substrate genes. In the absence of an assembled ESX-1 system, the conserved transcription factor, EspM, represses whiB6 expression by specifically binding the whiB6 promoter. Together, WhiB6 and EspM fine-tune the levels of ESX-1 substrates in response to the secretion system. The mechanisms underlying control of the ESX-1 system by EspM are unknown. Here, we conduct a structure and function analysis to investigate how EspM is regulated. Using biochemical approaches, we measured the formation of higher-order oligomers of EspM in vitro. We demonstrate that multimerization in vitro can be mediated through multiple domains of the EspM protein. Using a bacterial monohybrid system, we showed that EspM self-associates through multiple domains in Escherichia coli. Using this system, we performed a genetic screen to identify EspM variants that failed to self-associate. The screen yielded four EspM variants of interest, which we tested for activity in M. marinum. Our study revealed that the two helix-turn-helix domains are functionally distinct. Moreover, the helix bundle domain is required for wild-type multimerization in vitro. Our data support models where EspM monomers or hexamers contribute to the regulation of whiB6 expression. IMPORTANCE Pathogenic mycobacteria are bacteria that pose a large burden to human health globally. The ESX-1 secretion system is required for pathogenic mycobacteria to survive within and interact with the host. Proper function of the ESX-1 secretion system is achieved by tightly controlling the expression of secreted virulence factors, in part through transcriptional regulation. Here, we characterize the conserved transcription factor EspM, which regulates the expression of ESX-1 virulence factors. We define domains required for EspM to form multimers and bind DNA. These findings provide an initial characterization an ESX-1 transcription factor and provide insights into its mechanism of action.

Mycobacterium abscessus Mutants with a Compromised Functional Link between the Type VII ESX-3 System and an Iron Uptake Mechanism Reliant on an Unusual Mycobactin Siderophore

The opportunistic pathogen Mycobacterium abscessus subsp. abscessus (Mab) has become an emerging public health threat due to the increasing number of Mab-associated chronic pulmonary disease cases. Treatment requires multiple drug courses and is often combined with surgical resection. Cure rates are only ~50% due to treatment failure and comorbidities. Deeper understanding of the biology of Mab is required to illuminate potential avenues for the development of better therapeutics against Mab infections. The ESX-3 type VII protein secretion system of Mab has an important role in host inflammatory and pathological responses during infection. In this work, we demonstrate a functional link between ESX-3 and an iron uptake system based on an unusual mycobactin-type siderophore (designated MBT Ab) and exploit this link to implement a large screen for transposon mutants with an impaired ESX-3. Most mutants we identified carry insertions in genes encoding predicted ESX-3 secretion machinery components or potential ESX-3 substrates. The mutants overproduce MBT Ab, a trait consistent with an iron uptake defect. Our characterization of MBT Ab revealed structural features reminiscent of nocardial mycobactin-like compounds with cytotoxicity. This finding raises the possibility that MBT Ab may play roles in pathogenesis unlinked to iron homeostasis. The mutants generated herein will facilitate research to better understand the role of ESX-3 and its interplay with the siderophore system.

STREPTOMYCIN AND PYRIDOMYCIN DRUG MOLECULES PROVIDE THE MOST POTENTIAL INHIBITORS AGAINST RV3871 TO CURE TUBERCULOSIS

The ESX-1 secretion system of Mycobacterium tuberculosis delivers bacterial virulence factors to host cells during infection. The most abundant factor, the ESAT-6/CFP-10 dimer, is targeted for secretion via a C-terminal signal sequence on CFP-10 that is recognized by the cytosolic ATPase, Rv3871. ATPase that is a component of the ESAT-6/CFP-10 secretion system. ESX locus contains genes encoding conserved secretion machinery components termed EccCb1. these core components are required for ESAT-6/CFP-10 secretion [70, 71]. Rv3871 is a cytoplasmic protein connected with the Rv3870 and encoded by the EccCb1 gene. These proteins supply energy for the secretion process. Each of these ATPases is involved in targeting protein for ESX-1 secretion. EccCb1 binds a seven amino acid C-terminal signal peptide of CFT- 10, which is required for secretion of the ESAT-6/CFP-10 complex. In this we target the Rv3871 seven amino acid c-terminal region and block it through multiple drugs so, it cannot be activated by the ATPases and not to supply energy for the secretion protein during infection (Tuberculosis). In the present study we have comparatively studied different Rv3871 inhibitory antagonist drug molecule which could be a potential drug molecule to inhibit ATPases on the Rv3871 molecule that is responsible for the energy supply. We have 16 antagonistic drug molecules as shortlisted based on previous studies, that block the target site on the Rv3871 molecule. KEYWORDS: Tuberculosis/Drug target/Molecular Docking/Screening of antituberculosis drug.