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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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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.

Among the members of the large group of non-tuberculous mycobacteria (comprising more than 180 species), M. avium subsp. hominissuis (MAH) is the most significant causative agent of pulmonary infection in immunocompetent individuals as well as disseminated infection in immunocompromised hosts, e.g. human immunodeficiency virus (HIV)-positive patients. Due to increased incidence rate of mycobacteriosis, especially in HIV infection, much still need to be learnt about the MAH genetic control and virulence mechanisms. Deciphering the genome contents of the M. avium strain 104 (isolated from an AIDS patient with disseminated MAH disease) allowed to compare genome sequences of M. avium strains to gain insights into genomic diversity associated with variable hosts and environments. Comparative genome analysis of MAH strains isolated from patients with pulmonary and disseminated forms of mycobacteri-osis revealed differences in the structure of the genome, affecting the key virulence genes. This review provides current data on the genetic determinants of MAH virulence associated with the initial phase of infection. Several mycobacterial virulence-associated gene families, such as mce (mammalian cell entry), mmp (mycobacterial membrane proteins), pe/ppe and esx expressed by MAH during human infection are thought to be crucial for adhesion, entry, survival, and reproduction inside host macrophages. The genetic mechanisms of MAH survival in human macrophage cell culture as well as mice exposed to toxic effects of reactive oxygen, nitric oxide, bactericidal proteins (cathelicidin) are discussed. The MAH survival in the latency-like state is important for pathogen dissemination. Some genetic and phenotypic features of MAH (absence of a cord factor, presence of plasmids, potential to “switch” morphological types of colonies) are compared with M. tuberculosis. In addition, we summarized current state of MAH drug discovery, a role of MAH intrinsic multidrug resistance, genetic control, as well as mechanisms underlying formation of resistance to various groups of antibiotics in MAH strains.

BACKGROUND Mycolicibacterium fortuitum is an opportunistic pathogen associated with human and animal infection worldwide. Studies concerning this species are mainly represented by case reports, some of them addressing drug susceptibility with a focus on a specific geographic region, so there is a gap in relation to the global epidemiological scenario. OBJECTIVES We aimed determine the global epidemiological scenario of M. fortuitum and analyse its traits associated with pathogenicity. METHODS Based on publicly available genomes of M. fortuitum and a genome from Brazil (this study), we performed a genomic epidemiology analysis and in silico and in vitro characterisation of the resistome and virulome of this species. FINDINGS Three main clusters were defined, one including isolates from the environment, human and animal infections recovered over nearly a century. An apparent intrinsic resistome comprises mechanisms associated with macrolides, beta-lactams, aminoglycosides and antitubercular drugs such as rifampin. Besides, the virulome presented Type VII secretion systems (T7SS), including ESX-1, ESX-3, ESX-4 and ESX-4-bis, some of which play a role on the virulence of Mycobacteriaceae species. MAIN CONCLUSIONS Here, M. fortuitum was revealed as a reservoir of an expressive intrinsic resistome, as well as a virulome that may contribute to its success as a global opportunist pathogen.

Abstract Background Mycobacterium bovis Bacille Calmette-Guérin (BCG) is not only used as a vaccine against tuberculosis but also protects against leprosy and is used as part of bladder cancer treatment to induce a protective immune response. However, protection by BCG vaccination is not optimal. To improve vaccine efficacy, recombinant BCG expressing heterologous antigens has been put forward to elicit antigen-specific cellular and humoral responses. Cell surface localized or secreted antigens induce better immune responses than their cytosolic counterparts. Optimizing secretion of heterologous proteins or protein fragments holds therefore unexplored potential for improving the efficacy of recombinant BCG vaccine candidates. Secretion of heterologous antigens requires crossing the mycobacterial inner and outer membrane. Mycobacteria have specialized ESX or type VII secretion systems that enable translocation of proteins across both membranes. Probing this secretion system could therefore be a valid approach to surface localize heterologous antigens. Results We show that ESX-5 substrate LipY, a lipase, can be used as a carrier for heterologous secretion of an ovalbumin fragment (OVA). LipY contains a PE domain and a lipase domain, separated by a linker region. This linker domain is processed upon secretion. Fusion of the PE and linker domains of LipY to OVA enabled ESX-5-dependent secretion of the fusion construct LipY-OVA in M. marinum, albeit with low efficiency. Subsequent random mutagenesis of LipY-OVA and screening for increased secretion resulted in mutants with improved heterologous secretion. Detailed analysis identified two mutations in OVA that improved secretion, i.e. an L280P mutation and a protein-extending frameshift mutation. Finally, deletion of the linker domain of LipY enhanced secretion of LipY-OVA, although this mutation also reduced surface association. Further analysis in wild type LipY showed that the linker domain is required for surface association. Conclusion We show that the ESX-5 system can be used for heterologous secretion. Furthermore, minor mutations in the substrate can enhance secretion. Especially the C-terminal region seems to be important for this. The linker domain of LipY is involved in surface association. These findings show that non-biased screening approaches aid in optimization of heterologous secretion, which can contribute to heterologous vaccine development.

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Mobile learning under cloud environment, an amalgamation between mobile cloud computing and mobile learning has gained wide academic and commercial recognition during last few years. Although researches on MOOC and other means of Digital Learning is widely circulated, but research to enhance traditional mobile learning using newer types of ubiquitous and pervasive devices (e.g. Modular Object Oriented Dynamic Learning Environment or MOODLE) for collecting resources is yet to be widely circulated in the literature. The proposed work is expected to make learning more cost-effective, collaborative and practical for the learners using Personal Cloud environment. The work further expedites the proposed virtualization framework (through VMware vSphere Hypervisor- ESXi) and how it provides technical back-bone in which some pedagogical reforms may optimize knowledge transfer substantiating outcome based education (OBE). This solution can be beneficial for mass learners including poor and under privileged and will help in getting rid of digital divide. [ABSTRACT FROM AUTHOR]

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 EccB 5 and MycP 5 , 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 eccB 5 and a mycP 5 knockout in Mycobacterium marinum. To study the ESX complex containing this fusion protein in more detail, we replaced the original eccB 5 and mycP 5 of the Mycobacterium xenopi esx-5 locus, reconstituted in Mycobacterium smegmatis , with the chimeric gene. The EccB 5 -MycP 5 fusion construct also restored ESX-5 secretion under these double knockout conditions. Subsequent protein pulldowns on the central complex component EccC 5 showed that under these conditions, the EccB 5 -MycP 5 fusion was specifically copurified and a stable component of the ESX-5 complex. Based on our results, we can conclude that MycP 5 carries out its essential function in secretion in close proximity to EccB 5 , indicating that EccB 5 is the direct interaction partner of MycP 5. Image 1 • Mycosin protease (MycP) is an essential type VII secretion (T7S) membrane component. • MycP 5 transiently interacts with and stabilizes the ESX-5 membrane complex. • Fusing MycP 5 to the complex component EccB 5 does not impair ESX-5 functionality. • EccB 5 -MycP 5 fusion is a stable component of the ESX-5 membrane complex. • MycP 5 functions in close proximity to EccB 5. [ABSTRACT FROM AUTHOR]

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. [Display omitted] • APEX2 proximity labeling accurately identifies mycobacterial cell wall proteins • "Impermeable" N-hydroxysuccinimide reagents do not selectively tag surface proteins • Cytoplasmic or cell wall tagging of membrane proteins informs their topology • Cell wall tagging of Type VII secretion substrates supports role in export mechanism The cell wall of the bacterial pathogen Mycobacterium tuberculosis is a protective barrier, but also an obstacle to the flow of nutrients and other biomolecules. Jaisinghani et al. use APEX2-mediated proximity labeling in live cells to identify cell wall proteins and thereby inform the mechanism of poorly understood transport processes. [ABSTRACT FROM AUTHOR]

Abstract Background The ESX secretion system, also known as the Type VII secretion system, is mostly found in mycobacteria and plays important roles in nutrient acquisition and host pathogenicity. One of the five ESXs, ESX-3, is associated with mycobactin-mediated iron acquisition. Although the functions of some of the membrane-associated components of the ESX systems have been described, the role of by mycosin-3 remains elusive. The esx-3 gene cluster encoding ESX-3 in both Mycobacterium tuberculosis and Mycobacterium smegmatis has two promoters, suggesting the presence of two transcriptional units. Previous studies indicated that the two promoters only showed a difference in response under acid stress (pH 4.2). This study aimed to study the effect of a mycosin-3 deletion on the physiology of M. smegmatis and to assess the promoter activities in wildtype, mycosin-3 mutant and complementation strains. Results The gene mycP 3 was deleted from wildtype M. smegmatis via homologous recombination. The mycP 3 gene was complemented in the deletion mutant using each of the two intrinsic promoters from the M. smegmatis esx-3 gene cluster. The four strains were compared in term of bacterial growth and intracellular iron content. The two promoter activities were assessed under iron-rich, iron-deprived and iron-rescued conditions by assessing the mycP 3 expression level. Although the mycP 3 gene deletion did not significantly impact bacterial growth or intracellular iron levels in comparison to the wild-type and complemented strains, the two esx-3 promoters were shown to respond inversely to iron deprivation and iron rescue. Conclusion This finding correlates with the previously published data that the first promoter upstream of msmeg0615, is upregulated under low iron levels but downregulated under high iron levels. In addition, the second promoter, upstream of msmeg0620, behaves in an inverse fashion to the first promoter implying that the genes downstream may have additional roles when the iron levels are high.

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.

Over the long course of evolution from a probable environmental reservoir, the pathogen that we know today as Mycobacterium tuberculosis has become fully capable of adapting to the life inside host cells by evading and modifying their responses to infection. Factors contributing to the success of this pathogen are numerous and thanks to a large body of work accumulated over the past decades, we are closer to understanding the remarkable complexity of tuberculosis pathogenesis. The unique type VII secretion systems and various complex lipids of the cell envelope have emerged as some of the most important and most studied factors in this regard. This review attempts to summarize recent findings on these and other virulence factors, while discussing their evolution in different closely related tuberculosis-causing bacteria as well, with the aim of exploring the processes which led M. tuberculosis to becoming one of the deadliest infections agents. • Globally identified mycobacterial virulence factors discussed. • Phagosomal-rupture-mediated cytosolic pattern recognition increases innate signaling. • Virulence lipids summarized and broad function discussed. • Evolutionary aspects of virulence gene recovery discussed. [ABSTRACT FROM AUTHOR]

Objectives: 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. Materials and Methods: 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. Results: 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. Conclusion: 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. [ABSTRACT FROM AUTHOR]

Background: Mycobacterium tuberculosis is the causative agent of tuberculosis (TB). Bacille Calmette-Guerin (BCG) vaccine, is not effective in adults, therefore, many efforts have been made to produce an effective adult TB vaccine. The aim of this study was to develop a new tuberculosis DNA vaccine candidate encoding a recombinant HspX-PPE44-EsxV fusion antigen of M. tuberculosis. Methods: A fusion DNA segment consisting of HspX, linker, PPE44, linker, and EsxV, after codon optimization, was designed. The fusion DNA was cloned and its sequence confirmed. Then, expression of a recombinant pcDNA3.1 (+)/HspX-PPE44-EsxV plasmid in Chinese hamster ovary (CHO) cells was verified by RT-PCR and Western-blot analysis. Results: A 1968 bp band in RT-PCR and a 68 kDa band on Western-blot analysis confirmed transcription and expression of recombinant hspX-ppe44-esxV in eukaryotic cells. Conclusions: A recombinant DNA segment encoding the HspX-PPE44-EsxV fusion antigen of M. tuberculosis was constructed and considered to be tested as a new TB DNA vaccine candidate

Mycobacteria encode five type VII secretion system (T7SS) or ESX for nutrient acquisition and virulence. Mycosins are membrane-anchored components of ESX with serine protease activity but an unidentified substrate range. Establishing the substrate specificity of individual mycosins will help to elucidate individual ESX functions. Mycosin-1 and -3 orthologues from two environmental mycobacterial species, Mycobacterium smegmatis and Mycobacterium thermoresistibile, have been heterologously produced, but mycosins from Mycobacterium tuberculosis (Mtb) remain to be studied. Here we describe the successful production of Mtb mycosin-3 as a first step in investigating its structure and function.

Tuberculosis remains one of the deadliest diseases. Emergence of drug-resistant and multidrug-resistant M. tuberculosis strains makes treating tuberculosis increasingly challenging. In order to develop novel intervention strategies, detailed understanding of the molecular mechanisms behind the success of this pathogen is required. Here, we review recent literature to provide a systems level overview of the molecular and cellular components involved in divalent metal homeostasis and their role in regulating the three main virulence strategies of M. tuberculosis: immune modulation, dormancy and phagosomal rupture. We provide a visual and modular overview of these components and their regulation. Our analysis identified a single regulatory cascade for these three virulence strategies that respond to limited availability of divalent metals in the phagosome. [ABSTRACT FROM AUTHOR]

Significant exploitation and utilization of cloud computing in industry is come with and in the identical time vulnerable by unease regarding protection of data hold by cloud computing providers. One of the penalties of moving data processing and storage off business site is that organizations have fewer controls over their infrastructure. seeing that, cloud service (CS) providers must hope that the CS provider is capable to protect their data and infrastructure from both exterior and domestic attacks. Presently however, such hope can only rely on organizational procedures stated by the CS provider and cannot be remotely verified and validated by an external party. The central distinction between cloud computing and conventional enterprise internal Information Technology services is that the proprietor and the consumer of cloud Information Technology infrastructures are separated in cloud. This transform requires a safety responsibility severance in cloud computing. Cloud service providers (CSP) should safe the services they propose and cannot surpass the customers' authorities. Virtualization is a buildup utterance in the Information Technology world. With the assure to reduce the ever mounting infrastructure inside data centers connected to other important apprehensions such as ease of use and scalability, virtualization technology has been in advance recognition not only with IT experts yet also among administrators and executives as well. The progressively more growing rate of the approval of this technology has exposed these systems to new protection concerns which in recent history have been unnoticed or merely overlooked. This paper presents an in depth state of art gaze at present most old server virtualization explanations, as well as a writing study on different security matters found inside this virtualization technology. These problems can be practical to all the existing virtualization technologies accessible with no spotlight on a specific answer. Nevertheless, we do susceptibility investigation of two of the mainstream recognized virtualization answers: VMware ESX and Xen. to conclude, we illustrate some clarifications on how to progress the security of online banking and electronic commerce, using virtualization. [ABSTRACT FROM AUTHOR]

Bacteria export proteins across membranes using a range of transport machineries. Type VII secretion systems (T7SSs), originally described in mycobacteria, are now known to be widespread across diverse bacterial phyla. Recent studies have characterized secretion components and mechanisms of type VII secretion in pathogenic and environmental bacteria. A variety of functions have been attributed to T7SS substrates, including interactions with eukaryotes and with other bacteria. Here, we evaluate the growing body of knowledge on T7SSs, with focus on the nonmycobacterial systems, reviewing their phylogenetic distribution, structure and function in diverse settings. [ABSTRACT FROM AUTHOR]

Mycobacteria have a distinct secretion system, termed type VII (T7SS), which is encoded byparalogous chromosomal loci (ESX) and associated with pathogenesis, conjugation, and metal homeostasis. Evolution of paralogous gene families is of interest because duplication is an importantmechanismbywhich novel genes evolve, but there are potential conflicts between adaptive forces that stabilize duplications and those that enable evolution of new functions. Our objective was to delineate the adaptive forces underlying diversification of T7SS. Plasmid-borne ESXwere described recently, and we found evidence that the initial duplication and divergence of ESX systems occurred on plasmids and was driven by selection for advantageo usmutations. Plasmid conjugation has been linked to T7SS and type IV secretion systems (T4SS) inmycobacteria, and we discovered that T7SS and T4SS genes evolved in concert on the plasmids. We hypothesize that differentiation of plasmid ESX helps to prevent conjugation among cells harboring incompatible plasmids. Plasmid ESX appear to have been repurposed following migration to the chromosome, and there is evidence of positive selection driving further differentiation of chromosomal ESX. We hypothesize that ESX loci were initially stabilized on the chromosome by mediating their own transfer. These results emphasize the diverse adaptive paths underlying evolution of novelty, which in this case involved plasmid duplications, selection for advantageous mutations in the mobile and core genomes, migration of the loci between plasmids and chromosomes, and lateral transfer among chromosomes. We discuss further implications for the choice of model organism to study ESX functions in Mycobacterium tuberculosis. [ABSTRACT FROM AUTHOR]

Background: Mycobacterium tuberculosis is the causative agent of tuberculosis (TB). Bacille Calmette- Guerin (BCG) vaccine, is not effective in adults, therefore, many efforts have been made to produce an effective adult TB vaccine. The aim of this study was to develop a new tuberculosis DNA vaccine candidate encoding a recombinant HspX-PPE44-EsxV fusion antigen of M. tuberculosis. Methods: A fusion DNA segment consisting of HspX, linker, PPE44, linker, and EsxV, after codon optimization, was designed. The fusion DNA was cloned and its sequence confirmed. Then, expression of a recombinant pcDNA3.1 (+)/HspX-PPE44-EsxV plasmid in Chinese hamster ovary (CHO) cells was verified by RT-PCR and Western-blot analysis. Results: A 1968 bp band in RT-PCR and a 68 kDa band on Western-blot analysis confirmed transcription and expression of recombinant hspX-ppe44-esxVin eukaryotic cells. Conclusions: A recombinant DNA segment encoding the HspX-PPE44-EsxV fusion antigen of M. tuberculosis was constructed and considered to be tested as a new TB DNA vaccine candidate. [ABSTRACT FROM AUTHOR]

Background: The genome of Mycobacterium tuberculosis contains five copies of the ESX gene cluster, each encoding a dedicated protein secretion system. These ESX secretion systems have been defined as a novel Type VII secretion machinery, responsible for the secretion of proteins across the characteristic outer mycomembrane of the mycobacteria. Some of these secretion systems are involved in virulence and survival in M. tuberculosis; however they are also present in other non-pathogenic mycobacteria, and have been identified in some non-mycobacterial actinomycetes. Three components of the ESX gene cluster have also been found clustered in some gram positive monoderm organisms and are predicted to have preceded the ESX gene cluster. Results: This study used in silico and phylogenetic analyses to describe the evolution of the ESX gene cluster from the WXG-FtsK cluster of monoderm bacteria to the five ESX clusters present in M. tuberculosis and other slowgrowing mycobacteria. The ancestral gene cluster, ESX-4, was identified in several nonmycomembrane producing actinobacteria as well as the mycomembrane-containing Corynebacteriales in which the ESX cluster began to evolve and diversify. A novel ESX gene cluster, ESX-4EVOL, was identified in some non-mycobacterial actinomycetes and M. abscessus subsp. bolletii. ESX-4EVOL contains all of the conserved components of the ESX gene cluster and appears to be a precursor of the mycobacterial ESX duplications. Between two and seven ESX gene clusters were identified in each mycobacterial species, with ESX-2 and ESX-5 specifically associated with the slow growers. The order of ESX duplication in the mycobacteria is redefined as ESX-4, ESX-3, ESX-1 and then ESX-2 and ESX-5. Plasmid-encoded precursor ESX gene clusters were identified for each of the genomic ESX-3, -1, -2 and -5 gene clusters, suggesting a novel plasmid-mediated mechanism of ESX duplication and evolution. Conclusions: The influence of the various ESX gene clusters on vital biological and virulence-related functions has clearly influenced the diversification and success of the various mycobacterial species, and their evolution from the non-pathogenic fast-growing saprophytic to the slow-growing pathogenic organisms. [ABSTRACT FROM AUTHOR]

Background: The ESX-1 type VII secretion system is an important determinant of virulence in pathogenic mycobacteria, including Mycobacterium tuberculosis. This complicated molecular machine secretes folded proteins through the mycobacterial cell envelope to subvert the host immune response. Despite its important role in disease very little is known about the molecular architecture of the ESX-1 secretion system. Results: This study characterizes the structures of the soluble domains of two conserved core ESX-1 components - EccB1 and EccD1. The periplasmic domain of EccB1 consists of 4 repeat domains and a central domain, which together form a quasi 2-fold symmetrical structure. The repeat domains of EccB1 are structurally similar to a known peptidoglycan binding protein suggesting a role in anchoring the ESX-1 system within the periplasmic space. The cytoplasmic domain of EccD1 has a ubiquitin-like fold and forms a dimer with a negatively charged groove. Conclusions: These structures represent a major step towards resolving the molecular architecture of the entire ESX-1 assembly and may contribute to ESX-1 targeted tuberculosis intervention strategies. [ABSTRACT FROM AUTHOR]

Mycobacterium tuberculosis secretes multiple virulence factors during infection via the general Sec and Tat pathways, and via specialized ESX secretion systems, also referred to as type VII secretion systems. The ESX-1 secretion system is an important virulence determinant because deletion of ESX-1 leads to attenuation of M. tuberculosis . ESX-1 secreted protein B (EspB) contains putative PE (Pro-Glu) and PPE (Pro-Pro-Glu) domains, and a C-terminal domain, which is processed by MycP 1 protease during secretion. We determined the crystal structure of PE–PPE domains of EspB, which represents an all-helical, elongated molecule closely resembling the structure of the PE25–PPE41 heterodimer despite limited sequence similarity. Also, we determined the structure of full-length EspB, which does not have interpretable electron density for the C-terminal domain confirming that it is largely disordered. Comparative analysis of EspB in cell lysate and culture filtrates of M. tuberculosis revealed that mature secreted EspB forms oligomers. Electron microscopy analysis showed that the N-terminal fragment of EspB forms donut-shaped particles. These data provide a rationale for the future investigation of EspB’s role in M. tuberculosis pathogenesis. [ABSTRACT FROM AUTHOR]

Mycobacterium tuberculosis has evolved from a Mycobacterium canettii-like progenitor pool into one of the most successful and widespread human pathogens. The pathogenicity of M. tuberculosis is linked to its ability to secrete/export/release selected mycobacterial proteins, and it is also established that active release of mycobacterial antigens is a prerequisite for strong immune recognition. Recent research has enabled mycobacterial secretion systems and vesicle-based release of mycobacterial antigens to be elucidated, which together with host-related specificities constitute key variables that determine the outcome of infection. Here, we discuss recently discovered, novel aspects on the nature and the regulation of antigen release of the tuberculosis agent with particular emphasis on the biological characterization of mycobacteria-specific ESX/type VII secretion systems and their secreted proteins, belonging to the Esx, PE, and PPE categories. The importance of specific mycobacterial antigen release is probably best exemplified by the striking differences observed between the cellular events during infection with the ESX-1-deficient, attenuated Mycobacterium bovis BCG compared to the virulent M. tuberculosis, which are clearly important for design of more specific diagnostics and more efficient vaccines. [ABSTRACT FROM AUTHOR]

Summary Iron is an essential element to most life forms including mycobacterial species. However, in the oxidative atmosphere iron exists as insoluble salts. Free and soluble iron ions are scarce in both the extracellular and intracellular environment which makes iron assimilation very challenging to mycobacteria. Tuberculosis, caused by the pathogen, Mycobacterium tuberculosis , is one of the most infectious and deadly diseases in the world. Extensive studies regarding iron acquisition strategies have been documented in mycobacteria, including work on the mycobacterial iron chelators (siderophores), the iron-responsive regulon, and iron transport and utilization pathways. Under low iron conditions, expression of the genes encoding iron importers, exporters and siderophore biosynthetic enzymes is up-regulated significantly increasing the ability of the bacteria to acquire limited host iron. Disabling these proteins impairs the growth of mycobacteria under low iron conditions both in vitro and in vivo , and that of pathogenic mycobacteria in animal models. Drugs targeting siderophore-mediated iron transport could offer promising therapeutic options. However, the discovery and characterization of an alternative iron acquisition mechanism, the heme transport and utilization pathway, questions the effectiveness of the siderophore-centered therapeutic strategy. Links have been found between these two distinct iron acquisition mechanisms, thus, targeting a few candidate proteins or mechanisms may influence both pathways, leading to effective elimination of the bacteria in the host. [ABSTRACT FROM AUTHOR]

The PowerPlex ® ESI 16 Fast, ESI 17 Fast, ESX 16 Fast, and ESX 17 Fast Systems represent faster cycling versions (50 min or less) of the PowerPlex ® ESI and ESX Systems released by Promega in 2009 to accommodate the ENFSI and EDNAP groups’ call for new STR multiplexes for Europe. In addition to amplification of purified DNA samples, these new faster cycling systems allow for direct amplification from single-source blood and buccal samples deposited on FTA ® and nonFTA paper as well as from SwabSolution™ extracts of buccal swabs without the need for purification and quantitation. There are no changes to the autosomal primer pair sequences in the PowerPlex ® ESI Fast and ESX Fast Systems compared to the original multiplexes, and full concordance at all autosomal loci and amelogenin was observed with data generated previously with the original PowerPlex ® ESI and ESX Systems. This paper describes the developmental validation study performed on these new fast systems following guidelines issued by the Scientific Working Group on DNA Analysis Methods (SWGDAM) and those of the DNA Advisory Board (DAB). Validation data demonstrate that these systems are sensitive for detecting low levels of DNA while also being capable of generating robust profiles from the high amount of input DNA present in direct-amplification samples. These systems are also tolerant to both high concentrations of PCR inhibitors as well as to slight variations in the final concentration of master mix and primer pair present in the amplification reaction that might be encountered due to pipetting error. The results of this validation study demonstrate that these systems may be used on multiple thermal cyclers and capillary electrophoresis platforms. [ABSTRACT FROM AUTHOR]

Abstract: Mycobacteria use specialized ESX secretion systems to transport proteins across their cell membranes in order to manipulate their environment. In pathogenic Mycobacterium tuberculosis there are five paralogous ESX secretion systems, named ESX-1 through ESX-5. Each system includes a subtilisin-like protease (mycosin or MycP) as a core component essential for secretion. Here we report crystal structures of MycP1 and MycP3, the mycosins expressed by the ESX-1 and ESX-3 systems, respectively. In both mycosins the putative propeptide wraps around the catalytic domain and does not occlude the active site. The extensive contacts between the putative propeptide and catalytic domain, which include a disulfide bond, suggest that the N-terminal extension is an integral part of the active mycosin. The catalytic residues of MycP1 and MycP3 are located in a deep active site groove in contrast with an exposed active site in majority of subtilisins. We show that MycP1 specifically cleaves ESX-1 secretion-associated protein B (EspB) in vitro at residues Ala358 and Ala386. We also systematically characterize the specificity of MycP1 using peptide libraries, and show that it has evolved a narrow specificity relative to other subtilisins. Finally, comparison of the MycP1 and MycP3 structures suggest that both enzymes have stringent and different specificity profiles that result from the structurally distinct active site pockets, which could explain the system specific functioning of these proteases. [Copyright &y& Elsevier]

Mycobacteria, such as the major human pathogen Mycobacterium tuberculosis, have a highly unusual and characteristic diderm cell envelope that protects them against harmful conditions. Protein secretion across this hydrophobic barrier requires specialized secretion systems. Recently, a type VII secretion (T7S) pathway has been identified that fulfills this function. Pathogenic mycobacteria have up to five different T7S systems, some of which play a crucial role in virulence. The interactions between secreted substrates and host molecules are only starting to become clear and will help in furthering our understanding of the persistence of these enigmatic pathogens. In this review, we discuss current knowledge on the role of T7S systems in mycobacterial virulence. [ABSTRACT FROM AUTHOR]

Virtualization has the potential to revolutionise the way networking is taught in higher learning institutions. This article outlines, through the use ofa case study, the way in which VMware Workstation and VMware ESX Server are used so that each student in the class runs his or her own set of seven virtual servers, including a Linux router. The Linux router connects virtual networks within the context of student physical machines to one another as well as a lecturer virtual server, which runs on an ESX server In this way, a class of twenty two students can together run an enterprise-like network comprising of some 177 servers and implement Microsofi Active Directory sites and associated services in order to optimize a specific scenario-based replication topology. [ABSTRACT FROM AUTHOR]

Summary: Mycobacterium tuberculosis is an important pathogen that infects approximately one-third of the world’s population and kills almost two million people annually. An important aspect of M. tuberculosis physiology and pathogenesis is its ability to export proteins into and across the thick mycobacterial cell envelope, where they are ideally positioned to interact with the host. In addition to the specific proteins that are exported by M. tuberculosis, the systems through which these proteins are exported represent potential targets for future drug development. M. tuberculosis possesses two well-known and conserved export systems: the housekeeping Sec pathway and the Tat pathway. In addition, M. tuberculosis possesses specialized export systems including the accessory SecA2 pathway and five ESX pathways. Here we review the current understanding of each of these export systems, with a focus on M. tuberculosis, and discuss the contribution of each system to disease and physiology. [Copyright &y& Elsevier]

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Abstract: Small molecules that mimic the transcriptional activation domain of eukaryotic transcriptional activators have the potential to serve as effective inhibitors of transcriptional processes. Here we show that one class of transcriptional activation domain mimics, amphipathic isoxazolidines, can be converted into inhibitors of gene expression mediated by the transcriptional activator ESX through small structural modifications. Addition of the small molecules leads to decreased expression of the cell surface growth receptor ErbB2(Her2) in ErbB2-positive cancer cells and, correspondingly, decreased proliferation. [Copyright &y& Elsevier]

The transcription factor Elf3, which is one of over 25 Ets family members, is expressed in a wide variety of carcinomas and has been shown to promote the transcription of many genes implicated in cancer. To understand how the Elf3 gene is regulated at the transcriptional level, we probed its 5′-flanking region, and we report here the identification of both proximal and distal regions that regulate murine Elf3 promoter activity. In addition to mapping the transcription start site of the Elf3 gene, the work described in this study identifies four cis-regulatory elements in the proximal promoter region of the gene. These include a cis-regulatory element previously designated ESE, a κB site, a POU motif, and a CCAAT box. In addition, we demonstrate that a novel 94 bp region 2 kb upstream of the transcription start site significantly elevates Elf3 promoter activity in F9-differentiated cells, but not in the parental F9 embryonal carcinoma (EC) cells. This region appears to be largely responsible for the increase in Elf3 promoter activity that accompanies the differentiation of embryonal carcinoma cells. [Copyright &y& Elsevier]