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1.  Conservation of Structure and Protein-Protein Interactions Mediated by the Secreted Mycobacterial Proteins EsxA, EsxB, and EspA▿ †  
Journal of Bacteriology  2009;192(1):326-335.
Mycobacterium tuberculosis EsxA and EsxB proteins are founding members of the WXG100 (WXG) protein family, characterized by their small size (∼100 amino acids) and conserved WXG amino acid motif. M. tuberculosis contains 11 tandem pairs of WXG genes; each gene pair is thought to be coexpressed to form a heterodimer. The precise role of these proteins in the biology of M. tuberculosis is unknown, but several of the heterodimers are secreted, which is important for virulence. However, WXG proteins are not simply virulence factors, since nonpathogenic mycobacteria also express and secrete these proteins. Here we show that three WXG heterodimers have structures and properties similar to those of the M. tuberculosis EsxBA (MtbEsxBA) heterodimer, regardless of their host species and apparent biological function. Biophysical studies indicate that the WXG proteins from M. tuberculosis (EsxG and EsxH), Mycobacterium smegmatis (EsxA and EsxB), and Corynebacterium diphtheriae (EsxA and EsxB) are heterodimers and fold into a predominately α-helical structure. An in vivo protein-protein interaction assay was modified to identify proteins that interact specifically with the native WXG100 heterodimer. MtbEsxA and MtbEsxB were fused into a single polypeptide, MtbEsxBA, to create a biomimetic bait for the native heterodimer. The MtbEsxBA bait showed specific association with several esx-1-encoded proteins and EspA, a virulence protein secreted by ESX-1. The MtbEsxBA fusion peptide was also utilized to identify residues in both EsxA and EsxB that are important for establishing protein interactions with Rv3871 and EspA. Together, the results are consistent with a model in which WXG proteins perform similar biological roles in virulent and nonvirulent species.
doi:10.1128/JB.01032-09
PMCID: PMC2798242  PMID: 19854905
2.  Characterization of EssB, a protein required for secretion of ESAT-6 like proteins in Staphylococcus aureus  
BMC Microbiology  2012;12:219.
Background
Staphylococcus aureus secretes EsxA and EsxB, two small polypeptides of the WXG100 family of proteins. Genetic analyses have shown that production and secretion of EsxA and EsxB require an intact ESAT-6 Secretion System (ESS), a cluster of genes that is conserved in many Firmicutes and encompasses esxA and esxB . Here, we characterize EssB, one of the proteins encoded by the ESS cluster. EssB is highly conserved in Gram-positive bacteria and belongs to the Cluster of Orthologous Groups of protein COG4499 with no known function.
Results
By generating an internal deletion in essB , we demonstrate that EssB is required for secretion of EsxA. We use a polyclonal antibody to identify EssB and show that the protein fractionates with the plasma membrane of S. aureus . Yet, when produced in Escherichia coli, EssB remains mostly soluble and the purified protein assembles into a highly organized oligomer that can be visualized by electron microscopy. Production of truncated EssB variants in wild-type S. aureus confers a dominant negative phenotype on EsxA secretion.
Conclusions
The data presented here support the notion that EssB may oligomerize and interact with other membrane components to form the WXG100-specific translocon in S. aureus .
doi:10.1186/1471-2180-12-219
PMCID: PMC3489787  PMID: 23006124
ESAT-6 secretion; ESS; WXG100; EssB; Type 7 secretion; Staphylococcus aureus
3.  ESAT-6-Like Protein Secretion in Bacillus anthracis▿ † 
Journal of Bacteriology  2008;190(21):7004-7011.
Proteins of the WXG100 family represent the prototypical substrates of bacterial type VII secretion systems that typically encompass 100 residues, lack canonical signal peptides, and form helix-turn-helix hairpin structures with WXG positioned in the turn element. Bacillus anthracis encodes six WXG100 proteins, herein referred to as EsxB, EsxL, EsxP, EsxQ, EsxV, and EsxW. With the exception of EsxB, B. anthracis proteins harbor C-terminal extensions that are appended to canonical WXG domains. When cultured in liquid broth, B. anthracis secretes two substrates, EsxB and EsxW, into the extracellular environment. EsxB is required for the stability and secretion of EsxW; however, EsxW is dispensable for EsxB secretion. In agreement with the hypothesis that EsxB binding to substrates promotes recognition and secretion by the type VII pathway, EsxB is reported to interact with EsxB and EsxW. Unlike deletions in mycobacterial EsxB, deletion of five N- or C-terminal residues does not affect the ability of mutant B. anthracis EsxB to travel the type VII pathway and initiate secretion of EsxW. Translational fusion of ubiquitin to the N or C terminus of EsxB also had no effect, while ubiquitin insertion into the center turn abrogated secretion. Anthrax-infected guinea pigs mounted humoral immune responses to EsxB, EsxP, and EsxW, which suggests that B. anthracis activates the type VII secretion pathway during infection.
doi:10.1128/JB.00458-08
PMCID: PMC2580693  PMID: 18723613
4.  EsaD, a Secretion Factor for the Ess Pathway in Staphylococcus aureus▿  
Journal of Bacteriology  2011;193(7):1583-1589.
Staphylococcus aureus encodes the Sec-independent Ess secretion pathway, an ortholog of mycobacterial T7 secretion systems which is required for the virulence of this Gram-positive microbe. The Ess (ESX secretion) pathway was previously defined as a genomic cluster of eight genes, esxA, esaA, essA, essB, esaB, essC, esaC, and esxB. essABC encode membrane proteins involved in the stable expression of esxA, esxB, and esaC, genes specifying three secreted polypeptide substrates. esaB, which encodes a small cytoplasmic protein, represses the synthesis of EsaC but not that of EsxA and EsxB. Here we investigated a hitherto uncharacterized gene, esaD, located downstream of esxB. Expression of esaD is activated by mutations in esaB and essB. EsaD, the 617-amino-acid product of esaD, is positioned in the membrane and is also accessible to EsaD-specific antibodies on the bacterial surface. S. aureus mutants lacking esaD are defective in the secretion of EsxA. Following intravenous inoculation of mice, S. aureus esaD mutants generate fewer abscesses with a reduced bacterial load compared to wild-type parent strain Newman. The chromosomes of Listeria and Bacillus species with Ess pathways also harbor esaD homologues downstream of esxB, suggesting that the contributory role of EsaD in Ess secretion may be shared among Gram-positive pathogens.
doi:10.1128/JB.01096-10
PMCID: PMC3067666  PMID: 21278286
5.  WXG100 Protein Superfamily Consists of Three Subfamilies and Exhibits an α-Helical C-Terminal Conserved Residue Pattern 
PLoS ONE  2014;9(2):e89313.
Members of the WXG100 protein superfamily form homo- or heterodimeric complexes. The most studied proteins among them are the secreted T-cell antigens CFP-10 (10 kDa culture filtrate protein, EsxB) and ESAT-6 (6 kDa early secreted antigen target, EsxA) from Mycobacterium tuberculosis. They are encoded on an operon within a gene cluster, named as ESX-1, that encodes for the Type VII secretion system (T7SS). WXG100 proteins are secreted in a full-length form and it is known that they adopt a four-helix bundle structure. In the current work we discuss the evolutionary relationship between the homo- and heterodimeric WXG100 proteins, the basis of the oligomeric state and the key structural features of the conserved sequence pattern of WXG100 proteins. We performed an iterative bioinformatics analysis of the WXG100 protein superfamily and correlated this with the atomic structures of the representative WXG100 proteins. We find, firstly, that the WXG100 protein superfamily consists of three subfamilies: CFP-10-, ESAT-6- and sagEsxA-like proteins (EsxA proteins similar to that of Streptococcus agalactiae). Secondly, that the heterodimeric complexes probably evolved from a homodimeric precursor. Thirdly, that the genes of hetero-dimeric WXG100 proteins are always encoded in bi-cistronic operons and finally, by combining the sequence alignments with the X-ray data we identify a conserved C-terminal sequence pattern. The side chains of these conserved residues decorate the same side of the C-terminal α-helix and therefore form a distinct surface. Our results lead to a putatively extended T7SS secretion signal which combines two reported T7SS recognition characteristics: Firstly that the T7SS secretion signal is localized at the C-terminus of T7SS substrates and secondly that the conserved residues YxxxD/E are essential for T7SS activity. Furthermore, we propose that the specific α-helical surface formed by the conserved sequence pattern including YxxxD/E motif is a key component of T7SS-substrate recognition.
doi:10.1371/journal.pone.0089313
PMCID: PMC3935865  PMID: 24586681
6.  The homodimeric GBS1074 from Streptococcus agalactiae  
The homodimeric nature of the ESAT-6 homologue GBS1074 and the potential for fibre-like assemblies are revealed by the 2 Å resolution crystal structure.
ESAT-6 is a well characterized secreted protein from Mycobacterium tuberculosis and represents the archetype of the WXG100 family of proteins. Genes encoding ESAT-6 homologues have been identified in the genome of the human pathogen Streptococcus agalactiae; one of these genes, esxA, has been cloned and the recombinant protein has been crystallized. In contrast to M. tuberculosis ESAT-6, the crystal structure of GBS1074 reveals a homo­dimeric structure similar to homologous structures from Staphylococcus aureus and Helicobacter pylori. Intriguingly, GBS1074 forms elongated fibre-like assemblies in the crystal structure.
doi:10.1107/S1744309110036286
PMCID: PMC3001639  PMID: 21045286
ESAT-6; WXG100; GBS1074; four-helix bundle
7.  EsaC substrate for the ESAT-6 Secretion Pathway and its role in persistent infections of S. aureus 
Molecular microbiology  2008;69(3):736-746.
Summary
Staphylococcus aureus encodes the specialized secretion system Ess (ESAT-6 secretion system). The ess locus is a cluster of eight genes (esxAB, essABC, esaABC) of which esxA and esxB display homology to secreted ESAT-6 proteins of Mycobacterium tuberculosis. EsxA and EsxB require EssA, EssB and EssC for transport across the staphylococcal envelope. Herein, we examine the role of EsaB and EsaC and show that EsaB is a negative regulator of EsaC. Further, EsaC production is repressed when staphylococci are grown in broth and increased when staphylococci replicate in serum or infected hosts. EsaB is constitutively produced and remains in the cytoplasm whereas EsaC is secreted. This secretion requires an intact Ess pathway. Mutants lacking esaB or esaC display only a small defect in acute infection, but remarkably are unable to promote persistent abscesses during animal infection. Together, the data suggest a model whereby EsaB controls the production of effector molecules that are important for host pathogen interaction. One such effector, EsaC, is a secretion substrate of the Ess pathway and implements its pathogenic function during infection.
doi:10.1111/j.1365-2958.2008.06324.x
PMCID: PMC2597432  PMID: 18554323
WXG100 proteins; virulence; abscess formation
8.  Opposing roles of σB and σB-controlled SpoVG in the global regulation of esxA in Staphylococcus aureus 
BMC Microbiology  2012;12:17.
Background
The production of virulence factors in Staphylococcus aureus is tightly controlled by a complex web of interacting regulators. EsxA is one of the virulence factors that are excreted by the specialized, type VII-like Ess secretion system of S. aureus. The esxA gene is part of the σB-dependent SpoVG subregulon. However, the mode of action of SpoVG and its impact on other global regulators acting on esxA transcription is as yet unknown.
Results
We demonstrate that the transcription of esxA is controlled by a regulatory cascade involving downstream σB-dependent regulatory elements, including the staphylococcal accessory regulator SarA, the ArlRS two-component system and SpoVG. The esxA gene, preceding the ess gene cluster, was shown to form a monocistronic transcript that is driven by a σA promoter, whereas a putative σB promoter identified upstream of the σA promoter was shown to be inactive. Transcription of esxA was strongly upregulated upon either sarA or sigB inactivation, but decreased in agr, arlR and spoVG single mutants, suggesting that agr, ArlR and SpoVG are able to increase esxA transcription and relieve the repressing effect of the σB-controlled SarA on esxA.
Conclusion
SpoVG is a σB-dependent element that fine-tunes the expression of esxA by counteracting the σB-induced repressing activity of the transcriptional regulator SarA and activates esxA transcription.
doi:10.1186/1471-2180-12-17
PMCID: PMC3313859  PMID: 22272815
9.  The ESX System in Bacillus subtilis Mediates Protein Secretion 
PLoS ONE  2014;9(5):e96267.
Esat-6 protein secretion systems (ESX or Ess) are required for the virulence of several human pathogens, most notably Mycobacterium tuberculosis and Staphylococcus aureus. These secretion systems are defined by a conserved FtsK/SpoIIIE family ATPase and one or more WXG100 family secreted substrates. Gene clusters coding for ESX systems have been identified amongst many organisms including the highly tractable model system, Bacillus subtilis. In this study, we demonstrate that the B. subtilis yuk/yue locus codes for a nonessential ESX secretion system. We develop a functional secretion assay to demonstrate that each of the locus gene products is specifically required for secretion of the WXG100 virulence factor homolog, YukE. We then employ an unbiased approach to search for additional secreted substrates. By quantitative profiling of culture supernatants, we find that YukE may be the sole substrate that depends on the FtsK/SpoIIIE family ATPase for secretion. We discuss potential functional implications for secretion of a unique substrate.
doi:10.1371/journal.pone.0096267
PMCID: PMC4010439  PMID: 24798022
10.  EsxA might as a virulence factor induce antibodies in patients with Staphylococcus aureus infection 
Brazilian Journal of Microbiology  2013;44(1):267-271.
Staphylococcus aureus (S. aureus) is an important human pathogen, which commonly causes the acquired infectious diseases in the hospital and community. Effective and simple antibiotic treatment against S. aureus-related disease becomes increasingly difficult. Developing a safe and effective vaccine against S. aureus has become one of the world’s hot spots once again. The key issue of developing the vaccine of S. aureus is how to find an ideal key pathogenic gene of S. aureus. It was previously suggested that EsxA might be a very important factor in S. aureus abscess formation in mice, but clinical experimental evidence was lacking. We therefore expressed EsxA protein through prokaryotic expression system and purified EsxA protein by Ni-affinity chromatography. ELISA was used to detect the anti-EsxA antibodies in sera of 78 patients with S. aureus infection and results showed that the anti-EsxA antibodies were positive in the sera of 19 patients. We further analyzed the EsxA positive antibodies related strains by antimicrobial susceptibility assay and found that all of the corresponding strains were multi-drug resistant. Among those multi-drug resistant strains, 73.7% were resistant to MRSA. The results indicated EsxA is very important in the pathogenesis of S. aureus. We suggested that the EsxA is very valuable as vaccine candidate target antigens for prevention and control of S. aureus infection.
doi:10.1590/S1517-83822013005000019
PMCID: PMC3804208  PMID: 24159314
S. aureus; esxA; anti-EsxA antibodies; multi-drug resistant
11.  A β-Lactamase Based Reporter System for ESX Dependent Protein Translocation in Mycobacteria 
PLoS ONE  2012;7(4):e35453.
Protein secretion is essential for all bacteria in order to interact with their environment. Mycobacterium tuberculosis depends on protein secretion to subvert host immune response mechanisms. Both the general secretion system (Sec) and the twin-arginine translocation system (Tat) are functional in mycobacteria. Furthermore, a novel type of protein translocation system named ESX has been identified. In the genome of M. tuberculosis five paralogous ESX regions (ESX-1 to ESX-5) have been found. Several components of the ESX translocation apparatus have been identified over the last ten years. The ESX regions are composed of a basic set of genes for the translocation machinery and the main substrate - a heterodimer. The best studied of these heterodimers is EsxA (ESAT-6)/EsxB (CFP-10), which has been shown to be exported by ESX-1. EsxA/B is heavily involved in virulence of M. tuberculosis. EsxG/H is exported by ESX-3 and seems to be involved in an essential iron-uptake mechanism in M. tuberculosis. These findings make ESX-3 components high profile drug targets. Until now, reporter systems for determination of ESX protein translocation have not been developed. In order to create such a reporter system, a truncated β-lactamase (‘bla TEM-1) was fused to the N-terminus of EsxB, EsxG and EsxU, respectively. These constructs have then been tested in a β-lactamase (BlaS) deletion strain of Mycobacterium smegmatis. M. smegmatis ΔblaS is highly susceptible to ampicillin. An ampicillin resistant phenotype was conferred by translocation of Bla TEM-1-Esx fusion proteins into the periplasm. BlaTEM-1-Esx fusion proteins were not found in the culture filtrate suggesting that plasma membrane translocation and outer membrane translocation are two distinct steps in ESX secretion. Thus we have developed a powerful tool to dissect the molecular mechanisms of ESX dependent protein translocation and to screen for novel components of the ESX systems on a large scale.
doi:10.1371/journal.pone.0035453
PMCID: PMC3329429  PMID: 22530024
12.  Independent Loss of Immunogenic Proteins in Mycobacterium ulcerans Suggests Immune Evasion▿ †  
The highly immunogenic mycobacterial proteins ESAT-6, CFP-10, and HspX represent potential target antigens for the development of subunit vaccines and immunodiagnostic tests. Recently, the complete genome sequence revealed the absence of these coding sequences in Mycobacterium ulcerans, the causative agent of the emerging human disease Buruli ulcer. Genome reduction and the acquisition of a cytopathic and immunosuppressive macrolide toxin plasmid are regarded as crucial for the emergence of this pathogen from its environmental progenitor, Mycobacterium marinum. Earlier, we have shown the evolution of M. ulcerans into two distinct lineages. Here, we show that while the genome of M. marinum M contains two copies of the esxB-esxA gene cluster at different loci (designated MURD4 and MURD152), both copies are deleted from the genome of M. ulcerans strains belonging to the classical lineage. Members of the ancestral lineage instead retained some but disrupted most functional MURD4 or MURD152 copies, either by newly identified genomic insertion-deletion events or by conversions of functional genes to pseudogenes via point mutations. Thus, the esxA (ESAT-6), esxB (CFP-10), and hspX genes are located in hot-spot regions for genomic variation where functional disruption seems to be favored by selection pressure. Our detailed genomic analyses have identified a variety of independent genomic changes that have led to the loss of expression of functional ESAT-6, CFP-10, and HspX proteins. Loss of these immunodominant proteins helps the bacteria bypass the host's immunological response and may represent part of an ongoing adaptation of M. ulcerans to survival in host environments that are screened by immunological defense mechanisms.
doi:10.1128/CVI.00472-07
PMCID: PMC2292651  PMID: 18256209
13.  Solution Structure of the Mycobacterium tuberculosis EsxG·EsxH Complex 
The Journal of Biological Chemistry  2011;286(34):29993-30002.
Mycobacterium tuberculosis encodes five type VII secretion systems that are responsible for exporting a number of proteins, including members of the Esx family, which have been linked to tuberculosis pathogenesis and survival within host cells. The gene cluster encoding ESX-3 is regulated by the availability of iron and zinc, and secreted protein products such as the EsxG·EsxH complex have been associated with metal ion acquisition. EsxG and EsxH have previously been shown to form a stable 1:1 heterodimeric complex, and here we report the solution structure of the complex, which features a core four-helix bundle decorated at both ends by long, highly flexible, N- and C-terminal arms that contain a number of highly conserved residues. Despite clear similarities in the overall backbone fold to the EsxA·EsxB complex, the structure reveals some striking differences in surface features, including a potential protein interaction site on the surface of the EsxG·EsxH complex. EsxG·EsxH was also found to contain a specific Zn2+ binding site formed from a cluster of histidine residues on EsxH, which are conserved across obligate mycobacterial pathogens including M. tuberculosis and Mycobacterium leprae. This site may reflect an essential role in zinc ion acquisition or point to Zn2+-dependent regulation of its interaction with functional partner proteins. Overall, the surface features of both the EsxG·EsxH and the EsxA·EsxB complexes suggest functions mediated via interactions with one or more target protein partners.
doi:10.1074/jbc.M111.248732
PMCID: PMC3191040  PMID: 21730061
Bacteria; NMR; Protein Structure; Secretion; Zinc; EsxG/EsxH; Pathogenesis; Tuberculosis
14.  EspD Is Critical for the Virulence-Mediating ESX-1 Secretion System in Mycobacterium tuberculosis 
Journal of Bacteriology  2012;194(4):884-893.
ESAT-6 system 1 (ESX-1)-mediated secretion in Mycobacterium tuberculosis is dependent on proteins encoded by the cotranscribed espA-espC-espD gene cluster. While the roles of EspA and EspC with respect to the ESX-1 secretion system have been actively investigated, the function of EspD remains unknown. We show that EspD is secreted by M. tuberculosis, but unlike EspA and EsxA, its export does not exclusively require the ESX-1 system. Evidence for stabilization of cellular levels of EspA and EspC by EspD is presented, and depletion of EspD results in loss of EsxA secretion. Site-directed mutagenesis of EspD reveals that its role in the maintenance of cellular levels of EspA in M. tuberculosis is distinct from its facilitation of EsxA secretion. The same mutagenesis experiments have also shown that secretion of EspD is not required for the secretion of EsxA. Our findings highlight a critical and complex role for EspD in modulating the ESX-1 secretion system in M. tuberculosis.
doi:10.1128/JB.06417-11
PMCID: PMC3272943  PMID: 22155774
15.  EspA Acts as a Critical Mediator of ESX1-Dependent Virulence in Mycobacterium tuberculosis by Affecting Bacterial Cell Wall Integrity 
PLoS Pathogens  2010;6(6):e1000957.
Mycobacterium tuberculosis (Mtb) requires the ESX1 specialized protein secretion system for virulence, for triggering cytosolic immune surveillance pathways, and for priming an optimal CD8+ T cell response. This suggests that ESX1 might act primarily by destabilizing the phagosomal membrane that surrounds the bacterium. However, identifying the primary function of the ESX1 system has been difficult because deletion of any substrate inhibits the secretion of all known substrates, thereby abolishing all ESX1 activity. Here we demonstrate that the ESX1 substrate EspA forms a disulfide bonded homodimer after secretion. By disrupting EspA disulfide bond formation, we have dissociated virulence from other known ESX1-mediated activities. Inhibition of EspA disulfide bond formation does not inhibit ESX1 secretion, ESX1-dependent stimulation of the cytosolic pattern receptors in the infected macrophage or the ability of Mtb to prime an adaptive immune response to ESX1 substrates. However, blocking EspA disulfide bond formation severely attenuates the ability of Mtb to survive and cause disease in mice. Strikingly, we show that inhibition of EspA disulfide bond formation also significantly compromises the stability of the mycobacterial cell wall, as does deletion of the ESX1 locus or individual components of the ESX1 system. Thus, we demonstrate that EspA is a major determinant of ESX1-mediated virulence independent of its function in ESX1 secretion. We propose that ESX1 and EspA play central roles in the virulence of Mtb in vivo because they alter the integrity of the mycobacterial cell wall.
Author Summary
From studies of BCG, the tuberculosis vaccine, we know that Mycobacterium tuberculosis requires a specialized protein secretion system, ESX1, to cause disease in people. ESX1 is required for Mtb to co-opt the host cells in which the bacterium resides and it is thought that this explains its central role in virulence. However, other data suggests that ESX1 serves an important role in the bacterium itself, altering the organism's cell wall. It has been difficult to determine the relative significance of these ESX1-associated functions, however, because deletion of any piece of the apparatus completely abolishes all ESX1 activities. Here we use a simple approach to pinpoint the functionally significant target of one of the proteins secreted by ESX1, EspA. We mutate EspA such that the ESX1 system still secretes its substrates but the bacterium no longer causes disease. The attenuated EspA mutant has defects in its cell wall but not in its interactions with host cells in vitro. We propose that the ESX1 system and the proteins it secretes are important for Mtb to survive and cause disease in people because they act to ensure the integrity of the bacterial cell wall.
doi:10.1371/journal.ppat.1000957
PMCID: PMC2891827  PMID: 20585630
16.  Assessment of the Genetic Diversity of Mycobacterium tuberculosis esxA, esxH, and fbpB Genes among Clinical Isolates and Its Implication for the Future Immunization by New Tuberculosis Subunit Vaccines Ag85B-ESAT-6 and Ag85B-TB10.4 
The effort to develop a tuberculosis (TB) vaccine more effective than the widely used Bacille Calmette-Guérin (BCG) has led to the development of two novel fusion protein subunit vaccines: Ag85B-ESAT-6 and Ag85B-TB10.4. Studies of these vaccines in animal models have revealed their ability to generate protective immune responses. Yet, previous work on TB fusion subunit vaccine candidate, Mtb72f, has suggested that genetic diversity among M. tuberculosis strains may compromise vaccine efficacy. In this study, we sequenced the esxA, esxH, and fbpB genes of M. tuberculosis encoding ESAT-6, TB10.4, and Ag85B proteins, respectively, in a sample of 88 clinical isolates representing 57 strains from Ark, USA, and 31 strains from Turkey, to assess the genetic diversity of the two vaccine candidates. We found no DNA polymorphism in esxA and esxH genes in the study sample and only one synonymous single nucleotide change (C to A) in fbpB gene among 39 (44.3%) of the 88 strains sequenced. These data suggest that it is unlikely that the efficacy of Ag85B-ESAT-6 and Ag85B-TB10.4 vaccines will be affected by the genetic diversity of M. tuberculosis population. Future studies should include a broader pool of M. tuberculosis strains to validate the current conclusion.
doi:10.1155/2010/208371
PMCID: PMC2896655  PMID: 20617139
17.  ESAT-6 (EsxA) and TB10.4 (EsxH) Based Vaccines for Pre- and Post-Exposure Tuberculosis Vaccination 
PLoS ONE  2013;8(12):e80579.
The ESX systems from Mycobacterium tuberculosis are responsible for the secretion of highly immunogenic proteins of key importance for bacterial survival and growth. The two prototypic proteins, ESAT-6 (EsxA from ESX-1) and TB10.4 (EsxH from ESX-3) share a lot of characteristics regarding genome organization, size, antigenic properties, and vaccine potential but the two molecules clearly have very different roles in bacterial physiology. To further investigate the role of ESAT-6 and TB10.4 as preventive and post-exposure tuberculosis vaccines, we evaluated four different fusion-protein vaccines; H1, H4, H56 and H28, that differ only in these two components. We found that all of these vaccines give rise to protection in a conventional prophylactic vaccination model. In contrast, only the ESAT-6-containing vaccines resulted in significant protection against reactivation, when administered post-exposure. This difference in post-exposure activity did not correlate with a difference in gene expression during infection or a differential magnitude or quality of the vaccine-specific CD4 T cells induced by ESAT-6 versus TB10.4-containing vaccines. The post-exposure effect of the ESAT-6 based vaccines was found to be influenced by the infectious load at the time-point of vaccination and was abolished in chronically infected animals with high bacterial loads at the onset of vaccination. Our data demonstrate that there are specific requirements for the immune system to target an already established tuberculosis infection and that ESAT-6 has a unique potential in post-exposure vaccination strategies.
doi:10.1371/journal.pone.0080579
PMCID: PMC3861245  PMID: 24349004
18.  ESX-1 Secreted Virulence Factors Are Recognized by Multiple Cytosolic AAA ATPases in Pathogenic Mycobacteria 
Molecular microbiology  2009;73(5):950-962.
Summary
The ESX-1 secretion system of M. 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. However, the selection determinants for other ESX-1 substrates appear to be more complex. Some substrates, such as ESAT-6, are secreted despite lacking signal sequences. Furthermore, all substrates require targeting of the other ESX-1 secreted proteins, a distinguishing feature of this system. How ESX-1 substrates are selected and the basis for co-dependent secretion is unknown. Here we show that the EspC substrate interacts with Rv3868, a cytosolic AAA ATPase, through its C-terminus. Swapping the C-termini of EspC and CFP-10 revealed that these signals are functionally distinct, suggesting that the proteins are targeted via interactions with different ATPases. Surprisingly, biochemical purification experiments demonstrate that these substrates and ATPases form multi-protein complexes inside the cell and identified a new secreted substrate. By interfering with this protein interaction network, we have partially uncoupled co-dependent substrate secretion. Our results suggest that proper functioning of the ESX-1 pathway requires the interaction of multiple ESX-1 substrates and components prior to their secretion. Ultimately, understanding the details of ESX-1 targeting may allow for engineering of better vaccines to prevent tuberculosis.
doi:10.1111/j.1365-2958.2009.06821.x
PMCID: PMC3023814  PMID: 19682254
19.  Dissection of ESAT-6 System 1 of Mycobacterium tuberculosis and Impact on Immunogenicity and Virulence  
Infection and Immunity  2006;74(1):88-98.
The dedicated secretion system ESX-1 of Mycobacterium tuberculosis encoded by the extended RD1 region (extRD1) assures export of the ESAT-6 protein and its partner, the 10-kDa culture filtrate protein CFP-10, and is missing from the vaccine strains M. bovis BCG and M. microti. Here, we systematically investigated the involvement of each individual ESX-1 gene in the secretion of both antigens, specific immunogenicity, and virulence. ESX-1-complemented BCG and M. microti strains were more efficiently engulfed by bone-marrow-derived macrophages than controls, and this may account for the enhanced in vivo growth of ESX-1-carrying strains. Inactivation of gene pe35 (Rv3872) impaired expression of CFP-10 and ESAT-6, suggesting a role in regulation. Genes Rv3868, Rv3869, Rv3870, Rv3871, and Rv3877 encoding an ATP-dependent chaperone and translocon were essential for secretion of ESAT-6 and CFP-10 in contrast to ppe68 Rv3873 and Rv3876, whose inactivation did not impair secretion of ESAT-6. A strict correlation was found between ESAT-6 export and the generation of ESAT-6 specific T-cell responses in mice. Furthermore, ESAT-6 secretion and specific immunogenicity were almost always correlated with enhanced virulence in the SCID mouse model. Only loss of Rv3865 and part of Rv3866 did not affect ESAT-6 secretion or immunogenicity but led to attenuation. This suggests that Rv3865/66 represent a new virulence factor that is independent from ESAT-6 secretion. The present study has allowed us to identify new aspects of the extRD1 region of M. tuberculosis and to explore its role in the pathogenesis of tuberculosis.
doi:10.1128/IAI.74.1.88-98.2006
PMCID: PMC1346617  PMID: 16368961
20.  Heterologous Expression of Mycobacterial Esx Complexes in Escherichia coli for Structural Studies Is Facilitated by the Use of Maltose Binding Protein Fusions 
PLoS ONE  2013;8(11):e81753.
The expression of heteroligomeric protein complexes for structural studies often requires a special coexpression strategy. The reason is that the solubility and proper folding of each subunit of the complex requires physical association with other subunits of the complex. The genomes of pathogenic mycobacteria encode many small protein complexes, implicated in bacterial fitness and pathogenicity, whose characterization may be further complicated by insolubility upon expression in Escherichia coli, the most common heterologous protein expression host. As protein fusions have been shown to dramatically affect the solubility of the proteins to which they are fused, we evaluated the ability of maltose binding protein fusions to produce mycobacterial Esx protein complexes. A single plasmid expression strategy using an N-terminal maltose binding protein fusion to the CFP-10 homolog proved effective in producing soluble Esx protein complexes, as determined by a small-scale expression and affinity purification screen, and coupled with intracellular proteolytic cleavage of the maltose binding protein moiety produced protein complexes of sufficient purity for structural studies. In comparison, the expression of complexes with hexahistidine affinity tags alone on the CFP-10 subunits failed to express in amounts sufficient for biochemical characterization. Using this strategy, six mycobacterial Esx complexes were expressed, purified to homogeneity, and subjected to crystallization screening and the crystal structures of the Mycobacterium abscessus EsxEF, M. smegmatis EsxGH, and M. tuberculosis EsxOP complexes were determined. Maltose binding protein fusions are thus an effective method for production of Esx complexes and this strategy may be applicable for production of other protein complexes.
doi:10.1371/journal.pone.0081753
PMCID: PMC3843698  PMID: 24312350
21.  Host-Detrimental Role of Esx-1-Mediated Inflammasome Activation in Mycobacterial Infection 
PLoS Pathogens  2010;6(5):e1000895.
The Esx-1 (type VII) secretion system is a major virulence determinant of pathogenic mycobacteria, including Mycobacterium marinum. However, the molecular events and host-pathogen interactions underlying Esx-1-mediated virulence in vivo remain unclear. Here we address this problem in a non-lethal mouse model of M. marinum infection that allows detailed quantitative analysis of disease progression. M. marinum established local infection in mouse tails, with Esx-1-dependent formation of caseating granulomas similar to those formed in human tuberculosis, and bone deterioration reminiscent of skeletal tuberculosis. Analysis of tails infected with wild type or Esx-1-deficient bacteria showed that Esx-1 enhanced generation of proinflammatory cytokines, including the secreted form of IL-1β, suggesting that Esx-1 promotes inflammasome activation in vivo. In vitro experiments indicated that Esx-1-dependent inflammasome activation required the host NLRP3 and ASC proteins. Infection of wild type and ASC-deficient mice demonstrated that Esx-1-dependent inflammasome activation exacerbated disease without restricting bacterial growth, indicating a host-detrimental role of this inflammatory pathway in mycobacterial infection. These findings define an immunoregulatory role for Esx-1 in a specific host-pathogen interaction in vivo, and indicate that the Esx-1 secretion system promotes disease and inflammation through its ability to activate the inflammasome.
Author Summary
With ∼2 million people dying from tuberculosis every year, Mycobacterium tuberculosis represents the single most important bacterial pathogen globally. We use the closely related Mycobacterium marinum to study fundamental aspects of mycobacterial pathogenesis, likely to extend to human tuberculosis. The Esx-1 (type VII) secretion system is a major virulence determinant of pathogenic mycobacteria, including M. tuberculosis and M. marinum. However, a molecular explanation for Esx-1-mediated virulence in vivo has been lacking. Here we address this problem in a non-lethal mouse model of M. marinum infection that allows quantitative analysis of disease progression. M. marinum established local infection with important features of human tuberculosis, including formation of granulomas with caseating centers. Using a combination of bacterial and host mutants, we show that Esx-1-mediated activation of the host inflammasome increases inflammation without restricting bacterial growth, suggesting that activation of the inflammasome during mycobacterial infection is a manifestation of bacterial virulence rather than a manifestation of host response. These findings define a biological role for Esx-1 in a specific host-pathogen interaction in vivo, and imply that the Esx-1 secretion system has evolved specifically to promote host pathology.
doi:10.1371/journal.ppat.1000895
PMCID: PMC2865529  PMID: 20463815
22.  Molecular Assays for Determining Mycobacterium leprae Viability in Tissues of Experimentally Infected Mice 
Background
The inability of Mycobacterium leprae to grow on axenic media has necessitated specialized techniques in order to determine viability of this organism. The purpose of this study was to develop a simple and sensitive molecular assay for determining M. leprae viability directly from infected tissues.
Methodology/Principle Findings
Two M. leprae-specific quantitative reverse transcription PCR (qRT-PCR) assays based on the expression levels of esxA, encoding the ESAT-6 protein, and hsp18, encoding the heat shock 18 kDa protein, were developed and tested using infected footpad (FP) tissues of both immunocompetent and immunocompromised (athymic nu/nu) mice. In addition, the ability of these assays to detect the effects of anti-leprosy drug treatment on M. leprae viability was determined using rifampin and rifapentine, each at 10 mg/kg for 1, 5, or 20 daily doses, in the athymic nu/nu FP model. Molecular enumeration (RLEP PCR) and viability determinations (qRT-PCR) were performed via Taqman methodology on DNA and RNA, respectively, purified from ethanol-fixed FP tissue and compared with conventional enumeration (microscopic counting of acid fast bacilli) and viability assays (radiorespirometry, viability staining) which utilized bacilli freshly harvested from the contralateral FP. Both molecular and conventional assays demonstrated growth and high viability of M. leprae in nu/nu FPs over a 4 month infection period. In contrast, viability was markedly decreased by 8 weeks in immunocompetent mice. Rifapentine significantly reduced bacterial viability after 5 treatments, whereas rifampin required up to 20 treatments for the same efficacy. Neither drug was effective after a single treatment. In addition, host gene expression was monitored with the same RNA preparations.
Conclusions
hsp18 and esxA qRT-PCR are sensitive molecular indicators, reliably detecting viability of M. leprae in tissues without the need for bacterial isolation or immediate processing, making these assays applicable for in vivo drug screening and promising for clinical and field applications.
Author Summary
M. leprae, the causative agent of leprosy, cannot be grown on laboratory culture media. This characteristic, along with its extremely long generation time of 12–14 days, makes the study of the pathogenicity of this organism and the experimental testing of new drugs for the treatment of leprosy extremely difficult. We developed two M. leprae-specific quantitative reverse transcription PCR assays and tested their utility as biological markers of M. leprae viability in tissue specimens. These assays could detect high viability of bacilli growing in immunosuppressed mice as well as the inhibitory effects of anti-leprosy drug treatment, or of the host immune system in immunocompetent mice. The RNA preparations were also successfully used for detection of host gene expression. The application of these assays to various experimental models would benefit characterization of the infection or novel drug screening. Furthermore, because these assays utilize fixed tissues, their potential application to clinical and field settings could enable monitoring of M. leprae viability in conjunction with the host immune response during treatment.
doi:10.1371/journal.pntd.0002404
PMCID: PMC3750008  PMID: 24179562
23.  Mycobacterium tuberculosis MycP1 protease plays a dual role in regulation of ESX-1 secretion and virulence 
Cell host & microbe  2010;7(3):210-220.
SUMMARY
Mycobacterium tuberculosis uses the ESX-1 secretion system to deliver virulence proteins during infection of host cells. Here we report a novel post-transcriptional control mechanism of ESX-1 mediated by MycP1, a serine protease. We show that MycP1 is required for ESX-1 secretion and has unusual substrate specificity. Unexpectedly, inhibition of protease activity increases secretion of ESX-1 substrates. We demonstrate that EspB, an ESX-1 substrate required for secretion, is a target of MycP1 in vitro and in vivo. During macrophage infection, an inactive MycP1 protease mutant causes hyper-activation of ESX-1 stimulated innate signaling pathways. MycP1 is required for growth in mice during acute infection, while protease inhibition leads to attenuated virulence during chronic infection. As the key ESX-1 substrates ESAT-6 and CFP-10 are highly immunogenic, fine-tuning of their secretion by MycP1 may balance virulence and immune detection and be essential for successful maintenance of long-term M. tuberculosis infection.
doi:10.1016/j.chom.2010.02.006
PMCID: PMC3121311  PMID: 20227664
24.  Cloning, purification, crystallization and preliminary X-ray analysis of ESX-1-secreted protein regulator (EspR) from Mycobacterium tuberculosis  
ESX-1 secreted protein regulator (EspR, Rv3849) from M. tuberculosis has been purified and crystallized, and diffracted to 3.2 Å resolution at wavelength 0.97625 Å.
ESX-1-secreted protein regulator (EspR; Rv3849) is a key regulator in Mycobacterium tuberculosis that delivers bacterial proteins into the host cell during infection. EspR binds directly to the Rv3616c-Rv3614c promoter and activates transcription and secretes itself from the bacterial cell by the ESX-1 system. The three-dimensional structure of EspR will aid in understanding the mechanisms by which it binds to the Rv3616c-Rv3614c promoter and is involved in transcriptional activation. This study will significantly aid in the development of EspR-based therapeutics against M. tuberculosis. The full-length EspR gene from M. tuberculosis (H37Rv strain) was cloned and overexpressed as a soluble protein in Escherichia coli. The protein was purified by affinity chromatography using His-tagged protein followed by size-exclusion chromatography. EspR was crystallized using polyethylene glycol 3350 as precipitant. The crystals diffracted to 3.2 Å resolution using synchrotron radiation of wavelength 0.97625 Å. The crystal belonged to space group P3121 and contained three monomers in the asymmetric unit. Native and heavy-atom-derivatized data sets were collected from EspR crystals for use in ab initio structure-solution techniques.
doi:10.1107/S174430911004618X
PMCID: PMC3079979  PMID: 21206031
ESX-1-secreted protein regulator; Rv3849; Mycobacterium tuberculosis
25.  Secreted transcription factor controls Mycobacterium tuberculosis virulence 
Nature  2008;454(7205):717-721.
Bacterial pathogens trigger specialized virulence factor secretion systems on encountering host cells. The ESX-1 protein secretion system of Mycobacterium tuberculosis—the causative agent of the human disease tuberculosis—delivers bacterial proteins into host cells during infection and is critical for virulence, but how it is regulated is unknown. Here we show that EspR (also known as Rv3849) is a key regulator of ESX-1 that is required for secretion and virulence in mice. EspR activates transcription of an operon that includes three ESX-1 components, Rv3616c–Rv3614c, whose expression in turn promotes secretion of ESX-1 substrates. EspR directly binds to and activates the Rv3616c–Rv3614c promoter and, unexpectedly, is itself secreted from the bacterial cell by the ESX-1 system that it regulates. Efflux of the DNA-binding regulator results in reduced Rv3616c–Rv3614c transcription, and thus reduced ESX-1 secretion. Our results reveal a direct negative feedback loop that regulates the activity of a secretion system essential for virulence. As the virulence factors secreted by the ESX-1 system are highly antigenic, fine control of secretion may be critical to successful infection.
doi:10.1038/nature07219
PMCID: PMC2862998  PMID: 18685700

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