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1.  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
2.  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
3.  Secretion of atypical protein subtrates by the ESAT-6 Secretion System of Staphylococcus aureus 
Molecular microbiology  2013;90(4):734-743.
SUMMARY
Staphylococcus aureus encodes the specialized ESAT-6 Secretion System (ESS). EsxA and EsxB are secreted by the ESS pathway, and share sequence features of ESAT-6 and CFP-10 of the Type VII Secretion System (T7SS) of Mycobacterium tuberculosis. Unlike ESAT-6 and CFP-10, EsxA and EsxB do not interact. Instead, EsxB associates with a novel substrate, EsxD, and EsxA dimerizes with itself or EsxC (EsaC). Unlike EsxA and EsxB, EsxC and EsxD do not share obvious sequence features of WXG100 proteins nor PE/PPE and Esp families of proteins, all of which belong to the pfam EsxAB clan of mycobacterial T7SS. EsxD carries the C terminal motif YxxxD/E that has been proposed to target T7 substrates for secretion in mycobacteria. Here, we find that deletion, but not amino acid substitutions, in this motif prevent secretion of EsxA and EsxC but not EsxB or EsxD. This is unlike the genetic inactivation of esxA, esxB, esxC or esxD that leads to loss of secretion of all four substrates. Thus, substrate secretion can be uncoupled by deleting the last six amino acids of EsxD. The physical association of EsxC and EsxD with canonical WXG100 proteins suggests that these proteins belong to the EsxAB clan.
doi:10.1111/mmi.12395
PMCID: PMC3951145  PMID: 24033479
4.  Heterogeneity in ess transcriptional organization and variable contribution of the Ess/Type VII protein secretion system to virulence across closely related Staphylocccus aureus strains 
Molecular Microbiology  2014;93(5):928-943.
The Type VII protein secretion system, found in Gram-positive bacteria, secretes small proteins, containing a conserved W-x-G amino acid sequence motif, to the growth medium. Staphylococcus aureus has a conserved Type VII secretion system, termed Ess, which is dispensable for laboratory growth but required for virulence. In this study we show that there are unexpected differences in the organization of the ess gene cluster between closely related strains of S. aureus. We further show that in laboratory growth medium different strains of S. aureus secrete the EsxA and EsxC substrate proteins at different growth points, and that the Ess system in strain Newman is inactive under these conditions. Systematic deletion analysis in S. aureus RN6390 is consistent with the EsaA, EsaB, EssA, EssB, EssC and EsxA proteins comprising core components of the secretion machinery in this strain. Finally we demonstrate that the Ess secretion machinery of two S. aureus strains, RN6390 and COL, is important for nasal colonization and virulence in the murine lung pneumonia model. Surprisingly, however, the secretion system plays no role in the virulence of strain SA113 under the same conditions.
doi:10.1111/mmi.12707
PMCID: PMC4285178  PMID: 25040609
5.  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
6.  Characterization of Staphylococcus aureus EssB, an integral membrane component of the Type VII secretion system: atomic resolution crystal structure of the cytoplasmic segment 
Biochemical Journal  2012;449(Pt 2):469-477.
The Type VII protein translocation/secretion system, unique to Gram-positive bacteria, is a key virulence determinant in Staphylococcus aureus. We aim to characterize the architecture of this secretion machinery and now describe the present study of S. aureus EssB, a 52 kDa bitopic membrane protein essential for secretion of the ESAT-6 (early secretory antigenic target of 6 kDa) family of proteins, the prototypic substrate of Type VII secretion. Full-length EssB was heterologously expressed in Escherichia coli, solubilized from the bacterial membrane, purified to homogeneity and shown to be dimeric. A C-terminal truncation, EssB∆C, and two soluble fragments termed EssB-N and EssB-C, predicted to occur on either side of the cytoplasmic membrane, have been successfully purified in a recombinant form, characterized and, together with the full-length protein, used in crystallization trials. EssB-N, the 25 kDa N-terminal cytoplasmic fragment, gave well-ordered crystals and we report the structure, determined by SAD (single-wavelength anomalous diffraction) targeting an SeMet (selenomethionine) derivative, refined to atomic (1.05 Å; 1 Å=0.1 nm) resolution. EssB-N is dimeric in solution, but crystallizes as a monomer and displays a fold comprised of two globular domains separated by a cleft. The structure is related to that of serine/threonine protein kinases and the present study identifies that the Type VII secretion system exploits and re-uses a stable modular entity and fold that has evolved to participate in protein–protein interactions in a similar fashion to the catalytically inert pseudokinases.
doi:10.1042/BJ20121209
PMCID: PMC3526858  PMID: 23098276
early secretory antigenic target of 6 kDa system 1 (ESX-1); Gram-positive bacterium; protein kinase; protein secretion; pseudokinase; X-ray crystallography; BAP, biotin-acceptor peptide; BCG, Bacille Calmette–Guérin; BN-PAGE, Blue native PAGE; CV, column volume; DDM, dodecyl maltoside; DOPC, 1,2-dioleoyl-sn-glycero-3-phosphocholine; DTT, dithiothreitol; ESAT-6, early secreted antigenic target of 6 kDa; ESI–Q–TOF-MS, electrospray ionization–quadrupole–time-of-flight MS; ESX-1, ESAT-6 system 1; ess, ESX-1 secretion system; IPTG, isopropyl-β-D-thiogalactopyranoside; LB, Luria–Bertani; MALDI–TOF-MS, matrix-assisted laser-desorption ionization–time-of-flight MS; MWCO, molecular-mass cut-off; PEG3350, poly(ethylene glycol) 3350; rmsd, root-mean-square deviation; SAD, single-wavelength anomalous diffraction; SeMet, selenomethionine; SPR, surface plasmon resonance; TEV, tobacco etch virus; T7SS, Type VII secretion system
7.  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
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.  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
10.  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
11.  Structure of Staphylococcus aureus EsxA suggests a contribution to virulence by action as a transport chaperone and/or adaptor protein 
Journal of molecular biology  2008;383(3):603-614.
Staphylococcus aureus pathogenesis depends on a specialized protein secretion system, ESX-1, that delivers a range of virulence factors to assist infectivity. We report the characterization of two such factors, EsxA and EsxB; small acidic dimeric proteins carrying a distinctive WXG motif. EsxA crystallized in triclinic and monoclinic forms and high-resolution structures were determined. The asymmetric unit of each crystal form is a dimer. The EsxA subunit forms an elongated cylindrical structure created from side-by-side α-helices linked with a hairpin bend formed by the WXG motif. Approximately 25% of the solvent accessible surface area of each subunit is involved in interactions, predominantly hydrophobic, with the partner subunit. Secondary structure predictions suggest that EsxB displays a similar structure. The WXG motif helps to create a shallow cleft at each end of the dimer, forming a short β-sheet-like feature with an N-terminal segment of the partner subunit. Structural and sequence comparisons, exploiting biological data on related proteins found in Mycobacteria tuberculosis suggest that this family of proteins may contribute to pathogenesis by transporting protein cargo through the ESX-1 system exploiting a C-terminal secretion signal and / or are capable of acting as adaptor proteins to facilitate interactions with host receptor proteins.
doi:10.1016/j.jmb.2008.08.047
PMCID: PMC3465917  PMID: 18773907
adaptor protein; chaperone; helical bundle; secretion system; virulence factor
12.  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
13.  A Novel ESX-1 Locus Reveals that Surface-Associated ESX-1 Substrates Mediate Virulence in Mycobacterium marinum 
Journal of Bacteriology  2014;196(10):1877-1888.
EsxA (ESAT-6) and EsxB (CFP-10) are virulence factors exported by the ESX-1 system in mycobacterial pathogens. In Mycobacterium marinum, an established model for ESX-1 secretion in Mycobacterium tuberculosis, genes required for ESX-1 export reside at the extended region of difference 1 (RD1) locus. In this study, a novel locus required for ESX-1 export in M. marinum was identified outside the RD1 locus. An M. marinum strain bearing a transposon-insertion between the MMAR_1663 and MMAR_1664 genes exhibited smooth-colony morphology, was deficient for ESX-1 export, was nonhemolytic, and was attenuated for virulence. Genetic complementation revealed a restoration of colony morphology and a partial restoration of virulence in cell culture models. Yet hemolysis and the export of ESX-1 substrates into the bacteriological medium in vitro as measured by both immunoblotting and quantitative proteomics were not restored. We show that genetic complementation of the transposon insertion strain partially restored the translocation of EsxA and EsxB to the mycobacterial cell surface. Our findings indicate that the export of EsxA and EsxB to the cell surface, rather than secretion into the bacteriological medium, correlates with virulence in M. marinum. Together, these findings not only expand the known genetic loci required for ESX-1 secretion in M. marinum but also provide an explanation for the observed disparity between in vitro ESX-1 export and virulence.
doi:10.1128/JB.01502-14
PMCID: PMC4011007  PMID: 24610712
14.  The Architecture of EssB, an Integral Membrane Component of the Type VII Secretion System 
Structure(London, England:1993)  2013;21(4):595-603.
Summary
The membrane-bound EssB is an integral and essential component of the bacterial type VII secretion system that can contribute to pathogenicity. The architecture of Geobacillus thermodenitrificans EssB has been investigated by combining crystallographic and EPR spectroscopic methods. The protein forms a dimer that straddles the cytoplasmic membrane. A helical fold is observed for the C-terminal segment, which is positioned on the exterior of the membrane. This segment contributes most to dimer formation. The N-terminal segment displays a structure related to the pseudokinase fold and may contribute to function by recognizing substrates or secretion system partners. The remaining part of EssB may serve as an anchor point for the secretion apparatus, which is embedded in the cytoplasmic membrane with the C-terminal domain protruding out to interact with partner proteins or components of peptidoglycan.
Graphical Abstract
Highlights
► EssB, a membrane-bound component of the type VII secretion system, forms a dimer ► The cytoplasmic segment EssB-N is remarkably similar to pseudokinases ► The extracellular C-terminal domain displays a helical fold ► PELDOR spectroscopy enabled construction of a model of the complete dimer
The architecture of EssB, an essential component of the type VII secretion system, derived from crystallographic and EPR methods by Zoltner et al., reveals a few surprises: a protein that forms a dimer, an extracellular domain with a novel helical fold, and a cytoplasmic segment with remarkable similarity to pseudokinases.
doi:10.1016/j.str.2013.02.007
PMCID: PMC3694306  PMID: 23499020
15.  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
16.  Mycobacterial Esx-3 Requires Multiple Components for Iron Acquisition 
mBio  2014;5(3):e01073-14.
ABSTRACT
The type VII secretion systems are conserved across mycobacterial species and in many Gram-positive bacteria. While the well-characterized Esx-1 pathway is required for the virulence of pathogenic mycobacteria and conjugation in the model organism Mycobacterium smegmatis, Esx-3 contributes to mycobactin-mediated iron acquisition in these bacteria. Here we show that several Esx-3 components are individually required for function under low-iron conditions but that at least one, the membrane-bound protease MycP3 of M. smegmatis, is partially expendable. All of the esx-3 mutants tested, including the ΔmycP3ms mutant, failed to export the native Esx-3 substrates EsxHms and EsxGms to quantifiable levels, as determined by targeted mass spectrometry. Although we were able to restore low-iron growth to the esx-3 mutants by genetic complementation, we found a wide range of complementation levels for protein export. Indeed, minute quantities of extracellular EsxHms and EsxGms were sufficient for iron acquisition under our experimental conditions. The apparent separation of Esx-3 function in iron acquisition from robust EsxGms and EsxHms secretion in the ΔmycP3ms mutant and in some of the complemented esx-3 mutants compels reexamination of the structure-function relationships for type VII secretion systems.
IMPORTANCE
Mycobacteria have several paralogous type VII secretion systems, Esx-1 through Esx-5. Whereas Esx-1 is required for pathogenic mycobacteria to grow within an infected host, Esx-3 is essential for growth in vitro. We and others have shown that Esx-3 is required for siderophore-mediated iron acquisition. In this work, we identify individual Esx-3 components that contribute to this process. As in the Esx-1 system, most mutations that abolish Esx-3 protein export also disrupt its function. Unexpectedly, however, ultrasensitive quantitation of Esx-3 secretion by multiple-reaction-monitoring mass spectrometry (MRM-MS) revealed that very low levels of export were sufficient for iron acquisition under similar conditions. Although protein export clearly contributes to type VII function, the relationship is not absolute.
doi:10.1128/mBio.01073-14
PMCID: PMC4010830  PMID: 24803520
17.  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
18.  A Newly Discovered Mycobacterial Pathogen Isolated from Laboratory Colonies of Xenopus Species with Lethal Infections Produces a Novel Form of Mycolactone, the Mycobacterium ulcerans Macrolide Toxin  
Infection and Immunity  2005;73(6):3307-3312.
Mycobacterium ulcerans, the causative agent of Buruli ulcer, produces a macrolide toxin, mycolactone A/B, which is thought to play a major role in virulence. A disease similar to Buruli ulcer recently appeared in United States frog colonies following importation of the West African frog, Xenopus tropicalis. The taxonomic position of the frog pathogen has not been fully elucidated, but this organism, tentatively designated Mycobacterium liflandii, is closely related to M. ulcerans and Mycobacterium marinum, and as further evidence is gathered, it will most likely be considered a subspecies of one of these species. In this paper we show that M. liflandii produces a novel plasmid-encoded mycolactone, mycolactone E. M. liflandii contains all of the genes in the mycolactone cluster with the exception of that encoding CYP140A2, a putative p450 monooxygenase. Although the core lactone structure is conserved in mycolactone E, the fatty acid side chain differs from that of mycolactone A/B in the number of hydroxyl groups and double bonds. The cytopathic phenotype of mycolactone E is identical to that of mycolactone A/B, although it is less potent. To further characterize the relationship between M. liflandii and M. ulcerans, strains were analyzed for the presence of the RD1 region genes, esxA (ESAT-6) and esxB (CFP-10). The M. ulcerans genome strain has a deletion in RD1 and lacks these genes. The results of these studies show that M. liflandii contains both esxA and esxB.
doi:10.1128/IAI.73.6.3307-3312.2005
PMCID: PMC1111873  PMID: 15908356
19.  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
20.  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
21.  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
22.  High Levels of DegU-P Activate an Esat-6-Like Secretion System in Bacillus subtilis 
PLoS ONE  2013;8(7):e67840.
The recently discovered Type VII/Esat-6 secretion systems seem to be widespread among bacteria of the phyla Actinobacteria and Firmicutes. In some species they play an important role in pathogenic interactions with eukaryotic hosts. Several studies have predicted that the locus yukEDCByueBC of the non-pathogenic, Gram-positive bacterium Bacillus subtilis would encode an Esat-6-like secretion system (Ess). We provide here for the first time evidences for the functioning of this secretion pathway in an undomesticated B. subtilis strain. We show that YukE, a small protein with the typical features of the secretion substrates from the WXG100 superfamily is actively secreted to culture media. YukE secretion depends on intact yukDCByueBC genes, whose products share sequence or structural homology with known components of the S. aureus Ess. Biochemical characterization of YukE indicates that it exists as a dimer both in vitro and in vivo. We also show that the B. subtilis Ess essentially operates in late stationary growth phase in absolute dependence of phosphorylated DegU, the response regulator of the two-component system DegS-DegU. We present possible reasons that eventually have precluded the study of this secretion system in the B. subtilis laboratory strain 168.
doi:10.1371/journal.pone.0067840
PMCID: PMC3701619  PMID: 23861817
23.  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
24.  Computational Analysis of the ESX-1 Region of Mycobacterium tuberculosis: Insights into the Mechanism of Type VII Secretion System 
PLoS ONE  2011;6(11):e27980.
Type VII secretion system (T7SS) is a recent discovery in bacterial secretion systems. First identified in Mycobacterium tuberculosis, this secretion system has later been reported in organisms belonging to the Actinomycetales order and even to distant phyla like Firmicutes. The genome of M. tuberculosis H37Rv contains five gene clusters that have evolved through gene duplication events and include components of the T7SS secretion machinery. These clusters are called ESAT-6 secretion system (ESX) 1 through 5. Out of these, ESX-1 has been the most widely studied region because of its pathological importance. In spite of this, the overall mechanism of protein translocation through ESX-1 secretion machinery is not clearly understood. Specifically, the structural components contributing to the translocation through the mycomembrane have not been characterized yet. In this study, we have carried out a comprehensive in silico analysis of the genes known to be involved in ESX-1 secretion pathway and identified putative proteins having high probability to be associated with this particular pathway. Our study includes analysis of phylogenetic profiles, identification of domains, transmembrane helices, 3D folds, signal peptides and prediction of protein-protein associations. Based on our analysis, we could assign probable novel functions to a few of the ESX-1 components. Additionally, we have identified a few proteins with probable role in the initial activation and formation of mycomembrane translocon of ESX-1 secretion machinery. We also propose a probable working model of T7SS involving ESX-1 secretion pathway.
doi:10.1371/journal.pone.0027980
PMCID: PMC3227618  PMID: 22140496
25.  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

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