结核分枝杆菌ESX-1分泌蛋白EspB结构和功能分析

结核分枝杆菌作为肺结核病的病原菌,在人类中致死率远高于其他病原菌.结核分枝杆菌具有特殊的疏水性细胞壁结构,这种致密的细胞壁结构帮助结核分枝杆菌抵御外界环境压力和来自宿主细胞的毒素.同时,它利用特殊的分泌系统将体内的毒力蛋白输出体外,ESX-1分泌系统就是其中之一.结核分枝杆菌ESX-1系统在结核分枝杆菌进入宿主细胞吞噬小体、逃逸至细胞质以及杀死吞噬细胞这些过程中发挥重要作用.研究表明,在结核分枝杆菌内膜上存在一个由多亚基组成、旨在帮助结核分枝杆菌向外输送分泌蛋白的分泌装置.在这个分泌装置的帮助下,结核分枝杆菌重要的毒力蛋白ESAT-6跨内膜向外分泌,EspB也通过这个内膜上的分泌装置被转运至胞外.EspB存在于静置培养的结核分枝杆菌的胶囊层中,也可在振荡培养的结核分枝杆菌的培养液中被检测.通过X射线晶体衍射分析,我们解析了EspB的晶体结构,相比于其他同源结构,发现了EspB的不同构象,即EspB单体能够自组装成为七聚体的规则结构,联系其与毒力因子ESAT-6具有共分泌的特点,七聚体构象的发现为解释EspB在结核分枝杆菌向外分泌蛋白的过程中发挥的作用提供线索,即EspB具有锚定在结核分枝杆菌胶囊层中,作为运输ESAT-6的孔道而存在的可能.     

A Novel ESX-1 Locus Reveals that Surface-Associated ESX-1 Substrates Mediate Virulence in Mycobacterium marinum

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.     

The ESAT-6 Protein of Mycobacterium tuberculosis Interacts with Beta-2-Microglobulin (β2M) Affecting Antigen Presentation Function of Macrophage

ESAT-6, an abundantly secreted protein of Mycobacterium tuberculosis (M. tuberculosis) is an important virulence factor, inactivation of which leads to reduced virulence of M. tuberculosis. ESAT-6 alone, or in complex with its chaperone CFP-10 (ESAT-6:CFP-10), is known to modulate host immune responses; however, the detailed mechanisms are not well understood. The structure of ESAT-6 or ESAT-6:CFP-10 complex does not suggest presence of enzymatic or DNA-binding activities. Therefore, we hypothesized that the crucial role played by ESAT-6 in the virulence of mycobacteria could be due to its interaction with some host cellular factors. Using a yeast two-hybrid screening, we identified that ESAT-6 interacts with the host protein beta-2-microglobulin (β2M), which was further confirmed by other assays, like GST pull down, co-immunoprecipitation and surface plasmon resonance. The C-terminal six amino acid residues (90–95) of ESAT-6 were found to be essential for this interaction. ESAT-6, in complex with CFP-10, also interacts with β2M. We found that ESAT-6/ESAT-6:CFP-10 can enter into the endoplasmic reticulum where it sequesters β2M to inhibit cell surface expression of MHC-I-β2M complexes, resulting in downregulation of class I-mediated antigen presentation. Interestingly, the ESAT-6:β2M complex could be detected in pleural biopsies of individuals suffering from pleural tuberculosis. Our data highlight a novel mechanism by which M. tuberculosis may undermine the host adaptive immune responses to establish a successful infection. Identification of such novel interactions may help us in designing small molecule inhibitors as well as effective vaccine design against tuberculosis.     

A mycobacterium ESX-1-secreted virulence factor with unique requirements for export

Specialized secretion systems of pathogenic bacteria commonly transport multiple effectors that act in concert to control and exploit the host cell as a replication-permissive niche. Both the Mycobacterium marinum and the Mycobacterium tuberculosis genomes contain an extended region of difference 1 (extRD1) locus that encodes one such pathway, the early secretory antigenic target 6 (ESAT-6) system 1 (ESX-1) secretion apparatus. ESX-1 is required for virulence and for secretion of the proteins ESAT-6, culture filtrate protein 10 (CFP-10), and EspA. Here, we show that both Rv3881c and its M. marinum homolog, Mh3881c, are secreted proteins, and disruption of RD1 in either organism blocks secretion. We have renamed the Rv3881c/Mh3881c gene espB for ESX-1 substrate protein B. Secretion of M. marinum EspB (EspBM) requires both the Mh3879c and Mh3871 genes within RD1, while CFP-10 secretion is not affected by disruption of Mh3879c. In contrast, disruption of Mh3866 or Mh3867 within the extRD1 locus prevents CFP-10 secretion without effect on EspBM. Mutants that fail to secrete only EspBM or only CFP-10 are less attenuated in macrophages than mutants failing to secrete both substrates. EspBM physically interacts with Mh3879c; the M. tuberculosis homolog, EspBT, physically interacts with Rv3879c; and mutants of EspBM that fail to bind Mh3879c fail to be secreted. We also found interaction between Rv3879c and Rv3871, a component of the ESX-1 machine, suggesting a mechanism for the secretion of EspB. The results establish EspB as a substrate of ESX-1 that is required for virulence and growth in macrophages and suggests that the contribution of ESX-1 to virulence may arise from the secretion of multiple independent substrates.     

A unique Mycobacterium ESX-1 protein co-secretes with CFP-10/ESAT-6 and is necessary for inhibiting phagosome maturation

The ESX-1 secretion system plays a critical role in the virulence of Mycobacterium tuberculosis and M. marinum. To date, three proteins are known to be secreted by ESX-1 and necessary for virulence, two of which are CFP-10 and ESAT-6. The ESX-1 secretion and the virulence mechanisms are not well understood. In this study, we have examined the M. marinum secretomes and identified four proteins specific to ESX-1. Two of those are CFP-10 and ESAT-6, and the other two are novel: MM1553 (homologous to Rv3483c) and Mh3881c (homologous to Rv3881c). We have shown that Mh3881c, CFP-10 and ESAT-6 are co-dependent for secretion. Mh3881c is being cleaved at close to the C-terminus during secretion, and the C-terminal portion is critical to the co-dependent secretion, the ESAT-6 cellular levels, and interaction with ESAT-6. The co-dependent secretion is required for M. marinum intracellular growth in macrophages, where the Mh3881c C-terminal portion plays a critical role. The role of the co-dependent secretion in intracellular growth correlates with its role in inhibiting phagosome maturation. Both the secretion and the virulence defects of the Mh3881c mutant are complemented by Mh3881c or its M. tuberculosis homologue Rv3881c, suggesting that in M. tuberculosis, Rv3881c has similar functions.     

ESAT-6 from Mycobacterium tuberculosis dissociates from its putative chaperone CFP-10 under acidic conditions and exhibits membrane-lysing activity

The 6-kDa early secreted antigenic target ESAT-6 and the 10-kDa culture filtrate protein CFP-10 of Mycobacterium tuberculosis are secreted by the ESX-1 system into the host cell and thereby contribute to pathogenicity. Although different studies performed at the organismal and cellular levels have helped to explain ESX-1-associated phenomena, not much is known about how ESAT-6 and CFP-10 contribute to pathogenesis at the molecular level. In this study we describe the interaction of both proteins with lipid bilayers, using biologically relevant liposomal preparations containing dimyristoylphosphatidylcholine (DMPC), dimyristoylphosphatidylglycerol, and cholesterol. Using flotation gradient centrifugation, we demonstrate that ESAT-6 showed strong association with liposomes, and in particular with preparations containing DMPC and cholesterol, whereas the interaction of CFP-10 with membranes appeared to be weaker and less specific. Most importantly, binding to the biomembranes no longer occurred when the proteins were present as a 1:1 ESAT-6.CFP-10 complex. However, lowering of the pH resulted in dissociation of the protein complex and subsequent protein-liposome interaction. Finally, cryoelectron microscopy revealed that ESAT-6 destabilized and lysed liposomes, whereas CFP-10 did not. In conclusion, we propose that one of the main features of ESAT-6 in the infection process of M. tuberculosis is the interaction with biomembranes that occurs after dissociation from its putative chaperone CFP-10 under acidic conditions typically encountered in the phagosome.     

EspD is critical for the virulence-mediating ESX-1 secretion system in Mycobacterium tuberculosis

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.     

A protein linkage map of the ESAT-6 secretion system 1 (ESX-1) of Mycobacterium tuberculosis

Tuberculosis is a chronic infectious disease caused by bacteria of the Mycobacterium tuberculosis complex. One of the major contributors to virulence and intercellular spread of M. tuberculosis is the ESAT-6 secretion system 1 (ESX-1) that has been lost by the live vaccines Mycobacterium bovis BCG (Bacille Calmette Guérin) and Mycobacterium microti as a result of independent deletions. ESX-1 consists of at least 10 genes (Rv3868-Rv3877) encoding the T-cell antigens ESAT-6 and CFP-10 as well as AAA-ATPases, chaperones, and membrane proteins which probably form a novel export system. To better understand the mode of action of the ESX-1 proteins, as a prelude to drug development, we examined systematically the interactions between the various proteins using the two-hybrid system in Saccharomyces cerevisiae. Interestingly, ESAT-6 and CFP-10 formed both hetero- and homodimers. Moreover, Rv3866, Rv3868, and CFP-10 interacted with Rv3873 which also homodimerized. The data were summarized in a protein linkage map that is consistent with the model for the secretion apparatus and can be used as a basis to identify inhibitors of specific interactions.     

Signal Regulatory Protein α (SIRPα)+ Cells in the Adaptive Response to ESAT-6/CFP-10 Protein of Tuberculous Mycobacteria

Background

Early secretory antigenic target-6 (ESAT-6) and culture filtrate protein-10 (CFP-10) are co-secreted proteins of Mycobacterium tuberculosis complex mycobacteria (includes M. bovis, the zoonotic agent of bovine tuberculosis) involved in phagolysosome escape of the bacillus and, potentially, in the efficient induction of granulomas. Upon tuberculosis infection, multi-nucleate giant cells are elicited, likely as a response aimed at containing mycobacteria. In tissue culture models, signal regulatory protein (SIRP)α (also referred to as macrophage fusion receptor or CD172a) is essential for multi-nucleate giant cell formation.

Methodology/Principal Findings

In the present study, ESAT-6/CFP-10 complex and SIRPα interactions were evaluated with samples obtained from calves experimentally infected with M. bovis. Peripheral blood CD172a⁺ (SIRPα-expressing) cells from M. bovis-infected calves proliferated upon in vitro stimulation with ESAT-6/CFP-10 (either as a fusion protein or a peptide cocktail), but not with cells from animals receiving M. bovis strains lacking ESAT-6/CFP-10 (i.e, M. bovis BCG or M. bovis ΔRD1). Sorted CD172a⁺ cells from these cultures had a dendritic cell/macrophage morphology, bound fluorescently-tagged rESAT-6:CFP-10, bound and phagocytosed live M. bovis BCG, and co-expressed CD11c, DEC-205, CD44, MHC II, CD80/86 (a subset also co-expressed CD11b or CD8α). Intradermal administration of rESAT-6:CFP-10 into tuberculous calves elicited a delayed type hypersensitive response consisting of CD11c⁺, CD172a⁺, and CD3⁺ cells, including CD172a-expressing multi-nucleated giant cells.

Conclusions/Significance

These findings demonstrate the ability of ESAT-6/CFP-10 to specifically expand CD172a⁺ cells, bind to CD172a⁺ cells, and induce multi-nucleated giant cells expressing CD172a.     

ESX-1 secreted virulence factors are recognized by multiple cytosolic AAA ATPases in pathogenic mycobacteria

The ESX-1 secretion system of Mycobacterium tuberculosis delivers bacterial virulence factors to host cells during infection. The most abundant factor, the ESAT-6/CFP-10 dimer, is targeted for secretion via a C-terminal signal sequence on CFP-10 that is recognized by the cytosolic ATPase, Rv3871. 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.     

Phagosomal Rupture by Mycobacterium tuberculosis Results in Toxicity and Host Cell Death

Survival within macrophages is a central feature of Mycobacterium tuberculosis pathogenesis. Despite significant advances in identifying new immunological parameters associated with mycobacterial disease, some basic questions on the intracellular fate of the causative agent of human tuberculosis in antigen-presenting cells are still under debate. To get novel insights into this matter, we used a single-cell fluorescence resonance energy transfer (FRET)-based method to investigate the potential cytosolic access of M. tuberculosis and the resulting cellular consequences in an unbiased, quantitative way. Analysis of thousands of THP-1 macrophages infected with selected wild-type or mutant strains of the M. tuberculosis complex unambiguously showed that M. tuberculosis induced a change in the FRET signal after 3 to 4 days of infection, indicating phagolysosomal rupture and cytosolic access. These effects were not seen for the strains M. tuberculosisΔRD1 or BCG, both lacking the ESX-1 secreted protein ESAT-6, which reportedly shows membrane-lysing properties. Complementation of these strains with the ESX-1 secretion system of M. tuberculosis restored the ability to cause phagolysosomal rupture. In addition, control experiments with the fish pathogen Mycobacterium marinum showed phagolysosomal translocation only for ESX-1 intact strains, further validating our experimental approach. Most importantly, for M. tuberculosis as well as for M. marinum we observed that phagolysosomal rupture was followed by necrotic cell death of the infected macrophages, whereas ESX-1 deletion- or truncation-mutants that remained enclosed within phagolysosomal compartments did not induce such cytotoxicity. Hence, we provide a novel mechanism how ESX-1 competent, virulent M. tuberculosis and M. marinum strains induce host cell death and thereby escape innate host defenses and favor their spread to new cells. In this respect, our results also open new research directions in relation with the extracellular localization of M. tuberculosis inside necrotic lesions that can now be tackled from a completely new perspective.     

Patients with Tuberculosis Disease Have Mycobacterium tuberculosis-Specific CD8 T Cells with a Pro-Apoptotic Phenotype and Impaired Proliferative Capacity,Which Is Not Restored following Treatment

CD8 T cells play a critical role in control of chronic viral infections; however, the role of these cells in containing persistent bacterial infections, such as those caused by Mycobacterium tuberculosis (Mtb), is less clear. We assessed the phenotype and functional capacity of CD8 T cells specific for the immunodominant Mtb antigens CFP-10 and ESAT-6, in patients with pulmonary tuberculosis (TB) disease, before and after treatment, and in healthy persons with latent Mtb infection (LTBI). In patients with TB disease, CFP-10/ESAT-6-specific IFN-γ⁺ CD8 T cells had an activated, pro-apoptotic phenotype, with lower Bcl-2 and CD127 expression, and higher Ki67, CD57, and CD95 expression, than in LTBI. When CFP-10/ESAT-6-specific IFN-γ⁺ CD8 T cells were detectable, expression of distinct combinations of these markers was highly sensitive and specific for differentiating TB disease from LTBI. Successful treatment of disease resulted in changes of these markers, but not in restoration of CFP-10/ESAT-6-specific CD8 or CD4 memory T cell proliferative capacity. These data suggest that high mycobacterial load in active TB disease is associated with activated, short-lived CFP-10/ESAT-6-specific CD8 T cells with impaired functional capacity that is not restored following treatment. By contrast, LTBI is associated with preservation of long-lived CFP-10/ESAT-6-specific memory CD8 T cells that maintain high Bcl-2 expression and which may readily proliferate.     

Molecular features governing the stability and specificity of functional complex formation by Mycobacterium tuberculosis CFP-10/ESAT-6 family proteins

The Mycobacterium tuberculosis complex CFP-10/ESAT-6 family proteins play essential but poorly defined roles in tuberculosis pathogenesis. In this article we report the results of detailed spectroscopic studies of several members of the CFP-10/ESAT-6 family. This work shows that the CFP-10/ESAT-6 related proteins, Rv0287 and Rv0288, form a tight 1:1 complex, which is predominantly helical in structure and is predicted to closely resemble the complex formed by CFP-10 and ESAT-6. In addition, the Rv0287.Rv0288 complex was found to be significantly more stable to both chemical and temperature induced denaturation than CFP-10.ESAT-6. This approach demonstrated that neither Rv0287.Rv0288 nor the CFP-10.ESAT-6 complexes are destabilized at low pH (4.5), indicating that even in low pH environments, such as the mature phagosome, both Rv0287.Rv0288 and CFP-10.ESAT-6 undoubtedly function as complexes rather than individual proteins. Analysis of the structure of the CFP-10.ESAT-6 complex and optimized amino acid sequence alignments of M. tuberculosis CFP-10/ESAT-6 family proteins revealed that residues involved in the intramolecular contacts between helices are conserved across the CFP-10/ESAT-6 family, but not those involved in primarily intermolecular contacts. This analysis identified the molecular basis for the specificity and stability of complex formation between CFP-10/ESAT-6 family proteins, and indicates that the formation of functional complexes with key roles in pathogenesis will be limited to genome partners, or very closely related family members, such as Rv0287/Rv0288 and Rv3019c/Rv3020c.     

The ESX-3 Secretion System Is Necessary for Iron and Zinc Homeostasis in Mycobacterium tuberculosis

ESX-3 is one of the five type VII secretion systems encoded by the Mycobacterium tuberculosis genome. We recently showed the essentiality of ESX-3 for M. tuberculosis viability and proposed its involvement in iron and zinc metabolism. In this study we confirmed the role of ESX-3 in iron uptake and its involvement in the adaptation to low zinc environment in M. tuberculosis. Moreover, we unveiled functional differences between the ESX-3 roles in M. tuberculosis and M. smegmatis showing that in the latter ESX-3 is only involved in the adaptation to iron and not to zinc restriction. Finally, we also showed that in M. tuberculosis this secretion system is essential for iron and zinc homeostasis not only in conditions in which the concentrations of these metals are limiting but also in metal sufficient conditions.     

The ESAT-6 gene cluster of Mycobacterium tuberculosis and other high G+C Gram-positive bacteria

Background  

The genome of Mycobacterium tuberculosis H37Rv has five copies of a cluster of genes known as the ESAT-6 loci. These clusters contain members of the CFP-10 (lhp) and ESAT-6 (esat-6) gene families (encoding secreted T-cell antigens that lack detectable secretion signals) as well as genes encoding secreted, cell-wall-associated subtilisin-like serine proteases, putative ABC transporters, ATP-binding proteins and other membrane-associated proteins. These membrane-associated and energy-providing proteins may function to secrete members of the ESAT-6 and CFP-10 protein families, and the proteases may be involved in processing the secreted peptide.     

Combined Antigen-Specific Interferon-γ and Interleukin-2 Release Assay (FluoroSpot) for the Diagnosis of Mycobacterium tuberculosis Infection

Background

To evaluate interleukin (IL)-2 and interferon (IFN)-γ secreting T-cells in parallel for the differentiation of latent infection with Mycobacterium tuberculosis infection (LTBI) from active tuberculosis.

Methods

Following ex-vivo stimulation of peripheral blood mononuclear cells (PBMC) with M. tuberculosis-specific antigens early secretory antigenic target (ESAT)-6 and culture filtrate protein (CFP)-10, immune responses were assessed by enzyme-linked immunospot IFN-γ release assay (EliSpot-IGRA) and a novel dual cytokine detecting fluorescence-linked immunospot (FluoroSpot) in 18 patients with pulmonary tuberculosis, 10 persons with previously cured tuberculosis, 25 individuals with LTBI and 16 healthy controls.

Results

Correlation of IFN- γ⁺ spot-forming cells in EliSpot-IGRA and FluoroSpot were R² = 0.67 for ESAT-6 and R² = 0.73 for CFP-10. The number of IL-2^- IFN- γ⁺ producing cells was higher in patients with tuberculosis compared with past tuberculosis (CFP-10-induced p = 0.0068) or individuals with LTBI (ESAT-6-induced p = 0.0136). A cutoff value of >16 CFP-10-induced IFN-γ⁺ secreting cells/200.000 PBMC in the EliSpot-IGRA discriminated with highest sensitivity and specificity (89% and 76%, respectively). However, overlap in cytokine responses precludes distinction between the cohorts on an individual basis.

Conclusions

Combined analysis of IFN-γ and IL-2 secretion by antigen specific T-cells does not allow a reliable differentiation between different states of M. tuberculosis infection in clinical practice.     

Novel M tuberculosis antigen-specific T-cells are early markers of infection and disease progression

Background

Mycobacterium tuberculosis Region-of-Difference-1 gene products present opportunities for specific diagnosis of M. tuberculosis infection, yet immune responses to only two gene-products, Early Secretory Antigenic Target-6 (ESAT-6) and Culture Filtrate Protein-10 (CFP-10), have been comprehensively investigated.

Methods

T-cell responses to Rv3873, Rv3878 and Rv3879c were quantified by IFN-γ-enzyme-linked-immunospot (ELISpot) in 846 children with recent household tuberculosis exposure and correlated with kinetics of tuberculin skin test (TST) and ESAT-6/CFP-10-ELISpot conversion over six months and clinical outcome over two years.

Results

Responses to Rv3873, Rv3878, and Rv3879c were present in 20–25% of contacts at enrolment. Rv3873 and Rv3879c responses were associated with and preceded TST conversion (P = 0.02 and P = 0.04 respectively), identifying these antigens as early targets of cell-mediated immunity following M. tuberculosis exposure. Responses to Rv3873 were additionally associated with subsequent ESAT-6/CFP-10-ELISpot conversion (P = 0.04). Responses to Rv3873 and Rv3878 predicted progression to active disease (adjusted incidence rate ratio [95% CI] 3.06 [1.05,8.95; P = 0.04], and 3.32 [1.14,9.71; P = 0.03], respectively). Presence of a BCG-vaccination scar was associated with a 67% (P = 0.03) relative risk reduction for progression to active tuberculosis.

Conclusions

These RD1-derived antigens are early targets of cellular immunity following tuberculosis exposure and T-cells specific for these antigens predict progression to active tuberculosis suggesting diagnostic and prognostic utility.     

Functional analysis of early secreted antigenic target-6, the dominant T-cell antigen of Mycobacterium tuberculosis, reveals key residues involved in secretion, complex formation, virulence, and immunogenicity

Proteins of the 6-kDa early secreted antigenic target (ESAT-6) secretion system-1 of Mycobacterium tuberculosis are not only strongly involved in the anti-mycobacterial Th1-host immune response but are also key players for virulence. In this study, protein engineering together with bioinformatic, immunological, and virulence analyses allowed us to pinpoint regions of the ESAT-6 molecule that are critical for its biological activity in M. tuberculosis. Mutation of the Trp-Xaa-Gly motif, conserved in a wide variety of ESAT-6-like proteins, abolished complex formation with the partner protein CFP-10, induction of specific T-cell responses, and virulence. Replacement of conserved Leu residues interfered with secretion, coiled-coil formation, and virulence, whereas certain mutations at the extreme C terminus did not affect secretion but caused attenuation, possibly because of altered ESAT-6 targeting or trafficking. In contrast, the mutation of several residues on the outer surface of the four-helical bundle structure of the ESAT-6.CFP-10 complex showed much less effect. Construction of recombinant BCG expressing ESAT-6 with a C-terminal hexahistidine tag allowed us to co-purify ESAT-6 and CFP-10, experimentally confirming their strong interaction both in and outside of the mycobacterial cell. The strain induced potent, antigen-specific T-cell responses and intermediate in vivo growth in mice, suggesting that it remained immunogenic and biologically active despite the tag. Together with previous NMR data, the results of this study have allowed a biologically relevant model of the ESAT-6.CFP-10 complex to be constructed that is critical for understanding the structure-function relationship in tuberculosis pathogenesis.