ESX-1 dependent impairment of autophagic flux by Mycobacterium tuberculosis in human dendritic cells

Emerging evidence points to an important role of autophagy in the immune response mediated by dendritic cells (DC) against Mycobacterium tuberculosis (Mtb). Since current vaccination based on Bacillus Calmette-Guerin (BCG) is unable to stop the tuberculosis epidemic, a deeper comprehension of the alterations induced by Mtb in DC is essential for setting new vaccine strategies. Here, we compared the capacity of virulent (H37Rv) and avirulent (H37Ra) Mtb strains as well as BCG to modulate autophagy in human primary DC. We found that Mtb H37Rv impairs autophagy at the step of autophagosome-lysosome fusion. In contrast, neither Mtb H37Ra nor BCG strains were able to hamper autophagosome maturation. Both these attenuated strains have a functional inhibition of the 6kD early secreted antigenic target ESAT-6, an effector protein of the ESAT-6 Secretion System-1(ESX-1)/type VII secretion system. Notably, the ability to inhibit autophagy was fully restored in recombinant BCG and Mtb H37Ra strains in which ESAT-6 secretion was re-established by genetic complementation using either the ESX-1 region from Mtb (BCG::ESX-1) or the PhoP gene (Mtb H37Ra::PhoP), a regulator of ESAT-6 secretion. Importantly, the autophagic block induced by Mtb was overcome by rapamycin treatment leading to an increased interleukin-12 expression and, in turn, to an enhanced capacity to expand a Th1-oriented response. Collectively, our study demonstrated that Mtb alters the autophagic machinery through the ESX-1 system, and thereby opens new exciting perspectives to better understand the relationship between Mtb virulence and its ability to escape the DC-mediated immune response.     

Control of M. tuberculosis ESAT-6 secretion and specific T cell recognition by PhoP

Analysis of mycobacterial strains that have lost their ability to cause disease is a powerful approach to identify yet unknown virulence determinants and pathways involved in tuberculosis pathogenesis. Two of the most widely used attenuated strains in the history of tuberculosis research are Mycobacterium bovis BCG (BCG) and Mycobacterium tuberculosis H37Ra (H37Ra), which both lost their virulence during in vitro serial passage. Whereas the attenuation of BCG is due mainly to loss of the ESAT-6 secretion system, ESX-1, the reason why H37Ra is attenuated remained unknown. However, here we show that a point mutation (S219L) in the predicted DNA binding region of the regulator PhoP is involved in the attenuation of H37Ra via a mechanism that impacts on the secretion of the major T cell antigen ESAT-6. Only H37Ra "knock-ins" that carried an integrated cosmid with the wild-type phoP gene from M. tuberculosis H37Rv showed changes in colony morphology, increased virulence, ESAT-6 secretion, and induction of specific T cell responses, whereas other H37Ra constructs did not. This finding established a link between the PhoP regulator and ESAT-6 secretion that opens exciting new perspectives for elucidating virulence regulation 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.     

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.     

Early secreted antigen ESAT-6 of Mycobacterium tuberculosis promotes protective T helper 17 cell responses in a toll-like receptor-2-dependent manner

Despite its relatively poor efficacy, Bacillus Calmette-Guérin (BCG) has been used as a tuberculosis (TB) vaccine since its development in 1921. BCG induces robust T helper 1 (Th1) immune responses but, for many individuals, this is not sufficient for host resistance against Mycobacterium tuberculosis (M. tb) infection. Here we provide evidence that early secreted antigenic target protein 6 (ESAT-6), expressed by the virulent M. tb strain H37Rv but not by BCG, promotes vaccine-enhancing Th17 cell responses. These activities of ESAT-6 were dependent on TLR-2/MyD88 signalling and involved IL-6 and TGF-β production by dendritic cells. Thus, animals that were previously infected with H37Rv or recombinant BCG containing the RD1 region (BCG::RD1) exhibited improved protection upon re-challenge with virulent H37Rv compared with mice previously infected with BCG or RD1-deficient H37Rv (H37RvΔRD1). However, TLR-2 knockout (TLR-2^-/-) animals neither showed Th17 responses nor exhibited improved protection in response to immunization with H37Rv. Furthermore, H37Rv and BCG::RD1 infection had little effect on the expression of the anti-inflammatory microRNA-146a (miR146a) in dendritic cells (DCs), whereas BCG and H37RvΔRD1 profoundly induced its expression in DCs. Consistent with these findings, ESAT-6 had no effect on miR146a expression in uninfected DCs, but dramatically inhibited its upregulation in BCG-infected or LPS-treated DCs. Collectively, our findings indicate that, in addition to Th1 immunity induced by BCG, RD1/ESAT-6-induced Th17 immune responses are essential for optimal vaccine efficacy.     

Mycobacterium tuberculosis Rv2536 protein implicated in specific binding to human cell lines

The gene encoding the Mycobacterium tuberculosis Rv2536 protein is present in the Mycobacterium tuberculosis complex (as assayed by PCR) and transcribed (as determined by RT-PCR) in M. tuberculosis H37Rv, M. tuberculosis H37Ra, M. bovis BCG, and M. africanum strains. Rabbits immunized with synthetic polymer peptides from this protein produced antibodies specifically recognizing a 25-kDa band in mycobacterial sonicate. U937 and A549 cells were used in binding assays involving 20-amino-acid-long synthetic peptides covering the whole Rv2536 protein sequence. Peptide 11207 (161DVFSAVRADDSPTGEMQVAQY180) presented high specific binding to both types of cells; the binding was saturable and presented nanomolar affinity constants. Cross-linking assays revealed that this peptide specifically binds to 50 kDa U937 cell membrane and 45 kDa A549 cell membrane proteins.     

The Delta fbpA mutant derived from Mycobacterium tuberculosis H37Rv has an enhanced susceptibility to intracellular antimicrobial oxidative mechanisms, undergoes limited phagosome maturation and activates macrophages and dendritic cells

Mycobacterium tuberculosis H37Rv (Mtb) excludes phagocyte oxidase (phox) and inducible nitric oxide synthase (iNOS) while preventing lysosomal fusion in macrophages (MPhis). The antigen 85A deficient (Delta fbpA) mutant of Mtb was vaccinogenic in mice and the mechanisms of attenuation were compared with MPhis infected with H37Rv and BCG. Delta fbpA contained reduced amounts of trehalose 6, 6, dimycolate and induced minimal levels of SOCS-1 in MPhis. Blockade of oxidants enhanced the growth of Delta fbpA in MPhis that correlated with increased colocalization with phox and iNOS. Green fluorescent protein-expressing strains within MPhis or purified phagosomes were analysed for endosomal traffick with immunofluorescence and Western blot. Delta fbpA phagosomes were enriched for rab5, rab11, LAMP-1 and Hck suggesting enhanced fusion with early, recycling and late endosomes in MPhis compared with BCG or H37Rv. Delta fbpA phagosomes were thus more mature than H37Rv or BCG although, they failed to acquire rab7 and CD63 preventing lysosomal fusion. Finally, Delta fbpA infected MPhis and dendritic cells (DCs) showed an enhanced MHC-II and CD1d expression and primed immune T cells to release more IFN-gamma compared with those infected with BCG and H37Rv. Delta fbpA was thus more immunogenic in MPhis and DCs because of an enhanced susceptibility to oxidants and increased maturation.     

Differential release of tumor necrosis factor-α from murine peritoneal macrophages stimulated with virulent and avirulent species of mycobacteria

Abstract The ability of Mycobacterium tuberculosis H37Rv and H37Ra, M. bovis BCG and M. smegmatis to induce the secretion of tumor necrosis factor-α (TNF-α) by cultured murine peritoneal macrophages is inversely related to their virulence. The avirulent species of mycobacteria which were unable to persist in macrophages were capable of inducing significant levels of TNF-α compared to that formed in cultures infected with the virulent M. tuberculosis H37Rv. This difference was also associated with an inherent toxicity by live H37Rv for macrophage cultures. Heat-killed H37Rv was non-toxic and induced significant levels of TNF-α; in contrast, live and heat-killed suspensions of avirulent mycobacteria had an equivalent ability to trigger TNF-α secretion. The TNF-α response was dose-dependent, related directly to the percentage of infected cells, and peaked 6–12 h post-infection. An early and vigorous TNF-α response appears to be a marker of macrophage resistance, while the downregulation of this response seems associated with macrophage toxicity and unrestricted mycobacterial growth.     

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.     

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.     

Mycobacterium tuberculosis induces delayed lipid droplet accumulation in dendritic cells depending on bacterial viability and virulence

Tuberculosis remains a global health threat with high morbidity. Dendritic cells (DCs) participate in the acute and chronic inflammatory responses to Mycobacterium tuberculosis (Mtb) by directing the adaptive immune response and are present in lung granulomas. In macrophages, the interaction of lipid droplets (LDs) with mycobacteria-containing phagosomes is central to host-pathogen interactions. However, the data available for DCs are still a matter of debate. Here, we reported that bone marrow-derived DCs (BMDCs) were susceptible to Mtb infection and replication at similar rate to macrophages. Unlike macrophages, the analysis of gene expression showed that Mtb infection induced a delayed increase in lipid droplet-related genes and proinflammatory response. Hence, LD accumulation has been observed by high-content imaging in late periods. Infection of BMDCs with killed H37Rv demonstrated that LD accumulation depends on Mtb viability. Moreover, infection with the attenuated strains H37Ra and Mycobacterium bovis-BCG induced only an early transient increase in LDs, whereas virulent Mtb also induced delayed LD accumulation. In addition, infection with the BCG strain with the reintroduced virulence RD1 locus induced higher LD accumulation and bacterial replication when compared to parental BCG. Collectively, our data suggest that delayed LD accumulation in DCs is dependent on mycobacterial viability and virulence.     

Disruption of the ESX-5 system of Mycobacterium tuberculosis causes loss of PPE protein secretion, reduction of cell wall integrity and strong attenuation

The chromosome of Mycobacterium tuberculosis encodes five type VII secretion systems (ESX-1-ESX-5). While the role of the ESX-1 and ESX-3 systems in M. tuberculosis has been elucidated, predictions for the function of the ESX-5 system came from data obtained in Mycobacterium marinum, where it transports PPE and PE_PGRS proteins and modulates innate immune responses. To define the role of the ESX-5 system in M. tuberculosis, in this study, we have constructed five M. tuberculosis H37Rv ESX-5 knockout/deletion mutants, inactivating eccA(5), eccD(5), rv1794 and esxM genes or the ppe25-pe19 region. Whereas the Mtbrv1794ko displayed no obvious phenotype, the other four mutants showed defects in secretion of the ESX-5-encoded EsxN and PPE41, a representative member of the large PPE protein family. Strikingly, the MtbeccD(5) ko mutant also showed enhanced sensitivity to detergents and hydrophilic antibiotics. When the virulence of the five mutants was evaluated, the MtbeccD(5) ko and MtbΔppe25-pe19 mutants were found attenuated both in macrophages and in the severe combined immune-deficient mouse infection model. Altogether these findings indicate an essential role of ESX-5 for transport of PPE proteins, cell wall integrity and full virulence of M. tuberculosis, thereby opening interesting new perspectives for the study of this human pathogen.     

Evaluation of immunogenicity and protective efficacy against Mycobacterium tuberculosis infection elicited by recombinant Mycobacterium bovis BCG expressing human Interleukin-12p70 and Early Secretory Antigen Target-6 fusion protein

ESAT-6 protein of Mycobacterium tuberculosis is absent in Mycobacterium bovis BCG and Mycobacterium microti and has been demonstrated to stimulate strong cell-mediated immunity. IL-12 can play crucial roles in regulating IFN-γ production and Th1 effectors production. In this study, we constructed three rBCG vaccines that could express proteins of human IL-12p70 and/or ESAT-6 and evaluated their immunogenicity and protective efficacy in mice. Our experiments illustrated that the rBCG-IE (expressing a fusion protein of human IL-12p70 and ESAT-6) was capable of inducing stronger Th1 type cell-mediated immune responses than conventional BCG, or rBCG-I (expressing human IL-12p70), or rBCG-E (expressing ESAT-6). However, the results of protective experiments showed that rBCG-IE could only confer similar and even lower protective efficacy against M. tuberculosis H37Rv infection compared with BCG vaccine.     

Novel pyridopyrazine and pyrimidothiazine derivatives as FtsZ inhibitors

A series of pyridopyrazine and pyrimidothiazine derivatives have been synthesized and their activity against FtsZ from Mycobacterium tuberculosis (Mtb) and in vitro antibacterial activity against Mtb H(37)Ra and Mtb H(37)Rv are reported. Certain analogs described herein showed moderate to good inhibitory activity.