Functional and structural investigations of fibronectin-binding protein Apa from Mycobacterium tuberculosis

BackgroundAlanine and proline-rich protein (Apa) is a secreted antigen of Mycobacterium spp. which involves in stimulating immune responses and adhering to host cells by binding to fibronectin (Fn). Here, we report the crystal structure of Apa from Mycobacterium tuberculosis (Mtb) and its Fn-binding characteristics.MethodsThe crystal structure of Mtb Apa was determined at resolutions of 1.54 Å. The dissociation constants (K_D) of Apa and individual modules of Fn were determined by surface plasmon resonance and enzyme-linked immunosorbent assay. Site-directed mutagenesis was performed to investigate the putative Fn-binding motif of Apa.ResultsMtb Apa folds into a large seven-stranded anti-parallel β-sheet which is flanked by three α-helices. The binding affinity of Mtb Apa to individual Fn modules was assessed and the results indicated that the Mtb Apa binds to FnIII-4 and FnIII-5 of Fn CBD segment. Notably, structure analysis suggested that the previously proposed Fn-binding motif ²⁵⁸RWFV²⁶¹ is buried within the protein and may not be accessible to the binding counterpart.ConclusionsThe structural and Fn-binding characteristics we reported here provide molecular insights into the multifunctional protein Mtb Apa. FnIII-4 and FnIII-5 of CBD are the only two modules contributing to Apa-Fn interaction.General significanceThis is the first study to report the structure and Fn-binding characteristics of mycobacterial Apa. Since Apa plays a central role in stimulating immune responses and host cells adhesion, these results are of great importance in understanding the pathogenesis of mycobacterium. This information shall provide a guidance for the development of anti-mycobacteria regimen.     

ESX‐1 and phthiocerol dimycocerosates of Mycobacterium tuberculosis act in concert to cause phagosomal rupture and host cell apoptosis

Although phthiocerol dimycocerosates (DIM) are major virulence factors of Mycobacterium tuberculosis (Mtb), the causative agent of human tuberculosis, little is known about their mechanism of action. Localized in the outer membrane of mycobacterial pathogens, DIM are predicted to interact with host cell membranes. Interaction with eukaryotic membranes is a property shared with another virulence factor of Mtb, the early secretory antigenic target EsxA (also known as ESAT‐6). This small protein, which is secreted by the type VII secretion system ESX‐1 (T7SS/ESX‐1), is involved in phagosomal rupture and cell death induced by virulent mycobacteria inside host phagocytes. In this work, by the use of several knock‐out or knock‐in mutants of Mtb or Mycobacterium bovis BCG strains and different cell biological assays, we present conclusive evidence that ESX‐1 and DIM act in concert to induce phagosomal membrane damage and rupture in infected macrophages, ultimately leading to host cell apoptosis. These results identify an as yet unknown function for DIM in the infection process and open up a new research field for the study of the interaction of lipid and protein virulence factors of Mtb.     

Organelle membrane proteomics reveals differential influence of mycobacterial lipoglycans on macrophage phagosome maturation and autophagosome accumulation

The mycobacterial cell wall component lipoarabinomannan (LAM) has been described as one of the key virulence factors of Mycobacterium tuberculosis. Modification of the terminal arabinan residues of this lipoglycan with mannose caps in M. tuberculosis or with phosphoinositol caps in Mycobacterium smegmatis results in distinct host immune responses. Given that M. tuberculosis typically persists in the phagosomal vacuole after being phagocytosed by macrophages, we performed a proteomic analysis of that organelle after treatment of macrophages with LAMs purified from the two mycobacterial species. The quantitative changes in phagosomal proteins suggested a distinct role for mannose-capped LAM in modulating protein trafficking pathways that contribute to the arrest of phagosome maturation. Enlightened by our proteomic data, we performed further experiments to show that only the LAM from M. tuberculosis inhibits accumulation of autophagic vacuoles in the macrophage, suggesting a new function for this virulence-associated lipid.     

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.     

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.     

In vivo gene silencing identifies the Mycobacterium tuberculosis proteasome as essential for the bacteria to persist in mice

The success of Mycobacterium tuberculosis (Mtb) as a human pathogen relies on its ability to resist eradication by the immune system. The identification of mechanisms that enable Mtb to persist is key for finding ways to limit latent tuberculosis, which affects one-third of the world's population. Here we show that conditional gene silencing can be used to determine whether an Mtb gene required for optimal growth in vitro is also important for virulence and, if so, during which phase of an infection it is required. Application of this approach to the prcBA genes, which encode the core of the mycobacterial proteasome, revealed an unpredicted requirement of the core proteasome for the persistence of Mtb during the chronic phase of infection in mice. Proteasome depletion also attenuated Mtb in interferon-gamma-deficient mice, pointing to a function of the proteasome beyond defense against the adaptive immune response. Genes that are essential for growth in vitro, in vivo or both account for approximately 20% of Mtb's genome. Conditional gene silencing could therefore facilitate the validation of up to 800 potential Mtb drug targets and improve our understanding of host-pathogen dynamics.     

The timing of TNF and IFN-gamma signaling affects macrophage activation strategies during Mycobacterium tuberculosis infection

During most infections, the population of immune cells known as macrophages are key to taking up and killing bacteria as an integral part of the immune response. However, during infection with Mycobacterium tuberculosis (Mtb), host macrophages serve as the preferred environment for mycobacterial growth. Further, killing of Mtb by macrophages is impaired unless they become activated. Activation is induced by stimulation from bacterial antigens and inflammatory cytokines derived from helper T cells. The key macrophage-activating cytokines in Mtb infection are tumor necrosis factor-α (TNF) and interferon (IFN)-γ. Due to differences in cellular sources and secretion pathways for TNF and IFN-γ, the possibility of heterogeneous cytokine distributions exists, suggesting that the timing of macrophage activation from these signals may affect activation kinetics and thus impact the outcome of Mtb infection. Here we use a mathematical model to show that negative feedback from production of nitric oxide (the key mediator of mycobacterial killing) that typically optimizes macrophage responses to activating stimuli may reduce effective killing of Mtb. Statistical sensitivity analysis predicts that if TNF and IFN-γ signals precede infection, the level of negative feedback may have a strong effect on how effectively macrophages kill Mtb. However, this effect is relaxed when IFN-γ or TNF+IFN-γ signals are received coincident with infection. Under these conditions, the model suggests that negative feedback induces fast responses and an initial overshoot of nitric oxide production for given doses of TNF and IFN-γ, favoring killing of Mtb. Together, our results suggest that direct entry of macrophages into a granuloma site (and not distal to it) from lung vascular sources represents a preferred host strategy for mycobacterial control. We examine implications of these results in establishment of latent Mtb infection.     

Local production of tumor necrosis factor and IFN-gamma in tuberculous pleuritis

TNF and IFN-gamma are thought to be involved in the immune response to mycobacterial infection because they exhibit antimycobacterial effects in vitro. To investigate the roles of these cytokines in vivo at the site of disease activity in human tuberculosis, we evaluated local cytokine production in patients with tuberculous pleuritis. Both TNF and IFN-gamma were selectively concentrated 5- to 30-fold in pleural fluid, compared to blood of the same patients. Messenger RNA for both cytokines was detected in pleural tissue by in situ hybridization, suggesting that selective cytokine concentration is due to local cytokine production. Two Mycobacterium tuberculosis cell wall components, the protein-peptidoglycan complex and lipoarabinomannan, caused dose-dependent release of TNF by pleural fluid mononuclear cells and may constitute the stimuli for TNF production in the pleural space. In contrast to results obtained for TNF release, the protein-peptidoglycan complex, but not lipoarabinomannan, stimulated IFN-gamma release by pleural fluid mononuclear cells. The clinical manifestations of tuberculous pleuritis, such as fever, exudative pleural effusion, and tissue necrosis, may be due to the effects of elevated local TNF concentrations, produced in response to mycobacterial cell wall components.     

Intracellular signalling cascades regulating innate immune responses to Mycobacteria: branching out from Toll-like receptors

Toll-like receptors (TLRs) recognize Mycobacterium tuberculosis (Mtb) or Mtb components and initiate mononuclear phagocyte responses that influence both innate and adaptive immunity. Recent studies have revealed the intracellular signalling cascades involved in the TLR-initiated immune response to mycobacterial infection. Although both TLR2 and TLR4 have been implicated in host interactions with Mtb, the relationship between specific mycobacterial molecules and various signal transduction pathways is not well understood. This review will discuss recent studies indicating critical roles for mycobacteria and mycobacterial components in regulation of mitogen-activated protein kinases and related signal transduction pathways that govern the outcome of infection and antibacterial defence. To better understand the roles of infection-induced signalling cascades in molecular pathogenesis, future studies are needed to clarify mechanisms that integrate the multiple signalling pathways that are activated by engagement of TLRs by both individual mycobacterial molecules and whole mycobacteria. These efforts will allow for the development of novel diagnostic and therapeutic modalities for tuberculosis that targets the intracellular signalling pathways permitting the replication of this nefarious pathogen.     

Aggravated infection in mice co-administered with Mycobacterium tuberculosis and the 27-kDa lipoprotein

We have previously reported that mice immunized with the mycobacterial 27-kDa lipoprotein were more susceptible to Mycobacterium tuberculosis (Mtb) challenge. We also showed that 27-kDa lipoprotein abrogated the protection afforded by the BCG vaccine when administrated together, suggesting that the 27-kDa lipoprotein may modulate the course of experimental mycobacterial infection. In this study, we address the role of the 27-kDa lipoprotein in modulating the immune response to mycobacteria. Our results show that co-administration of BALB/c mice with Mtb and the 27-kDa lipoprotein (Mtb+27kDa), but not its non-acylated form, increases the susceptibility of mice to Mtb infection. Significantly lower DTH reaction and splenocyte proliferation to PPD stimulation were also observed in Mtb+27kDa-infected mice compared to Mtb-infected mice. Furthermore, during infection, splenocytes and purified T cells lost their ability to proliferate in response to concanavalin A stimulation more rapidly in the Mtb+27kDa-infected mice, which was accompanied by high IFN-gamma and NO production, but low TNF-alpha secretion levels. Addition of L-NMMA, anti-IFN-gamma and anti-TNF-alpha antibodies restored in vitro proliferative responses of T cells from Mtb+27kDa-infected mice. Short-term L-NMMA treatment of Mtb+27kDa-infected mice prevented the 27-kDa-mediated immunosuppression and increase in susceptibility to Mtb. Altogether, these data suggest that the 27-kDa lipoprotein plays a role in Mtb infection by inducing increased suppression of the immune response due to elevated NO production.     

Importance of differential identification of <Emphasis Type="Italic">Mycobacterium tuberculosis</Emphasis> strains for understanding differences in their prevalence,treatment efficacy,and vaccine development

Tuberculosis (TB), caused by Mycobacterium tuberculosis (Mtb), remains a serious global health problem in the 21^st century because of its high mortality. Mtb is an extremely successful human-adapted pathogen that displays a multifactorial ability to control the host immune response and to evade killing by drugs, resulting in the breakdown of BCG vaccine-conferred anti-TB immunity and development of multidrug-resistant (MDR) and extensively drug-resistant (XDR) Mtb. Although genetic components of the genomes of the Mtb complex strains are highly conserved, showing over 99% similarity to other bacterial genera, recently accumulated evidence suggests that the genetic diversity of the Mtb complex strains has implications for treatment outcomes, development of MDR/XDR Mtb, BCG vaccine efficacy, transmissibility, and epidemiological outbreaks. Thus, new insights into the pathophysiological features of the Mtb complex strains are required for development of novel vaccines and for control of MDR/XDR Mtb infection, eventually leading to refinement of treatment regimens and the health care system. Many studies have focused on the differential identification of Mtb complex strains belonging to different lineages because of differences in their virulence and geographical dominance. In this review, we discuss the impact of differing genetic characteristics among Mtb complex strains on vaccine efficacy, treatment outcome, development of MDR/XDR Mtb strains, and epidemiological outbreaks by focusing on the best-adapted human Mtb lineages. We further explore the rationale for differential identification of Mtb strains for more effective control of TB in clinical and laboratory settings by scrutinizing current diagnostic methods.     

Mycobacterial lipoarabinomannan: an extraordinary lipoheteroglycan with profound physiological effects

Detailed structural and functional studies over the last decade have led to current recognition of the mycobacterial lipoarabinomannan (LAM) as a phosphatidylinositol anchored lipoglycan with diverse biological activities. Fatty acylation has been demonstrated to be essential for LAM to maintain its functional integrity although the focus has largely been on the arabinan motifs and the terminal capping function. It has recently been shown that the mannose caps may be involved not only in attenuating host immune response, but also in mediating the binding of mycobacteria to and subsequent entry into macrophages. This may further be linked to an intracellular trafficking pathway through which LAM is thought to be presented by CD1 to subsets of T-cells. The implication of LAM as major histocompatibility complex (MHC)-independent T-cell epitope and the ensuing immune response is an area of intensive studies. Another recent focus of research is the biosynthesis of arabinan which has been shown to be inhibitable by the anti- tuberculosis drug, ethambutol. The phenomenon of truncated LAM as synthesized by ethambutol resistant strains provides an invaluable handle for dissecting the array of arabinosyltransferases involved, as well as generating much needed structural variants for further structural and functional studies. It is hoped that with more systematic investigations based on clinical isolates and human cell lines, the true significance of LAM in the immunopathogenesis of tuberculosis and leprosy can eventually be explained.      

Identification of the Mycobacterium marinum Apa antigen O-mannosylation sites reveals important glycosylation variability with the M. tuberculosis Apa homologue

The 45/47kDa Apa, an immuno-dominant antigen secreted by Mycobacterium tuberculosis is O-mannosylated at multiple sites. Glycosylation of Apa plays a key role in colonization and invasion of the host cells by M. tuberculosis through interactions of Apa with the host immune system C-type lectins. Mycobacterium marinum (M.ma) a fish pathogen, phylogenetically close to M. tuberculosis, induces a granulomatous response with features similar to those described for M. tuberculosis in human. Although M.ma possesses an Apa homologue, its glycosylation status is unknown, and whether this represents a crucial element in the pathophysiology induced by M.ma remains to be addressed. To this aim, we have identified two concanavalin A-reactive 45/47kDa proteins from M.ma, which have been further purified by a two-step anion exchange chromatography process. Advanced liquid chromatography-nanoESI mass spectrometry-based proteomic analyses of peptides, derived from either tryptic digestion alone or in combination with the Asp-N endoproteinase, established that M.ma Apa possesses up to seven distinct O-mannosylated sites with mainly single mannose substitutions, which can be further extended at the Ser/Thr/Pro rich region near the N-terminus. This opens the way to further studies focussing on the involvement and biological functions of Apa O-mannosylation using the M.ma/zebrafish model.     

New structural insights into the molecular deciphering of mycobacterial lipoglycan binding to C-type lectins: lipoarabinomannan glycoform characterization and quantification by capillary electrophoresis at the subnanomole level

Lipoarabinomannans are key molecules of the mycobacterial envelopes involved in many steps of tuberculosis immunopathogenesis. Several of the biological activities of lipoarabinomannans are mediated by their ability to bind human C-type lectins, such as the macrophage mannose receptor, the mannose-binding protein and the surfactant proteins A and D. The lipoarabinomannan mannooligosaccharide caps have been demonstrated to be involved in the binding to the lectin carbohydrate recognition domains. We report an original analytical approach, based on capillary electrophoresis monitored by laser-induced fluorescence, allowing the absolute quantification, in nanomole quantities of lipoarabinomannan, of the number of mannooligosaccharide units per lipoarabinomannan molecule. Moreover, this analytical approach was successful for the glycosidic linkage determination of the mannooligosaccharide motifs and has been applied to the comparative analysis of parietal and cellular lipoarabinomannans of Mycobacterium bovis BCG and Mycobacterium tuberculosis H37Rv, H37Ra and Erdman strains. Significant differences were observed in the amounts of the various mannooligosaccharide units between lipoarabinomannans of different strains and between parietal and cellular lipoarabinomannans of the same strain. Nevertheless, no relationship was found between the number of mannooligosaccharide caps and the virulence of the corresponding strain. The results of the present study should help us to gain more understanding of the molecular basis of lipoarabinomannan discrimination in the process of binding to C-type lectins.     

结核分枝杆菌对宿主巨噬细胞死亡方式的调控

结核分枝杆菌（Mycobacterium tuberculosis,Mtb）感染后能抑制宿主巨噬细胞（M西）的免疫反应,并在其中生存、复制。研究表明Mtb减毒株感染主要诱导宿主Mφ凋亡,凋亡能抑制胞内Mtb的活力;而Mtb毒力株感染能抑制凋亡的完成,诱导Mφ坏死,最终导致Mtb扩散、感染临近细胞。通过对Mtb感染诱导宿主Mφ不同死亡方式的讨论,进一步认识Mtb的致病机制。     

Exploitation of Mycobacterium tuberculosis Reporter Strains to Probe the Impact of Vaccination at Sites of Infection

Mycobacterium tuberculosis (Mtb) remains a major public health problem, with an effective vaccine continuing to prove elusive. Progress in vaccination strategies has been hampered by a lack of appreciation of the bacterium''s response to dynamic changes in the host immune environment. Here, we utilize reporter Mtb strains that respond to specific host immune stresses such as hypoxia and nitric oxide (hspX′::GFP), and phagosomal maturation (rv2390c′::GFP), to investigate vaccine-induced alterations in the environmental niche during experimental murine infections. While vaccination undoubtedly decreased bacterial burden, we found that it also appeared to accelerate Mtb''s adoption of a phenotype better equipped to survive in its host. We subsequently utilized a novel replication reporter strain of Mtb to demonstrate that, in addition to these alterations in host stress response, there is a decreased percentage of actively replicating Mtb in vaccinated hosts. This observation was supported by the differential sensitivity of recovered bacteria to the front-line drug isoniazid. Our study documents the natural history of the impact that vaccination has on Mtb''s physiology and replication and highlights the value of reporter Mtb strains for probing heterogeneous Mtb populations in the context of a complex, whole animal model.     

Mycobacterium tuberculosis LprA is a lipoprotein agonist of TLR2 that regulates innate immunity and APC function

TLR2 recognizes components of Mycobacterium tuberculosis (Mtb) and initiates responses by APCs that influence both innate and adaptive immunity. Mtb lipoproteins are an important class of TLR2 ligand, but only two, LpqH and LprG, have been characterized to date. In this study, we characterize a third Mtb lipoprotein, LprA, and determine its effects on host macrophages and dendritic cells. LprA is a cell wall-associated lipoprotein with no homologs outside the slow-growing mycobacteria. Using Mycobacterium smegmatis as an expression host, we purified 6x His-tagged LprA both with and without its acyl modifications. Acylated LprA had agonist activity for both human and murine TLR2 and induced expression of TNF-alpha, IL-10, and IL-12. LprA also induced dendritic cell maturation as shown by increased expression of CD40, CD80, and class II MHC (MHC-II). In macrophages, prolonged (24 h) incubation with LprA decreased IFN-gamma-induced MHC-II Ag processing and presentation, consistent with an observed decrease in MHC-II expression (macrophage viability was not affected and apoptosis was not induced by LprA). Reduced MHC-II Ag presentation may represent a negative feedback mechanism for control of inflammation that may be subverted by Mtb for immune evasion. Thus, Mtb LprA is a TLR2 agonist that induces cytokine responses and regulates APC function.     

The innate immune response in human tuberculosis

Mycobacterium tuberculosis (Mtb) infection can be cleared by the innate immune system before the initiation of an adaptive immune response. This innate protection requires a variety of robust cell autonomous responses from many different host immune cell types. However, Mtb has evolved strategies to circumvent some of these defences. In this mini‐review, we discuss these host–pathogen interactions with a focus on studies performed in human cells and/or supported by human genetics studies (such as genome‐wide association studies).