热休克蛋白HSP70和gp96在抗病毒感染中的作用

热休克蛋白(HSP)是一组在进化上高度保守、具有重要生理功能的蛋白质家族,是生物在应激条件下产生的一种非特异性防御产物,在调节免疫应答和抗病毒反应中起重要作用。现简要介绍HSP70、gp96(HSP96,GRP94)这两种HSP与病毒感染的关系及在抗病毒感染中的作用。     

Effects of Mycobacterium tuberculosis ESAT-6/CFP-10 fusion protein on the autophagy function of mouse macrophages

Autophagy plays specific roles in host innate and adaptive immune responses to numerous intracellular pathogens, including Mycobacterium tuberculosis. The ESAT-6 and CFP-10 proteins are secreted by M. tuberculosis and play important roles in pathogenesis. We hypothesized that these two proteins may affect the autophagy function of host macrophages during infection with M. tuberculosis, thereby shaping the immune reaction toward the pathogen. Interestingly, we found that rapamycin-induced autophagy of macrophages infected with M. tuberculosis H37Rv enhanced localization of mycobacteria with autophagosomes and lysosomes. Ectopic expression of the ESAT-6/CFP-10 fusion in macrophages dramatically inhibited autophagosome formation, and M. tuberculosis survival inside infected macrophages was significantly affected as well. Further, M. tuberculosis viability was increased by the fusion protein. Expression levels of autophagy-related genes (ATG), especially atg8, also decreased (p<0.05). These results suggested that ESAT-6 and CFP-10 proteins play significant roles in autophagy formation in M. tuberculosis infection and that autophagosome formation is regulated through the expression of ATG.     

Bacterial heat shock proteins promote CD91-dependent class I MHC cross-presentation of chaperoned peptide to CD8+ T cells by cytosolic mechanisms in dendritic cells versus vacuolar mechanisms in macrophages

APCs process mammalian heat shock protein (HSP):peptide complexes to present HSP-chaperoned peptides on class I MHC (MHC-I) molecules to CD8(+) T cells. HSPs are also expressed in prokaryotes and chaperone microbial peptides, but the ability of prokaryotic HSPs to contribute chaperoned peptides for Ag presentation is unknown. Our studies revealed that exogenous bacterial HSPs (Escherichia coli DnaK and Mycobacterium tuberculosis HSP70) delivered an extended OVA peptide for processing and MHC-I presentation by both murine macrophages and dendritic cells. HSP-enhanced MHC-I peptide presentation occurred only if peptide was complexed to the prokaryotic HSP and was dependent on CD91, establishing CD91 as a receptor for prokaryotic as well as mammalian HSPs. Inhibition of cytosolic processing mechanisms (e.g., by transporter for Ag presentation deficiency or brefeldin A) blocked HSP-enhanced peptide presentation in dendritic cells but not macrophages. Thus, prokaryotic HSPs deliver chaperoned peptide for alternate MHC-I Ag processing and cross-presentation via cytosolic mechanisms in dendritic cells and vacuolar mechanisms in macrophages. Prokaryotic HSPs are a potential source of microbial peptide Ags during phagocytic processing of bacteria during infection and could potentially be incorporated in vaccines to enhance presentation of peptides to CD8(+) T cells.     

果蝇免疫研究进展及其感染分枝杆菌的免疫特征

耐药性、持续感染以及与HIV病毒的共感染等诸多因素导致一度得到控制的结核病死灰复燃, 有效控制日益严峻的结核病迫切需要深入认识其致病菌——结核分枝杆菌Mycobacterium tuberculosis的基础生物学特性, 以及宿主相应的免疫控制机理。目前尚无一个动物模型能够同时回答这些关键问题, 而利用多种动物模型有望从不同角度回答上述问题, 普遍认为果蝇Drosophila 是比较理想的研究结核病天然免疫的简易模式动物之一。本文综述了果蝇免疫研究的最新进展, 包括免疫途径及其新成员与负调控子, 重点总结了用海分枝菌杆菌M. marinum、偶发分枝杆菌M. fortuitum和耻垢分枝杆菌M. smegmatis等分枝杆菌感染果蝇的新发现, 其中包括感染期间不诱导抗菌肽表达, 多个宿主因子(如CD36家族成员和ESCRT)参与了应答, 鉴定出具有杀灭分支杆菌作用的β-己糖酰胺酶, 感染期间能量代谢相关基因差异表达等。这些工作为利用果蝇模型快速筛选治疗结核病的新药物靶标和药物先导物提供了思路。     

Protein kinase E of Mycobacterium tuberculosis has a role in the nitric oxide stress response and apoptosis in a human macrophage model of infection

Mycobacterium tuberculosis , an intracellular pathogen, inhibits macrophage apoptosis to support survival and replication inside the host cell. We provide evidence that the functional serine/threonine kinase, PknE, is important for survival of M. tuberculosis that enhances macrophage viability by inhibiting apoptosis. A promoter of PknE identified in this study was shown to respond to nitric oxide stress. Deletion of pknE in virulent M. tuberculosis , H37Rv, resulted in a strain that has increased resistance to nitric oxide donors and increased sensitivity to reducing agents. The deletion mutant created by specialized transduction induced enhanced apoptosis while inhibiting necrosis. The pknE mutant also modifies the innate immune response as shown by the marked decline in the pro-inflammatory cytokines in a macrophage model of infection. These findings suggest a novel mechanism, by which PknE senses nitric oxide stress and prevents apoptosis by interfering with host signalling pathways.     

Genome-wide screen for Mycobacterium tuberculosis genes that regulate host immunity

In spite of its highly immunogenic properties, Mycobacterium tuberculosis (Mtb) establishes persistent infection in otherwise healthy individuals, making it one of the most widespread and deadly human pathogens. Mtb's prolonged survival may reflect production of microbial factors that prevent even more vigorous immunity (quantitative effect) or that divert the immune response to a non-sterilizing mode (qualitative effect). Disruption of Mtb genes has produced a list of several dozen candidate immunomodulatory factors. Here we used robotic fluorescence microscopy to screen 10,100 loss-of-function transposon mutants of Mtb for their impact on the expression of promoter-reporter constructs for 12 host immune response genes in a mouse macrophage cell line. The screen identified 364 candidate immunoregulatory genes. To illustrate the utility of the candidate list, we confirmed the impact of 35 Mtb mutant strains on expression of endogenous immune response genes in primary macrophages. Detailed analysis focused on a strain of Mtb in which a transposon disrupts Rv0431, a gene encoding a conserved protein of unknown function. This mutant elicited much more macrophage TNFα, IL-12p40 and IL-6 in vitro than wild type Mtb, and was attenuated in the mouse. The mutant list provides a platform for exploring the immunobiology of tuberculosis, for example, by combining immunoregulatory mutations in a candidate vaccine strain.     

Heat shock proteins protect platyfish (Xiphophorus maculatus) from Yersinia ruckeri induced mortality

The significant disadvantages accompanied with the use of antibiotics in aquaculture, emphasize the need for developing alternative disease control strategies, like novel vaccine approaches and immunostimulating measures. Several studies have already pointed out the ability of heat shock proteins (HSPs) to modulate innate and adaptive immune responses, what makes them potent candidates for the development of a new disease prevention method. In this study, the use of self and non-self heat shock proteins as a new prophylactic treatment against bacterial diseases in freshwater aquaculture was investigated. Therefore, an infection model was developed with platyfish as a host for Yersinia ruckeri infections. In this infection model, the effect of different treatments with HSPs on the survival of the fish after bacterial infection was tested: non-lethal heat shock, intracoelomal injection with two recombinant bacterial HSPs, GroEL and DnaK, and a combination of a non-lethal heat shock and an injection with bacterial HSPs. The results show that a non-lethal heat shock could not protect fish against a subsequent infection with Y. ruckeri. However, when the fish received an injection with bacterial HSPs, Y. ruckeri induced mortality was reduced. This effect became significant when the administration of bacterial HSPs was combined with a non-lethal heat shock. These data suggest a possible role for heat shock proteins as an immunostimulating treatment in fish against bacterial infections.     

Cellular heat shock factor 1 positively regulates human immunodeficiency virus-1 gene expression and replication by two distinct pathways

Human immunodeficiency virus-1 (HIV-1) infection leads to changes in cellular gene expression, which in turn tend to modulate viral gene expression and replication. Cellular heat shock proteins (HSPs) are induced upon heat shock, UV irradiation and microbial or viral infections. We have reported earlier Nef-dependent induction of HSP40 leading to increased HIV-1 gene expression; however, the mechanism of induction remained to be elucidated. As expression of HSPs is regulated by heat shock factors (HSFs), we have now studied the role of HSF1 not only in Nef-dependent HSP40 induction but also in HIV-1 gene expression. Our results show that HSF1 is also induced during HIV-1 infection and it positively regulates HIV-1 gene expression by two distinct pathways. First, along with Nef it activates HSP40 promoter which in turn leads to increased HIV-1 gene expression. Second, HSF1 directly interacts with newly identified HSF1 binding sequence on HIV-1 LTR promoter and induces viral gene expression and replication. Thus, the present work not only identifies a molecular basis for HSF1-mediated enhancement of viral replication but also provides another example of how HIV-1 uses host cell machinery for its successful replication in the host.     

Conserved Immune Recognition Hierarchy of Mycobacterial PE/PPE Proteins during Infection in Natural Hosts

The Mycobacterium tuberculosis genome contains two large gene families encoding proteins of unknown function, characterized by conserved N-terminal proline and glutamate (PE and PPE) motifs. The presence of a large number of PE/PPE proteins with repetitive domains and evidence of strain variation has given rise to the suggestion that these proteins may play a role in immune evasion via antigenic variation, while emerging data suggests that some family members may play important roles in mycobacterial pathogenesis. In this study, we examined cellular immune responses to a panel of 36 PE/PPE proteins during human and bovine infection. We observed a distinct hierarchy of immune recognition, reflected both in the repertoire of PE/PPE peptide recognition in individual cows and humans and in the magnitude of IFN-γ responses elicited by stimulation of sensitized host cells. The pattern of immunodominance was strikingly similar between cattle that had been experimentally infected with Mycobacterium bovis and humans naturally infected with clinical isolates of M. tuberculosis. The same pattern was maintained as disease progressed throughout a four-month course of infection in cattle, and between humans with latent as well as active tuberculosis. Detailed analysis of PE/PPE responses at the peptide level suggests that antigenic cross-reactivity amongst related family members is a major determinant in the observed differences in immune hierarchy. Taken together, these results demonstrate that a subset of PE/PPE proteins are major targets of the cellular immune response to tuberculosis, and are recognized at multiple stages of infection and in different disease states. Thus this work identifies a number of novel antigens that could find application in vaccine development, and provides new insights into PE/PPE biology.     

Enhanced mortality despite control of lung infection in mice aerogenically infected with a Mycobacterium tuberculosis mce1 operon mutant

Mycobacterium tuberculosis causes a variety of host clinical outcomes. We previously showed that M. tuberculosis disrupted in an operon called mce1 proliferates unchecked in BALB/c mouse lungs. The observed outcome could be attributed either to the mutant bacterial burden or to the host immunopathologic response. To differentiate these possibilities, we studied the outcomes of infection in a mouse strain (C57BL/6) less susceptible to M. tuberculosis than BALB/c. We found that the mutant infection reached a plateau in the lungs at a rate similar to that of the wild type. All mice infected with the mutant, but only half of the groups of mice infected with the wild type or complemented strain, died by 40 weeks (p<0.05). At 12-21 weeks of infection, histological examination of the lungs of mice infected with the mutant showed a diffuse pattern of lymphocyte infiltration, while that of mice infected with the other strains exhibited a nodular cellular infiltration pattern. Surprisingly, the number of bacilli recovered from the lungs was similar in all three groups. These observations suggest that rather than the bacterial burden, products of the mce1 operon may directly or indirectly modulate the host immune response that is protective to both the tubercle bacilli and the host.     

Baculovirus IE2 Stimulates the Expression of Heat Shock Proteins in Insect and Mammalian Cells to Facilitate Its Proper Functioning

Baculoviruses have gained popularity as pest control agents and for protein production in insect systems. These viruses are also becoming popular for gene expression, tissue engineering and gene therapy in mammalian systems. Baculovirus infection triggers a heat shock response, and this response is crucial for its successful infection of host insect cells. However, the viral protein(s) or factor(s) that trigger this response are not yet clear. Previously, we revealed that IE2-an early gene product of the baculovirus-could form unique nuclear bodies for the strong trans-activation of various promoters in mammalian cells. Here, we purified IE2 nuclear bodies from Vero E6 cells and investigated the associated proteins by using mass spectrometry. Heat shock proteins (HSPs) were found to be one of the major IE2-associated proteins. Our experiments show that HSPs are greatly induced by IE2 and are crucial for the trans-activation function of IE2. Interestingly, blocking both heat shock protein expression and the proteasome pathway preserved the IE2 protein and its nuclear body structure, and revived its function. These observations reveal that HSPs do not function directly to assist the formation of the nuclear body structure, but may rather protect IE2 from proteasome degradation. Aside from functional studies in mammalian cells, we also show that HSPs were stimulated and required to determine IE2 protein levels, in insect cells infected with baculovirus. Upon inhibiting the expression of heat shock proteins, baculovirus IE2 was substantially suppressed, resulting in a significantly suppressed viral titer. Thus, we demonstrate a unique feature in that IE2 can function in both insect and non-host mammalian cells to stimulate HSPs, which may be associated with IE2 stabilization and lead to the protection of the its strong gene activation function in mammalian cells. On the other hand, during viral infection in insect cells, IE2 could also strongly stimulate HSPs and ultimately affect viral replication.     

5'-Adenosinephosphosulphate reductase (CysH) protects Mycobacterium tuberculosis against free radicals during chronic infection phase in mice

A major obstacle to tuberculosis (TB) control is the problem of chronic TB infection (CTBI). Here we report that 5'-adenosinephosphosulphate reductase (CysH), an enzyme essential for the production of reduced-sulphur-containing metabolites, is critical for Mycobacterium tuberculosis (Mtb) survival in chronic infection phase in mice. Disruption of cysH rendered Mtb auxotrophic for cysteine and methionine, and attenuated virulence in BALB/c and C57BL/6 immunocompetent mice. The mutant and wild-type Mtb replicated similarly during the acute phase of infection, but the mutant showed reduced viability during the persistent phase of the infection. The cysH mutant caused disease and death after 4-7 weeks of infection in four different groups of mice - Rag1(-/-), NOS2(-/-), gp91phox(-/-) NOS2(-/-) and gp91phox(-/-) mice given aminoguanidine [to suppress the effects of nitric oxide synthase 2 (NOS2)]- indicating minimal metabolic effect on the cysH mutant survival in these mice. The cysH mutant was also susceptible to peroxynitrite and hydrogen peroxide in vitro. These results show that CysH is important for Mtb protection during the chronic infection phase, and that resistance to nitrosative and oxidative stress may be the mechanism of this protection. Thus, this metabolic gene of an intracellular pathogen could have a secondary role in protection against the host immune response. Finally the lack of an endogenous human orthologue of cysH and its possible role in defence against adaptive immunity renders CysH an attractive enzyme for further studies as a target for therapeutics active against CTBI.     

IL17A augments autophagy in Mycobacterium tuberculosis-infected monocytes from patients with active tuberculosis in association with the severity of the disease

During mycobacterial infection, macroautophagy/autophagy, a process modulated by cytokines, is essential for mounting successful host responses. Autophagy collaborates with human immune responses against Mycobacterium tuberculosis (Mt) in association with specific IFNG secreted against the pathogen. However, IFNG alone is not sufficient to the complete bacterial eradication, and other cytokines might be required. Actually, induction of Th1 and Th17 immune responses are required for protection against Mt. Accordingly, we showed that IL17A and IFNG expression in lymphocytes from tuberculosis patients correlates with disease severity. Here we investigate the role of IFNG and IL17A during autophagy in monocytes infected with Mt H37Rv or the mutant MtΔRD1. Patients with active disease were classified as high responder (HR) or low responder (LR) according to their T cell responses against Mt. IL17A augmented autophagy in infected monocytes from HR patients through a mechanism that activated MAPK1/ERK2-MAPK3/ERK1 but, during infection of monocytes from LR patients, IL17A had no effect on the autophagic response. In contrast, addition of IFNG to infected monocytes, increased autophagy by activating MAPK14/p38 α both in HR and LR patients. Interestingly, proteins codified in the RD1 region did not interfere with IFNG and IL17A autophagy induction. Therefore, in severe tuberculosis patients' monocytes, IL17A was unable to augment autophagy because of a defect in the MAPK1/3 signaling pathway. In contrast, both IFNG and IL17A increased autophagy levels in patients with strong immunity to Mt, promoting mycobacterial killing. Our findings might contribute to recognize new targets for the development of novel therapeutic tools to fight the pathogen.     

Bacterial heat shock proteins enhance class II MHC antigen processing and presentation of chaperoned peptides to CD4+ T cells

APCs process heat shock protein (HSP):peptide complexes to present HSP-chaperoned peptides on class I MHC molecules, but the ability of HSPs to contribute chaperoned peptides for class II MHC (MHC-II) Ag processing and presentation is unclear. Our studies revealed that exogenous bacterial HSPs (Escherichia coli DnaK and Mycobacterium tuberculosis HSP70) delivered an extended OVA peptide for processing and MHC-II presentation, as detected by T hybridoma cells. Bacterial HSPs enhanced MHC-II presentation only if peptide was complexed to the HSP, suggesting that the key HSP function was enhanced delivery or processing of chaperoned peptide Ag rather than generalized enhancement of APC function. HSP-enhanced processing was intact in MyD88 knockout cells, which lack most TLR signaling, further suggesting the effect was not due to TLR-induced induction of accessory molecules. Bacterial HSPs enhanced uptake of peptide, which may contribute to increased MHC-II presentation. In addition, HSPs enhanced binding of peptide to MHC-II molecules at pH 5.0 (the pH of vacuolar compartments), but not at pH 7.4, indicating another mechanism for enhancement of MHC-II Ag processing. Bacterial HSPs are a potential source of microbial peptide Ags during phagocytic processing of bacteria during infection and could potentially be incorporated in vaccines to enhance presentation of peptides to CD4+ T cells.     

Mycobacterial secretion systems ESX-1 and ESX-5 play distinct roles in host cell death and inflammasome activation

During infection of humans and animals, pathogenic mycobacteria manipulate the host cell causing severe diseases such as tuberculosis and leprosy. To understand the basis of mycobacterial pathogenicity, it is crucial to identify the molecular virulence mechanisms. In this study, we address the contribution of ESX-1 and ESX-5--two homologous type VII secretion systems of mycobacteria that secrete distinct sets of immune modulators--during the macrophage infection cycle. Using wild-type, ESX-1- and ESX-5-deficient mycobacterial strains, we demonstrate that these secretion systems differentially affect subcellular localization and macrophage cell responses. We show that in contrast to ESX-1, the effector proteins secreted by ESX-5 are not required for the translocation of Mycobacterium tuberculosis or Mycobacterium marinum to the cytosol of host cells. However, the M. marinum ESX-5 mutant does not induce inflammasome activation and IL-1β activation. The ESX-5 system also induces a caspase-independent cell death after translocation has taken place. Importantly, by means of inhibitory agents and small interfering RNA experiments, we reveal that cathepsin B is involved in both the induction of cell death and inflammasome activation upon infection with wild-type mycobacteria. These results reveal distinct roles for two different type VII secretion systems during infection and shed light on how virulent mycobacteria manipulate the host cell in various ways to replicate and spread.     

Infection biology of a novel alpha-crystallin of Mycobacterium tuberculosis: Acr2

Heat shock proteins assist the survival of Mycobacterium tuberculosis (MTB) but also provide a signal to the immune response. The gene most strongly induced by heat shock in MTB is Rv0251c, which encodes Acr2, a novel member of the alpha-crystallin family of molecular chaperones. The expression of acr2 increased within 1 h after infection of monocytes or macrophages, reaching a peak of 18- to 55-fold by 24 h. Inhibition of superoxide action reduced the intracellular increase in acr2. Despite this contribution to the stress response of MTB, the gene for acr2 appears dispensable; a deletion mutant (Deltaacr2) was unimpaired in log phase growth and persisted in IFN-gamma-activated human macrophages. Acr2 protein was strongly recognized by cattle with early primary Mycobacterium bovis infection and by healthy MTB-sensitized people. Within the latter group, those with recent exposure to infectious tuberculosis had, on average, 2.6 times the frequency of Acr2-specific IFN-gamma-secreting T cells than those with more remote exposure (p = 0.009). These data show that, by its up-regulation early after entry to cells, Acr2 gives away the presence of MTB to the immune response. The demonstration that there is infection stage-specific immunity to tuberculosis has implications for vaccine design.     

Heat shock response to vaccinia virus infection.

We have investigated the induction of heat shock proteins (HSPs) in mice infected with vaccinia virus. Vaccinia virus replicates to high levels in the ovaries of infected mice and causes a significant inhibition of host cell DNA, RNA, and protein synthesis. Many HSPs are constitutively expressed in murine ovarian tissue at low levels, consistent with their obligatory role in normal physiological events. In contrast with these events, HSP expression was augmented in virus-infected mouse ovaries 6 days postinfection. In particular, there was a dramatic increase in the expression of a protein identified as the inducible 72-kDa HSP. Analysis of cellular mRNA confirmed this protein to be the major mouse inducible HSP70 and demonstrated its presence within virus-infected cells. Hence, we have demonstrated the expression of stress proteins during poxvirus infection in vivo.     

GPI7-mediated glycosylphosphatidylinositol anchoring regulates appressorial penetration and immune evasion during infection of Magnaporthe oryzae

Glycosylphosphatidylinositol (GPI) anchoring plays key roles in many biological processes by targeting proteins to the cell wall; however, its roles are largely unknown in plant pathogenic fungi. Here, we reveal the roles of the GPI anchoring in Magnaporthe oryzae during plant infection. The GPI-anchored proteins were found to highly accumulate in appressoria and invasive hyphae. Disruption of GPI7, a GPI anchor-pathway gene, led to a significant reduction in virulence. The Δgpi7 mutant showed significant defects in penetration and invasive growth. This mutant also displayed defects of the cell wall architecture, suggesting GPI7 is required for cell wall biogenesis. Removal of GPI-anchored proteins in the wild-type strain by hydrofluoric acid (HF) pyridine treatment exposed both the chitin and β-1,3-glucans to the host immune system. Exposure of the chitin and β-1,3-glucans was also observed in the Δgpi7 mutant, indicating GPI-anchored proteins are required for immune evasion. The GPI anchoring can regulate subcellular localization of the Gel proteins in the cell wall for appressorial penetration and abundance of which for invasive growth. Our results indicate the GPI anchoring facilitates the penetration of M. oryzae into host cells by affecting the cell wall integrity and the evasion of host immune recognition.     

Inactivation of the Mycobacterium tuberculosis fadB4 gene results in increased virulence in host cell and mice

The genome of Mycobacterium tuberculosis encodes many proteins involved in fatty acid metabolism, a subset of which are required for virulence. The Mycobacterium tuberculosis fadB4 gene, which shares strong similarity with oxidoreductases and fatty acid synthases, is up-regulated during infection of macrophages and is predicted to protect the bacterium from the hostile environment of the host cell. In order to determine if fadB4 plays a role in the virulence of M. tuberculosis, we constructed a M. tuberculosis mutant in which the fadB4 had been disrupted (DeltafadB4). Surprisingly, DeltafadB4, grew more rapidly in host cells compared to wild-type M. tuberculosis or the DeltafadB4 or the gene-disrupted strain complemented with fadB4. In addition, macrophages infected with DeltafadB4 displayed reduced secretion of the cytokine TNF-alpha, suggesting a role for the FadB4 protein in influencing the pro-inflammatory host response to M. tuberculosis. After infection of mice, DeltafadB4 demonstrated an increased replication at early time-points post-infection compared to the growth of wild-type M. tuberculosis. This increased capacity of DeltafadB4 to replicate in vivo was reflected in the decreased time to death of immuno-deficient RAG-1(-/-) mice infected with M. tuberculosis lacking the fadB4 gene. Therefore fadB4 is part of the family of genes whose expression serves to regulate the virulence of M. tuberculosis within the host.