InvA protein is a Nudix hydrolase required for infection by pathogenic Leptospira in cell lines and animals

Leptospirosis caused by pathogenic species of the genus Leptospira is a re-emerging zoonotic disease, which affects a wide variety of host species and is transmitted by contaminated water. The genomes of several pathogenic Leptospira species contain a gene named invA, which contains a Nudix domain. However, the function of this gene has never been characterized. Here, we demonstrated that the invA gene was highly conserved in protein sequence and present in all tested pathogenic Leptospira species. The recombinant InvA protein of pathogenic L. interrogans strain Lai hydrolyzed several specific dinucleoside oligophosphate substrates, reflecting the enzymatic activity of Nudix in Leptospira species. Pathogenic leptospires did not express this protein in media but temporarily expressed it at early stages (within 60 min) of infection of macrophages and nephric epithelial cells. Comparing with the wild type, the invA-deficient mutant displayed much lower infectivity and a significantly reduced survival rate in macrophages and nephric epithelial cells. Moreover, the invA-deficient leptospires presented an attenuated virulence in hamsters, caused mild histopathological damage, and were transmitted in lower numbers in the urine, compared with the wild-type strain. The invA revertant, made by complementing the invA-deficient mutant with the invA gene, reacquired virulence similar to the wild type in vitro and in vivo. The LD(50) in hamsters was 1000-fold higher for the invA-deficient mutant than for the invA revertant and wild type. These results demonstrate that the InvA protein is a Nudix hydrolase, and the invA gene is essential for virulence in pathogenic Leptospira species.     

A cytoplasmic RNA virus generates functional viral small RNAs and regulates viral IRES activity in mammalian cells

The roles of virus-derived small RNAs (vsRNAs) have been studied in plants and insects. However, the generation and function of small RNAs from cytoplasmic RNA viruses in mammalian cells remain unexplored. This study describes four vsRNAs that were detected in enterovirus 71-infected cells using next-generation sequencing and northern blots. Viral infection produced substantial levels (>10⁵ copy numbers per cell) of vsRNA1, one of the four vsRNAs. We also demonstrated that Dicer is involved in vsRNA1 generation in infected cells. vsRNA1 overexpression inhibited viral translation and internal ribosomal entry site (IRES) activity in infected cells. Conversely, blocking vsRNA1 enhanced viral yield and viral protein synthesis. We also present evidence that vsRNA1 targets stem-loop II of the viral 5′ untranslated region and inhibits the activity of the IRES through this sequence-specific targeting. Our study demonstrates the ability of a cytoplasmic RNA virus to generate functional vsRNA in mammalian cells. In addition, we also demonstrate a potential novel mechanism for a positive-stranded RNA virus to regulate viral translation: generating a vsRNA that targets the IRES.     

Interactome-wide Analysis Identifies End-binding Protein 1 as a Crucial Component for the Speck-like Particle Formation of Activated Absence in Melanoma 2 (AIM2) Inflammasomes

Inflammasomes are cytoplasmic receptors that can recognize intracellular pathogens or danger signals and are critical for interleukin 1β production. Although several key components of inflammasome activation have been identified, there has not been a systematic analysis of the protein components found in the stimulated complex. In this study, we used the isobaric tags for relative and absolute quantification approach to systemically analyze the interactomes of the NLRP3, AIM2, and RIG-I inflammasomes in nasopharyngeal carcinoma cells treated with specific stimuli of these interactomes (H₂O₂, poly (dA:dT), and EBV noncoding RNA, respectively). We identified a number of proteins that appeared to be involved in the interactomes and also could be precipitated with anti-apoptosis-associated speck-like protein containing caspase activation and recruitment domain antibodies after stimulation. Among them, end binding protein 1 was an interacting component in all three interactomes. Silencing of end binding protein 1 expression by small interfering RNA inhibited the activation of the three inflammasomes, as indicated by reduced levels of interleukin 1β secretion. We confirmed that end binding protein 1 directly interacted with AIM2 and ASC in vitro and in vivo. Most importantly, fluorescence confocal microscopy showed that end binding protein 1 was required for formation of the speck-like particles that represent activation of the AIM2 inflammasome. In nasopharyngeal carcinoma tissues, immunohistochemical staining showed that end binding protein 1 expression was elevated and significantly correlated with AIM2 and ASC expression in nasopharyngeal carcinoma tumor cells. In sum, we profiled the interactome components of three inflammasomes and show for the first time that end binding protein 1 is crucial for the speck-like particle formation that represents activated inflammasomes.Nasopharyngeal carcinoma (NPC)¹ is a malignancy of the head and the neck that is highly prevalent in Southern China and Southeast Asia (1). Both environmental and genetic risk factors are considered to be important for the development of NPC (2, 3); among them, Epstein-Barr virus (EBV) infection of the epithelium is the most important known factor (1). In addition to the EBV-encoded oncoprotein-mediated blockade of intracellular mechanisms in EBV-associated tumors (1), chronic inflammation is considered to be an important oncogenic factor in NPC (4). Interleukin 1 beta (IL-1β), which is an inflammatory cytokine that has oncogenic effects in many tumors (5), can be detected in NPC tumor tissues (6, 7). IL-1β secretion is mediated by cytosolic protein complexes called inflammasomes, which induce IL-1β secretion by activating catalytic caspase 1 (8). However, no previous study has examined inflammasome components in NPC tumor cells or the mechanisms of inflammasome regulation in NPC.Inflammasomes are cytoplasmic receptors that act in innate immunity to recognize intracellular pathogen-associated molecular pattern (PAMP) or danger signal-associated molecular pattern (DAMP). A number of inflammasomes have been identified in recent years (9), and they can be classified into different subgroups according to their recognizing PAMP. These groups include the NOD-like receptors (NLR), which sense intracellular pathogens (e.g. bacteria, fungi, and parasites) and activate pro-caspase 1 with or without an adaptive protein called apoptosis-associated speck-like protein containing caspase activation and recruitment domain (ASC) (10). Activated caspase 1 then induces IL-1β secretion through direct cleavage of pro-IL-1β (8). Among the NLR family members, the NLRP3 inflammasome recognizes both pathogens and danger signals such as ATP or reactive oxygen species (ROS) generation (11, 12). Members of the two other subgroups, absence in melanoma 2 (AIM2) and retinoic acid-inducible gene I (RIG-I), sense cytoplasmic double-strand DNA and 5′-triphoshphate RNA, respectively, and then recruit ASC to activate pro-caspase 1 (13, 14). Although inflammasomes are important for pathogen defense in immune cells, recent studies have shown that inflammasomes also participate in tumorigenesis in colon cancer and melanoma (15–17). A previous report showed that EBV noncoding RNAs (EBERs) are recognized by RIG-I and activate signaling to induce type I IFN in EBV-infected B lymphocytes (18). This report is consistent with our recent unpublished observation that RIG-I is activated by EBERs in NPC cells. We additionally show that NLRP3 is triggered by tumor microenvironmental factors, such as ATP and ROS, and the clinical drug cisplatin; AIM2 recognizes EBV genomic DNA and is activated by irradiation in NPC cells. Although these inflammasomes play important role in NPC, the regulation and the interactome of these inflammasome complexes are not fully understood.On activation by PAMP or DAMP, the activated inflammasomes tend to aggregate in the cytosol as speck-like particles (13). Biochemical and cell biological data have indicated that the core components of the inflammasome comprise the receptor, ASC, and pro-caspase 1, but an increasing number of proteins have been identified as interacting with these complexes. For example, heat-shock protein 90 (HSP90) is essential for the function of the NLRP3 and RIG-I inflammasomes (19, 20). NLRC5, another member of the NLR family, is involved in the NLRP3 inflammasome and is required for its activity (21). Rac1, a small Rho GTPase family member, is reportedly required for NLRP3 inflammasome activation during C. pneumoniae infection (22). The S. Typhimurium effector, SopE, activates caspase 1 through Rac1 activity (23), whereas Yersinia bacteria prevent caspase 1 activation by inhibiting Rac1 activity via the effector protein, YopE (24). Notably, Rac1 regulates cytoskeletal rearrangement (25), suggesting that cytoskeletal components may participate in inflammasome activation.End-binding protein 1 (EB1), an adenomatous polyposis coli (APC)-binding protein, regulates microtubule polymerization by recruiting the plus-end tracking protein (+TIP) complex to the plus end of microtubules (26). The interaction of EB1 and the +TIP complex depends on the C-terminal (CT) domain of EB1, whereas the calponin homology (CH) domain of EB1 binds to the microtubule (26). Many studies have shown that EB1 participates in different biological processes, including mitosis, migration and signal transduction (27–29), and also that it plays an oncogenic role in cancer by affecting cell growth or migration (30, 31). However, although EB1 is known to be a cytoskeletal component that is regulated by the small GTPase, RhoA (28), its role in inflammasome activation has not yet been explored.Here, we used the isobaric tags for relative and absolute quantification (iTRAQ) approach to systemically analyze the interactomes of the NLRP3, AIM2, and RIG-I inflammasomes in NPC cell lines treated with their specific stimuli, H₂O₂, poly (dA:dT), and EBER, respectively. We characterized the interactomes of the NLRP3, AIM2, and RIG-I inflammasomes in NPC cells by proteomic analysis, and report for the first time that EB1 can directly bind to the AIM2 inflammasome and is essential for speck-like particle formation in NPC cells. Finally, we suggest some possible mechanisms for EB1-associated AIM2 inflammasome activation via microtubule polymerization and RhoA activity.     

Inactivation of the fliY gene encoding a flagellar motor switch protein attenuates mobility and virulence of Leptospira interrogans strain Lai

Background  

Pathogenic Leptospira species cause leptospirosis, a zoonotic disease of global importance. The spirochete displays active rotative mobility which may contribute to invasion and diffusion of the pathogen in hosts. FliY is a flagellar motor switch protein that controls flagellar motor direction in other microbes, but its role in Leptospira, and paricularly in pathogenicity remains unknown.     

Identification of novel HLA-DR1-restricted epitopes from the hepatitis B virus envelope protein in mice expressing HLA-DR1 and vaccinated human subjects

Helper T lymphocytes that control CD8(+) T-cell and antibody responses are key elements for the resolution of infection by the hepatitis B virus and for the development of effective immunological memory after hepatitis B vaccination. We have used H-2 class II-deficient mice that express the human MHC class II molecule, HLA-DR1, to identify novel hepatitis B virus envelope-derived T helper epitopes. We confirmed the immunogenicity of a previously described HLA-DR1-restricted epitope, and identified three novel epitopes. CD4(+) T-cell immune responses against these epitopes were detected in peripheral blood mononuclear cells from HLA-DR1(+) individuals vaccinated against hepatitis B. We showed that subjects receiving the currently available hepatitis B vaccines do not develop cross-reactive T helper responses against one of the novel epitopes which are structurally variable between different hepatitis B virus subtypes. These findings highlight the need for developing vaccines against a wider range of viral subtypes, and establish humanized mice as a convenient tool for identifying new immunogenic epitopes from pathogens.     

Critical involvement of the ATM-dependent DNA damage response in the apoptotic demise of HIV-1-elicited syncytia

DNA damage can activate the oncosuppressor protein ataxia telangiectasia mutated (ATM), which phosphorylates the histone H2AX within characteristic DNA damage foci. Here, we show that ATM undergoes an activating phosphorylation in syncytia elicited by the envelope glycoprotein complex (Env) of human immunodeficiency virus-1 (HIV-1) in vitro. This was accompanied by aggregation of ATM in discrete nuclear foci that also contained phospho-histone H2AX. DNA damage foci containing phosphorylated ATM and H2AX were detectable in syncytia present in the brain or lymph nodes from patients with HIV-1 infection, as well as in a fraction of blood leukocytes, correlating with viral status. Knockdown of ATM or of its obligate activating factor NBS1 (Nijmegen breakage syndrome 1 protein), as well as pharmacological inhibition of ATM with KU-55933, inhibited H2AX phosphorylation and prevented Env-elicited syncytia from undergoing apoptosis. ATM was found indispensable for the activation of MAP kinase p38, which catalyzes the activating phosphorylation of p53 on serine 46, thereby causing p53 dependent apoptosis. Both wild type HIV-1 and an HIV-1 mutant lacking integrase activity induced syncytial apoptosis, which could be suppressed by inhibiting ATM. HIV-1-infected T lymphoblasts from patients with inactivating ATM or NBS1 mutations also exhibited reduced syncytial apoptosis. Altogether these results indicate that apoptosis induced by a fusogenic HIV-1 Env follows a pro-apoptotic pathway involving the sequential activation of ATM, p38MAPK and p53.     

Modulation of P2Z/P2X(7) receptor activity in macrophages infected with Chlamydia psittaci

Given the role that extracellular ATP (ATP(o))-mediated apoptosis may play in inflammatory responses and in controlling mycobacterial growth in macrophages, we investigated whether ATP(o) has any effect on the viability of chlamydiae in macrophages and, conversely, whether the infection has any effect on susceptibility to ATP(o)-induced killing via P2Z/P2X(7) purinergic receptors. Apoptosis of J774 macrophages could be selectively triggered by ATP(o), because other purine/pyrimidine nucleotides were ineffective, and it was inhibited by oxidized ATP, which irreversibly inhibits P2Z/P2X(7) purinergic receptors. Incubation with ATP(o) but not other extracellular nucleotides inhibits the growth of intracellular chlamydiae, consistent with previous observations on ATP(o) effects on growth of intracellular mycobacteria. However, chlamydial infection for 1 day also inhibits ATP(o)-mediated apoptosis, which may be a mechanism to partially protect infected cells against the immune response. Infection by Chlamydia appears to protect cells by decreasing the ability of ATP(o) to permeabilize macrophages to small molecules and by abrogating a sustained Ca(2+) influx previously associated with ATP(o)-induced apoptosis.     

A role for mitogen-activated protein kinase(Erk1/2) activation and non-selective pore formation in P2X7 receptor-mediated thymocyte death

Extracellular ATP (ATPe) binds to P2X7 receptors (P2X7R) expressed on the surface of cells of hematopoietic lineage, including murine thymocytes. Activation of P2X7R by ATPe results in the opening of cation-specific channels, and prolonged ATPe exposure leads to the formation of non-selective pores enabling transmembrane passage of solutes up to 900 Da. In the presence of ATPe, P2X7R-mediated thymocyte death is due primarily to necrosis/lysis and not apoptosis, as measured by the release of lactate dehydrogenase indicative of a loss of plasma membrane integrity. The present study is focused on the identification of P2X7R signaling mediators in ATP-induced thymocyte necrosis/lysis. Thus, extracellular signal-regulated protein kinase 1/2 (Erk1/2) phosphorylation was found to be required for cell lysis, and both events were independent of ATP-induced calcium influx. P2X7R-dependent thymocyte death involved the chronological activation of Src family tyrosine kinase(s), phosphatidylinositol 3-kinase, the mitogen-activated protein (MAP) kinase(Erk1/2) module, and the proteasome. Although independent of this signaling cascade, non-selective pore formation may modulate ATP-mediated thymocyte death. These results therefore suggest a role for both activation of MAP kinase(Erk1/2) and non-selective pore opening in P2X7R-induced thymocyte death.