Type I Interferon Response Dysregulates Host Iron Homeostasis and Enhances Candida glabrata Infection

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Differential Regulation of Type I Interferon and Epidermal Growth Factor Pathways by a Human Respirovirus Virulence Factor

A number of paramyxoviruses are responsible for acute respiratory infections in children, elderly and immuno-compromised individuals, resulting in airway inflammation and exacerbation of chronic diseases like asthma. To understand the molecular pathogenesis of these infections, we searched for cellular targets of the virulence protein C of human parainfluenza virus type 3 (hPIV3-C). We found that hPIV3-C interacts directly through its C-terminal domain with STAT1 and GRB2, whereas C proteins from measles or Nipah viruses failed to do so. Binding to STAT1 explains the previously reported capacity of hPIV3-C to block type I interferon signaling, but the interaction with GRB2 was unexpected. This adaptor protein bridges Epidermal Growth Factor (EGF) receptor to MAPK/ERK pathway, a signaling cascade recently found to be involved in airway inflammatory response. We report that either hPIV3 infection or transient expression of hPIV3-C both increase cellular response to EGF, as assessed by Elk1 transactivation and phosphorylation levels of ERK1/2, 40S ribosomal subunit protein S6 and translation initiation factor 4E (eIF4E). Furthermore, inhibition of MAPK/ERK pathway with U0126 prevented viral protein expression in infected cells. Altogether, our data provide molecular basis to explain the role of hPIV3-C as a virulence factor and determinant of pathogenesis and demonstrate that Paramyxoviridae have evolved a single virulence factor to block type I interferon signaling and to boost simultaneous cellular response to growth factors.     

Coverage of Related Pathogenic Species by Multivalent and Cross-Protective Vaccine Design: Arenaviruses as a Model System

Summary: The arenaviruses are a family of negative-sense RNA viruses that cause severe human disease ranging from aseptic meningitis to hemorrhagic fever syndromes. There are currently no FDA-approved vaccines for the prevention of arenavirus disease, and therapeutic treatment is limited to the use of ribavirin and/or immune plasma for a subset of the pathogenic arenaviruses. The considerable genetic variability observed among the seven arenaviruses that are pathogenic for humans illustrates one of the major challenges for vaccine development today, namely, to overcome pathogen heterogeneity. Over the past 5 years, our group has tested several strategies to overcome pathogen heterogeneity, utilizing the pathogenic arenaviruses as a model system. Because T cells play a prominent role in protective immunity following arenavirus infection, we specifically focused on the development of human vaccines that would induce multivalent and cross-protective cell-mediated immune responses. To facilitate our vaccine development and testing, we conducted large-scale major histocompatibility complex (MHC) class I and class II epitope discovery on murine, nonhuman primate, and human backgrounds for each of the pathogenic arenaviruses, including the identification of protective HLA-restricted epitopes. Finally, using the murine model of lymphocytic choriomeningitis virus infection, we studied the phenotypic characteristics associated with immunodominant and protective T cell epitopes. This review summarizes the findings from our studies and discusses their application to future vaccine design.     

Tetrahymena: An Alternative Model Host for Evaluating Virulence of Aeromonas Strains

An easier assessment model would be helpful for high-throughput screening of Aeromonas virulence. The previous study indicated the potential of Tetrahymena as a permissive model to examine virulence of Aeromonas hydrophila. Here our aim was to assess virulence of Aeromonas spp. using two model hosts, a zebrafish assay and Tetrahymena-Aeromonas co-culture, and to examine whether data from the Tetrahymena thermophila model reflects infections in the well-established animal model. First, virulence of 39 Aeromonas strains was assessed by determining the 50% lethal dose (LD₅₀) in zebrafish. LD₅₀ values ranging from 1.3×10² to 3.0×10⁷ indicated that these strains represent a high to moderate degree of virulence and could be useful to assess virulence in the Tetrahymena model. In Tetrahymena-Aeromonas co-culture, we evaluated the virulence of Aeromonas by detecting relative survival of Aeromonas and Tetrahymena. An Aeromonas isolate was considered virulent when its relative survival was greater than 60%, while the Aeromonas isolate was considered avirulent if its relative survival was below 40%. When relative survival of T. thermophila was lower than 40% after co-culture with an Aeromonas isolate, the bacterial strain was regarded as virulent. In contrast, the strain was classified as avirulent if relative survival of T. thermophila was greater than 50%. Encouragingly, data from the 39 Aeromonas strains showed good correlation in zebrafish and Tetrahymena-Aeromonas co-culture models. The results provide sufficient data to demonstrate that Tetrahymena can be a comparable alternative to zebrafish for determining the virulence of Aeromonas isolates.     

A Versatile Vector for In Vivo Monitoring of Type I Interferon Induction and Signaling

Development of reporter systems for in vivo examination of IFN-β induction or signaling of type I interferon (IFN-I) pathways is of great interest in order to characterize biological responses to different inducers such as viral infections. Several reporter mice have been developed to monitor the induction of both pathways in response to different agonists. However, alternative strategies that do not require transgenic mice breeding have to date not been reported. In addition, detection of these pathways in vivo in animal species other than mice has not yet been addressed. Herein we describe a simple method based on the use of an adeno-associated viral vector (AAV8-3xIRF-ISRE-Luc) containing an IFN-β induction and signaling-sensitive promoter sequence controlling the expression of the reporter gene luciferase. This vector is valid for monitoring IFN-I responses in vivo elicited by diverse stimuli in different organs. Intravenous administration of the vector in C57BL/6 mice and Syrian hamsters was able to detect activation of the IFN pathway in the liver upon systemic treatment with different pro-inflammatory agents and infection with Newcastle disease virus (NDV). In addition, intranasal instillation of AAV8-3xIRF-ISRE-Luc showed a rapid and transient IFN-I response in the respiratory tract of mice infected with the influenza A/PR8/34 virus lacking the NS1 protein. In comparison, this response was delayed and exacerbated in mice infected with influenza A/PR/8 wild type virus. In conclusion, the AAV8-3xIRF-ISRE-Luc vector offers the possibility of detecting IFN-I activation in response to different stimuli and in different animal models with no need for reporter transgenic animals.     

A Novel Serine/Threonine Protein Kinase Homologue of Pseudomonas aeruginosa Is Specifically Inducible within the Host Infection Site and Is Required for Full Virulence in Neutropenic Mice

A genetic locus of Pseudomonas aeruginosa was identified that is highly and specifically inducible during infection of neutropenic mice. This locus, ppkA, encodes a protein that is highly homologous to eukaryote-type serine/threonine protein kinases. A ppkA null mutant strain shows reduced virulence in neutropenic mice compared to the wild type. Overexpression of the PpkA protein greatly inhibited the growth of Escherichia coli or P. aeruginosa. However, a single amino acid change at the catalytic site of the kinase domain eliminated the toxic effect of PpkA on bacterial cells, suggesting that the kinase domain of PpkA is functional within bacterial cells.     

Identification of Host Cytosolic Sensors and Bacterial Factors Regulating the Type I Interferon Response to Legionella pneumophila

Legionella pneumophila is a gram-negative bacterial pathogen that replicates in host macrophages and causes a severe pneumonia called Legionnaires'' Disease. The innate immune response to L. pneumophila remains poorly understood. Here we focused on identifying host and bacterial factors involved in the production of type I interferons (IFN) in response to L. pneumophila. It was previously suggested that the delivery of L. pneumophila DNA to the host cell cytosol is the primary signal that induces the type I IFN response. However, our data are not easily reconciled with this model. We provide genetic evidence that two RNA-sensing proteins, RIG-I and MDA5, participate in the IFN response to L. pneumophila. Importantly, these sensors do not seem to be required for the IFN response to L. pneumophila DNA, whereas we found that RIG-I was required for the response to L. pneumophila RNA. Thus, we hypothesize that bacterial RNA, or perhaps an induced host RNA, is the primary stimulus inducing the IFN response to L. pneumophila. Our study also identified a secreted effector protein, SdhA, as a key suppressor of the IFN response to L. pneumophila. Although viral suppressors of cytosolic RNA-sensing pathways have been previously identified, analogous bacterial factors have not been described. Thus, our results provide new insights into the molecular mechanisms by which an intracellular bacterial pathogen activates and also represses innate immune responses.