TNFR1 signaling converging on FGF14 controls neuronal hyperactivity and sickness behavior in experimental cerebral malaria.

BACKGROUND: Excess tumor necrosis factor (TNF) is implicated in the pathogenesis of hyperinflammatory experimental cerebral malaria (eCM), including gliosis, increased levels of fibrin(ogen) in the brain, behavioral changes, and mortality. However, the role of TNF in eCM within the brain parenchyma, particularly directly on neurons, remains underdefined. Here, we investigate electrophysiological consequences of eCM on neuronal excitability and cell signaling mechanisms that contribute to observed phenotypes. METHODS: The split-luciferase complementation assay (LCA) was used to investigate cell signaling mechanisms downstream of tumor necrosis factor receptor 1 (TNFR1) that could contribute to changes in neuronal excitability in eCM. Whole-cell patch-clamp electrophysiology was performed in brain slices from eCM mice to elucidate consequences of infection on CA1 pyramidal neuron excitability and cell signaling mechanisms that contribute to observed phenotypes. Involvement of identified signaling molecules in mediating behavioral changes and sickness behavior observed in eCM were investigated in vivo using genetic silencing. RESULTS: Exploring signaling mechanisms that underlie TNF-induced effects on neuronal excitability, we found that the complex assembly of fibroblast growth factor 14 (FGF14) and the voltage-gated Na+ (Nav) channel 1.6 (Nav1.6) is increased upon tumor necrosis factor receptor 1 (TNFR1) stimulation via Janus Kinase 2 (JAK2). On account of the dependency of hyperinflammatory experimental cerebral malaria (eCM) on TNF, we performed patch-clamp studies in slices from eCM mice and showed that Plasmodium chabaudi infection augments Nav1.6 channel conductance of CA1 pyramidal neurons through the TNFR1-JAK2-FGF14-Nav1.6 signaling network, which leads to hyperexcitability. Hyperexcitability of CA1 pyramidal neurons caused by infection was mitigated via an anti-TNF antibody and genetic silencing of FGF14 in CA1. Furthermore, knockdown of FGF14 in CA1 reduced sickness behavior caused by infection. CONCLUSIONS: FGF14 may represent a therapeutic target for mitigating consequences of TNF-mediated neuroinflammation.

Role of Sca2 and RickA in the Dissemination of Rickettsia parkeri in Amblyomma maculatum.

The Gram-negative obligate intracellular bacterium Rickettsia parkeri is an emerging tick-borne human pathogen. Recently, R. parkeri Sca2 and RickA have been implicated in adherence and actin-based motility in vertebrate host cell infection models; however, the rickettsia-derived factors essential to tick infection are unknown. Using R. parkeri mutants lacking functional Sca2 or RickA to compare actin polymerization, replication, and cell-to-cell spread in vitro, similar phenotypes in tick and mammalian cells were observed. Specifically, actin polymerization in cultured tick cells is controlled by the two separate proteins in a time-dependent manner. To assess the role of Sca2 and RickA in dissemination in the tick host, Rickettsia-free Amblyomma maculatum, the natural vector of R. parkeri, was exposed to wild-type, R. parkeri rickA::tn, or R. parkeri sca2::tn bacteria, and individual tick tissues, including salivary glands, midguts, ovaries, and hemolymph, were analyzed at 12 h and after continued bloodmeal acquisition for 3 or 7 days postexposure. Initially, ticks exposed to wild-type R. parkeri had the highest rickettsial load across all organs; however, rickettsial loads decreased and wild-type rickettsiae were cleared from the ovaries at 7 days postexposure. In contrast, ticks exposed to R. parkeririckA::tn or R. parkerisca2::tn had comparatively lower rickettsial loads, but bacteria persisted in all organs for 7 days. These data suggest that while RickA and Sca2 function in actin polymerization in tick cells, the absence of these proteins did not change dissemination patterns within the tick vector.

Involvement of ATP synthase ß subunit in chikungunya virus entry into insect cells.

Chikungunya virus (CHIKV), the virus responsible for the disease chikungunya fever in humans, is transmitted by Aedes mosquitoes. While significant progress has been made in understanding the process by which CHIKV enters into mammalian cells, far less progress has been made in understanding the CHIKV entry process in insect cells. This study sought to identify mosquito-cell-expressed CHIKV-binding proteins through a combination of virus overlay protein binding assays (VOPBA) and mass spectroscopy. A 50-kDa CHIKV-binding protein was identified as the ATP synthase ß subunit (ATPSß). Co-immunoprecipitation studies confirmed the interaction, and colocalization analysis showed cell-surface and intracellular co-localization between CHIKV and ATPSß. Both antibody inhibition and siRNA-mediated downregulation experiments targeted to ATPSß showed a significant reduction in viral entry and virus production. These results suggest that ATPSß is a CHIKV-binding protein capable of mediating the entry of CHIKV into insect cells.

Voltage dependent anion channel is redistributed during Japanese encephalitis virus infection of insect cells.

Despite the availability of an effective vaccine, Japanese encephalitis remains a significant cause of morbidity and mortality in many parts of Asia. Japanese encephalitis is caused by the Japanese encephalitis virus (JEV), a mosquito transmitted flavivirus. Many of the details of the virus replication cycle in mosquito cells remain unknown. This study sought to determine whether GRP78, a well-characterized flavivirus E protein interacting protein, interacted with JEV E protein in insect cells, and whether this interaction was mediated at the cell surface. GRP78 was shown to interact with JEV E protein by coimmunoprecipitation, and was additionally shown to interact with voltage dependent anion protein (VDAC) through the same methodology. Antibody inhibition experiments showed that neither GRP78 nor VDAC played a role in JEV internalization to insect cells. Interestingly, VDAC was shown to be significantly relocalized in response to JEV infection, and significant levels of colocalization between VDAC and GRP78 and VDAC and ribosomal L28 protein were seen in JEV infected but not uninfected cells. This is the first report of relocalization of VDAC in response to JEV infection and suggests that this may be a part of the JEV replication strategy in insect cells.

VEEV TC-83 Triggers Dysregulation of the Tryptophan-Kynurenine Pathway in the Central Nervous System That Correlates with Cognitive Impairment in Tg2576 Mice.

Neurodegenerative diseases are chronic conditions affecting the central nervous system (CNS). Alzheimer's disease (AD) is a neurodegenerative disorder characterized by the accumulation of amyloid beta in the limbic and cortical brain regions. AD is presumed to result from genetic abnormalities or environmental factors, including viral infections, which may have deleterious, long-term effects. In this study, we demonstrate that the Venezuelan equine encephalitis virus (VEEV) commonly induces neurodegeneration and long-term neurological or cognitive sequelae. Notably, the effects of VEEV infection can persistently influence gene expression in the mouse brain, suggesting a potential link between the observed neurodegenerative outcomes and long-term alterations in gene expression. Additionally, we show that alphavirus encephalitis exacerbates the neuropathological profile of AD through crosstalk between inflammatory and kynurenine pathways, generating a range of metabolites with potent effects. Using a mouse model for ß-amyloidosis, Tg2576 mice, we found that cognitive deficits and brain pathology were more severe in Tg2576 mice infected with VEEV TC-83 compared to mock-infected controls. Thus, during immune activation, the kynurenine pathway plays a more active role in the VEEV TC-83-infected cells, leading to increases in the abundance of transcripts related to the kynurenine pathway of tryptophan metabolism. This pathway generates several metabolites with potent effects on neurotransmitter systems as well as on inflammation, as observed in VEEV TC-83-infected animals.

Arbovirus infection increases the risk for the development of neurodegenerative disease pathology in the murine model.

Alzheimer's disease is classified as a progressive disorder resulting from protein misfolding, also known as proteinopathies. Proteinopathies include synucleinopathies triggered by misfolded amyloid α-synuclein, tauopathies triggered by misfolded tau, and amyloidopathies triggered by misfolded amyloid of which Alzheimer's disease (ß-amyloid) is most prevalent. Most neurodegenerative diseases (>90%) are not due to dominantly inherited genetic causes. Instead, it is thought that the risk for disease is a complicated interaction between inherited and environmental risk factors that, with age, drive pathology that ultimately results in neurodegeneration and disease onset. Since it is increasingly appreciated that encephalitic viral infections can have profoundly detrimental neurological consequences long after the acute infection has resolved, we tested the hypothesis that viral encephalitis exacerbates the pathological profile of protein-misfolding diseases. Using a robust, reproducible, and well-characterized mouse model for ß-amyloidosis, Tg2576, we studied the contribution of alphavirus-induced encephalitis (TC-83 strain of VEEV to model alphavirus encephalitis viruses) on the progression of neurodegenerative pathology. We longitudinally evaluated neurological, neurobehavioral, and cognitive levels, followed by a post-mortem analysis of brain pathology focusing on neuroinflammation. We found more severe cognitive deficits and brain pathology in Tg2576 mice inoculated with TC-83 than in their mock controls. These data set the groundwork to investigate sporadic Alzheimer's disease and treatment interventions for this infectious disease risk factor.

Edwardsiella ictaluri T3SS effector EseN is a phosphothreonine lyase that inactivates ERK1/2, p38, JNK, and PDK1 and modulates cell death in infected macrophages.

IMPORTANCE: This work has global significance in the catfish industry, which provides food for increasing global populations. E. ictaluri is a leading cause of disease loss, and EseN is an important player in E. ictaluri virulence. The E. ictaluri T3SS effector EseN plays an essential role in establishing infection, but the specific role EseN plays is not well characterized. EseN belongs to a family of phosphothreonine lyase effectors that specifically target host mitogen activated protein kinase (MAPK) pathways important in regulating host responses to infection. No phosphothreonine lyase equivalents are known in eukaryotes, making this family of effectors an attractive target for indirect narrow-spectrum antibiotics. Targeting of major vault protein and PDK1 kinase by EseN has not been reported in EseN homologs in other pathogens and may indicate unique functions of E. ictaluri EseN. EseN targeting of PDK1 is particularly interesting in that it is linked to an extraordinarily diverse group of cellular functions.

The role of cofeeding arthropods in the transmission of Rickettsia felis.

Rickettsia felis is an emerging etiological agent of rickettsioses worldwide. The cosmopolitan cat flea (Ctenocephalides felis) is the primary vector of R. felis, but R. felis has also been reported in other species of hematophagous arthropods including ticks and mosquitoes. Canines can serve as a bacteremic host to infect fleas under laboratory conditions, yet isolation of R. felis from the blood of a vertebrate host in nature has not been realized. Cofeeding transmission is an efficient mechanism for transmitting rickettsiae between infected and uninfected fleas; however, the mechanism of transmission among different orders and classes of arthropods is not known. The potential for R. felis transmission between infected fleas and tick (Dermacentor variabilis) and mosquito (Anopheles quadrimaculatus) hosts was examined via cofeeding bioassays. Donor cat fleas infected with R. felis transmitted the agent to naïve D. variabilis nymphs via cofeeding on a rat host. Subsequent transstadial transmission of R. felis from the engorged nymphs to the adult ticks was observed with reduced prevalence in adult ticks. Using an artificial host system, An. quadrimaculatus exposed to a R. felis-infected blood meal acquired rickettsiae and maintained infection over 12 days post-exposure (dpe). Similar to ticks, mosquitoes were able to acquire R. felis while cofeeding with infected cat fleas on rats infection persisting in the mosquito for up to 3 dpe. The results indicate R. felis-infected cat fleas can transmit rickettsiae to both ticks and mosquitoes via cofeeding on a vertebrate host, thus providing a potential avenue for the diversity of R. felis-infected arthropods in nature.

Cocaine Self-Administration Influences Central Nervous System Immune Responses in Male HIV-1 Transgenic Rats.

Cocaine use increases the neurotoxic severity of human immunodeficiency virus-1 (HIV-1) infection and the development of HIV-associated neurocognitive disorders (HAND). Among the studied cellular mechanisms promoting neurotoxicity in HIV-1 and cocaine use, central nervous system (CNS) immunity, such as neuroimmune signaling and reduced antiviral activity, are risk determinants; however, concrete evidence remains elusive. In the present study, we tested the hypothesis that cocaine self-administration by transgenic HIV-1 (HIV-1Tg) rats promotes CNS inflammation. To test this hypothesis, we measured cytokine, chemokine, and growth factor protein levels in the frontal cortex (fCTX) and caudal striatum (cSTR). Our results demonstrated that cocaine self-administration significantly increased fCTX inflammation in HIV-1Tg rats, but not in the cSTR. Accordingly, we postulate that cocaine synergizes with HIV-1 proteins to increase neuroinflammation in a region-selective manner, including the fCTX. Given the fCTX role in cognition, this interaction may contribute to the hyperimmunity and reduced antiviral activity associated with cocaine-mediated enhancement of HAND.

Comparative Analysis of Infection by Rickettsia rickettsii Sheila Smith and Taiaçu Strains in a Murine Model.

Rocky Mountain spotted fever (RMSF) is a life-threatening tick-borne disease caused by Rickettsia rickettsii, which is widely distributed throughout the Americas. Over 4000 cases of RMSF are recorded annually in the United States, while only around 100 cases are reported in Brazil. Conversely, while case fatality rates in the United States oscillate around 5%, in Brazil they can surpass 70%, suggesting that differences in tick vectoring capacity, population sensitivity, and/or variability in virulence of the rickettsial strains may exist. In this study, we compared the susceptibility of C3H/HeN mice to two highly virulent strains of R. rickettsii, one from the United States (Sheila Smith) and the other from Brazil (Taiaçu). Animals inoculated with the Taiaçu strain succumbed to infection earlier and exhibited severe histological lesions in both liver and spleen sooner than mice infected with the Sheila Smith strain. These differences in survival and signs of the disease are not related to a greater proliferation of the Taiaçu strain, as there were no significant differences in the rickettsial load in mice tissues inoculated with either strain. The present study is the first step to experimentally assess differences in fatality rates of RMSF in two different regions of the American continent.

Dengue 2 infection of HepG2 liver cells results in endoplasmic reticulum stress and induction of multiple pathways of cell death.

BACKGROUND: A number of studies have implicated the direct involvement of the liver in dengue virus (DENV) infection, and it has been widely shown that liver cells subsequently undergo apoptosis. The mechanism by which liver cells undergo apoptosis in response to DENV infection remains unclear. To provide further information on the mechanism of apoptosis in DENV infected liver cells, HepG2 cells were infected with DENV 2 and analyzed for the induction of ER stress, apoptosis and autophagy. RESULTS: In response to DENV infection, HepG2 cells showed the induction of both the ER resident unfolded protein response as well as the Noxa/PUMA stress response pathways. Proteolytic activation of caspases 4, 7, 8 and 9 was observed as well as changes in mitochondrial transmembrane potential. Increased monodansylcadaverine staining was observed in DENV infected cells, consistent with the previously reported induction of autophagy. CONCLUSIONS: These results are consistent with a model in which the induction of multiple ER stress pathways is coupled with the induction of multiple cell death pathways as a mechanism to ensure the removal of infected liver cells from the system.

Identification and characterization of prohibitin as a receptor protein mediating DENV-2 entry into insect cells.

Dengue is transmitted primarily by mosquitoes of the Aedes genus. Despite a number of studies, no insect dengue virus receptor protein has been clearly identified and characterized. Using a number of separation methodologies and virus overlay protein binding assays we identified a 35kDa protein that segregated with susceptibility to dengue serotype 2 (DENV-2) infection in two mosquito species and two mosquito cell lines. Mass spectroscopy identified the protein to be prohibitin, a strongly conserved and ubiquitously expressed protein in eukaryotic cells. Antibody mediated inhibition of infection and siRNA mediated knockdown of prohibitin expression significantly reduced infection levels and subsequent virus production in both Aedes aegypti and Aedes albopictus cell lines. Confocal microscopy showed a significant degree of intracellular colocalization between prohibitin and DENV-2 E protein, and coimmunoprecipitation confirmed that prohibitin interacts with dengue E. Prohibitin is the first characterized insect cell expressed dengue virus receptor protein.