Proximity-dependent mapping of the HCMV US28 interactome identifies RhoGEF signaling as a requirement for efficient viral reactivation.

Human cytomegalovirus (HCMV) encodes multiple putative G protein-coupled receptors (GPCRs). US28 functions as a viral chemokine receptor and is expressed during both latent and lytic phases of virus infection. US28 actively promotes cellular migration, transformation, and plays a major role in mediating viral latency and reactivation; however, knowledge about the interaction partners involved in these processes is still incomplete. Herein, we utilized a proximity-dependent biotinylating enzyme (TurboID) to characterize the US28 interactome when expressed in isolation, and during both latent (CD34+ hematopoietic progenitor cells) and lytic (fibroblasts) HCMV infection. Our analyses indicate that the US28 signalosome converges with RhoA and EGFR signal transduction pathways, sharing multiple mediators that are major actors in processes such as cellular proliferation and differentiation. Integral members of the US28 signaling complex were validated in functional assays by immunoblot and small-molecule inhibitors. Importantly, we identified RhoGEFs as key US28 signaling intermediaries. In vitro latency and reactivation assays utilizing primary CD34+ hematopoietic progenitor cells (HPCs) treated with the small-molecule inhibitors Rhosin or Y16 indicated that US28 -RhoGEF interactions are required for efficient viral reactivation. These findings were recapitulated in vivo using a humanized mouse model where inhibition of RhoGEFs resulted in a failure of the virus to reactivate. Together, our data identifies multiple new proteins in the US28 interactome that play major roles in viral latency and reactivation, highlights the utility of proximity-sensor labeling to characterize protein interactomes, and provides insight into targets for the development of novel anti-HCMV therapeutics.

Human Cytomegalovirus Host Interactions: EGFR and Host Cell Signaling Is a Point of Convergence Between Viral Infection and Functional Changes in Infected Cells.

Viruses have evolved diverse strategies to manipulate cellular signaling pathways in order to promote infection and/or persistence. Human cytomegalovirus (HCMV) possesses a number of unique properties that allow the virus to alter cellular events required for infection of a diverse array of host cell types and long-term persistence. Of specific importance is infection of bone marrow derived and myeloid lineage cells, such as peripheral blood monocytes and CD34+ hematopoietic progenitor cells (HPCs) because of their essential role in dissemination of the virus and for the establishment of latency. Viral induced signaling through the Epidermal Growth Factor Receptor (EGFR) and other receptors such as integrins are key control points for viral-induced cellular changes and productive and latent infection in host organ systems. This review will explore the current understanding of HCMV strategies utilized to hijack cellular signaling pathways, such as EGFR, to promote the wide-spread dissemination and the classic life-long herpesvirus persistence.

The Differentiation of Human Cytomegalovirus Infected-Monocytes Is Required for Viral Replication.

Viral dissemination is a key mechanism responsible for persistence and disease following human cytomegalovirus (HCMV) infection. Monocytes play a pivotal role in viral dissemination to organ tissue during primary infection and following reactivation from latency. For example, during primary infection, infected monocytes migrate into tissues and differentiate into macrophages, which then become a source of viral replication. In addition, because differentiated macrophages can survive for months to years, they provide a potential persistent infection source in various organ systems. We broadly note that there are three phases to infection and differentiation of HCMV-infected monocytes: (1) Virus enters and traffics to the nucleus through a virus receptor ligand engagement event that activates a unique signalsome that initiates the monocyte-to-macrophage differentiation process. (2) Following initial infection, HCMV undergoes a "quiescence-like state" in monocytes lasting for several weeks and promotes monocyte differentiation into macrophages. While, the initial event is triggered by the receptor-ligand engagement, the long-term cellular activation is maintained by chronic viral-mediated signaling events. (3) Once HCMV infected monocytes differentiate into macrophages, the expression of immediate early viral (IE) genes is detectable, followed by viral replication and long term infectious viral particles release. Herein, we review the detailed mechanisms of each phase during infection and differentiation into macrophages and discuss the biological significance of the differentiation of monocytes in the pathogenesis of HCMV.

Cross-Species Functional Conservation and Possible Origin of the N-Terminal Specificity Domain of Mitochondrial Presequences.

Plants have two endosymbiotic organelles, chloroplast and mitochondrion. Although they have their own genomes, proteome assembly in these organelles depends on the import of proteins encoded by the nuclear genome. Previously, we elucidated the general design principles of chloroplast and mitochondrial targeting signals, transit peptide, and presequence, respectively, which are highly diverse in primary structure. Both targeting signals are composed of N-terminal specificity domain and C-terminal translocation domain. Especially, the N-terminal specificity domain of mitochondrial presequences contains multiple arginine residues and hydrophobic sequence motif. In this study we investigated whether the design principles of plant mitochondrial presequences can be applied to those in other eukaryotic species. We provide evidence that both presequences and import mechanisms are remarkably conserved throughout the species. In addition, we present evidence that the N-terminal specificity domain of presequence might have evolved from the bacterial TAT (twin-arginine translocation) signal sequence.

Immunization with a recombinant antigen composed of conserved blocks from TSA56 provides broad genotype protection against scrub typhus.

Scrub typhus is an acute febrile disease caused by Orientia tsutsugamushi infection. Despite the wide range of approaches explored during the last seventy years, an effective prophylactic vaccine is not yet available. Here, we developed a novel recombinant antigen derived from conserved regions of 56 kDa type-specific antigen (TSA56), a major outer membrane protein responsible for genetic heterogeneity and antigenicity, and evaluated it as a protective vaccine antigen. Our findings demonstrate that immunization with conserved blocks of TSA56 (cTSA56) not only provides protective immunity against lethal challenges with the homologous genotype, but also confers significantly better protection against heterologous genotypes than TSA56. Adoptive transfer of CD4+ or CD8+ T cells from immunized mice provided significantly enhanced protection against lethal challenge, whereas immune B cells failed to do so, indicating that cellular immunity against the conserved epitopes plays a protective role. Moreover, immunization with a 10-mer peptide mixture, screened from CD8+ T cell epitopes within the conserved region of TSA56, provided enhanced protection against lethal challenge with O. tsutsugamushi. Therefore, this novel recombinant antigen is a promising candidate for scrub typhus vaccine against a wide range of O. tsutsugamushi genotypes.

A Type I Interferon and IL-10 Induced by Orientia tsutsugamushi Infection Suppresses Antigen-Specific T Cells and Their Memory Responses.

Despite the various roles of type I interferon (type I IFN) responses during bacterial infection, its specific effects in vivo have been poorly characterized in scrub typhus caused by Orientia tsutsugamushi infection. Here, we show that type I IFNs are primarily induced via intracellular nucleic acids sensors, including RIG-I/MAVS and cGAS/STING pathways, during O. tsutsugamushi invasion. However, type I IFN signaling did not significantly affect pathogenesis, mortality, or bacterial burden during primary infection in vivo, when assessed in a mice model lacking a receptor for type I IFNs (IFNAR KO). Rather, it significantly impaired the induction of antigen-specific T cells and reduced memory T cell responses. IFNAR KO mice that recovered from primary infection showed stronger antigen-specific T cell responses, especially Th1, and more efficiently controlled bacteremia during secondary infection than wild type mice. Enhanced IL-10 expression by macrophages in the presence of type I IFN signaling might play a significant role in the suppression of antigen-specific T cell responses as neutralization or knock-out (KO) of IL-10 increased T cell responses in vitro. Therefore, induction of the type I IFN/IL-10 axis by O. tsutsugamushi infection might play a significant role in the suppression of T cell responses and contribute to the short longevity of cell-mediated immunity, often observed in scrub typhus patients.

Longevity of antibody and T-cell responses against outer membrane antigens of Orientia tsutsugamushi in scrub typhus patients.

Scrub typhus, caused by Orientia tsutsugamushi infection, has been a serious public health issue in the Asia-Pacific region, with rising incidence and sporadic outbreaks. However, human protective immunity against specific antigens has been poorly characterized for this bacterium. In addition, immunity produced in early vaccine trials or even after natural infections, did not last long and had poor cross-reactivity among various genotypes. Here, we systematically investigated the kinetics and magnitude of specific adaptive immunity against two membrane antigens, 56 kDa type-specific antigen (TSA56) and surface cell antigen A (ScaA), that are involved in bacterial adhesion and invasion of the host in 64 recovered scrub typhus patients. Antibody responses to the bacterial antigens in patients were generally short-lived and waned to baseline levels 2 years after recovery. The anti-TSA56 IgG responses were predominantly composed of the IgG1 and IgG3 subclasses and persisted for up to 1 year after recovery, whereas IgG specific to ScaA primarily consisted of more transient IgG1, with limited responses by other subclasses. Cellular immunity, including CD4 and CD8 T-cells specific to membrane antigens, also rapidly declined from 1 year after infection, as measured by enzyme-linked immunospot (ELISPOT) assays and flow cytometry. The short longevity of antigen-specific adaptive immunity might be attributable to limited memory responses, as observed in earlier vaccine studies using whole bacterial antigens. Finally, we identified HLA-A*0201-restricted and highly conserved CD8 T-cell epitopes in the TSA56 antigen, which may be valuable tools for assessing cellular immunity against O. tsutsugamushi and developing an effective scrub typhus vaccine.

Immunological dynamics associated with rapid virological response during the early phase of type I interferon therapy in patients with chronic hepatitis C.

Type I interferons (IFNs) play an important role in antiviral immunity as well as immunopathogenesis of diverse chronic viral infections. However, the precise mechanisms regulating the multifaceted effects of type I IFNs on the immune system and pathological inflammation still remain unclear. In order to assess the immunological dynamics associated with rapid viral clearance in chronic hepatitis C patients during the acute phase of type I IFN therapy, we analyzed multiple parameters of virological and immunological responses in a cohort of 59 Korean hepatitis C patients who received pegylated IFN-α and ribavirin (IFN/RBV). Most of the Korean patients had favorable alleles in the IFN-λ loci for responsiveness to IFN/RBV (i.e., C/C in rs12979860, T/T in rs8099917, and TT/TT in rs368234815). Rapid virological response (RVR) was determined mainly by the hepatitis C virus genotype. Among the cytokines analyzed, higher plasma levels of IL-17A and FGF were observed in non-RVR patients infected with viral genotype 1 and IP-10 was consistently elevated in RVR group infected with genotype 2 during the early phase of antiviral therapy. In addition, these three cytokines were correlated each other, suggesting a functional linkage of the cytokines in antiviral responses during IFN/RBV therapy. A low baseline frequencies of regulatory T cells and γδ T cells, but high level of group 2 innate lymphoid cells, in peripheral bloods were also significantly associated with the RVR group, implicating a potential role of the cellular immunity during the early phase of IFN/RBV therapy. Therefore, the immunological programs established by chronic hepatitis C and rapid disruption of the delicate balance by exogenous type I IFN might be associated with the subsequent virological outcomes in chronic hepatitis C patients.

Diversification of Orientia tsutsugamushi genotypes by intragenic recombination and their potential expansion in endemic areas.

BACKGROUND: Scrub typhus is a mite-borne febrile disease caused by O. tsutsugamushi infection. Recently, emergence of scrub typhus has attracted considerable attention in several endemic countries in Asia and the western Pacific. In addition, the antigenic diversity of the intracellular pathogen has been a serious obstacle for developing effective diagnostics and vaccine. METHODOLOGY/PRINCIPAL FINDINGS: To understand the evolutionary pathway of genotypic diversification of O. tsutsugamushi and the environmental factors associated with the epidemiological features of scrub typhus, we analyzed sequence data, including spatiotemporal information, of the tsa56 gene encoding a major outer membrane protein responsible for antigenic variation. A total of 324 tsa56 sequences covering more than 85% of its open reading frame were analyzed and classified into 17 genotypes based on phylogenetic relationship. Extensive sequence analysis of tsa56 genes using diverse informatics tools revealed multiple intragenic recombination events, as well as a substantially higher mutation rate than other house-keeping genes. This suggests that genetic diversification occurred via frequent point mutations and subsequent genetic recombination. Interestingly, more diverse bacterial genotypes and dominant vector species prevail in Taiwan compared to other endemic regions. Furthermore, the co-presence of identical and sub-identical clones of tsa56 gene in geographically distant areas implies potential spread of O. tsutsugamushi genotypes. CONCLUSIONS/SIGNIFICANCE: Fluctuation and diversification of vector species harboring O. tsutsugamushi in local endemic areas may facilitate genetic recombination among diverse genotypes. Therefore, careful monitoring of dominant vector species, as well as the prevalence of O. tsutsugamushi genotypes may be advisable to enable proper anticipation of epidemiological changes of scrub typhus.

Constitutive activation of T cells by γ2-herpesviral GPCR through the interaction with cellular CXCR4.

Members of the herpesviral family use multiple strategies to hijack infected host cells and exploit cellular signaling for their pathogenesis and latent infection. Among the most intriguing weapons in the arsenal of pathogenic herpesviruses are the constitutively active virally-encoded G protein-coupled receptors (vGPCRs). Even though vGPCRs contribute to viral pathogenesis such as immune evasion and proliferative disorders, the molecular details of how vGPCRs continuously activate cellular signaling are largely unknown. Here, we report that the vGPCR of Herpesvirus saimiri (HVS), an oncogenic γ2-herpesvirus, constitutively activates T cells via a heteromeric interaction with cellular CXCR4. Constitutive T cell activation also occurs with expression of the vGPCR of Kaposi's sarcoma-associated herpesvirus (KSHV), but not the vGPCR of Epstein-Barr virus. Expression of HVS vGPCR down-regulated the surface expression of CXCR4 but did not induce the degradation of the chemokine receptor, suggesting that vGPCR/CXCR4 signaling continues in cytosolic compartments. The physical association of vGPCR with CXCR4 was demonstrated by proximity ligation assay as well as immunoprecipitation. Interestingly, the constitutive activation of T cells by HVS vGPCR is independent of proximal T cell receptor (TCR) signaling molecules, such as TCRß, Lck, and ZAP70, whereas CXCR4 silencing by shRNA abolished T cell activation by vGPCRs of HVS and KSHV. Furthermore, previously identified inactive vGPCR mutants failed to interact with CXCR4. These findings on the positive cooperativity of vGPCR with cellular CXCR4 in T cell activation extend our current understanding of the molecular mechanisms of vGPCR function and highlight the importance of heteromerization for GPCR activity.

Generation of protective immunity against Orientia tsutsugamushi infection by immunization with a zinc oxide nanoparticle combined with ScaA antigen.

BACKGROUND: Zinc oxide nanoparticle (ZNP) has been applied in various biomedical fields. Here, we investigated the usage of ZNP as an antigen carrier for vaccine development by combining a high affinity peptide to ZNP. RESULTS: A novel zinc oxide-binding peptide (ZBP), FPYPGGDA, with high affinity to ZNP (K a  = 2.26 × 106 M-1) was isolated from a random peptide library and fused with a bacterial antigen, ScaA of Orientia tsutsugamushi, the causative agent of scrub typhus. The ZNP/ZBP-ScaA complex was efficiently phagocytosed by a dendritic cell line, DC2.4, in vitro and significantly enhanced anti-ScaA antibody responses in vivo compared to control groups. In addition, immunization with the ZNP/ZBP-ScaA complex promoted the generation of IFN-γ-secreting T cells in an antigen-dependent manner. Finally, we observed that ZNP/ZBP-ScaA immunization provided protective immunity against lethal challenge of O. tsutsugamushi, indicating that ZNP can be used as a potent adjuvant when complexed with ZBP-conjugated antigen. CONCLUSIONS: ZNPs possess good adjuvant potential as a vaccine carrier when combined with an antigen having a high affinity to ZNP. When complexed with ZBP-ScaA antigen, ZNPs could induce strong antibody responses as well as protective immunity against lethal challenges of O. tsutsugamushi. Therefore, application of ZNPs combined with a specific soluble antigen could be a promising strategy as a novel vaccine carrier system.

Comparative and kinetic analysis of viral shedding and immunological responses in MERS patients representing a broad spectrum of disease severity.

Despite the ongoing spread of MERS, there is limited knowledge of the factors affecting its severity and outcomes. We analyzed clinical data and specimens from fourteen MERS patients treated in a hospital who collectively represent a wide spectrum of disease severity, ranging from mild febrile illness to fatal pneumonia, and classified the patients into four groups based on severity and mortality. Comparative and kinetic analyses revealed that high viral loads, weak antibody responses, and lymphopenia accompanying thrombocytopenia were associated with disease mortality, whereas persistent and gradual increases in lymphocyte responses might be required for effective immunity against MERS-CoV infection. Leukocytosis, primarily due to increased neutrophils and monocytes, was generally observed in more severe and fatal cases. The blood levels of cytokines such as IL-10, IL-15, TGF-ß, and EGF were either positively or negatively correlated with disease mortality. Robust induction of various chemokines with differential kinetics was more prominent in patients that recovered from pneumonia than in patients with mild febrile illness or deceased patients. The correlation of the virological and immunological responses with disease severity and mortality, as well as their responses to current antiviral therapy, may have prognostic significance during the early phase of MERS.

Spread of Mutant Middle East Respiratory Syndrome Coronavirus with Reduced Affinity to Human CD26 during the South Korean Outbreak.

UNLABELLED: The newly emerging Middle East respiratory syndrome coronavirus (MERS-CoV) causes a severe respiratory infection with a high mortality rate (~35%). MERS-CoV has been a global threat due to continuous outbreaks in the Arabian peninsula and international spread by infected travelers since 2012. From May to July 2015, a large outbreak initiated by an infected traveler from the Arabian peninsula swept South Korea and resulted in 186 confirmed cases with 38 deaths (case fatality rate, 20.4%). Here, we show the rapid emergence and spread of a mutant MERS-CoV with reduced affinity to the human CD26 receptor during the South Korean outbreak. We isolated 13 new viral genomes from 14 infected patients treated at a hospital and found that 12 of these genomes possess a point mutation in the receptor-binding domain (RBD) of viral spike (S) protein. Specifically, 11 of these genomes have an I529T mutation in RBD, and 1 has a D510G mutation. Strikingly, both mutations result in reduced affinity of RBD to human CD26 compared to wild-type RBD, as measured by surface plasmon resonance analysis and cellular binding assay. Additionally, pseudotyped virus bearing an I529T mutation in S protein showed reduced entry into host cells compared to virus with wild-type S protein. These unexpected findings suggest that MERS-CoV adaptation during human-to-human spread may be driven by host immunological pressure such as neutralizing antibodies, resulting in reduced affinity to host receptor, and thereby impairs viral fitness and virulence, rather than positive selection for a better affinity to CD26. IMPORTANCE: Recently, a large outbreak initiated by an MERS-CoV-infected traveler from the Middle East swept South Korea and resulted in 186 confirmed cases with 38 deaths. This is the largest outbreak outside the Middle East, and it raised strong concerns about the possible emergence of MERS-CoV mutations. Here, we isolated 13 new viral genomes and found that 12 of them possess a point mutation in the receptor-binding domain of viral spike protein, resulting in reduced affinity to the human cognate receptor, CD26, compared to the wild-type virus. These unexpected findings suggest that MERS-CoV adaptation in humans may be driven by host immunological pressure.

Inhibition of eukaryotic translation by tetratricopeptide-repeat proteins of Orientia tsutsugamushi.

Orientia tsutsugamushi, an obligate intracellular bacterium, is the causative agent of scrub typhus. The genome of Orientia tsutsugamushi has revealed multiple ORFs encoding tetratricopeptide-repeat (TPR) proteins. The TPR protein family has been shown to be involved in a diverse spectrum of cellular functions such as cell cycle control, transcription, protein transport, and protein folding, especially in eukaryotic cells. However, little is known about the function of the TPR proteins in O. tsutsugamushi. To investigate the potential role of TPR proteins in host-pathogen interaction, two oriential TPR proteins were expressed in E. coli and applied for GSTpull down assay. DDX3, a DEAD-box containing RNA helicase, was identified as a specific eukaryotic target of the TPR proteins. Since the RNA helicase is involved in multiple RNA-modifying processes such as initiation of translation reaction, we performed in vitro translation assay in the presence of GST-TPR fusion proteins by using rabbit reticulocyte lysate system. The TPR proteins inhibited in vitro translation of a reporter luciferase in a dose dependent manner whereas the GST control proteins did not. These results suggested TPR proteins of O. tsutsugamushi might be involved in the modulation of eukaryotic translation through the interaction with DDX3 RNA helicase after secretion into host cytoplasm.

Immunization with an autotransporter protein of Orientia tsutsugamushi provides protective immunity against scrub typhus.

BACKGROUND: Scrub typhus is an acute febrile disease caused by Orientia tsutsugamushi infection. Recently, the rapid increase of scrub typhus incidence in several countries within the endemic region has become a serious public health issue. Despite the wide range of preventative approaches that have been attempted in the past 70 years, all have failed to develop an effective prophylactic vaccine. Currently, the selection of the proper antigens is one of the critical barriers to generating cross-protective immunity against antigenically-variable strains of O. tsutsugamushi. METHODOLOGY/PRINCIPAL FINDINGS: We examined the potential role of ScaA protein, an autotransporter protein of O. tsutsugamushi, in bacterial pathogenesis and evaluated the protective attributes of ScaA immunization in lethal O. tsutsugamushi infection in mice. Our findings demonstrate that ScaA functions as a bacterial adhesion factor, and anti-ScaA antibody significantly neutralizes bacterial infection of host cells. In addition, immunization with ScaA not only provides protective immunity against lethal challenges with the homologous strain, but also confers significant protection against heterologous strains when combined with TSA56, a major outer membrane protein of O. tsutsugamushi. CONCLUSIONS/SIGNIFICANCE: Immunization of ScaA proteins provides protective immunity in mice when challenged with the homologous strain and significantly enhanced protective immunity against infection with heterologous strains. To our knowledge, this is the most promising result of scrub typhus vaccination trials against infection of heterologous strains in mouse models thus far.

Multiple Orientia tsutsugamushi ankyrin repeat proteins interact with SCF1 ubiquitin ligase complex and eukaryotic elongation factor 1 α.

BACKGROUND: Orientia tsutsugamushi, the causative agent of scrub typhus, is an obligate intracellular bacterium. Previously, a large number of genes that encode proteins containing eukaryotic protein-protein interaction motifs such as ankyrin-repeat (Ank) domains were identified in the O. tsutsugamushi genome. However, little is known about the Ank protein function in O. tsutsugamushi. METHODOLOGY/PRINCIPAL FINDINGS: To characterize the function of Ank proteins, we investigated a group of Ank proteins containing an F-box-like domain in the C-terminus in addition to the Ank domains. All nine selected ank genes were expressed at the transcriptional level in host cells infected with O. tsutsugamushi, and specific antibody responses against three Ank proteins were detected in the serum from human patients, indicating an active expression of the bacterial Ank proteins post infection. When ectopically expressed in HeLa cells, the Ank proteins of O. tsutsugamushi were consistently found in the nucleus and/or cytoplasm. In GST pull-down assays, multiple Ank proteins specifically interacted with Cullin1 and Skp1, core components of the SCF1 ubiquitin ligase complex, as well as the eukaryotic elongation factor 1 α (EF1α). Moreover, one Ank protein co-localized with the identified host targets and induced downregulation of EF1α potentially via enhanced ubiquitination. The downregulation of EF1α was observed consistently in diverse host cell types infected with O. tsutsugamushi. CONCLUSION/SIGNIFICANCE: These results suggest that conserved targeting and subsequent degradation of EF1α by multiple O. tsutsugamushi Ank proteins could be a novel bacterial strategy for replication and/or pathogenesis during mammalian host infection.

Kaposi's sarcoma-associated herpesvirus K7 modulates Rubicon-mediated inhibition of autophagosome maturation.

Autophagy is an important innate safeguard mechanism for protecting an organism against invasion by pathogens. We have previously discovered that Kaposi's sarcoma-associated herpesvirus (KSHV) evades this host defense through tight suppression of autophagy by targeting multiple steps of autophagy signal transduction. Here, we report that KSHV K7 protein interacts with Rubicon autophagy protein and inhibits the autophagosome maturation step by blocking Vps34 enzymatic activity, further highlighting how KSHV deregulates autophagy-mediated host immunity for its life cycle.

Autophagy protein Rubicon mediates phagocytic NADPH oxidase activation in response to microbial infection or TLR stimulation.

Phagocytosis and autophagy are two important and related arms of the host's first-line defense against microbial invasion. Rubicon is a RUN domain containing cysteine-rich protein that functions as part of a Beclin-1-Vps34-containing autophagy complex. We report that Rubicon is also an essential, positive regulator of the NADPH oxidase complex. Upon microbial infection or Toll-like-receptor 2 (TLR2) activation, Rubicon interacts with the p22phox subunit of the NADPH oxidase complex, facilitating its phagosomal trafficking to induce a burst of reactive oxygen species (ROS) and inflammatory cytokines. Consequently, ectopic expression or depletion of Rubicon profoundly affected ROS, inflammatory cytokine production, and subsequent antimicrobial activity. Rubicon's actions in autophagy and in the NADPH oxidase complex are functionally and genetically separable, indicating that Rubicon functions in two ancient innate immune machineries, autophagy and phagocytosis, depending on the environmental stimulus. Rubicon may thus be pivotal to generating an optimal intracellular immune response against microbial infection.

The autophagy regulator Rubicon is a feedback inhibitor of CARD9-mediated host innate immunity.

Assembly of a scaffold consisting of CARD9, BCL10, and MALT1 (CBM complex) is critical for effective signaling by multiple pattern recognition receptors (PRRs) including Dectin and RIG-I. The RUN domain Beclin-1-interacting cysteine-rich-containing Rubicon protein associates constitutively with the Beclin-UVRAG-Vps34 complex under normal conditions to regulate autophagy. Rubicon also interacts with the phagocytic NADPH-oxidase complex upon TLR stimulation to induce potent antimicrobial responses. Here, we show Rubicon is a physiological feedback inhibitor of CBM-mediated PRR signaling, preventing unbalanced proinflammatory responses. Upon Dectin-1- or RIG-I-mediated activation, Rubicon dynamically exchanges binding partners from 14-3-3ß to CARD9 in a stimulation-specific and phosphorylation-dependent manner, disassembling the CBM signaling complex and ultimately terminating PRR-induced cytokine production. Remarkably, Rubicon's actions in the autophagy complex, phagocytosis complex, and CBM complex are functionally and genetically separable. Rubicon thus differentially targets signaling complexes, depending on environmental stimuli, and may function to coordinate various immune responses against microbial infection.

Global gene expression profile of Orientia tsutsugamushi.

Orientia tsutsugamushi, an obligate intracellular bacterium, is the causative agent of Scrub typhus. The control mechanisms for bacterial gene expression are largely unknown. Here, the global gene expression of O. tsutsugamushi within eukaryotic cells was examined using a microarray and proteomic approaches for the first time. These approaches identified 643 genes, corresponding to approximately 30% of the genes encoded in the genome. The majority of expressed genes belonged to several functional categories including protein translation, protein processing/secretion, and replication/repair. We also searched the conserved sequence blocks (CSBs) in the O. tsutsugamushi genome which is unique in that up to 40% of its genome consists of dispersed repeated sequences. Although extensive shuffling of genomic sequences was observed between two different strains, 204 CSBs, covering 48% of the genome, were identified. When combining the data of CSBs and global gene expression, the CSBs correlates well with the location of expressed genes, suggesting the functional conservation between gene expression and genomic location. Finally, we compared the gene expression of the bacteria-infected fibroblasts and macrophages using microarray analysis. Some major changes were the downregulation of genes involved in translation, protein processing and secretion, which correlated with the reduction in bacterial translation rates and growth within macrophages.