SHARPIN controls the development of regulatory T cells.

SHARPIN is an essential component of the linear ubiquitin chain assembly complex (LUBAC) complex that controls signalling pathways of various receptors, including the tumour necrosis factor receptor (TNFR), Toll-like receptor (TLR) and antigen receptor, in part by synthesis of linear, non-degrading ubiquitin chains. Consistent with SHARPIN's function in different receptor pathways, the phenotype of SHARPIN-deficient mice is complex, including the development of inflammatory systemic and skin diseases, the latter of which depend on TNFR signal transduction. Given the established function of SHARPIN in primary and malignant B cells, we hypothesized that SHARPIN might also regulate T-cell receptor (TCR) signalling and thereby control T-cell biology. Here, we focus primarily on the role of SHARPIN in T cells, specifically regulatory T (Treg) cells. We found that SHARPIN-deficient (Sharpin(cpdm/cpdm) ) mice have significantly reduced numbers of FOXP3(+) Treg cells in lymphoid organs and the peripheral blood. Competitive reconstitution of irradiated mice with mixed bone marrow from wild-type and SHARPIN-deficient mice revealed an overall reduced thymus population with SHARPIN-deficient cells with almost complete loss of thymic Treg development. Consistent with this cell-intrinsic function of SHARPIN in Treg development, TCR stimulation of SHARPIN-deficient thymocytes revealed reduced activation of nuclear factor-κB and c-Jun N-terminal kinase, establishing a function of SHARPIN in TCR signalling, which may explain the defective Treg development. In turn, in vitro generation and suppressive activity of mature SHARPIN-deficient Treg cells were comparable to wild-type cells, suggesting that maturation, but not function, of SHARPIN-deficient Treg cells is impaired. Taken together, these findings show that SHARPIN controls TCR signalling and is required for efficient generation of Treg cells in vivo, whereas the inhibitory function of mature Treg cells appears to be independent of SHARPIN.

A phospho-tyrosine-based signaling module using SPOP, CSK, and LYN controls TLR-induced IRF activity.

Toll-like receptors (TLRs) recognize pathogen- and host-derived factors and control immune responses via the adaptor protein MyD88 and members of the interferon regulatory transcription factor (IRF) family. IRFs orchestrate key effector functions, including cytokine release, cell differentiation, and, under certain circumstances, inflammation pathology. Here, we show that IRF activity is generically controlled by the Src kinase family member LYN, which phosphorylates all TLR-induced IRFs at a conserved tyrosine residue, resulting in K48-linked polyubiquitination and proteasomal degradation of IRFs. We further show that LYN activity is controlled by the upstream kinase C-terminal Src kinase (CSK), whose activity, in turn, is controlled by the adaptor protein SPOP, which serves as molecular bridge to recruit CSK into the TLR signaling complex and to activate CSK catalytic activity. Consistently, deletion of SPOP or CSK results in increased LYN activity, LYN-directed IRF degradation, and inhibition of IRF transcriptional activity. Together, the data reveal a key regulatory mechanism for IRF family members controlling TLR biology.

Tyrosine-phosphorylated Ehrlichia chaffeensis and Ehrlichia canis tandem repeat orthologs contain a major continuous cross-reactive antibody epitope in lysine-rich repeats.

A small subset of major immunoreactive proteins have been identified in Ehrlichia chaffeensis and Ehrlichia canis, including three molecularly and immunologically characterized pairs of immunoreactive tandem repeat protein (TRP) orthologs with major continuous species-specific epitopes within acidic tandem repeats (TR) that stimulate strong antibody responses during infection. In this study, we identified a fourth major immunoreactive TR-containing ortholog pair and defined a major cross-reactive epitope in homologous nonidentical 24-amino-acid lysine-rich TRs. Antibodies from patients and dogs with ehrlichiosis reacted strongly with recombinant TR regions, and epitopes were mapped to the N-terminal TR region (18 amino acids) in E. chaffeensis and the complete TR (24 amino acids) in E. canis. Two less-dominant epitopes were mapped to adjacent glutamate/aspartate-rich and aspartate/tyrosine-rich regions in the acidic C terminus of E. canis TRP95 but not in E. chaffeensis TRP75. Major immunoreactive proteins in E. chaffeensis (75-kDa) and E. canis (95-kD) whole-cell lysates and supernatants were identified with TR-specific antibodies. Consistent with other ehrlichial TRPs, the TRPs identified in ehrlichial whole-cell lysates and the recombinant proteins migrated abnormally slow electrophoretically a characteristic that was demonstrated with the positively charged TR and negatively charged C-terminal domains. E. chaffeensis TRP75 and E. canis TRP95 were immunoprecipitated with anti-pTyr antibody, demonstrating that they are tyrosine phosphorylated during infection of the host cell.

Ehrlichia chaffeensis TRP120 interacts with a diverse array of eukaryotic proteins involved in transcription, signaling, and cytoskeleton organization.

Ehrlichia chaffeensis is an obligately intracellular bacterium that exhibits tropism for mononuclear phagocytes and survives by evading host cell defense mechanisms. Recently, molecular interactions between E. chaffeensis 47-kDa tandem repeat (TR) protein (TRP47) and the eukaryotic host cell have been described. In this investigation, yeast (Saccharomyces cerevisiae) two-hybrid analysis demonstrated that E. chaffeensis-secreted tandem repeat protein 120 (TRP120) interacts with a diverse group of host cell proteins associated with major biological processes, including transcription and regulation, cell signaling, protein trafficking, and actin cytoskeleton organization. Twelve target proteins with the highest frequency of interaction with TRP120 were confirmed by cotransformation in yeast. Host targets, including human immunoglobulin lambda locus (IGL), cytochrome c oxidase subunit II (COX2), Golgi-associated gamma adaptin ear-containing ARF binding protein 1 (GGA1), polycomb group ring finger 5 (PCGF5), actin gamma 1 (ACTG1), and unc-13 homolog D (UNC13D; Caenorhabditis elegans), colocalized strongly with TRP120 in HeLa cells and with E. chaffeensis dense-cored morulae and areas adjacent to morulae in the host cytoplasm. The TR domain of TRP120 interacted only with PCGF5, indicating that distinct TRP120 domains contribute to specific host target interactions and that multiple domains are required to reconstitute TRP120 interactions with other host targets. Three previously defined molecular interactions between TRP47 and host proteins, PCGF5, IGLL1, and CAP1, were also associated with TRP120, demonstrating that molecular cross talk occurs between Ehrlichia TRPs and host targets. These findings further support the role of TRPs as effectors that reprogram the host cell.

Ehrlichia chaffeensis TRP120 binds a G+C-rich motif in host cell DNA and exhibits eukaryotic transcriptional activator function.

Ehrlichia chaffeensis is an obligately intracellular bacterium that modulates host cell gene transcription in the mononuclear phagocyte, but the host gene targets and mechanisms involved in transcriptional modulation are not well-defined. In this study, we identified a novel tandem repeat DNA-binding domain in the E. chaffeensis 120-kDa tandem repeat protein (TRP120) that directly binds host cell DNA. TRP120 was observed by immunofluorescent microscopy in the nucleus of E. chaffeensis-infected host cells and was detected in nuclear extracts by Western immunoblotting with TRP120-specific antibody. The TRP120 binding sites and associated host cell target genes were identified using high-throughput deep sequencing (Illumina) of immunoprecipitated DNA (chromatin immunoprecipitation and high-throughput DNA sequencing). Multiple em motif elicitation (MEME) analysis of the most highly enriched TRP120-bound sequences revealed a G+C-rich DNA motif, and recombinant TRP120 specifically bound synthetic oligonucleotides containing the motif. TRP120 target gene binding sites were mapped most frequently to intersecting regions (intron/exon; 49%) but were also identified in upstream regulatory regions (25%) and downstream locations (26%). Genes targeted by TRP120 were most frequently associated with transcriptional regulation, signal transduction, and apoptosis. TRP120 targeted inflammatory chemokine genes, CCL2, CCL20, and CXCL11, which were strongly upregulated during E. chaffeensis infection and were also upregulated by direct transfection with recombinant TRP120. This study reveals that TRP120 is a novel DNA-binding protein that is involved in a host gene transcriptional regulation strategy.

An Ehrlichia chaffeensis tandem repeat protein interacts with multiple host targets involved in cell signaling, transcriptional regulation, and vesicle trafficking.

Ehrlichia chaffeensis is an obligately intracellular bacterium that exhibits tropism for mononuclear phagocytes forming cytoplasmic membrane-bound microcolonies called morulae. To survive and replicate within phagocytes, E. chaffeensis exploits the host cell by modulating a number of host cell processes, but the ehrlichial effector proteins involved are unknown. In this study, we determined that p47, a secreted, differentially expressed, tandem repeat (TR) protein, interacts with multiple host proteins associated with cell signaling, transcriptional regulation, and vesicle trafficking. Yeast two-hybrid analysis revealed that p47 interacts with polycomb group ring finger 5 (PCGF5) protein, Src protein tyrosine kinase FYN (FYN), protein tyrosine phosphatase non-receptor type 2 (PTPN2), and adenylate cyclase-associated protein 1 (CAP1). p47 interaction with these proteins was further confirmed by coimmunoprecipitation assays and colocalization in HeLa cells transfected with p47-green fluorescent fusion protein (AcGFP1-p47). Moreover, confocal microscopy demonstrated p47-expressing dense-cored (DC) ehrlichiae colocalized with PCGF5, FYN, PTPN2, and CAP1. An amino-terminally truncated form of p47 containing TRs interacted only with PCGF5 and not with FYN, PTPN2, and CAP1, indicating differences in p47 domains that are involved in these interactions. These results demonstrate that p47 is involved in a complex network of interactions involving numerous host cell proteins. Furthermore, this study provides a new insight into the molecular and functional distinction of DC ehrlichiae, as well as the effector proteins involved in facilitating ehrlichial survival in mononuclear phagocytes.

Nuclear translocated Ehrlichia chaffeensis ankyrin protein interacts with a specific adenine-rich motif of host promoter and intronic Alu elements.

Ehrlichiae are obligately intracellular bacteria that reside and replicate in phagocytes by circumventing host cell defenses and modulating cellular processes, including host cell gene transcription. However, the mechanisms by which ehrlichiae influence host gene transcription have largely remained undetermined. Numerous ankyrin and tandem repeat-containing proteins associated with host-pathogen interactions have been identified in Ehrlichia species, but their roles in pathobiology are unknown. In this study, we determined by confocal immunofluorescence microscopy and by immunodetection in purified nuclear extracts that the ankyrin repeat-containing protein p200 is translocated to the nuclei of Ehrlichia-infected monocytes. Chromatin immunoprecipitation (ChIP) with DNA sequencing revealed an Ehrlichia chaffeensis p200 interaction located within host promoter and intronic Alu-Sx elements, the most abundant repetitive elements in the human genome. A specific adenine-rich (mid-A-stretch) motif within Alu-Sx elements was identified using electrophoretic mobility shift and NoShift assays. Whole-genome analysis with ChIP and DNA microarray analysis (ChIP-chip) determined that genes (n = 456) with promoter Alu elements primarily related to transcription, apoptosis, ATPase activity, and structural proteins associated with the nucleus and membrane-bound organelles were the primary targets of p200. Several p200 target genes (encoding tumor necrosis factor alpha, Stat1, and CD48) associated with ehrlichial pathobiology were strongly upregulated during infection, as determined by quantitative PCR. This is the first study to identify a nuclear translocation of bacterially encoded protein by E. chaffeensis and to identify a specific binding motif and genes that are primary targets of a novel molecular strategy to reprogram host cell gene expression to promote survival of the pathogen.

Patrolling monocytes promote the pathogenesis of early lupus-like glomerulonephritis.

Systemic lupus erythematosus (SLE) is a complex autoimmune disease with genetic and environmental contributions. Hallmarks of the disease are the appearance of immune complexes (IC) containing autoreactive Abs and TLR-activating nucleic acids, whose deposition in kidney glomeruli is suspected to promote tissue injury and glomerulonephritis (GN). Here, using a mouse model based on the human SLE susceptibility locus TNFAIP3-interacting protein 1 (TNIP1, also known as ABIN1), we investigated the pathogenesis of GN. We found that GN was driven by TLRs but, remarkably, proceeded independently of ICs. Rather, disease in 3 different mouse models and patients with SLE was characterized by glomerular accumulation of patrolling monocytes (PMos), a cell type with an emerging key function in vascular inflammation. Consistent with such function in GN, monocyte-specific deletion of ABIN1 promoted kidney disease, whereas selective elimination of PMos provided protection. In contrast to GN, PMo elimination did not protect from reduced survival or disease symptoms such as IC generation and splenomegaly, suggesting that GN and other inflammatory processes are governed by distinct pathogenic mechanisms. These data identify TLR-activated PMos as the principal component of an intravascular process that contributes to glomerular inflammation and kidney injury.

CadA negatively regulates Escherichia coli O157:H7 adherence and intestinal colonization.

Adherence of pathogenic Escherichia coli strains to intestinal epithelia is essential for infection. For enterohemorrhagic E. coli (EHEC) serotype O157:H7, we have previously demonstrated that multiple factors govern this pathogen's adherence to HeLa cells (A. G. Torres and J. B. Kaper, Infect. Immun. 71:4985-4995, 2003). One of these factors is CadA, a lysine decarboxylase, and this protein has been proposed to negatively regulate virulence in several enteric pathogens. In the case of EHEC strains, CadA modulates expression of the intimin, an outer membrane adhesin involved in pathogenesis. Here, we inactivated cadA in O157:H7 strain 86-24 to investigate the role of this gene in EHEC adhesion to tissue-cultured monolayers, global gene expression patterns, and colonization of the infant rabbit intestine. The cadA mutant did not possess lysine decarboxylation activity and was hyperadherent to tissue-cultured cells. Adherence of the cadA mutant was nearly twofold greater than that of the wild type, and the adherence phenotype was independent of pH, lysine, or cadaverine in the media. Additionally, complementation of the cadA defect reduced adherence back to wild-type levels, and it was found that the mutation affected the expression of the intimin protein. Disruption of the eae gene (intimin-encoding gene) in the cadA mutant significantly reduced its adherence to tissue-cultured cells. However, adherence of the cadA eae double mutant was greater than that of an 86-24 eae mutant, suggesting that the enhanced adherence of the cadA mutant is not entirely attributable to enhanced expression of intimin in this background. Gene array analysis revealed that the cadA mutation significantly altered EHEC gene expression patterns; expression of 1,332 genes was downregulated and that of 132 genes was upregulated in the mutant compared to the wild-type strain. Interestingly, the gene expression variation shows an EHEC-biased gene alteration including intergenic regions. Two putative adhesins, flagella and F9 fimbria, were upregulated in the cadA mutant, suggestive of their association with adherence in the absence of the Cad regulatory mechanism. In the infant rabbit model, the cadA mutant outcompeted the wild-type strain in the ileum but not in the cecum or mid-colon, raising the possibility that CadA negatively regulates EHEC pathogenicity in a tissue-specific fashion.

Complete Genome Sequence of Ehrlichia muris Strain AS145T, a Model Monocytotropic Ehrlichia Strain.

We report here the complete genome sequence of Ehrlichia muris strain AS145(T), which was isolated from a wild mouse in 1983 in Japan. E. muris establishes persistent infections in laboratory mice and is widely used as a surrogate pathogen in a murine model of ehrlichiosis.

Molecular basis of antibody mediated immunity against Ehrlichia chaffeensis involves species-specific linear epitopes in tandem repeat proteins.

Humoral immune mechanisms are an important component of protective immunity to Ehrlichia species. However, the molecular basis of antibody mediated immunity is not completely defined, and the role of most molecularly characterized major immunoreactive proteins is unknown. In previous studies, we mapped major species-specific continuous epitopes in three surface exposed and secreted tandem repeat proteins (TRP32, TRP47 and TRP120). In this study, we report that protection is provided by antibodies against these molecularly defined TRP epitopes using in vitro and in vivo models. Protection was demonstrated in vitro after prophylactic and therapeutic administration of epitope-specific anti-TRP antibodies, suggesting that the protective mechanisms involve extracellular and intracellular antibody-mediated effects. In vivo passive transfer of individual epitope-specific TRP sera significantly reduced the ehrlichial load and splenomegaly, and protected mice against lethal infection. Moreover, the combination of antibodies to all three TRPs provided enhanced reduction in ehrlichial load similar to that of Ehrlichia chaffeensis immune sera. IgG1 was the predominant antibody isotype in the epitope-specific TRP mouse sera. These results demonstrate that antibodies against linear epitopes in TRP32, TRP47 and TRP120 are protective during E. chaffeensis infection and involves extracellular and intracellular antibody-mediated mechanisms.

Ehrlichia chaffeensis transcriptome in mammalian and arthropod hosts reveals differential gene expression and post transcriptional regulation.

BACKGROUND: Human monocytotropic ehrlichiosis is an emerging life-threatening zoonosis caused by obligately intracellular bacterium, Ehrlichia chaffeensis. E. chaffeensis is transmitted by the lone star tick, Amblyomma americanum, and replicates in mononuclear phagocytes in mammalian hosts. Differences in the E. chaffeensis transcriptome in mammalian and arthropod hosts are unknown. Thus, we determined host-specific E. chaffeensis gene expression in human monocyte (THP-1) and in Amblyomma and Ixodes tick cell lines (AAE2 and ISE6) using a whole genome microarray. METHODOLOGY/PRINCIPAL FINDINGS: The majority (∼80%) of E. chaffeensis genes were expressed during infection in human and tick cells. There were few differences observed in E. chaffeensis gene expression between the vector Amblyomma and non-vector Ixodes tick cells, but extensive host-specific and differential gene expression profiles were detected between human and tick cells, including higher transcriptional activity in tick cells and identification of gene subsets that were differentially expressed in the two hosts. Differentially and host-specifically expressed ehrlichial genes encoded major immunoreactive tandem repeat proteins (TRP), the outer membrane protein (OMP-1) family, and hypothetical proteins that were 30-80 amino acids in length. Consistent with previous observations, high expression of p28 and OMP-1B genes was detected in human and tick cells, respectively. Notably, E. chaffeensis genes encoding TRP32 and TRP47 were highly upregulated in the human monocytes and expressed as proteins; however, although TRP transcripts were expressed in tick cells, the proteins were not detected in whole cell lysates demonstrating that TRP expression was post transcriptionally regulated. CONCLUSIONS/SIGNIFICANCE: Ehrlichia gene expression is highly active in tick cells, and differential gene expression among a wide variety of host-pathogen associated genes occurs. Furthermore, we demonstrate that genes associated with host-pathogen interactions are differentially expressed and regulated by post transcriptional mechanisms.