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.

A Multimode nonlinear optical imaging technique based on the combination of multichannel mode and Lambda mode is developed to investigate human dermis. Our findings show that this technique not only improves the image contrast of the structural proteins of extracellular matrix (ECM) but also provides an image-guided spectral analysis method to identify both cellular and ECM intrinsic components including collagen, elastin, NAD(P)H and flavin. By the combined use of multichannel mode and Lambda mode in tandem, the obtained in-depth two photon-excited fluorescence (TPEF) and second-harmonic generation (SHG) imaging and TPEF/SHG signals depth-dependence decay can offer a sensitive tool for obtaining quantitative tissue structural and biochemical information. These results suggest that the technique has the potential to provide more accurate information for determining tissue physiological and pathological states.

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.

Ehrlichia chaffeensis and E. canis have a small subset of tandem repeat (TR)-containing proteins that elicit strong host immune responses and are associated with host-pathogen interactions. In a previous study, we molecularly characterized a highly conserved 19-kDa major immunoreactive protein (gp19) of E. canis and identified the corresponding TR-containing ortholog variable-length PCR target (VLPT) protein in E. chaffeensis. In this study, the native 32-kDa VLPT protein was identified and the immunodeterminants defined in order to further understand the molecular basis of the host immune response to E. chaffeensis. Synthetic and/or recombinant polypeptides corresponding to various regions of VLPT were used to localize major antibody epitopes to the TR-containing region. Major antibody epitopes were identified in three nonidentical repeats (R2, R3, and R4), which reacted strongly with antibodies in sera from an E. chaffeensis-infected dog and human monocytotropic ehrlichiosis patients. VLPT-R3 and VLPT-R2 reacted most strongly with antibody, and the epitope was further localized to a nearly identical proximal 17-amino-acid region common between these repeats that was species specific. The epitope in R4 was distinct from that of R2 and R3 and was found to have conformational dependence. VLPT was detected in supernatants from infected cells, indicating that the protein was secreted. VLPT was localized on both reticulate and dense-core cells, and it was found extracellularly in the morula fibrillar matrix and associated with the morula membrane.Ehrlichia chaffeensis is a tick-transmitted, obligately intracellular bacterium which causes human monocytotropic ehrlichiosis (HME), an emerging life-threatening disease in humans, and also causes mild to severe disease in canines (28). Recently, a number of studies demonstrated that humoral immunity plays an essential role in host defenses against ehrlichial pathogens (10,33,34,36). Furthermore, a small subset of E. chaffeensis proteins, many of which contain tandem repeats (TRs), appear to be the primary targets of the humoral immune response and are considered to be the major immunoreactive proteins (4,5,15,30). However, the characteristics of the immunodeterminants that shape the humoral immune response to Ehrlichia species are not fully defined, nor has their role in protective immunity been determined.Major immunoreactive proteins of E. chaffeensis include 30). Some of these proteins (200-, 120-, 47-, and 28-kDa proteins) have been identified and molecularly characterized, including the corresponding orthologs in Ehrlichia canis respectively) (7,16,20,25,27,(38)(39)(40). Most recently, a strongly acidic 19-kDa major immunoreactive protein (gp19) of E. canis was identified. The gp19 gene has the same relative chromosomal location as and substantial amino acid homology in a C-terminal cysteine-tyrosine-rich domain to the previously reported variable-length PCR target (VLPT) protein identified in E. chaffeensis (19). The VLPT gene has 90-bp TRs that vary in number (2 t...

Diseases caused by viruses especially by white spot syndrome virus (WSSV) are the greatest challenge to worldwide shrimp aquaculture. The innate immunity of shrimp has attracted extensive attention, but no factor involved in the virus resistance has been reported. Here we report for the first time the identification of an antiviral gene from shrimp Penaeus monodon. A differential cDNA (designated as PmAV) cloned from virus-resistant shrimp P. monodon by differential display (DD) was found to have an open reading frame (ORF) encoding a 170 amino acid peptide with a C-type lectin-like domain (CTLD). The PmAV gene was expressed in Escherichia coli and the protein was purified. Recombinant PmAV protein displayed a strong antiviral activity in inhibiting virus-induced cytopathic effect in fish cell in vitro. Moreover, native PmAV protein was isolated from shrimp hemolymph by immuno-affinity chromatography and confirmed by Western blot. No agglutination activity was observed both in recombinant and native PmAV protein. Immunohistological study showed that PmAV protein was located mainly in the cytoplasm, and not bound to the shrimp WSSV. It implies that the antiviral mechanism of PmAV protein is not by inhibiting the attachment of virus to target host cell. The discovery of PmAV gene might provide a clue to elucidate the innate immunity of marine invertebrates and would be helpful to shrimp viral disease control.

Ehrlichia chaffeensis invades and survives in phagocytes by modulating host cell processes and evading innate defenses, but the mechanisms are not fully defined. Recently we have determined that E. chaffeensis tandem repeat proteins (TRPs) are type 1 secreted effectors involved in functionally diverse interactions with host targets, including components of the evolutionarily conserved Wnt signaling pathways. In this study, we demonstrated that induction of host canonical and noncanonical Wnt pathways by E. chaffeensis TRP effectors stimulates phagocytosis and promotes intracellular survival. After E. chaffeensis infection, canonical and noncanonical Wnt signalings were significantly stimulated during early stages of infection (1 to 3 h) which coincided with dephosphorylation and nuclear translocation of ␤-catenin, a major canonical Wnt signal transducer, and NFATC1, a noncanonical Wnt transcription factor. In total, the expression of ϳ44% of Wnt signaling target genes was altered during infec- Ehrlichia chaffeensis is an obligately intracellular bacterium responsible for the emerging life-threatening human zoonosis human monocytotropic ehrlichiosis (HME) (1). E. chaffeensis selectively infects mononuclear phagocytes and resides in early-endosome-like membrane-bound vacuoles (1). The mechanisms by which E. chaffeensis enters host cells, establishes persistent infection, and avoids host defenses are not completely understood but occur through functionally relevant host-pathogen interactions involving secreted ehrlichial tandem repeat protein (TRP) effectors that are posttranslationally modified by ubiquitin (Ub) and the small ubiquitin-like modifier (SUMO) (2-5). E. chaffeensis TRPs interact with a diverse group of human proteins associated with major cellular processes, including transcription, translation, protein trafficking, cell signaling, cytoskeleton organization, and apoptosis, indicating that they play a role in manipulating these important cellular processes to facilitate infection (6-8).E. chaffeensis TRPs were first recognized as antigens that elicit strong protective antibody responses during infection that are directed at continuous species-specific epitopes in tandem repeat regions (9-12). Subsequently, our understanding of the functional role of TRPs as effectors in pathobiology has been advanced through studies that have defined specific TRP-host protein and DNA interactions (4, 13). Notably, E. chaffeensis TRP120 and TRP32 interact with numerous host proteins and genes associated with the canonical and noncanonical Wnt signaling pathways. One of TRP32-interacting targets, deleted-in-azoospermia-associated protein 2 (DAZAP2), is a highly conserved protein that modulates gene transcription driven by Wnt/␤-catenin signaling effector T-cell factors (TCFs), and knockdown of host DAZAP2 by small interfering RNA (siRNA) reduced the E. chaffeensis load in infected cells (7,14). Several other TRP120-interacting host proteins, such as AT-rich interactive domain 1B (ARID1B), lysine (K)-specific demethylase...

Ehrlichia chaffeensis is an obligately intracellular Gram-negative bacterium that selectively infects mononuclear phagocytes. We recently reported that E. chaffeensis utilizes a type 1 secretion (T1S) system to export tandem repeat protein (TRP) effectors and demonstrated that these effectors interact with a functionally diverse array of host proteins. By way of these interactions, TRP effectors modulate host cell functions; however, the molecular basis of these interactions and their roles in ehrlichial pathobiology are not well defined. In this study, we describe the first bacterial protein posttranslational modification (PTM) by the small ubiquitin-like modifier (SUMO). The E. chaffeensis T1S effector TRP120 is conjugated to SUMO at a carboxy-terminal canonical consensus SUMO conjugation motif in vitro and in human cells. In human cells, TRP120 was selectively conjugated with SUMO2/3 isoforms. Disruption of TRP120 SUMOylation perturbed interactions with known host proteins, through predicted SUMO interaction motif-dependent and -independent mechanisms. E. chaffeensis infection did not result in dramatic changes in the global host SUMOylated protein profile, but a robust colocalization of predominately SUMO1 with ehrlichial inclusions was observed. Inhibiting the SUMO pathway with a small-molecule inhibitor had a significant impact on E. chaffeensis replication and recruitment of the TRP120-interacting protein polycomb group ring finger protein 5 (PCGF5) to the inclusion, indicating that the SUMO pathway is critical for intracellular survival. This study reveals the novel exploitation of the SUMO pathway by Ehrlichia, which facilitates effector-eukaryote interactions necessary to usurp the host and create a permissive intracellular niche. Ehrlichia chaffeensis, the etiologic agent of the life-threatening tick-borne zoonosis human monocytotropic ehrlichiosis (HME), is an obligately intracellular Gram-negative bacterium that selectively infects mononuclear phagocytes and replicates in cytoplasmic vacuoles resembling endosomes (1-3). The mechanisms through which E. chaffeensis directs internalization, establishes intracellular infection, and avoids innate and adaptive host defenses are not well understood. However, we identified a group of type 1 secretion (T1S) system ehrlichial tandem repeat protein (TRP) effectors, similar to the repeats-in-toxin family of exoproteins, that are involved in novel molecular interactions with a large group of functionally diverse host cell proteins and host cell DNA (4-9).E. chaffeensis TRP120 is a major immunoreactive protein that is found on the surfaces of infectious dense-cored ehrlichiae and is expressed in both arthropod and mammalian cells (10, 11). A single major linear epitope (22 amino acids) in the tandem repeat region of TRP120 has been identified that elicits protective antibodies (12). Following T1S, TRP120 crosses the ehrlichial vacuole membrane through an unknown mechanism, similar to the Chlamydia trachomatis protein effector CPAF (13), and is present in the host...

Ehrlichia chaffeensis preferentially targets mononuclear phagocytes and survives through a strategy of subverting innate immune defenses, but the mechanisms are unknown. We have shown E. chaffeensis type 1 secreted tandem repeat protein (TRP) effectors are involved in diverse molecular pathogen-host interactions, such as the TRP120 interaction with the Notch receptor-cleaving metalloprotease ADAM17. In the present study, we demonstrate E. chaffeensis, via the TRP120 effector, activates the canonical Notch signaling pathway to promote intracellular survival. We found that nuclear translocation of the transcriptionally active Notch intracellular domain (NICD) occurs in response to E. chaffeensis or recombinant TRP120, resulting in upregulation of Notch signaling pathway components and target genes notch1, adam17, hes, and hey. Significant differences in canonical Notch signaling gene expression levels (>40%) were observed during early and late stages of infection, indicating activation of the Notch pathway. We linked Notch pathway activation specifically to the TRP120 effector, which directly interacts with the Notch metalloprotease ADAM17. Using pharmacological inhibitors and small interfering RNAs (siRNAs) against γ-secretase enzyme, Notch transcription factor complex, Notch1, and ADAM17, we demonstrated that Notch signaling is required for ehrlichial survival. We studied the downstream effects and found that E. chaffeensis TRP120-mediated activation of the Notch pathway causes inhibition of the extracellular signal-regulated kinase 1/2 (ERK1/2) and p38 mitogen-activated protein kinase (MAPK) pathways required for PU.1 and subsequent Toll-like receptor 2/4 (TLR2/4) expression. This investigation reveals a novel mechanism whereby E. chaffeensis exploits the Notch pathway to evade the host innate immune response for intracellular survival.