RESUMEN
Rickettsial pathogens including Ehrlichia canis and Anaplasma platys are bacteria that cause parasitic infections in dogs such as canine monocytic ehrlichiosis (CME) and canine cyclic thrombocytopenia (CCT), respectively affecting mortality and morbidity worldwide. An accurate, sensitive, and rapid method to diagnose these agents is essential for effective treatment. In this study, a recombinase polymerase amplification (RPA) coupled with CRISPR-Cas12a methods was established to detect E. canis and A. platys infection in dogs based on the 16S rRNA. The optimal condition for DNA amplification by RPA was 37 °C for 20 min, followed by CRISPR-Cas12a digestion at 37 °C for one hour. A combination of RPA and the cas12a detection method did not react with other pathogens and demonstrated strong sensitivity, detecting as low as 100 copies of both E. canis and A. platys. This simultaneous detection method was significantly more sensitive than conventional PCR. The RPA-assisted cas12a assay provides specific, sensitive, rapid, simple and appropriate detection of rickettsial agents in canine blood at the point-of-care for diagnostics, disease prevention and surveillance.
Asunto(s)
Anaplasmosis , Enfermedades de los Perros , Ehrlichiosis , Perros , Animales , Ehrlichia canis/genética , Anaplasmosis/diagnóstico , Anaplasmosis/epidemiología , Anaplasmosis/genética , Sistemas CRISPR-Cas , Recombinasas/genética , Tailandia , ARN Ribosómico 16S/genética , Ehrlichiosis/diagnóstico , Ehrlichiosis/veterinaria , Ehrlichiosis/genética , Enfermedades de los Perros/diagnóstico , Enfermedades de los Perros/epidemiologíaRESUMEN
The intracellular cholesterol transport protein Niemann-Pick type C1 (NPC1) and lipid-raft protein flotillin (FLOT) are required for cholesterol uptake by the obligatory intracellular bacterium Anaplasma phagocytophilum and for infection, and each protein localizes to membrane-bound inclusions containing replicating bacteria. Here, we found striking localization of FLOT2 in NPC1-lined vesicles and a physical interaction between FLOT2 and NPC1. This interaction was cholesterol dependent, as a CRAC (cholesterol recognition/interaction amino acid cholesterol-binding) domain mutant of FLOT2 did not interact with NPC1, and the cholesterol-sequestering agent methyl-ß-cyclodextrin reduced the interaction. The stomatin-prohibitin-flotillin-HflC/K domain of FLOT2, FLOT21-183, was sufficient for the unique FLOT2 localization and interaction with NPC1. NPC1, FLOT2, and FLOT21-183 trafficked to the lumen of Anaplasma inclusions. A loss-of-function mutant, NPC1P691S (mutation in the sterol-sensing domain), did not colocalize or interact with FLOT2 or with Anaplasma inclusions and inhibited infection. Ezetimibe is a drug that blocks cholesterol absorption in the small intestine by inhibiting plasma membrane Niemann-Pick C1-like 1 interaction with FLOTs. Ezetimibe blocked the interaction between NPC1 and FLOT2 and inhibited Anaplasma infection. Ezetimibe did not directly inhibit Anaplasma proliferation but inhibited host membrane lipid and cholesterol traffic to the bacteria in the inclusion. These data suggest that Anaplasma hijacks NPC1 vesicles containing cholesterol bound to FLOT2 to deliver cholesterol into Anaplasma inclusions to assimilate cholesterol for its proliferation. These results provide insights into mechanisms of intracellular cholesterol transport and a potential approach to inhibit Anaplasma infection by blocking cholesterol delivery into the lumen of bacterial inclusions. IMPORTANCE Cholesterol influences membrane fluidity and forms membrane microdomains called lipid rafts that serve as organizing centers for the assembly of signaling molecules. Flotillin (FLOT) is a cholesterol-binding lipid-raft protein. The cholesterol-binding membrane glycoprotein Niemann-Pick type C1 (NPC1) is critical for managing cellular cholesterol level and its intracellular transport, and mutation of the gene encoding NPC1 causes the fatal cholesterol storage disease, Niemann-Pick disease, type C. Both FLOT and NPC1 are trafficked to inclusions created by the cholesterol-dependent bacterium Anaplasma phagocytophilum and required for cholesterol uptake by this bacterium for replication. Our novel findings that FLOT2 interacts physically with NPC1 and resides inside both bacterial inclusions and NPC1-containing vesicles underscore the important role for FLOT2 in infection, the intracellular transport of cholesterol in NPC1 vesicles, and cholesterol homeostasis. Both NPC1-FLOT2 interaction and A. phagocytophilum infection can be inhibited by ezetimibe, suggesting possible pharmacological intervention of intracellular cholesterol hijacking by Anaplasma.
Asunto(s)
Anaplasma phagocytophilum/crecimiento & desarrollo , Anaplasma phagocytophilum/metabolismo , Colesterol/metabolismo , Ehrlichiosis/microbiología , Ezetimiba/farmacología , Proteínas de la Membrana/metabolismo , Proteína Niemann-Pick C1/metabolismo , Anaplasma phagocytophilum/efectos de los fármacos , Anaplasma phagocytophilum/genética , Transporte Biológico , Membrana Celular/efectos de los fármacos , Membrana Celular/genética , Membrana Celular/metabolismo , Ehrlichiosis/genética , Ehrlichiosis/metabolismo , Interacciones Huésped-Patógeno , Humanos , Cuerpos de Inclusión/genética , Cuerpos de Inclusión/metabolismo , Cuerpos de Inclusión/microbiología , Proteínas de la Membrana/genética , Proteína Niemann-Pick C1/genética , Unión Proteica , Transporte de ProteínasRESUMEN
Anaplasma phagocytophilum (Ap), agent of human anaplasmosis, is an intracellular bacterium that causes the second most common tick-borne illness in North America. To address the lack of a genetic system for these pathogens, we used random Himar1 transposon mutagenesis to generate a library of Ap mutants capable of replicating in human promyelocytes (HL-60 cells). Illumina sequencing identified 1195 non-randomly distributed insertions. As the density of mutants was non-saturating, genes without insertions were either essential for Ap, or spared randomly. To resolve this question, we applied a biostatistical method for prediction of essential genes. Since the chances that a transposon was inserted into genomic TA dinucleotide sites should be the same for all loci, we used a Markov chain Monte Carlo model to estimate the probability that a non-mutated gene was essential for Ap. Predicted essential genes included those coding for structural ribosomal proteins, enzymes involved in metabolism, components of the type IV secretion system, antioxidant defense molecules and hypothetical proteins. We have used an in silico post-genomic approach to predict genes with high probability of being essential for replication of Ap in HL-60 cells. These results will help target genes to investigate their role in the pathogenesis of human anaplasmosis.
Asunto(s)
Anaplasma phagocytophilum/genética , ADN Bacteriano/genética , Ehrlichiosis , Genes Esenciales/genética , Células Precursoras de Granulocitos , Línea Celular , Elementos Transponibles de ADN/genética , Ehrlichiosis/genética , Ehrlichiosis/microbiología , Biblioteca de Genes , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Cadenas de MarkovRESUMEN
Infection with obligatory intracellular bacteria is difficult to treat, as intracellular targets and delivery methods of therapeutics are not well known. Ehrlichia translocated factor-1 (Etf-1), a type IV secretion system (T4SS) effector, is a primary virulence factor for an obligatory intracellular bacterium, Ehrlichia chaffeensis In this study, we developed Etf-1-specific nanobodies (Nbs) by immunizing a llama to determine if intracellular Nbs block Etf-1 functions and Ehrlichia infection. Of 24 distinct anti-Etf-1 Nbs, NbD7 blocked mitochondrial localization of Etf-1-GFP in cotransfected cells. NbD7 and control Nb (NbD3) bound to different regions of Etf-1. Size-exclusion chromatography showed that the NbD7 and Etf-1 complex was more stable than the NbD3 and Etf-1 complex. Intracellular expression of NbD7 inhibited three activities of Etf-1 and E. chaffeensis: up-regulation of mitochondrial manganese superoxide dismutase, reduction of intracellular reactive oxygen species, and inhibition of cellular apoptosis. Consequently, intracellular NbD7 inhibited Ehrlichia infection, whereas NbD3 did not. To safely and effectively deliver Nbs into the host cell cytoplasm, NbD7 was conjugated to cyclized cell-permeable peptide 12 (CPP12-NbD7). CPP12-NbD7 effectively entered mammalian cells and abrogated the blockade of cellular apoptosis caused by E. chaffeensis and inhibited infection by E. chaffeensis in cell culture and in a severe combined-immunodeficiency mouse model. Our results demonstrate the development of an Nb that interferes with T4SS effector functions and intracellular pathogen infection, along with an intracellular delivery method for this Nb. This strategy should overcome current barriers to advance mechanistic research and develop therapies complementary or alternative to the current broad-spectrum antibiotic.
Asunto(s)
Ehrlichia chaffeensis/efectos de los fármacos , Ehrlichiosis/tratamiento farmacológico , Anticuerpos de Dominio Único/farmacología , Sistemas de Secreción Tipo IV/genética , Animales , Apoptosis/genética , Subgrupos de Linfocitos B/inmunología , Ehrlichia chaffeensis/genética , Ehrlichia chaffeensis/inmunología , Ehrlichia chaffeensis/patogenicidad , Ehrlichiosis/genética , Ehrlichiosis/inmunología , Ehrlichiosis/patología , Interacciones Huésped-Patógeno/genética , Interacciones Huésped-Patógeno/inmunología , Humanos , Ratones , Especies Reactivas de Oxígeno/metabolismo , Anticuerpos de Dominio Único/inmunología , Sistemas de Secreción Tipo IV/antagonistas & inhibidores , Sistemas de Secreción Tipo IV/inmunología , Factores de VirulenciaRESUMEN
Ehrlichia species are obligatory intracellular bacteria transmitted by arthropods, and some of these species cause febrile diseases in humans and livestock. Genome sequencing has only been performed with cultured Ehrlichia species, and the taxonomic status of such ehrlichiae has been estimated by core genome-based phylogenetic analysis. However, many uncultured ehrlichiae exist in nature throughout the world, including Japan. This study aimed to conduct a molecular-based taxonomic and ecological characterization of uncultured Ehrlichia species or genotypes from ticks in Japan. We first surveyed 616 Haemaphysalis ticks by p28-PCR screening and analyzed five additional housekeeping genes (16S rRNA, groEL, gltA, ftsZ, and rpoB) from 11 p28-PCR-positive ticks. Phylogenetic analyses of the respective genes showed similar trees but with some differences. Furthermore, we found that V1 in the V1-V9 regions of Ehrlichia 16S rRNA exhibited the greatest variability. From an ecological viewpoint, the amounts of ehrlichiae in a single tick were found to equal approx. 6.3E+3 to 2.0E+6. Subsequently, core-partial-RGGFR-based phylogenetic analysis based on the concatenated sequences of the five housekeeping loci revealed six Ehrlichia genotypes, which included potentially new Ehrlichia species. Thus, our approach contributes to the taxonomic profiling and ecological quantitative analysis of uncultured or unidentified Ehrlichia species or genotypes worldwide.
Asunto(s)
Proteínas Bacterianas/genética , Biodiversidad , ADN Bacteriano/genética , Ehrlichia/fisiología , Ehrlichiosis/diagnóstico , Garrapatas/microbiología , Animales , ADN Bacteriano/análisis , Ehrlichiosis/genética , Ehrlichiosis/parasitología , Humanos , Japón , FilogeniaRESUMEN
Inflammasomes are an important innate immune host defense against intracellular microbial infection. Activation of inflammasomes by microbial or host ligands results in cleavage of caspase-1 (canonical pathway) or caspase-11 (noncanonical pathway), release of interleukin (IL)-1ß, IL-18, high mobility group box 1 (HMGB1), and inflammatory cell death known as pyroptosis. Ehrlichia are obligate, intracellular, gram-negative bacteria that lack lipopolysaccharide but cause potentially life-threatening monocytic ehrlichiosis in humans and mice that is characterized by liver injury followed by sepsis and multiorgan failure. Employing murine models of mild and fatal ehrlichiosis caused by infection with mildly and highly virulent Ehrlichia muris (EM) and Ixodes ovatus Ehrlichia (IOE), respectively, we have previously shown that IOE infection triggers type I interferon (IFN-I) response and deleterious caspase-11 activation in liver tissues, which promotes liver injury and sepsis. In this study, we examined the contribution of IFN-I signaling in hepatocytes (HCs) to Ehrlichia-induced liver injury. Compared to EM infection, we found that IOE enter and replicate in vitro cultured primary murine HCs and induce secretion of IFNß and several chemokines, including regulated upon activation, normal T-cell expressed, and secreted (RANTES), monocyte chemoattractant protein 1 (MCP1), monokine induced by gamma (MIG)/chemokine (C-X-C motif) ligand 9 (CXCL9), macrophage inflammatory protein 1 alpha (MIP1α), keratinocyte-derived chemokine (KC), and granulocyte-macrophage colony-stimulating factor (GM-CSF). Notably, in vitro stimulation of uninfected and Ehrlichia-infected HCs with recombinant IFNß triggered activation of caspase-1/11, cytosolic translocation of HMGB1, and enhanced autophagy and intracellular bacterial replication. Secretion of HMGB1 by IOE-infected HCs was dependent on caspase-11. Primary HCs from IOE- but not EM-infected mice also expressed active caspase-1/11. Conclusion: HC-specific IFN-I signaling may exacerbate liver pathology during infection with obligate intracellular Ehrlichia by promoting bacterial replication and detrimental caspase-11-mediated inflammasome activation.
Asunto(s)
Ehrlichia/inmunología , Ehrlichiosis/inmunología , Hepatocitos/metabolismo , Inflamasomas/inmunología , Interferón Tipo I/inmunología , Animales , Linfocitos T CD8-positivos/inmunología , Caspasa 1/genética , Caspasa 1/inmunología , Ehrlichiosis/genética , Femenino , Proteína HMGB1/genética , Proteína HMGB1/inmunología , Interferón Tipo I/genética , Interferón gamma/inmunología , Interleucina-18/inmunología , Interleucina-1beta/inmunología , Ixodes/microbiología , Ratones , Ratones Endogámicos C57BL , Receptor de Interferón alfa y beta/genética , Receptor de Interferón alfa y beta/metabolismoRESUMEN
Ehrlichia chaffeensis (E. chaffeensis) exploits evolutionarily conserved Notch and Wnt host cell signaling pathways to downregulate innate immune host defenses and promote infection. The multifunctional E. chaffeensis TRP120 effector which has HECT E3 ubiquitin ligase activity, interacts with the host nuclear tumor suppressor F-BOX and WD domain repeating-containing 7 (FBW7). FBW7 is the substrate recognition subunit of the Skp1-cullin-1-FBOX E3 ubiquitin (Ub) ligase complex (SCF) known to negatively regulate a network of oncoproteins (Notch, cyclin E, c-Jun, MCL1 and cMYC). In this study, we demonstrate that TRP120 and FBW7 colocalize strongly in the nucleus by confocal immunofluorescent microscopy and interactions between TRP120 and FBW7 FBOX and WD40 domains were demonstrated by ectopic expression and co-immunoprecipitation. Although FBW7 gene expression increased during E. chaffeensis infection, FBW7 levels significantly decreased (>70%) by 72 h post infection. Moreover, an iRNA knockdown of FBW7 coincided with increased E. chaffeensis infection and levels of Notch intracellular domain (NICD), phosphorylated c-Jun, MCL-1 and cMYC, which are negatively regulated by FBW7. An increase in FBW7 K48 ubiquitination was detected during infection by co-IP, and FBW7 degradation was inhibited in infected cells treated with the proteasomal inhibitor bortezomib. Direct TRP120 ubiquitination of native and recombinant FBW7 was demonstrated in vitro and confirmed by ectopic expression of TRP120 HECT Ub ligase catalytic site mutant. This study identifies the tumor suppressor, FBW7, as a TRP120 HECT E3 Ub ligase substrate, and demonstrates that TRP120 ligase activity promotes ehrlichial infection by degrading FBW7 to maintain stability of Notch and other oncoproteins involved in cell survival and apoptosis.
Asunto(s)
Ehrlichia chaffeensis/metabolismo , Ehrlichiosis/genética , Proteína 7 que Contiene Repeticiones F-Box-WD/metabolismo , Apoptosis/fisiología , Proteínas Bacterianas/metabolismo , Proteínas de Ciclo Celular/metabolismo , Ehrlichia chaffeensis/genética , Ehrlichia chaffeensis/fisiología , Ehrlichiosis/metabolismo , Proteínas F-Box/metabolismo , Proteína 7 que Contiene Repeticiones F-Box-WD/genética , Interacciones Huésped-Patógeno , Humanos , Proteínas Oncogénicas/genética , Unión Proteica/fisiología , Células THP-1 , Ubiquitina-Proteína Ligasas/metabolismo , UbiquitinaciónRESUMEN
The carbohydrate Galα1-3Galß1-(3)4GlcNAc-R (α-Gal) is produced in all mammals except for humans, apes and old world monkeys that lost the ability to synthetize this carbohydrate. Therefore, humans can produce high antibody titers against α-Gal. Anti-α-Gal IgE antibodies have been associated with tick-induced allergy (i.e. α-Gal syndrome) and anti-α-Gal IgG/IgM antibodies may be involved in protection against malaria, leishmaniasis and Chagas disease. The α-Gal on tick salivary proteins plays an important role in the etiology of the α-Gal syndrome. However, whether ticks are able to produce endogenous α-Gal remains currently unknown. In this study, the Ixodes scapularis genome was searched for galactosyltransferases and three genes were identified as potentially involved in the synthesis of α-Gal. Heterologous gene expression in α-Gal-negative cells and gene knockdown in ticks confirmed that these genes were involved in α-Gal synthesis and are essential for tick feeding. Furthermore, these genes were shown to play an important role in tick-pathogen interactions. Results suggested that tick cells increased α-Gal levels in response to Anaplasma phagocytophilum infection to control bacterial infection. These results provided the molecular basis of endogenous α-Gal production in ticks and suggested that tick galactosyltransferases are involved in vector development, tick-pathogen interactions and possibly the etiology of α-Gal syndrome in humans.
Asunto(s)
Anaplasma phagocytophilum/patogenicidad , Proteínas de Artrópodos/metabolismo , Galactosiltransferasas/metabolismo , Ixodes/microbiología , alfa-Galactosidasa/genética , alfa-Galactosidasa/metabolismo , Secuencia de Aminoácidos , Animales , Línea Celular Tumoral , Vectores de Enfermedades , Ehrlichiosis/genética , Ehrlichiosis/metabolismo , Genoma/genética , Células HL-60 , Interacciones Huésped-Patógeno/genética , HumanosRESUMEN
Eukaryotic proteome diversity exceeds that encoded within individual genes, and results in part from alternative splicing events of pre-messenger RNA. The diversity of these splicing events can shape the outcome in development and differentiation of normal tissues, and is important in pathogenic circumstances such as cancer and some heritable conditions. A role for alternative splicing of eukaryotic genes in response to viral and intracellular bacterial infections has only recently been recognized, and plays an important role in providing fitness for microbial survival, while potentially enhancing pathogenicity. Anaplasma phagocytophilum survives within mammalian neutrophils by reshaping transcriptional programs that govern cellular functions. We applied next generation RNAseq to ATRA-differentiated HL-60 cells established to possess transcriptional and functional responses similar to A. phagocytophilum-infected human neutrophils. This demonstrated an increase in transcripts with infection and high proportion of alternatively spliced transcript events (ASEs) for which predicted gene ontology processes were in part distinct from those identified by evaluation of single transcripts or gene-level analyses alone. The alternative isoforms are not on average shorter, and no alternative splicing in genes encoding spliceosome components is noted. Although not evident at gene-level analyses, individual spliceosome transcripts that impact nearly all spliceosome components were significantly upregulated. How the distinct GO processes predicted by ASEs are regulated by infection and whether they are relevant to fitness or pathogenicity of A. phagocytophilum should be addressed in more detailed studies.
Asunto(s)
Empalme Alternativo , Anaplasma phagocytophilum/fisiología , Ehrlichiosis/genética , Ehrlichiosis/microbiología , Células Mieloides/metabolismo , Transcriptoma , Diferenciación Celular/genética , Biología Computacional/métodos , Ehrlichiosis/metabolismo , Perfilación de la Expresión Génica , Ontología de Genes , Células HL-60 , Humanos , Células Mieloides/citología , Empalmosomas/metabolismo , Activación TranscripcionalRESUMEN
Ehrlichia chaffeensis has a group of well-characterized type I secreted tandem repeat protein (TRP) effectors that have moonlighting capabilities. TRPs modulate various cellular processes, reprogram host gene transcription as nucleomodulins, function as ubiquitin ligases, and directly activate conserved host cell signaling pathways to promote E. chaffeensis infection. One TRP-interacting host target is polycomb group ring finger protein 5 (PCGF5), a member of the polycomb group (PcG) protein family and a component of the polycomb repressive complex 1 (PRC1). The current study demonstrates that during early infection, PCGF5 strongly colocalizes with TRP120 in the nucleus and later dramatically redistributes to the ehrlichial vacuole along with other PCGF isoforms. Ectopic expression and immunoprecipitation of TRP120 confirmed the interaction of TRP120 with multiple different PCGF isoforms. At 48 h postinfection, a dramatic redistribution of PCGF isoforms from the nucleus to the ehrlichial vacuole was observed, which also temporally coincided with proteasomal degradation of PCGF isoforms and TRP120 expression on the vacuole. A decrease in PRC1-mediated repressive chromatin mark and an altered transcriptional activity in PRC1-associated Hox genes primarily from HOXB and HOXC clusters were observed along with the degradation of PCGF isoforms, suggesting disruption of the PRC1 in E. chaffeensis-infected cells. Notably, small interfering RNA (siRNA)-mediated knockdown of PCGF isoforms resulted in significantly increased E. chaffeensis infection. This study demonstrates a novel strategy in which E. chaffeensis manipulates PRC complexes through interactions between TRP120 and PCGF isoforms to promote infection.
Asunto(s)
Ehrlichia chaffeensis/fisiología , Ehrlichiosis/metabolismo , Ehrlichiosis/microbiología , Interacciones Huésped-Patógeno , Proteínas del Grupo Polycomb/metabolismo , Proteínas de Ciclo Celular/metabolismo , Núcleo Celular/metabolismo , Rastreo Celular , Ehrlichiosis/genética , Genes Homeobox , Histonas/metabolismo , Interacciones Huésped-Patógeno/genética , Humanos , Macrófagos/metabolismo , Macrófagos/microbiología , Proteínas del Grupo Polycomb/genética , Isoformas de Proteínas , ProteolisisRESUMEN
This study investigated the frequency of infection by Anaplasma platys and Ehrlichia canis in dogs submitted to animal health centers in Campo Grande, state of Mato Grosso do Sul, Brazil. E. canis and A. platys showed infection frequencies of 55.75% and 16.96%, respectively. The identity of the two species was confirmed by DNA sequencing.
Asunto(s)
Anaplasma/aislamiento & purificación , Anaplasmosis/epidemiología , Enfermedades de los Perros/epidemiología , Ehrlichia canis/aislamiento & purificación , Ehrlichiosis/veterinaria , Anaplasma/genética , Anaplasmosis/genética , Animales , Brasil/epidemiología , Enfermedades de los Perros/genética , Perros , Ehrlichia canis/genética , Ehrlichiosis/epidemiología , Ehrlichiosis/genética , Reacción en Cadena de la Polimerasa/veterinaria , Análisis de Secuencia de ADN/veterinariaRESUMEN
In a study in Costa Rica 314 serum samples from dogs throughout all seven provinces were tested using a commercial kit for the detection of circulating antibodies against Anaplasma spp., Borrelia burgdorferi sensu lato and Ehrlichia canis, and of circulating antigen of Dirofilaria immitis. A total of 6.4% (20/314) and 38.2% (120/314) were positive for Anaplasma spp. (An) and E. canis (Ec) antibodies. Overall, 8.0% (25/314) were positive for D. immitis (Di) antigen. One single dog reacted positive with B. burgdorferi s.l. (Bb) antigen (0.3%, 1/314). E. canis positive dogs were detected in all provinces (highest percentages in Guanacaste, Puntarenas [both significantly different compared to the overall] and Limón). Guanacaste and Puntarenas also showed the highest prevalences of Anaplasma spp. (both significantly different compared to the overall). The highest prevalence of D. immitis was detected in Puntarenas (significantly different compared to the overall). Double pathogen exposure (Ec plus An; Ec plus Di; Ec plus Bb) were recorded in 8.9% (28/314). Two dogs showed a triple pathogen exposure (0.6%, 2/314; An, Ec and Di). There was a significant difference between male (11.5%, 18/156) and female (4.4%, 7/158) animals for D. immitis positive results. There was also a significant difference between breed and no breed dogs regarding the characteristics of a general positive test, as well as seropositivity to the single pathogens of Anaplasma spp., E. canis and D. immitis. Finally there was a significant difference in the presence of clinical signs again regarding the characteristics of a general positive test, as well as seropositivity to Anaplasma spp., E. canis and D. immitis. Practitioners in Costa Rica should be aware of the canine vector-borne diseases mentioned as dogs are at risk of becoming infected. Concerning the positive B. burgdorferi s.l. dog, an autochthonous occurrence cannot be confirmed due to a history of adoption and an unusual tattoo number. Veterinary advice to protect dogs and limit transmission of vector-borne pathogens, also to humans, by using prophylactic measures is strongly recommended.
Asunto(s)
Anaplasmosis/epidemiología , Dirofilariasis/epidemiología , Enfermedades de los Perros/epidemiología , Ehrlichiosis/veterinaria , Enfermedad de Lyme/veterinaria , Factores de Edad , Anaplasma/aislamiento & purificación , Anaplasmosis/genética , Anaplasmosis/microbiología , Animales , Anticuerpos Antibacterianos/sangre , Antígenos Helmínticos/sangre , Grupo Borrelia Burgdorferi/aislamiento & purificación , Costa Rica/epidemiología , Dirofilaria immitis/aislamiento & purificación , Dirofilariasis/genética , Dirofilariasis/parasitología , Enfermedades de los Perros/genética , Enfermedades de los Perros/microbiología , Enfermedades de los Perros/parasitología , Perros , Ehrlichia canis/aislamiento & purificación , Ehrlichiosis/epidemiología , Ehrlichiosis/genética , Ehrlichiosis/microbiología , Femenino , Enfermedad de Lyme/epidemiología , Enfermedad de Lyme/genética , Enfermedad de Lyme/microbiología , Masculino , Prevalencia , Factores de Riesgo , Estudios Seroepidemiológicos , Factores SexualesRESUMEN
ABSTRACT This study investigated the frequency of infection by Anaplasma platys and Ehrlichia canis in dogs submitted to animal health centers in Campo Grande, state of Mato Grosso do Sul, Brazil. E. canis and A. platys showed infection frequencies of 55.75% and 16.96%, respectively. The identity of the two species was confirmed by DNA sequencing.
Asunto(s)
Animales , Perros , Ehrlichiosis/veterinaria , Ehrlichia canis/aislamiento & purificación , Enfermedades de los Perros/epidemiología , Anaplasma/aislamiento & purificación , Anaplasmosis/epidemiología , Brasil/epidemiología , Reacción en Cadena de la Polimerasa/veterinaria , Ehrlichiosis/genética , Ehrlichiosis/epidemiología , Análisis de Secuencia de ADN/veterinaria , Ehrlichia canis/genética , Enfermedades de los Perros/genética , Anaplasma/genética , Anaplasmosis/genéticaRESUMEN
OBJECTIVE: Bernese Mountain Dogs (BMD) have a higher prevalence of Borrelia burgdorferi sensu lato (Bbsl) antibodies than other breeds, but it is not known whether this is the case for other pathogens. Therefore, the aim of the study was to determine the frequency and level of specific antibodies against members of the Bbsl group, Anaplasma phagocytophilum (Ap), and Leptospira (L.) interrogans serovars in BMD and compare the results with those found in dogs of other breeds. MATERIALS AND METHODS: A total of 171 healthy BMD and 57 healthy control dogs of other breeds were included in the study. Controls were large dogs (> 30 kg) with long, dark hair coats. A two-tiered testing method consisting of computerized kinetic enzyme-linked immunosorbent assay (KELA) and Western blotting was used for detection of antibodies against Bbsl, an immunofluorescence assay (IFA) was used for detection of antibodies against Ap, and microscopic agglutination test (MAT) for antibodies to 18 different serovars of L. interrogans. RESULTS: The prevalence of anti-Bbsl antibodies was significantly higher in BMD (43.3%) than in controls (17.5%) (p < 0.001). Antibodies to Bbsl attributable to vaccination were excluded from the calculation of prevalence. Antibodies to Ap were found in 50.3% of BMD, whereas only 24.6% of the controls dogs were tested positive for Ap (p < 0.001). Antibody titers of the 18 different serovars of L. interrogans antibodies did not differ significantly between BMD and control dogs except for L. copenhageni antibody titers which were higher in BMD. Significantly higher antibody titers to L. canicola (p = 0.003), L. copenhageni (p = 0.005), L. grippothyphosa (p = 0.029) and L. vanderhoedoni (p = 0.035) were seen in BMD compared to control dogs. CONCLUSION AND CLINICAL RELEVANCE: BMD had a higher prevalence of anti-Bbsl, anti-L. copenhageni and anti-Ap antibodies than control dogs. Significantly higher antibody titers against L. canicola (p = 0.003), L. copenhageni (p = 0.005), L. grippothyphosa (p = 0.029) and L. vanderhoedoni (p = 0.035) were seen in BMD compared with control dogs, but the reason for this and potential clinical implications are not known.
Asunto(s)
Anaplasma phagocytophilum/inmunología , Anticuerpos Antibacterianos/sangre , Borrelia burgdorferi/inmunología , Enfermedades de los Perros/epidemiología , Leptospira interrogans/inmunología , Animales , Cruzamiento , Enfermedades de los Perros/genética , Enfermedades de los Perros/microbiología , Perros , Ehrlichiosis/epidemiología , Ehrlichiosis/genética , Ehrlichiosis/veterinaria , Femenino , Alemania/epidemiología , Leptospirosis/epidemiología , Leptospirosis/genética , Leptospirosis/veterinaria , Enfermedad de Lyme/epidemiología , Enfermedad de Lyme/genética , Enfermedad de Lyme/veterinaria , Masculino , PrevalenciaRESUMEN
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 ß-catenin, a major canonical Wnt signal transducer, and NFATC1, a noncanonical Wnt transcription factor. In total, the expression of â¼44% of Wnt signaling target genes was altered during infection. Knockdown of TRP120-interacting Wnt pathway components/regulators and other critical components, such as Wnt5a ligand, Frizzled 5 receptor, ß-catenin, nuclear factor of activated T cells (NFAT), and major signaling molecules, resulted in significant reductions in the ehrlichial load. Moreover, small-molecule inhibitors specific for components of canonical and noncanonical (Ca(2+) and planar cell polarity [PCP]) Wnt pathways, including IWP-2, which blocks Wnt secretion, significantly decreased ehrlichial infection. TRPs directly activated Wnt signaling, as TRP-coated microspheres triggered phagocytosis which was blocked by Wnt pathway inhibitors, demonstrating a key role of TRP activation of Wnt pathways to induce ehrlichial phagocytosis. These novel findings reveal that E. chaffeensis exploits canonical and noncanonical Wnt pathways through TRP effectors to facilitate host cell entry and promote intracellular survival.
Asunto(s)
Ehrlichia chaffeensis/fisiología , Ehrlichiosis/inmunología , Interacciones Huésped-Patógeno , Proteínas Wnt/inmunología , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Ehrlichia chaffeensis/genética , Ehrlichia chaffeensis/crecimiento & desarrollo , Ehrlichiosis/genética , Ehrlichiosis/microbiología , Humanos , Fagocitosis , Proteínas Wnt/genética , Vía de Señalización WntRESUMEN
Anaplasma phagocytophilum, the causative agent of Human Granulocytic Anaplasmosis (HGA), is an obligately intracellular α-proteobacterium that is transmitted by Ixodes spp ticks. However, the pathogen is not transovarially transmitted between tick generations and therefore needs to survive in both a mammalian host and the arthropod vector to complete its life cycle. To adapt to different environments, pathogens rely on differential gene expression as well as the modification of proteins and other molecules. Random transposon mutagenesis of A. phagocytophilum resulted in an insertion within the coding region of an o-methyltransferase (omt) family 3 gene. In wild-type bacteria, expression of omt was up-regulated during binding to tick cells (ISE6) at 2 hr post-inoculation, but nearly absent by 4 hr p.i. Gene disruption reduced bacterial binding to ISE6 cells, and the mutant bacteria that were able to enter the cells were arrested in their replication and development. Analyses of the proteomes of wild-type versus mutant bacteria during binding to ISE6 cells identified Major Surface Protein 4 (Msp4), but also hypothetical protein APH_0406, as the most differentially methylated. Importantly, two glutamic acid residues (the targets of the OMT) were methyl-modified in wild-type Msp4, whereas a single asparagine (not a target of the OMT) was methylated in APH_0406. In vitro methylation assays demonstrated that recombinant OMT specifically methylated Msp4. Towards a greater understanding of the overall structure and catalytic activity of the OMT, we solved the apo (PDB_ID:4OA8), the S-adenosine homocystein-bound (PDB_ID:4OA5), the SAH-Mn2+ bound (PDB_ID:4PCA), and SAM- Mn2+ bound (PDB_ID:4PCL) X-ray crystal structures of the enzyme. Here, we characterized a mutation in A. phagocytophilum that affected the ability of the bacteria to productively infect cells from its natural vector. Nevertheless, due to the lack of complementation, we cannot rule out secondary mutations.
Asunto(s)
Anaplasma phagocytophilum/enzimología , Ehrlichiosis/microbiología , Ixodes/microbiología , Metiltransferasas/metabolismo , Garrapatas/microbiología , Animales , Ehrlichiosis/genética , Ixodes/inmunología , Metiltransferasas/genética , Activación Transcripcional , Regulación hacia ArribaRESUMEN
Anaplasma phagocytophilum is an emerging zoonotic pathogen that causes human granulocytic anaplasmosis. These intracellular bacteria establish infection by affecting cell function in both the vertebrate host and the tick vector, Ixodes scapularis. Previous studies have characterized the tick transcriptome and proteome in response to A. phagocytophilum infection. However, in the postgenomic era, the integration of omics datasets through a systems biology approach allows network-based analyses to describe the complexity and functionality of biological systems such as host-pathogen interactions and the discovery of new targets for prevention and control of infectious diseases. This study reports the first systems biology integration of metabolomics, transcriptomics, and proteomics data to characterize essential metabolic pathways involved in the tick response to A. phagocytophilum infection. The ISE6 tick cells used in this study constitute a model for hemocytes involved in pathogen infection and immune response. The results showed that infection affected protein processing in endoplasmic reticulum and glucose metabolic pathways in tick cells. These results supported tick-Anaplasma co-evolution by providing new evidence of how tick cells limit pathogen infection, while the pathogen benefits from the tick cell response to establish infection. Additionally, ticks benefit from A. phagocytophilum infection by increasing survival while pathogens guarantee transmission. The results suggested that A. phagocytophilum induces protein misfolding to limit the tick cell response and facilitate infection but requires protein degradation to prevent ER stress and cell apoptosis to survive in infected cells. Additionally, A. phagocytophilum may benefit from the tick cell's ability to limit bacterial infection through PEPCK inhibition leading to decreased glucose metabolism, which also results in the inhibition of cell apoptosis that increases infection of tick cells. These results support the use of this experimental approach to systematically identify cell pathways and molecular mechanisms involved in tick-pathogen interactions. Data are available via ProteomeXchange with identifier PXD002181.
Asunto(s)
Anaplasma phagocytophilum/fisiología , Ehrlichiosis/veterinaria , Metabolómica/métodos , Proteómica/métodos , Garrapatas/microbiología , Animales , Línea Celular , Ehrlichiosis/genética , Ehrlichiosis/metabolismo , Retículo Endoplásmico/genética , Retículo Endoplásmico/metabolismo , Glucosa/metabolismo , Interacciones Huésped-Patógeno , Redes y Vías Metabólicas , Biología de Sistemas/métodosRESUMEN
Anaplasma phagocytophilum causes granulocytic anaplasmosis, an emerging disease of humans and domestic animals. The obligate intracellular bacterium uses its invasins OmpA, Asp14, and AipA to infect myeloid and non-phagocytic cells. Identifying the domains of these proteins that mediate binding and entry, and determining the molecular basis of their interactions with host cell receptors would significantly advance understanding of A. phagocytophilum infection. Here, we identified the OmpA binding domain as residues 59 to 74. Polyclonal antibody generated against a peptide spanning OmpA residues 59 to 74 inhibited A. phagocytophilum infection of host cells and binding to its receptor, sialyl Lewis x (sLe(x)-capped P-selectin glycoprotein ligand 1. Molecular docking analyses predicted that OmpA residues G61 and K64 interact with the two sLe(x) sugars that are important for infection, α2,3-sialic acid and α1,3-fucose. Amino acid substitution analyses demonstrated that K64 was necessary, and G61 was contributory, for recombinant OmpA to bind to host cells and competitively inhibit A. phagocytophilum infection. Adherence of OmpA to RF/6A endothelial cells, which express little to no sLe(x) but express the structurally similar glycan, 6-sulfo-sLe(x), required α2,3-sialic acid and α1,3-fucose and was antagonized by 6-sulfo-sLe(x) antibody. Binding and uptake of OmpA-coated latex beads by myeloid cells was sensitive to sialidase, fucosidase, and sLe(x) antibody. The Asp14 binding domain was also defined, as antibody specific for residues 113 to 124 inhibited infection. Because OmpA, Asp14, and AipA each contribute to the infection process, it was rationalized that the most effective blocking approach would target all three. An antibody cocktail targeting the OmpA, Asp14, and AipA binding domains neutralized A. phagocytophilum binding and infection of host cells. This study dissects OmpA-receptor interactions and demonstrates the effectiveness of binding domain-specific antibodies for blocking A. phagocytophilum infection.
Asunto(s)
Anaplasma phagocytophilum , Proteínas de la Membrana Bacteriana Externa , Ehrlichiosis , Simulación del Acoplamiento Molecular , Sustitución de Aminoácidos , Anaplasma phagocytophilum/química , Anaplasma phagocytophilum/genética , Anaplasma phagocytophilum/metabolismo , Anaplasma phagocytophilum/patogenicidad , Animales , Anticuerpos Antibacterianos/química , Proteínas de la Membrana Bacteriana Externa/química , Proteínas de la Membrana Bacteriana Externa/genética , Proteínas de la Membrana Bacteriana Externa/metabolismo , Células CHO , Cricetinae , Cricetulus , Ehrlichiosis/genética , Ehrlichiosis/metabolismo , Células HL-60 , Humanos , Mutación Missense , Unión Proteica , Estructura Terciaria de ProteínaRESUMEN
Ehrlichia species are intracellular bacteria that cause fatal ehrlichiosis, mimicking toxic shock syndrome in humans and mice. Virulent ehrlichiae induce inflammasome activation leading to caspase-1 cleavage and IL-18 secretion, which contribute to development of fatal ehrlichiosis. We show that fatal infection triggers expression of inflammasome components, activates caspase-1 and caspase-11, and induces host-cell death and secretion of IL-1ß, IL-1α, and type I interferon (IFN-I). Wild-type and Casp1(-/-) mice were highly susceptible to fatal ehrlichiosis, had overwhelming infection, and developed extensive tissue injury. Nlrp3(-/-) mice effectively cleared ehrlichiae, but displayed acute mortality and developed liver injury similar to wild-type mice. By contrast, Ifnar1(-/-) mice were highly resistant to fatal disease and had lower bacterial burden, attenuated pathology, and prolonged survival. Ifnar1(-/-) mice also had improved protective immune responses mediated by IFN-γ and CD4(+) Th1 and natural killer T cells, with lower IL-10 secretion by T cells. Importantly, heightened resistance of Ifnar1(-/-) mice correlated with improved autophagosome processing, and attenuated noncanonical inflammasome activation indicated by decreased activation of caspase-11 and decreased IL-1ß, compared with other groups. Our findings demonstrate that IFN-I signaling promotes host susceptibility to fatal ehrlichiosis, because it mediates ehrlichia-induced immunopathology and supports bacterial replication, perhaps via activation of noncanonical inflammasomes, reduced autophagy, and suppression of protective CD4(+) T cells and natural killer T-cell responses against ehrlichiae.
Asunto(s)
Ehrlichia/inmunología , Ehrlichiosis/inmunología , Inmunidad Celular , Inflamasomas/inmunología , Interferón Tipo I/inmunología , Lipopolisacáridos , Células TH1/inmunología , Animales , Caspasas/genética , Caspasas/inmunología , Caspasas Iniciadoras , Ehrlichiosis/genética , Ehrlichiosis/patología , Interferón Tipo I/genética , Interferón gamma/genética , Interferón gamma/inmunología , Interleucina-10/genética , Interleucina-10/inmunología , Interleucina-1alfa/genética , Interleucina-1beta/inmunología , Ratones , Ratones Noqueados , Receptor de Interferón alfa y beta/genética , Receptor de Interferón alfa y beta/inmunología , Transducción de Señal/genética , Transducción de Señal/inmunología , Células TH1/patologíaRESUMEN
Canine monocytic ehrlichiosis (CME) is a common tick-borne disease caused by the rickettsial bacterium Ehrlichia canis (Rickettsiales: Anaplasmataceae). In view of the different stages and variable clinical signs of CME, which can overlap with those of other infections, a conclusive diagnosis can more readily be obtained by combining clinical and hematological evaluations with molecular diagnostic methods. In this study, a loop-mediated isothermal amplification (LAMP) assay targeting the p30 gene of E. canis was developed. The assay was developed using DNA extracted from E. canis-infected cultures of the macrophage cell line DH82 and samples from dogs testing positive for E. canis DNA by PCR. The LAMP assay was compared to a p30-based PCR assay, using DNA extracted from EDTA-anticoagulated blood samples of 137 dogs from an endemic region in Brazil. The LAMP assay was sensitive enough to detect a single copy of the target gene, and identified 74 (54.0%) E. canis DNA-positive samples, while the p30 PCR assay detected 50 positive samples (36.5%) among the field samples. Agreement between the two assays was observed in 42 positive and 55 negative samples. However, 32 positive samples that were not detected by the PCR assay were identified by the LAMP assay, while eight samples identified as E. canis-positive by PCR showed negative results in LAMP. The developed E. canis LAMP assay showed the potential to maximize the use of nucleic acid tests in a veterinary clinical laboratory, and to improve the diagnosis of CME.