RESUMO
The microRNAs (miRNAs) are important regulators of gene expression. In this study, we provide evidence for the first time to show that rickettsial pathogen Anaplasma phagocytophilum infection results in the down-regulation of tick microRNA-133 (miR-133), to induce Ixodes scapularis organic anion transporting polypeptide (isoatp4056) gene expression critical for this bacterial survival in the vector and for its transmission to the vertebrate host. Transfection studies with recombinant constructs containing transcriptional fusions confirmed binding of miR-133 to isoatp4056 mRNA. Treatment with miR-133 inhibitor resulted in increased bacterial burden and isoatp4056 expression in ticks and tick cells. In contrast, treatment with miR-133 mimic or pre-mir-133 resulted in dramatic reduction in isoatp4056 expression and bacterial burden in ticks and tick cells. Moreover, treatment of ticks with pre-mir-133 affected vector-mediated A. phagocytophilum infection of murine host. These results provide novel insights to understand impact of modulation of tick miRNAs on pathogen colonization in the vector and their transmission to infect the vertebrate host.
Assuntos
Anaplasma phagocytophilum/genética , Interações Hospedeiro-Patógeno/genética , Ixodes/genética , MicroRNAs/genética , Anaplasma phagocytophilum/patogenicidade , Animais , Apoptose , Vetores de Doenças , Regulação da Expressão Gênica/genética , Genes Essenciais/genética , Humanos , Insetos Vetores/genética , Ixodes/patogenicidade , Camundongos , Transportadores de Ânions Orgânicos/genética , Peptídeos/genética , Transcriptoma/genéticaRESUMO
Ixodes scapularis is a medically important tick that transmits several microbes to humans, including rickettsial pathogen Anaplasma phagocytophilum. In nature, these ticks encounter several abiotic factors including changes in temperature, humidity, and light. Many organisms use endogenously generated circadian pathways to encounter abiotic factors. In this study, we provide evidence for the first time to show that A. phagocytophilum modulates the arthropod circadian gene for its transmission to the vertebrate host. We noted a circadian oscillation in the expression of arthropod clock, bmal1, period and timeless genes when ticks or tick cells were exposed to alternate 12 h light: 12 h dark conditions. Moreover, A. phagocytophilum significantly modulates the oscillation pattern of expression of these genes. In addition, increased levels of clock and bmal1 and decreased expression of Toll and JAK/STAT pathway immune genes such as pelle and jak, respectively, were noted during A. phagocytophilum transmission from ticks to the vertebrate host. RNAi-mediated knockdown of clock gene expression in ticks resulted in the reduced expression of jak and pelle that increased bacterial transmission from ticks to the murine host. Furthermore, clock-deficient ticks fed late and had less engorgement weights. These results indicate an important role for circadian modulation of tick gene expression that is critical for arthropod blood feeding and transmission of pathogens from vector to the vertebrate host.
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Artrópodes , Ixodes , Rickettsia , Fatores de Transcrição ARNTL/metabolismo , Animais , Humanos , Ixodes/genética , Ixodes/metabolismo , Janus Quinases/metabolismo , Camundongos , Fatores de Transcrição STAT/metabolismo , Transdução de Sinais , Vertebrados/metabolismoRESUMO
Reactive oxygen species (ROS) that are induced upon pathogen infection plays an important role in host defence. The rickettsial pathogen Anaplasma phagocytophilum, which is primarily transmitted by Ixodes scapularis ticks in the United States, has evolved many strategies to escape ROS and survive in mammalian cells. However, little is known on the role of ROS in A. phagocytophilum infection in ticks. Our results show that A. phagocytophilum and hemin induce activation of l-tryptophan pathway in tick cells. Xanthurenic acid (XA), a tryptophan metabolite, supports A. phagocytophilum growth in tick cells through inhibition of tryptophan dioxygenase (TDO) activity leading to reduced l-kynurenine levels that subsequently affects build-up of ROS. However, hemin supports A. phagocytophilum growth in tick cells by inducing TDO activity leading to increased l-kynurenine levels and ROS production. Our data reveal that XA and kynurenic acid (KA) chelate hemin. Furthermore, treatment of tick cells with 3-hydroxyl l-kynurenine limits A. phagocytophilum growth in tick cells. RNAi-mediated knockdown of kynurenine aminotransferase expression results in increased ROS production and reduced A. phagocytophilum burden in tick cells. Collectively, these results suggest that l-tryptophan pathway metabolites influence A. phagocytophilum survival by affecting build up of ROS levels in tick cells.
Assuntos
Anaplasma phagocytophilum/metabolismo , Ixodes/microbiologia , Triptofano/metabolismo , Animais , Hemina/metabolismo , Hemina/farmacologia , Interações Hospedeiro-Patógeno , Hidrolases/genética , Hidrolases/metabolismo , Ixodes/genética , Ixodes/metabolismo , Ácido Cinurênico/metabolismo , Ácido Cinurênico/farmacologia , Cinurenina/análogos & derivados , Cinurenina/metabolismo , Cinurenina/farmacologia , NADP/biossíntese , NADP/metabolismo , Interferência de RNA , Espécies Reativas de Oxigênio/metabolismo , Transaminases/genética , Transaminases/metabolismo , Triptofano Oxigenase/antagonistas & inibidores , Triptofano Oxigenase/metabolismo , Regulação para Cima , Xanturenatos/metabolismo , Xanturenatos/farmacologiaRESUMO
Dengue virus (DENV) is a mosquito-borne flavivirus that causes dengue fever in humans, worldwide. Using in vitro cell lines derived from Aedes albopictus and Aedes aegypti, the primary vectors of DENV, we report that DENV2/DENV3-infected cells secrete extracellular vesicles (EVs), including exosomes, containing infectious viral RNA and proteins. A full-length DENV2 genome, detected in arthropod EVs, was infectious to naïve mosquito and mammalian cells, including human-skin keratinocytes and blood endothelial cells. Cryo-electron microscopy showed mosquito EVs with a size range from 30 to 250 nm. Treatments with RNase A, Triton X-100, and 4G2 antibody-bead binding assays showed that infectious DENV2-RNA and proteins are contained inside EVs. Viral plaque formation and dilution assays also showed securely contained infectious viral RNA and proteins in EVs are transmitted to human cells. Up-regulated HSP70 upon DENV2 infection showed no role in viral replication and transmission through EVs. In addition, qRT-PCR and immunoblotting results revealed that DENV2 up-regulates expression of a mosquito tetraspanin-domain-containing glycoprotein, designated as Tsp29Fb, in A. aegypti mosquitoes, cells, and EVs. RNAi-mediated silencing and antibody blocking of Tsp29Fb resulted in reduced DENV2 loads in both mosquito cells and EVs. Immunoprecipitation showed Tsp29Fb to directly interact with DENV2 E-protein. Furthermore, treatment with GW4869 (exosome-release inhibitor) affected viral burden, direct interaction of Tsp29Fb with E-protein and EV-mediated transmission of viral RNA and proteins to naïve human cells. In summary, we report a very important finding on EV-mediated transmission of DENV2 from arthropod to mammalian cells through interactions with an arthropod EVs-enriched marker Tsp29Fb.
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Vírus da Dengue , Dengue , Vesículas Extracelulares , Proteínas de Insetos , Proteínas do Envelope Viral , Aedes , Animais , Linhagem Celular , Dengue/genética , Dengue/metabolismo , Dengue/transmissão , Vírus da Dengue/genética , Vírus da Dengue/metabolismo , Vírus da Dengue/patogenicidade , Vesículas Extracelulares/genética , Vesículas Extracelulares/metabolismo , Vesículas Extracelulares/virologia , Células Endoteliais da Veia Umbilical Humana , Humanos , Proteínas de Insetos/genética , Proteínas de Insetos/metabolismo , Camundongos , Domínios Proteicos , Proteínas do Envelope Viral/genética , Proteínas do Envelope Viral/metabolismoRESUMO
Molecular determinants and mechanisms of arthropod-borne flavivirus transmission to the vertebrate host are poorly understood. In this study, we show for the first time that a cell line from medically important arthropods, such as ticks, secretes extracellular vesicles (EVs) including exosomes that mediate transmission of flavivirus RNA and proteins to the human cells. Our study shows that tick-borne Langat virus (LGTV), a model pathogen closely related to tick-borne encephalitis virus (TBEV), profusely uses arthropod exosomes for transmission of viral RNA and proteins to the human- skin keratinocytes and blood endothelial cells. Cryo-electron microscopy showed the presence of purified arthropod/neuronal exosomes with the size range of 30 to 200 nm in diameter. Both positive and negative strands of LGTV RNA and viral envelope-protein were detected inside exosomes derived from arthropod, murine and human cells. Detection of Nonstructural 1 (NS1) protein in arthropod and neuronal exosomes further suggested that exosomes contain viral proteins. Viral RNA and proteins in exosomes derived from tick and mammalian cells were secured, highly infectious and replicative in all tested evaluations. Treatment with GW4869, a selective inhibitor that blocks exosome release affected LGTV loads in both arthropod and mammalian cell-derived exosomes. Transwell-migration assays showed that exosomes derived from infected-brain-microvascular endothelial cells (that constitute the blood-brain barrier) facilitated LGTV RNA and protein transmission, crossing of the barriers and infection of neuronal cells. Neuronal infection showed abundant loads of both tick-borne LGTV and mosquito-borne West Nile virus RNA in exosomes. Our data also suggest that exosome-mediated LGTV viral transmission is clathrin-dependent. Collectively, our results suggest that flaviviruses uses arthropod-derived exosomes as a novel means for viral RNA and protein transmission from the vector, and the vertebrate exosomes for dissemination within the host that may subsequently allow neuroinvasion and neuropathogenesis.
Assuntos
Vírus da Encefalite Transmitidos por Carrapatos/patogenicidade , Encefalite Transmitida por Carrapatos/transmissão , Exossomos/virologia , Modelos Biológicos , Neurônios/virologia , RNA Viral/metabolismo , Proteínas Virais/metabolismo , Animais , Vetores Artrópodes/citologia , Vetores Artrópodes/ultraestrutura , Vetores Artrópodes/virologia , Linhagem Celular , Células Cultivadas , Córtex Cerebral/citologia , Córtex Cerebral/patologia , Córtex Cerebral/ultraestrutura , Córtex Cerebral/virologia , Chlorocebus aethiops , Técnicas de Cocultura , Microscopia Crioeletrônica , Embrião de Mamíferos/citologia , Vírus da Encefalite Transmitidos por Carrapatos/fisiologia , Vírus da Encefalite Transmitidos por Carrapatos/ultraestrutura , Encefalite Transmitida por Carrapatos/patologia , Encefalite Transmitida por Carrapatos/virologia , Endotélio Vascular/citologia , Endotélio Vascular/patologia , Endotélio Vascular/ultraestrutura , Endotélio Vascular/virologia , Exossomos/ultraestrutura , Interações Hospedeiro-Parasita , Interações Hospedeiro-Patógeno , Humanos , Ixodes/citologia , Ixodes/ultraestrutura , Ixodes/virologia , Queratinócitos/citologia , Queratinócitos/patologia , Queratinócitos/ultraestrutura , Queratinócitos/virologia , Camundongos , Camundongos Endogâmicos C57BL , Neurônios/citologia , Neurônios/patologia , Neurônios/ultraestruturaRESUMO
Trace elements such as copper and cobalt have been associated with virus-host interactions. However, studies to show the effect of conjugation of copper(II) or cobalt(III) metal centers to thiosemicarbazone ligand(s) derived from either food additives or mosquito repellent such as 2-acetylethiazole or citral, respectively, on Zika virus (ZIKV) or dengue virus (serotype 2; DENV2) infections have not been explored. In this study, we show that four compounds comprising of thiosemicarbazone ligand derived from 2-acetylethiazole viz., (E)-N-ethyl-2-[1-(thiazol-2-yl)ethylidene]hydrazinecarbothioamide (acetylethTSC) (compound 1), a copper(II) complex with acetylethTSC as a ligand (compound 2), a thiosemicarbazone ligand-derived from citral (compound 3) and a cobalt(III) complex with a citral-thiosemicarbazone ligand (compound 4) increased DENV2 and ZIKV replication in both mosquito C6/36 cells and human keratinocytes (HaCaT cells). Treatment of both cell lines with compounds 2 or 4 showed increased dengue viral titers at all three tested doses. Enhanced dengue viral plaque formation was also noted at the tested dose of 100µM, suggesting higher production of infectious viral particles. Treatment with the compounds 2 or 4 enhanced ZIKV and DENV2 RNA levels in HeLa cell line and primary cultures of mouse bone marrow derived dendritic cells. Also, pre- or post treatments with conjugated compounds 2 or 4 showed higher loads of ZIKV or DENV2 envelope (E) protein in HaCaT cells. No changes in loads of E-protein were found in ZIKV-infected C6/36 cells, when compounds were treated after infection. In addition, we tested bis(1,10-phenanthroline)copper(II) chloride ([Cu(phen)2]Cl2, (compound 5) and tris(1,10-phenanthroline)cobalt(III) chloride ([Co(phen)3]Cl3, (compound 6) that also showed enhanced DENV2 loads. Also, we found that copper(II) chloride dehydrate (CuCl2·2H2O) or cobalt(II) chloride hexahydrate (CoCl2·6H2O) alone had no effects as "free" cations. Taken together, these findings suggest that use of Cu(II) or Co(III) conjugation to organic compounds, in insect repellents and/or food additives could enhance DENV2/ZIKV loads in human cells and perhaps induce pathogenesis in infected individuals or individuals pre-exposed to such conjugated complexes. IMPORTANCE: Mosquito-borne diseases are of great concern to the mankind. Use of chemicals/repellents against mosquito bites and transmission of microbes has been the topic of interest for many years. Here, we show that thiosemicarbazone ligand(s) derived from 2-acetylethiazole or citral or 1,10-phenanthroline upon conjugation with copper(II) or cobalt(III) metal centers enhances dengue virus (serotype 2; DENV2) and/or Zika virus (ZIKV) infections in mosquito, mouse and human cells. Enhanced ZIKV/DENV2 capsid mRNA or envelope protein loads were evident in mosquito cells and human keratinocytes, when treated with compounds before/after infections. Also, treatment with copper(II) or cobalt(III) conjugated compounds increased viral titers and number of plaque formations. These studies suggest that conjugation of compounds in repellents/essential oils/natural products/food additives with copper(II) or cobalt(III) metal centers may not be safe, especially in tropical and subtropical places, where several dengue infection cases and deaths are reported annually or in places with increased ZIKV caused microcephaly.
Assuntos
Cobalto , Complexos de Coordenação , Cobre , Vírus da Dengue/metabolismo , Queratinócitos/virologia , Carga Viral/efeitos dos fármacos , Zika virus/metabolismo , Animais , Chlorocebus aethiops , Cobalto/química , Cobalto/farmacologia , Complexos de Coordenação/química , Complexos de Coordenação/farmacologia , Cobre/química , Cobre/farmacologia , Culicidae , Células HeLa , Humanos , Queratinócitos/metabolismo , Queratinócitos/patologia , Células Vero , Proteínas do Envelope ViralRESUMO
Semaphorin 7A (Sema7A) is a membrane-associated/secreted protein that plays an essential role in connecting the vertebrate neuronal and immune systems. However, the role of Sema7A has not been elucidated in viral pathogenesis. In this study, we show that abrogation of Sema7A protects mice from lethal West Nile virus (WNV) infection. Mice lacking Sema7A showed increased survival, reduced viral burden, and less blood-brain barrier permeability upon WNV infection. Increased Sema7A levels were evident in murine tissues, as well as in murine cortical neurons and primary human macrophages upon WNV infection. Treatment with Sema7A Ab blocked WNV infection in both of these cell types. Furthermore, Sema7A positively regulates the production of TGF-ß1 and Smad6 to facilitate WNV pathogenesis in mice. Collectively, these data elucidate the role of Sema7A in shared signaling pathways used by the immune and nervous systems during viral pathogenesis that may lead to the development of Sema7A-blocking therapies for WNV and possibly other flaviviral infections.
Assuntos
Antígenos CD/fisiologia , Semaforinas/fisiologia , Transdução de Sinais/imunologia , Proteína Smad6/fisiologia , Fator de Crescimento Transformador beta1/fisiologia , Vírus do Nilo Ocidental/imunologia , Vírus do Nilo Ocidental/patogenicidade , Animais , Linhagem Celular , Células Cultivadas , Córtex Cerebral/imunologia , Córtex Cerebral/metabolismo , Córtex Cerebral/virologia , Modelos Animais de Doenças , Feminino , Células HEK293 , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Replicação Viral/imunologiaRESUMO
Exosomes/extracellular vesicles (EVs) are essential for the successful transmission of flaviviruses from vector to vertebrate host. Arthropod-EVs are envisioned as important target for blocking the transmission of vector-borne viral diseases. In this study, we show that the selective inhibition of EVs secretion by sphingomyelinase inhibitor, GW4869 significantly reduces vector efficiency and competence in acquiring and transmitting tick-borne flaviviruses. We show that GW4869 reduces EVs release from Langat Virus (LGTV)-infected Ixodes scapularis adult tick salivary glands (SGs). GW4869 treatment showed reduced dissemination of LGTV in SGs and other tissues within ticks. Decreased release of SG-EVs directly correlated with reduced tick blood-feeding efficiency, engorgement weights, and reduction in LGTV acquisition/transmission. Our data indicates that LGTV infection significantly improves molting/fitness, and survival efficiency of ticks and GW4869 alone affects the repletion rates of blood-feeding naïve-ticks. Overall, we provide evidence for GW4869 potential use as therapeutic agent in the tick control and prevention of tick-borne flaviviral transmission.
RESUMO
The invasive Asian longhorned tick Haemaphysalis longicornis that vectors and transmits several animal pathogens is significantly expanding in the United States. Recent studies report that these ticks also harbor human pathogens including Borrelia burgdorferi sensu lato, Babesia microti, and Anaplasma phagocytophilum. Therefore, studies that address the interactions of these ticks with human pathogens are important. In this study, we report the characterization of H. longicornis organic anion-transporting polypeptides (OATPs) in interactions of these ticks with A. phagocytophilum. Using OATP-signature sequence, we identified six OATPs in the H. longicornis genome. Bioinformatic analysis revealed that H. longicornis OATPs are closer to other tick orthologs rather than to mammalian counterparts. Quantitative real-time PCR analysis revealed that OATPs are highly expressed in immature stages when compared to mature stages of these ticks. In addition, we noted that the presence of A. phagocytophilum upregulates a specific OATP in these ticks. We also noted that exogenous treatment of H. longicornis with xanthurenic acid, a tryptophan metabolite, influenced OATP expression in these ticks. Immunoblotting analysis revealed that antibody generated against Ixodes scapularis OATP cross-reacted with H. longicornis OATP. Furthermore, treatment of H. longicornis with OATP antibody impaired colonization of A. phagocytophilum in these ticks. These results not only provide evidence that the OATP-tryptophan pathway is important for A. phagocytophilum survival in H. longicornis ticks but also indicate OATP as a promising candidate for the development of a universal anti-tick vaccine to target this bacterium and perhaps other rickettsial pathogens of medical importance.
Assuntos
Anaplasma phagocytophilum , Borrelia burgdorferi , Borrelia , Ixodes , Transportadores de Ânions Orgânicos , Animais , Humanos , Haemaphysalis longicornis , Anaplasma phagocytophilum/genética , Triptofano , Ixodes/microbiologia , Anticorpos/metabolismo , Transportadores de Ânions Orgânicos/genética , Borrelia burgdorferi/metabolismo , Mamíferos/metabolismoRESUMO
Several flaviviruses compromise the blood-brain barrier integrity, infect the central nervous system, and elicit neuroinvasion to successfully cause neuropathogenesis in the vertebrate host. Therefore, understanding the pathway(s) and mechanism(s) to block the transmission and/or dissemination of flaviviruses and perhaps other neuroinvasive viruses is considered as an important area of research. Moreover, studies that address mechanism(s) of neuroinvasion by flaviviruses are limited. In this chapter, we discuss detailed methods to isolate exosomes or extracellular vesicles (EVs) from mouse and human N2a cells, primary cultures of murine cortical neurons, and mouse brain tissue. Two different methods including differential ultracentrifugation and density gradient exosome (DG-Exo) isolation are described for the preparation of exosomes/EVs from N2a cells and cortical neurons. In addition, we discuss the detailed DG-Exo method for the isolation of exosomes from murine brain tissue. Studies on neuronal exosomes will perhaps enhance our understanding of the mechanism of neuroinvasion by these deadly viruses.
Assuntos
Exossomos , Vesículas Extracelulares , Vírus do Nilo Ocidental , Animais , Camundongos , Humanos , Neurônios , EncéfaloRESUMO
Anaplasma phagocytophilum modulates various cell signaling pathways in mammalian cells for its survival. In this study, we report that A. phagocytophilum modulates tick tryptophan pathway to activate arthropod p38 MAP kinase for the survival of both this bacterium and its vector host. Increased level of tryptophan metabolite, xanthurenic acid (XA), was evident in A. phagocytophilum-infected ticks and tick cells. Lower levels of cell death markers and increased levels of total and phosphorylated p38 MAPK was noted in A. phagocytophilum-infected ticks and tick cells. Treatment with XA increased phosphorylated p38 MAPK levels and reduced cell death in A. phagocytophilum-infected tick cells. Furthermore, treatment with p38 MAPK inhibitor affected bacterial replication, decreased phosphorylated p38 MAPK levels and increased tick cell death. However, XA reversed these effects. Taken together, we provide evidence that rickettsial pathogen modulates arthropod tryptophan and p38 MAPK pathways to inhibit cell death for its survival in ticks.
RESUMO
Human anaplasmosis caused by Anaplasma phagocytophilum is one of the most common tick-borne diseases in the United States. The black-legged ticks, Ixodes scapularis, vector and transmit this bacterium to humans. In this study, we provide evidence that targeting I. scapularis membrane-bound organic anion transporting polypeptide 4056 (IsOATP4056) with an anti-vector vaccine affects transmission of A. phagocytophilum from ticks to the vertebrate host. Anaplasma phagocytophilum induces expression of IsOATP4056 in ticks and tick cells. Increased membrane localization of IsOATP4056 was evident in A. phagocytophilum-infected tick cells. Treatment with high dose (10 µg/ml) but not low dose (5 µg/ml) of EL-6 antibody that targets the largest extracellular loop of IsOATP4056 showed cytotoxic effects in tick cells but not in human keratinocyte cell line (HaCaT). Passive immunization, tick-mediated transmission and in vitro studies performed with mice ordered from two commercial vendors and with tick cells showed that EL-6 antibody not only impairs A. phagocytophilum transmission from ticks to the murine host but also aids in the reduction in the bacterial loads within engorged ticks and in tick cells by activation of arthropod Toll pathway. Furthermore, reduced molting efficiency was noted in ticks fed on EL-6 antibody-immunized mice. Collectively, these results provide a good candidate for the development of anti-tick vaccine to target the transmission of A. phagocytophilum and perhaps other tick-borne pathogens of medical importance.
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Subolesin is a conserved molecule in both hard and soft ticks and is considered as an effective candidate molecule for the development of anti-tick vaccine. Previous studies have reported the role of subolesin in blood feeding, reproduction, development, and gene expression in hard ticks. However, studies addressing the role of subolesin in soft ticks are limited. In this study, we report that subolesin is not only important in soft tick Ornithodoros turicata americanus blood feeding but also in the regulation of innate immune gene expression in these ticks. We identified and characterized several putative innate immune genes including Toll, Lysozyme precursor (Lp), fibrinogen-domain containing protein (FDP), cystatin and ML-domain containing protein (MLD) in O. turicata americanus ticks. Quantitative real-time polymerase chain reaction analysis revealed the expression of these genes in both O. turicata americanus salivary glands and midgut and in all developmental stages of these soft ticks. Significantly increased expression of fdp was noted in salivary glands and midgut upon O. turicata americanus blood feeding. Furthermore, RNAi-mediated knockdown of O. turicata americanus subolesin expression affected blood feeding and innate immune gene expression in these ticks. Significant downregulation of toll, lp, fdp, cystatin, and mld transcripts was evident in sub-dsRNA-treated ticks when compared to the levels noted in mock-dsRNA-treated control. Collectively, our study not only reports identification and characterization of various innate immune genes in O. turicata americanus ticks but also provides evidence on the role of subolesin in blood feeding and innate immune gene expression in these medically important ticks.
Assuntos
Argasidae , Cistatinas , Ornithodoros , Vacinas , Animais , Ornithodoros/genética , Vacinas/genética , Expressão Gênica , Cistatinas/genética , Imunidade InataRESUMO
Tick microbiota can be targeted for the control of tick-borne diseases such as human granulocytic anaplasmosis (HGA) caused by model pathogen, Anaplasma phagocytophilum. Frankenbacteriosis is inspired by Frankenstein and defined here as paratransgenesis of tick symbiotic/commensal bacteria to mimic and compete with tick-borne pathogens. Interactions between A. phagocytophilum and symbiotic Sphingomonas identified by metaproteomics analysis in Ixodes scapularis midgut showed competition between both bacteria. Consequently, Sphingomonas was selected for frankenbacteriosis for the control of A. phagocytophilum infection and transmission. The results showed that Franken Sphingomonas producing A. phagocytophilum major surface protein 4 (MSP4) mimic pathogen and reduce infection in ticks by competition and interaction with cell receptor components of infection. Franken Sphingomonas-MSP4 transovarial and trans-stadial transmission suggests that tick larvae with genetically modified Franken Sphingomonas-MSP4 could be produced in the laboratory and released in the field to compete and replace the wildtype populations with associated reduction in pathogen infection/transmission and HGA disease risks.
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BACKGROUND: Cell adhesion, an integral part of D. discoideum development, is important for morphogenesis and regulated gene expression in the multicellular context and is required to trigger cell-differentiation. G-protein linked adenylyl cyclase pathways are crucially involved and a mutant lacking the aggregation specific adenylyl cyclase ACA does not undergo multicellular development. RESULTS: Here, we have investigated the role of cyclase-associated protein (CAP), an important regulator of cell polarity and F-actin/G-actin ratio in the aca- mutant. We show that ectopic expression of GFP-CAP improves cell polarization, streaming and aggregation in aca- cells, but it fails to completely restore development. Our studies indicate a requirement of CAP in the ACA dependent signal transduction for progression of the development of unicellular amoebae into multicellular structures. The reduced expression of the cell adhesion molecule DdCAD1 together with csA is responsible for the defects in aca- cells to initiate multicellular development. Early development was restored by the expression of GFP-CAP that enhanced the DdCAD1 transcript levels and to a lesser extent the csA mRNA levels. CONCLUSIONS: Collectively, our data shows a novel role of CAP in regulating cell adhesion mechanisms during development that might be envisioned to unravel the functions of mammalian CAP during animal embryogenesis.
Assuntos
Adenilil Ciclases/deficiência , Proteínas do Citoesqueleto/biossíntese , Dictyostelium/metabolismo , Proteínas de Protozoários/biossíntese , Adesão Celular , Polaridade Celular , Proteínas do Citoesqueleto/genética , Proteínas do Citoesqueleto/metabolismo , Dictyostelium/citologia , Dictyostelium/enzimologia , Proteínas de Fluorescência Verde/biossíntese , Estrutura Terciária de Proteína , Transporte Proteico , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo , Proteínas Recombinantes de Fusão/biossíntese , Transcrição GênicaRESUMO
BACKGROUND: The maintenance of Borrelia burgdorferi in its complex tick-mammalian enzootic life cycle is dependent on the organism's adaptation to its diverse niches. To this end, the RpoN-RpoS regulatory pathway in B. burgdorferi plays a central role in microbial survival and Lyme disease pathogenesis by up- or down-regulating the expression of a number of virulence-associated outer membrane lipoproteins in response to key environmental stimuli. Whereas a number of studies have reported on the expression of RpoS and its target genes, a more comprehensive understanding of when activation of the RpoN-RpoS pathway occurs, and when induction of the pathway is most relevant to specific stage(s) in the life cycle of B. burgdorferi, has been lacking. RESULTS: Herein, we examined the expression of rpoS and key lipoprotein genes regulated by RpoS, including ospC, ospA, and dbpA, throughout the entire tick-mammal infectious cycle of B. burgdorferi. Our data revealed that transcription of rpoS, ospC, and dbpA is highly induced in nymphal ticks when taking a blood meal. The RpoN-RpoS pathway remains active during the mammalian infection phase, as indicated by the sustained transcription of rpoS and dbpA in B. burgdorferi within mouse tissues following borrelial dissemination. However, dbpA transcription levels in fed larvae and intermolt larvae suggested that an additional layer of control likely is involved in the expression of the dbpBA operon. Our results also provide further evidence for the downregulation of ospA expression during mammalian infection, and the repression of ospC at later phases of mammalian infection by B. burgdorferi. CONCLUSION: Our study demonstrates that the RpoN-RpoS regulatory pathway is initially activated during the tick transmission of B. burgdorferi to its mammalian host, and is sustained during mammalian infection.
Assuntos
Proteínas de Bactérias , Borrelia burgdorferi/fisiologia , Regulação Bacteriana da Expressão Gênica , Doença de Lyme/microbiologia , RNA Polimerase Sigma 54 , Fator sigma , Animais , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Borrelia burgdorferi/genética , Borrelia burgdorferi/metabolismo , Perfilação da Expressão Gênica , Interações Hospedeiro-Parasita , Lipoproteínas/genética , Camundongos , Ninfa/microbiologia , RNA Polimerase Sigma 54/genética , RNA Polimerase Sigma 54/metabolismo , Fator sigma/genética , Fator sigma/metabolismo , Carrapatos/microbiologiaRESUMO
In the enterobacterial species Escherichia coli and Salmonella enterica, expression of horizontally acquired genes with a higher than average AT content is repressed by the nucleoid-associated protein H-NS. A classical example of an H-NS-repressed locus is the bgl (aryl-beta,D-glucoside) operon of E. coli. This locus is "cryptic," as no laboratory growth conditions are known to relieve repression of bgl by H-NS in E. coli K12. However, repression can be relieved by spontaneous mutations. Here, we investigated the phylogeny of the bgl operon. Typing of bgl in a representative collection of E. coli demonstrated that it evolved clonally and that it is present in strains of the phylogenetic groups A, B1, and B2, while it is presumably replaced by a cluster of ORFans in the phylogenetic group D. Interestingly, the bgl operon is mutated in 20% of the strains of phylogenetic groups A and B1, suggesting erosion of bgl in these groups. However, bgl is functional in almost all B2 isolates and, in approximately 50% of them, it is weakly expressed at laboratory growth conditions. Homologs of bgl genes exist in Klebsiella, Enterobacter, and Erwinia species and also in low GC-content Gram-positive bacteria, while absent in E. albertii and Salmonella sp. This suggests horizontal transfer of bgl genes to an ancestral Enterobacterium. Conservation and weak expression of bgl in isolates of phylogenetic group B2 may indicate a functional role of bgl in extraintestinal pathogenic E. coli.
Assuntos
Proteínas de Bactérias/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/genética , Evolução Molecular , Óperon , Proteínas de Bactérias/genética , Proteínas de Ligação a DNA/genética , Regulação para Baixo , Enterobacteriaceae/classificação , Enterobacteriaceae/genética , Escherichia coli/classificação , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Transferência Genética Horizontal , FilogeniaRESUMO
Aim: Targeting the modes of pathogen shedding/transmission via exosomes or extracellular vesicles has been envisioned as the best approach to control vector-borne diseases. This study is focused on altering exosomes stability to affect the pathogen transmission from infected to naïve recipient cells. Methods: In this study, neuronal or arthropod exosomes were treated at different temperatures or with different salts or pH conditions to analyze their ability and efficiency in the transmission of tick-borne Langat virus (LGTV) from infected to naïve recipient cells. Results: Quantitative real-time PCR (qRT-PCR) and immunoblotting analyses revealed that treatment of neuronal or tick exosomes at warmer temperatures of 37 °C or 23 °C, respectively, or with sulfate salts such as Magnesium or Ammonium sulfates or with highly alkaline pH of 9 or 11.5, dramatically reduced transmission of LGTV via infectious exosomes (human or tick cells-derived) to human neuronal (SH-SY5Y) cells or skin keratinocytes (HaCaT cells), respectively. Conclusion: Overall, this study suggests that exosome-mediated viral transmission of vector-borne pathogens to the vertebrate host or the viral dissemination and replication within or between the mammalian host can be reduced by altering the ability of exosomes with basic changes in temperatures, salts or pH conditions.
RESUMO
Bacterial endosymbionts are abundantly found in both hard and soft ticks. Occidentia massiliensis, a rickettsial endosymbiont, was first identified in the soft tick Ornithodoros sonrai collected from Senegal and later was identified in a hard tick Africaniella transversale. In this study, we noted the presence of Occidentia species, designated as Occidentia-like species, in a soft tick O. turicata americanus. Sequencing and phylogenetic analyses of the two genetic markers, 16S rRNA and groEL confirmed the presence of Occidentia-like species in O. turicata americanus ticks. The Occidentia-like species was noted to be present in all developmental stages of O. turicata americanus and in different tick tissues including ovaries, synganglion, guts and salivary gland. The levels of Occidentia-like species 16S rRNA transcripts were noted to be significantly higher in ovaries than in a gut tissue. In addition, Occidentia-like species groEL expression was noted to be significantly higher in tick synganglion than in ovaries and gut tissues. Furthermore, levels of Occidentia-like species 16S rRNA transcripts increased significantly upon O. turicata americanus blood feeding. Taken together, our study not only shows that Occidentia-like species is present in O. turicata americanus but also suggests that this bacterium may play a role in tick-bacteria interactions.
Assuntos
Argasidae , Ornithodoros , Animais , Argasidae/genética , Ornithodoros/genética , RNA Ribossômico 16S/genética , Filogenia , SenegalRESUMO
Ixodes scapularis ticks acquire several pathogens from reservoir animals and transmit them to humans. Development of an animal model to study acquisition/transmission dynamics of these pathogens into and from ticks, respectively, is challenging due to the fact that in nature ticks feed for a longer duration and on multiple vertebrate hosts. To understand the complex nature of pathogen acquisition/transmission, it is essential to set up a successful tick blood feeding method on a suitable vertebrate host. In this study, we provide evidence that murine model can be successfully used to study acquisition dynamics of Langat virus (LGTV), a member of tick-borne flaviviruses. Mice were inoculated intraperitoneally with LGTV that showed detectable viral loads in blood, skin, and other tissues including the brain. Both larval and nymphal ticks that were allowed to feed on the murine host successfully acquired LGTV loads. Also, we found that after molting, LGTV was transstadially transmitted from larval to nymphal stage. In addition, we noted that LGTV down-regulated IsSMase expression in all groups of ticks possibly for its survival in its vector host. Taken together, we provide evidence for the use of murine model to not only study acquisition dynamics of LGTV but also to study changes in tick gene expression during acquisition of arboviruses into ticks.