RESUMO
19ISP is a nucleoside-modified mRNA-lipid nanoparticle vaccine that targets 19 Ixodes scapularis proteins. We demonstrate that adult I. scapularis have impaired fecundity when allowed to engorge on 19ISP-immunized rabbits. 19ISP, therefore, has the potential to interrupt the tick reproductive cycle, without triggering some of the other effects associated with acquired tick resistance. This may lead to the development of new strategies to reduce I. scapularis populations in endemic areas.
Assuntos
Ixodes , Animais , Coelhos , Ixodes/genética , RNA Mensageiro/genética , Vacinação , FertilidadeRESUMO
Guinea pigs repeatedly exposed to Ixodes scapularis develop acquired resistance to the ticks (ATR). The molecular mechanisms of ATR have not been fully elucidated, and partially involves immune responses to proteins in tick saliva. In this study, we examined the metabolome of sera of guinea pigs during the development of ATR. Induction of components of the tyrosine metabolic pathway, including hydroxyphenyllactic acid (HPLA), were associated with ATR. We therefore administered HPLA to mice, an animal that does not develop ATR, and exposed the animals to I. scapularis. We also administered nitisinone, a known inhibitor of tyrosine degradation, to another group of mice. The mortality of I. scapularis that fed on mice given HPLA or nitisinone was 26 % and 72 % respectively, compared with 2 % mortality among ticks that fed on control animals. These data indicate that tick bites alter the guinea pig metabolome, and that the tyrosine metabolism pathway can potentially be targeted for I. scapularis control.
Assuntos
Ixodes , Animais , Camundongos , Cobaias , Ixodes/fisiologia , Saliva , TirosinaRESUMO
Guinea pigs repeatedly exposed to Ixodes scapularis develop acquired resistance to the ticks (ATR). The molecular mechanisms of ATR have not been fully elucidated, and partially involve immune responses to proteins in tick saliva. In this study, we examined the metabolome of sera of guinea pigs during the development of ATR. Induction of components of the tyrosine metabolic pathway, including hydroxyphenyllactic acid (HPLA), were associated with ATR. We therefore administered HPLA to mice, an animal that does not develop ATR, and exposed the animals to I. scapularis . We also administered nitisinone, a known inhibitor of tyrosine degradation, to another group of mice. The mortality of I. scapularis that fed on mice given HPLA or nitisinone was 26% and 72% respectively, compared with 2% mortality among ticks that fed on control animals. These data indicate that metabolic changes that occur after tick bites contribute to ATR.
RESUMO
Acquired resistance to ticks can develop when animals are repeatedly exposed to ticks. Recently, acquired resistance to Ixodes scapularis was induced in guinea pigs immunized with an mRNA-lipid nanoparticle vaccine (19ISP) encoding 19 I. scapularis proteins. Here, we evaluated specific mRNAs present in 19ISP to identify critical components associated with resistance to ticks. A lipid nanoparticle containing 12 mRNAs which included all the targets within 19ISP that elicited strong humoral responses in guinea pigs, was sufficient to induce robust resistance to ticks. Lipid nanoparticles containing fewer mRNAs or a single mRNA were not able to generate strong resistance to ticks. All lipid nanoparticles containing salp14 mRNA, however, were associated with increased redness at the tick bite site - which is the first manifestation of acquired resistance to ticks. This study demonstrates that more than one I. scapularis target within 19ISP is required for resistance to ticks, and that additional targets may also play a role in this process.
Assuntos
Ixodes , Doença de Lyme , Animais , Cobaias , RNA Mensageiro , Ixodes/genéticaRESUMO
BACKGROUND: Ixodes scapularis is the predominant tick vector of Borrelia burgdorferi, the agent of Lyme disease, in the USA. Molecular interactions between the tick and B. burgdorferi orchestrate the migration of spirochetes from the midgut to the salivary glands-critical steps that precede transmission to the vertebrate host. Over the last decade, research efforts have invoked a potential role for the tick microbiome in modulating tick-pathogen interactions. RESULTS: Using multiple strategies to perturb the microbiome composition of B. burgdorferi-infected nymphal ticks, we observe that changes in the microbiome composition do not significantly influence B. burgdorferi migration from the midgut, invasion of salivary glands, or transmission to the murine host. We also show that within 24 and 48 h of the onset of tick feeding, B. burgdorferi spirochetes are within the peritrophic matrix and epithelial cells of the midgut in preparation for exit from the midgut. CONCLUSIONS: This study highlights two aspects of tick-spirochete interactions: (1) environmental bacteria associated with the tick do not influence spirochete transmission to the mammalian host and (2) the spirochete may utilize an intracellular exit route during migration from the midgut to the salivary glands, a strategy that may allow the spirochete to distance itself from microbiota in the midgut lumen effectively. This may explain in part, the inability of environment-acquired midgut microbiota to significantly influence spirochete transmission. Unraveling a molecular understanding of this exit strategy will be critical to gain new insights into the biology of the spirochete and the tick. Video Abstract.
Assuntos
Borrelia burgdorferi , Ixodes , Doença de Lyme , Microbiota , Animais , Borrelia burgdorferi/genética , Ixodes/microbiologia , Doença de Lyme/microbiologia , Mamíferos , Camundongos , Ninfa/microbiologiaRESUMO
As hematophagous parasites, many tick species are important vectors of medical and veterinary disease agents. Proteins found in tick saliva and midgut have been used with some success in immunizations of animal hosts against feeding ticks, and whole saliva has been used effectively in this capacity against Ixodes scapularis, the primary vector of tickborne pathogens in the United States. Tick saliva is a complex substance containing hundreds of proteins, and the identification of specific protective antigens is ongoing. We performed a series of experiments immunizing guinea pigs with extracts prepared from midgut or attachment cement collected from adult female I. scapularis followed by challenge with nymphs of the same species. Midgut extract did not induce protective immunity, while immunization with cement extract resulted in partial protection of hosts as evidenced by premature tick detachment and 34-41% reduction in tick engorgement weights. Proteomic characterization of I. scapularis cement was performed, demonstrating that the cement extract was compositionally different from tick saliva, and vitellogenin-like lipoproteins were the most abundant proteins in cement extract (>40%). Cement was also heavily enriched with lysozymes and defensins, including those originating from both the mammalian host as well as ticks. These results demonstrate that I. scapularis cement contains immunogenic components capable of stimulating host resistance against tick feeding. Because the cement is present at the tick-host interface for an extended period of time during the feeding process, these antigens present auspicious candidates for further evaluation and potential inclusion in an anti-tick vaccine.
RESUMO
Malaria is caused when Plasmodium sporozoites are injected along with saliva by an anopheline mosquito into the dermis of a vertebrate host. Arthropod saliva has pleiotropic effects that can influence local host responses, pathogen transmission, and exacerbation of the disease. A mass spectrometry screen identified mosquito salivary proteins that are associated with Plasmodium sporozoites during saliva secretions. In this study, we demonstrate that one of these salivary antigens, Anopheles gambiae sporozoite-associated protein (AgSAP), interacts directly with Plasmodium falciparum and Plasmodium berghei sporozoites. AgSAP binds to heparan sulfate and inhibits local inflammatory responses in the skin. The silencing of AgSAP in mosquitoes reduces their ability to effectively transmit sporozoites to mice. Moreover, immunization with AgSAP decreases the Plasmodium burden in mice that are bitten by Plasmodium-infected mosquitoes. These data suggest that AgSAP facilitates early Plasmodium infection in the vertebrate host and serves as a target for the prevention of malaria. IMPORTANCE Malaria is a vector-borne disease caused by Plasmodium sporozoites. When an anopheline mosquito bites its host, it releases Plasmodium sporozoites as well as saliva components. Mosquito proteins have the potential to serve as antigens to prevent or influence malaria without directly targeting the pathogen. This may help set a new paradigm for vaccine development. In this study, we have elucidated the role of a novel salivary antigen, named Anopheles gambiae sporozoite-associated protein (AgSAP). The results presented here show that AgSAP interacts with Plasmodium falciparum and Plasmodium berghei sporozoites and modulates local inflammatory responses in the skin. Furthermore, our results show that AgSAP is a novel mosquito salivary antigen that influences the early stages of Plasmodium infection in the vertebrate host. Individuals living in countries where malaria is endemic generate antibodies against AgSAP, which indicates that AgSAP can serve as a biomarker for disease prevalence and epidemiological analysis.
Assuntos
Anopheles/imunologia , Proteínas de Insetos/imunologia , Malária/parasitologia , Mosquitos Vetores/imunologia , Plasmodium berghei/fisiologia , Plasmodium falciparum/fisiologia , Proteínas e Peptídeos Salivares/imunologia , Animais , Anopheles/genética , Anopheles/parasitologia , Feminino , Humanos , Proteínas de Insetos/genética , Malária/imunologia , Malária/transmissão , Camundongos , Camundongos Endogâmicos C57BL , Mosquitos Vetores/genética , Mosquitos Vetores/parasitologia , Plasmodium berghei/genética , Plasmodium falciparum/genética , Proteínas e Peptídeos Salivares/genética , Esporozoítos/genética , Esporozoítos/fisiologiaRESUMO
Guinea pigs exposed to multiple infestations with Ixodes scapularis ticks develop acquired resistance to ticks, which is also known as tick immunity. The I. scapularis salivary components that contribute to tick immunity are likely multifactorial. An anticoagulant that inhibits factor Xa, named Salp14, is present in tick saliva and is associated with partial tick immunity. A tick bite naturally releases tick saliva proteins into the vertebrate host for several days, which suggests that the mode of antigen delivery may influence the genesis of tick immunity. We therefore utilized Salp14 as a model antigen to examine tick immunity using mRNA lipid nanoparticles (LNPs), plasmid DNA, or recombinant protein platforms. salp14 containing mRNA-LNPs vaccination elicited erythema at the tick bite site after tick challenge that occurred earlier, and that was more pronounced, compared with DNA or protein immunizations. Humoral and cellular responses associated with tick immunity were directed towards a 25 amino acid region of Salp14 at the carboxy terminus of the protein, as determined by antibody responses and skin-testing assays. This study demonstrates that the model of antigen delivery, also known as the vaccine platform, can influence the genesis of tick immunity in guinea pigs. mRNA-LNPs may be useful in helping to elicit erythema at the tick bite site, one of the most important early hallmarks of acquired tick resistance. mRNA-LNPs containing tick genes is a useful platform for the development of vaccines that can potentially prevent selected tick-borne diseases.
Assuntos
Ixodes , Proteínas e Peptídeos Salivares/imunologia , Vacinas/imunologia , Animais , DNA , Cobaias , Lipossomos , Nanopartículas , RNA Mensageiro , Proteínas e Peptídeos Salivares/administração & dosagemRESUMO
Ixodes scapularis ticks transmit many pathogens that cause human disease, including Borrelia burgdorferi. Acquired resistance to I. scapularis due to repeated tick exposure has the potential to prevent tick-borne infectious diseases, and salivary proteins have been postulated to contribute to this process. We examined the ability of lipid nanoparticlecontaining nucleoside-modified mRNAs encoding 19 I. scapularis salivary proteins (19ISP) to enhance the recognition of a tick bite and diminish I. scapularis engorgement on a host and thereby prevent B. burgdorferi infection. Guinea pigs were immunized with a 19ISP mRNA vaccine and subsequently challenged with I. scapularis. Animals administered 19ISP developed erythema at the bite site shortly after ticks began to attach, and these ticks fed poorly, marked by early detachment and decreased engorgement weights. 19ISP immunization also impeded B. burgdorferi transmission in the guinea pigs. The effective induction of local redness early after I. scapularis attachment and the inability of the ticks to take a normal blood meal suggest that 19ISP may be used either alone or in conjunction with traditional pathogen-based vaccines for the prevention of Lyme disease, and potentially other tick-borne infections.
Assuntos
Ixodes , Doença de Lyme , Animais , Cobaias , Lipossomos , Doença de Lyme/metabolismo , Doença de Lyme/prevenção & controle , Nanopartículas , RNA Mensageiro , Vacinação , Vacinas Sintéticas , Vacinas de mRNARESUMO
In many regions where ticks negatively impact public health or economic production, multiple medically important tick species may have overlapping geographic distribution, and in North America, this includes members of Ixodes, Dermacentor, and Amblyomma genera. Acquired tick resistance is the process by which some animals develop an immune response against feeding ticks after one or more exposures. This form of immunity can restrict the ability of ticks to feed and may inhibit transmission of pathogens. Likewise, many proteins present in tick saliva are conserved among tick species, and prior studies have reported cross-protective host immunity against certain combinations of ticks. In this study, we used a guinea pig model to assess whether host resistance against Ixodes scapularis could confer protection against two other medically important tick vectors, Dermacentor variabilis and Amblyomma americanum. Tick challenges using nymphs were used to induce host resistance against a primary species, followed by additional challenge using a secondary tick species. Tick attachment to hosts and engorgement weights were reduced significantly for D. variabilis and A. americanum feeding on I. scapularis-sensitized hosts. Reciprocally, I. scapularis engorgement weights were reduced to a lesser extent, and attachment was unaffected when feeding on hosts sensitized with either D. variabilis or A. americanum. These results indicate that immunity against I. scapularis could potentially be exploited for use in an anti-tick vaccine targeting multiple tick species and their associated pathogens.
Assuntos
Vetores Aracnídeos/imunologia , Suscetibilidade a Doenças/imunologia , Cobaias , Ixodes/imunologia , Doenças dos Roedores/parasitologia , Infestações por Carrapato/veterinária , Animais , Ciência dos Animais de Laboratório , Doenças dos Roedores/imunologia , Infestações por Carrapato/imunologiaRESUMO
Tick-borne diseases pose a global medical problem. As transmission of tick-borne pathogens to their hosts occurs during tick feeding, development of vaccines thwarting this process could potentially prevent transmission of multiple tick-borne pathogens. The idea of tick vaccines is based on the phenomenon of acquired tick immunity, rejection of ticks feeding on hosts which were repeatedly infested by ticks. Recently, we demonstrated that saliva of the blacklegged tick Ixodes scapularis, which is the main vector of tick-borne pathogens in northeast USA, is sufficient for induction of tick immunity in the guinea pig model and that immunity directed against tick glycoproteins is important in this phenomenon. Nevertheless, immunity elicited against individual tick salivary antigens, which have been identified and tested so far, provided only modest tick rejection. We therefore now tested fractions of tick saliva produced by liquid chromatography for their ability to induce tick immunity in the guinea pig model. Immunization with all individual fractions elicited antibodies that reacted with tick saliva, however only some fractions displayed the ability to induce robust protective tick immunity. Mass spectrometry analysis led to identification of 24 proteins present only in saliva fractions which were able to induce tick immunity, suggesting suitable candidates for development of a tick vaccine.
Assuntos
Ixodes , Animais , Cromatografia Líquida , Glicoproteínas , Cobaias , SalivaRESUMO
Ixodes scapularis vectors several pathogens including Borrelia burgdorferi, the agent of Lyme disease. Nymphal and larval stages, and the pathogens transmitted by I. scapularis are maintained in a zoonotic cycle involving rodent reservoir hosts, predominantly Peromyscus leucopus. Humans are not reservoir hosts, however, accidental encounters of infected ticks with humans, results in pathogen transmission to the human host. Laboratory models of non-reservoir hosts such as guinea pigs develop a strong immune response to tick salivary proteins and reject ticks upon repeated tick infestations. Anecdotal and scientific evidence suggests that humans that get frequent tick bites might also develop resistance to ticks. Mus musculus, the laboratory model of natural host, does not develop resistance to I. scapularis upon repeated tick infestations. Addressing this dichotomy in vector-host interaction, we present data that suggest that the salivary transcriptome and proteome composition is different in mouse and guinea pig-fed I. scapularis, and that these differences might contribute to differences in host immune responses. These findings reveal a new insight into vector-host interactions and offer a functional paradigm to better understand the phenomenon of acquired tick-resistance.
Assuntos
Expressão Gênica , Especificidade de Hospedeiro , Ixodes/genética , Proteoma , Proteínas e Peptídeos Salivares/genética , Animais , Borrelia burgdorferi , Reservatórios de Doenças/microbiologia , Feminino , Cobaias , Camundongos , Camundongos Endogâmicos C3H , Camundongos Endogâmicos C57BL , Ninfa/fisiologia , Saliva/química , Glândulas Salivares , Infestações por CarrapatoRESUMO
The insect immune deficiency (IMD) pathway resembles the tumour necrosis factor receptor network in mammals and senses diaminopimelic-type peptidoglycans present in Gram-negative bacteria. Whether unidentified chemical moieties activate the IMD signalling cascade remains unknown. Here, we show that infection-derived lipids 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol (POPG) and 1-palmitoyl-2-oleoyl diacylglycerol (PODAG) stimulate the IMD pathway of ticks. The tick IMD network protects against colonization by three distinct bacteria, that is the Lyme disease spirochete Borrelia burgdorferi and the rickettsial agents Anaplasma phagocytophilum and A. marginale. Cell signalling ensues in the absence of transmembrane peptidoglycan recognition proteins and the adaptor molecules Fas-associated protein with a death domain (FADD) and IMD. Conversely, biochemical interactions occur between x-linked inhibitor of apoptosis protein (XIAP), an E3 ubiquitin ligase, and the E2 conjugating enzyme Bendless. We propose the existence of two functionally distinct IMD networks, one in insects and another in ticks.
Assuntos
Artrópodes/imunologia , Síndromes de Imunodeficiência/imunologia , Síndromes de Imunodeficiência/veterinária , Ixodes/imunologia , Lipídeos/efeitos adversos , Lipídeos/imunologia , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Anaplasma marginale/imunologia , Anaplasma marginale/patogenicidade , Anaplasma phagocytophilum/imunologia , Anaplasma phagocytophilum/patogenicidade , Animais , Artrópodes/metabolismo , Borrelia burgdorferi/imunologia , Borrelia burgdorferi/patogenicidade , Proteínas de Transporte , Modelos Animais de Doenças , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Escherichia coli/genética , Proteína de Domínio de Morte Associada a Fas , Inativação Gênica , Células HEK293 , Humanos , Ixodes/metabolismo , Doença de Lyme/imunologia , Fosfatidilgliceróis/imunologia , RNA Interferente Pequeno/metabolismo , Proteínas Recombinantes , Transdução de Sinais , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Enzimas de Conjugação de Ubiquitina/genética , Enzimas de Conjugação de Ubiquitina/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Proteínas Inibidoras de Apoptose Ligadas ao Cromossomo X/metabolismoRESUMO
Borrelia miyamotoi is a relapsing fever spirochete in Ixodes ticks that has been recently identified as a human pathogen causing hard tick-borne relapsing fever (HTBRF) across the Northern Hemisphere. No validated serologic test exists, and current serologic assays have low sensitivity in early HTBRF. To examine the humoral immune response against B. miyamotoi, we infected C3H/HeN mice with B. miyamotoi strain LB-2001 expressing variable small protein 1 (Vsp1) and demonstrated that spirochetemia was cleared after 3 d, coinciding with anti-Vsp1 IgM production. Clearance was also observed after passive transfer of immune sera to infected SCID mice. Next, we showed that anti-Vsp1 IgG eliminates Vsp1-expressing B. miyamotoi, selecting for spirochetes expressing a variable large protein (VlpC2) resistant to anti-Vsp1. The viability of Asian isolate B. miyamotoi HT31, expressing Vlp15/16 and Vlp18, was also unaffected by anti-Vsp1. Finally, in nine HTBRF patients, we demonstrated IgM reactivity to Vsp1 in two and against Vlp15/16 in four â¼1 wk after these patients tested positive for B. miyamotoi by PCR. Our data show that B. miyamotoi is able to express various variable major proteins (VMPs) to evade humoral immunity and that VMPs are antigenic in humans. We propose that serologic tests based on VMPs are of additional value in diagnosing HTBRF.
Assuntos
Anticorpos Antibacterianos/imunologia , Formação de Anticorpos , Proteínas da Membrana Bacteriana Externa/imunologia , Lipoproteínas/imunologia , Febre Recorrente/imunologia , Sequência de Aminoácidos , Animais , Anticorpos Antibacterianos/sangue , Sequência de Bases , Borrelia/imunologia , Feminino , Humanos , Imunização Passiva , Imunoglobulina G/sangue , Imunoglobulina G/imunologia , Imunoglobulina M/sangue , Imunoglobulina M/imunologia , Camundongos , Camundongos Endogâmicos C3H , Camundongos SCID , Estrutura Terciária de ProteínaRESUMO
Arthopods such as Ixodes scapularis ticks serve as vectors for many human pathogens. The arthropod gut presents a pivotal microbial entry point and determines pathogen colonization and survival. We show that the gut microbiota of I. scapularis, a major vector of the Lyme disease spirochete Borrelia burgdorferi, influence spirochete colonization of ticks. Perturbing the gut microbiota of larval ticks reduced Borrelia colonization, and dysbiosed larvae displayed decreased expression of the transcription factor signal transducer and activator of transcription (STAT). Diminished STAT expression corresponded to lower expression of peritrophin, a key glycoprotein scaffold of the glycan-rich mucus-like peritrophic matrix (PM) that separates the gut lumen from the epithelium. The integrity of the I. scapularis PM was essential for B. burgdorferi to efficiently colonize the gut epithelium. These data elucidate a functional link between the gut microbiota, STAT-signaling, and pathogen colonization in the context of the gut epithelial barrier of an arthropod vector.
Assuntos
Vetores Aracnídeos/microbiologia , Borrelia burgdorferi/crescimento & desenvolvimento , Proteínas de Transporte/genética , Mucosa Intestinal/microbiologia , Ixodes/microbiologia , Larva/microbiologia , Fatores de Transcrição STAT/genética , Animais , Borrelia burgdorferi/patogenicidade , Proteínas de Transporte/antagonistas & inibidores , Proteínas de Transporte/metabolismo , Disbiose/microbiologia , Regulação da Expressão Gênica , Interações Hospedeiro-Patógeno , Humanos , Microbiota/genética , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Fatores de Transcrição STAT/metabolismo , Transdução de SinaisRESUMO
Ixodes scapularis, the black-legged tick, vectors several human pathogens including Borrelia burgdorferi, the agent of Lyme disease in North America. Pathogen transmission to the vertebrate host occurs when infected ticks feed on the mammalian host to obtain a blood meal. Efforts to understand how the tick confronts host hemostatic mechanisms and imbibes a fluid blood meal have largely focused on the anticoagulation strategies of tick saliva. The blood meal that enters the tick gut remains in a fluid state for several days during the process of feeding, and the role of the tick gut in maintaining the blood-meal fluid is not understood. We now demonstrate that the tick gut produces a potent inhibitor of thrombin, a key enzyme in the mammalian coagulation cascade. Chromatographic fractionation of engorged tick gut proteins identified one predominant thrombin inhibitory activity associated with an approximately 18 kDa protein, henceforth referred to as Ixophilin. The ixophilin gene was preferentially transcribed in the guts of feeding nymphs. Expression began after 24 hours of feeding, coincident with the flow of host blood into the tick gut. Immunity against Ixophilin delayed tick feeding, and decreased feeding efficiency significantly. Surprisingly, immunity against Ixophilin resulted in increased Borrelia burgdorferi transmission to the host, possibly due to delayed feeding and increased transmission opportunity. These observations illuminate the potential drawbacks of targeting individual tick proteins in a functional suite. They also underscore the need to identify the "anticoagulome" of the tick gut, and to prioritize a critical subset of anticoagulants that could be targeted to efficiently thwart tick feeding, and block pathogen transmission to the vertebrate host.
Assuntos
Proteínas de Artrópodes/farmacologia , Trato Gastrointestinal/química , Ixodes/química , Trombina/antagonistas & inibidores , Sequência de Aminoácidos , Animais , Proteínas de Artrópodes/química , Proteínas de Artrópodes/genética , Proteínas de Artrópodes/imunologia , Proteínas de Artrópodes/isolamento & purificação , Feminino , Expressão Gênica , Humanos , Ixodes/genética , Camundongos , Dados de Sequência Molecular , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/farmacologia , Alinhamento de SequênciaRESUMO
BACKGROUND: Generation of active procoagulant cofactor factor Va (FVa) and its subsequent association with the enzyme activated factor X (FXa) to form the prothrombinase complex is a pivotal initial event in blood coagulation and has been the subject of investigative effort, speculation, and controversy. The current paradigm assumes that FV activation is initiated by limited proteolysis by traces of (meizo) thrombin. METHODS AND RESULTS: Recombinant tick salivary protein TIX-5 was produced and anticoagulant properties were studied with the use of plasma, whole blood, and purified systems. Here, we report that TIX-5 specifically inhibits FXa-mediated FV activation involving the B domain of FV and show that FXa activation of FV is pivotal for plasma and blood clotting. Accordingly, tick feeding is impaired on TIX-5 immune rabbits, displaying the in vivo importance of TIX-5. CONCLUSIONS: Our data elucidate a unique molecular mechanism by which ticks inhibit the host's coagulation system. From our data, we propose a revised blood coagulation scheme in which direct FXa-mediated FV activation occurs in the initiation phase during which thrombin-mediated FV activation is restrained by fibrinogen and inhibitors.
Assuntos
Anticoagulantes/farmacologia , Proteínas de Artrópodes/farmacologia , Coagulação Sanguínea/efeitos dos fármacos , Fator V/metabolismo , Fator Xa/metabolismo , Proteínas e Peptídeos Salivares/farmacologia , Animais , Anticoagulantes/sangue , Anticoagulantes/química , Proteínas de Artrópodes/química , Proteínas de Artrópodes/genética , Coagulação Sanguínea/fisiologia , Testes de Coagulação Sanguínea , Relação Dose-Resposta a Droga , Fator V/antagonistas & inibidores , Inibidores do Fator Xa , Comportamento Alimentar , Fibrinogênio/metabolismo , Humanos , Ixodes/química , Ixodes/genética , Ixodes/fisiologia , Mutagênese , Estrutura Terciária de Proteína , Coelhos , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/farmacologia , Proteínas e Peptídeos Salivares/química , Proteínas e Peptídeos Salivares/genética , Ressonância de Plasmônio de Superfície , Trombina/metabolismoRESUMO
The Lyme disease agent Borrelia burgdorferi is primarily transmitted to vertebrates by Ixodes ticks. The classical and alternative complement pathways are important in Borrelia eradication by the vertebrate host. We recently identified a tick salivary protein, designated P8, which reduced complement-mediated killing of Borrelia. We now discover that P8 interferes with the human lectin complement cascade, resulting in impaired neutrophil phagocytosis and chemotaxis and diminished Borrelia lysis. Therefore, P8 was renamed the tick salivary lectin pathway inhibitor (TSLPI). TSLPI-silenced ticks, or ticks exposed to TSLPI-immune mice, were hampered in Borrelia transmission. Moreover, Borrelia acquisition and persistence in tick midguts was impaired in ticks feeding on TSLPI-immunized, B. burgdorferi-infected mice. Together, our findings suggest an essential role for the lectin complement cascade in Borrelia eradication and demonstrate how a vector-borne pathogen co-opts a vector protein to facilitate early mammalian infection and vector colonization.
Assuntos
Borrelia burgdorferi/patogenicidade , Lectina de Ligação a Manose da Via do Complemento , Proteínas de Insetos/imunologia , Ixodes/microbiologia , Doença de Lyme/transmissão , Sequência de Aminoácidos , Animais , Borrelia burgdorferi/imunologia , Ensaios de Migração Celular , Clonagem Molecular , Complexo de Ataque à Membrana do Sistema Complemento/imunologia , Feminino , Inativação Gênica , Hemólise/imunologia , Humanos , Imunização Passiva , Imunoterapia Ativa , Proteínas de Insetos/farmacologia , Larva/microbiologia , Doença de Lyme/imunologia , Doença de Lyme/microbiologia , Camundongos , Camundongos Endogâmicos C3H , Dados de Sequência Molecular , Neutrófilos/efeitos dos fármacos , Neutrófilos/imunologia , Ninfa/microbiologia , Fagocitose , Coelhos , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/farmacologia , Saliva/imunologia , Saliva/microbiologia , Proteínas e Peptídeos Salivares/imunologia , Proteínas e Peptídeos Salivares/farmacologia , Alinhamento de SequênciaRESUMO
Repeated exposure of rabbits and other animals to ticks results in acquired resistance or immunity to subsequent tick bites and is partially elicited by antibodies directed against tick antigens. In this study we demonstrate the utility of a yeast surface display approach to identify tick salivary antigens that react with tick-immune serum. We constructed an Ixodes scapularis nymphal salivary gland yeast surface display library and screened the library with nymph-immune rabbit sera and identified five salivary antigens. Four of these proteins, designated P8, P19, P23 and P32, had a predicted signal sequence. We generated recombinant (r) P8, P19 and P23 in a Drosophila expression system for functional and immunization studies. rP8 showed anti-complement activity and rP23 demonstrated anti-coagulant activity. Ixodes scapularis feeding was significantly impaired when nymphs were fed on rabbits immunized with a cocktail of rP8, rP19 and rP23, a hall mark of tick-immunity. These studies also suggest that these antigens may serve as potential vaccine candidates to thwart tick feeding.
Assuntos
Antígenos/isolamento & purificação , Imunização/métodos , Ixodes/imunologia , Animais , Antígenos/sangue , Antígenos/imunologia , Imunidade , Ninfa , Biblioteca de Peptídeos , Coelhos , Glândulas Salivares/imunologia , Carrapatos/imunologia , LevedurasRESUMO
Fucosylated structures participate in a wide range of pathological processes in eukaryotes and prokaryotes. The impact of fucose on microbial pathogenesis, however, has been less appreciated in arthropods of medical relevance. Thus, we used the tick-borne bacterium Anaplasma phagocytophilum- the agent of human granulocytic anaplasmosis to understand these processes. Here we show that A. phagocytophilum uses alpha1,3-fucose to colonize ticks. We demonstrate that A. phagocytophilum modulates the expression of alpha1,3-fucosyltransferases and gene silencing significantly reduces colonization of tick cells. Acquisition but not transmission of A. phagocytophilum was affected when alpha1,3-fucosyltransferases were silenced during tick feeding. Our results uncover a novel mechanism of pathogen colonization in arthropods. Decoding mechanisms of pathogen invasion in ticks might expedite the development of new strategies to interfere with the life cycle of A. phagocytophilum.