RESUMEN
Vector-borne diseases are a leading cause of death worldwide and pose a substantial unmet medical need. Pathogens binding to host extracellular proteins (the "exoproteome") represents a crucial interface in the etiology of vector-borne disease. Here, we used bacterial selection to elucidate host-microbe interactions in high throughput (BASEHIT)-a technique enabling interrogation of microbial interactions with 3,324 human exoproteins-to profile the interactomes of 82 human-pathogen samples, including 30 strains of arthropod-borne pathogens and 8 strains of related non-vector-borne pathogens. The resulting atlas revealed 1,303 putative interactions, including hundreds of pairings with potential roles in pathogenesis, including cell invasion, tissue colonization, immune evasion, and host sensing. Subsequent functional investigations uncovered that Lyme disease spirochetes recognize epidermal growth factor as an environmental cue of transcriptional regulation and that conserved interactions between intracellular pathogens and thioredoxins facilitate cell invasion. In summary, this interactome atlas provides molecular-level insights into microbial pathogenesis and reveals potential host-directed targets for next-generation therapeutics.
Asunto(s)
Interacciones Huésped-Patógeno , Humanos , Animales , Enfermedad de Lyme/microbiología , Enfermedades Transmitidas por Vectores , Interacciones Microbiota-Huesped , Borrelia burgdorferi/patogenicidad , Borrelia burgdorferi/metabolismoRESUMEN
Ticks transmit a diverse array of microbes to vertebrate hosts, including human pathogens, which has led to a human-centric focus in this vector system. Far less is known about pathogens of ticks themselves. Here, we discover that a toxin in blacklegged ticks (Ixodes scapularis) horizontally acquired from bacteria-called domesticated amidase effector 2 (dae2)-has evolved to kill mammalian skin microbes with remarkable efficiency. Secreted into the saliva and gut of ticks, Dae2 limits skin-associated staphylococci in ticks while feeding. In contrast, Dae2 has no intrinsic ability to kill Borrelia burgdorferi, the tick-borne Lyme disease bacterial pathogen. These findings suggest ticks resist their own pathogens while tolerating symbionts. Thus, just as tick symbionts can be pathogenic to humans, mammalian commensals can be harmful to ticks. Our study underscores how virulence is context-dependent and bolsters the idea that "pathogen" is a status and not an identity.
Asunto(s)
Bacterias/metabolismo , Factores Inmunológicos/metabolismo , Ixodes/fisiología , Piel/microbiología , Simbiosis , Animales , Antibacterianos/farmacología , Biocatálisis , Pared Celular/metabolismo , Conducta Alimentaria , Femenino , Tracto Gastrointestinal/metabolismo , Interacciones Huésped-Patógeno , Ratones , Modelos Moleculares , Peptidoglicano/metabolismo , Filogenia , Saliva/metabolismo , Glándulas Salivales/metabolismo , Staphylococcus epidermidis/fisiología , Homología Estructural de Proteína , Especificidad por Sustrato , Regulación hacia ArribaRESUMEN
The immune deficiency (IMD) pathway directs host defense in arthropods upon bacterial infection. In Pancrustacea, peptidoglycan recognition proteins sense microbial moieties and initiate nuclear factor-κB-driven immune responses. Proteins that elicit the IMD pathway in non-insect arthropods remain elusive. Here, we show that an Ixodes scapularis homolog of croquemort (Crq), a CD36-like protein, promotes activation of the tick IMD pathway. Crq exhibits plasma membrane localization and binds the lipid agonist 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol. Crq regulates the IMD and jun N-terminal kinase signaling cascades and limits the acquisition of the Lyme disease spirochete B. burgdorferi. Additionally, nymphs silenced for crq display impaired feeding and delayed molting to adulthood due to a deficiency in ecdysteroid synthesis. Collectively, we establish a distinct mechanism for arthropod immunity outside of insects and crustaceans.
Asunto(s)
Artrópodos , Infecciones Bacterianas , Borrelia burgdorferi , Ixodes , Enfermedad de Lyme , Animales , Ixodes/microbiología , Borrelia burgdorferi/genética , FN-kappa B , Enfermedad de Lyme/microbiologíaRESUMEN
Ticks exist across diverse environments and transmit numerous pathogens. Due to their long and unique life cycles, these arthropods likely evolved robust epigenetic mechanisms that provide sustainable responses and buffers against extreme environmental conditions. Herein, we highlight how the study of the epigenetic basis of tick biology and vectorial capacity will enrich our knowledge of tick-borne infections.
Asunto(s)
Vectores Artrópodos/crecimiento & desarrollo , Transmisión de Enfermedad Infecciosa , Epigénesis Genética , Interacciones Huésped-Patógeno , Enfermedades por Picaduras de Garrapatas/epidemiología , Garrapatas/fisiología , Animales , Vectores Artrópodos/clasificación , Vectores Artrópodos/genética , Humanos , Enfermedades por Picaduras de Garrapatas/genética , Enfermedades por Picaduras de Garrapatas/transmisión , Garrapatas/microbiología , Garrapatas/parasitología , Garrapatas/virologíaRESUMEN
BACKGROUND: Lyme disease is the most prevalent vector-borne disease in the US, yet its host factors are poorly understood and diagnostic tests are limited. We evaluated patients in a large health system to uncover cholesterol's role in the susceptibility, severity, and machine learning-based diagnosis of Lyme disease. METHODS: A longitudinal health system cohort comprised 1 019 175 individuals with electronic health record data and 50 329 with linked genetic data. Associations of blood cholesterol level, cholesterol genetic scores comprising common genetic variants, and burden of rare loss-of-function (LoF) variants in cholesterol metabolism genes with Lyme disease were investigated. A portable machine learning model was constructed and tested to predict Lyme disease using routine lipid and clinical measurements. RESULTS: There were 3832 cases of Lyme disease. Increasing cholesterol was associated with greater risk of Lyme disease and hypercholesterolemia was more prevalent in Lyme disease cases than in controls. Cholesterol genetic scores and rare LoF variants in CD36 and LDLR were associated with Lyme disease risk. Serological profiling of cases revealed parallel trajectories of rising cholesterol and immunoglobulin levels over the disease course, including marked increases in individuals with LoF variants and high cholesterol genetic scores. The machine learning model predicted Lyme disease solely using routine lipid panel, blood count, and metabolic measurements. CONCLUSIONS: These results demonstrate the value of large-scale genetic and clinical data to reveal host factors underlying infectious disease biology, risk, and prognosis and the potential for their clinical translation to machine learning diagnostics that do not need specialized assays.
Asunto(s)
Hipercolesterolemia , Enfermedad de Lyme , Humanos , Enfermedad de Lyme/diagnóstico , Enfermedad de Lyme/epidemiología , Colesterol , Pronóstico , Aprendizaje AutomáticoRESUMEN
The E3 ubiquitin ligase X-linked inhibitor of apoptosis (XIAP) acts as a molecular rheostat for the immune deficiency (IMD) pathway of the tick Ixodes scapularis How XIAP activates the IMD pathway in response to microbial infection remains ill defined. Here, we identified the XIAP enzymatic substrate p47 as a positive regulator of the I. scapularis IMD network. XIAP polyubiquitylates p47 in a lysine 63-dependent manner and interacts with the p47 ubiquitin-like (UBX) module. p47 also binds to Kenny (IKKγ/NEMO), the regulatory subunit of the inhibitor of nuclear factor (NF)- κB kinase complex. Replacement of the amino acid lysine to arginine within the p47 linker region completely abrogated molecular interactions with Kenny. Furthermore, mitigation of p47 transcription levels through RNA interference in I. scapularis limited Kenny accumulation, reduced phosphorylation of IKKß (IRD5), and impaired cleavage of the NF-κB molecule Relish. Accordingly, disruption of p47 expression increased microbial colonization by the Lyme disease spirochete Borrelia burgdorferi and the rickettsial agent Anaplasma phagocytophilum Collectively, we highlight the importance of ticks for the elucidation of paradigms in arthropod immunology. Manipulating immune signaling cascades within I. scapularis may lead to innovative approaches to reducing the burden of tick-borne diseases.
Asunto(s)
Ixodes/inmunología , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas Adaptadoras Transductoras de Señales/fisiología , Anaplasma , Animales , Proteínas de Artrópodos/metabolismo , Proteínas de Artrópodos/fisiología , Borrelia burgdorferi , Drosophila , Técnicas de Inactivación de Genes , Ixodes/microbiología , Ixodes/fisiología , FN-kappa B/metabolismo , Dominios Proteicos , Proteína Inhibidora de la Apoptosis Ligada a X/metabolismo , Proteína Inhibidora de la Apoptosis Ligada a X/fisiologíaRESUMEN
Vector-borne diseases cause over 700,000 deaths annually and represent 17% of all infectious illnesses worldwide. This public health menace highlights the importance of understanding how arthropod vectors, microbes and their mammalian hosts interact. Currently, an emphasis of the scientific enterprise is at the vector-host interface where human pathogens are acquired and transmitted. At this spatial junction, arthropod effector molecules are secreted, enabling microbial pathogenesis and disease. Extracellular vesicles manipulate signaling networks by carrying proteins, lipids, carbohydrates and regulatory nucleic acids. Therefore, they are well positioned to aid in cell-to-cell communication and mediate molecular interactions. This Review briefly discusses exosome and microvesicle biogenesis, their cargo, and the role that nanovesicles play during pathogen spread, host colonization and disease pathogenesis. We then focus on the role of extracellular vesicles in dictating microbial pathogenesis and host immunity during transmission of vector-borne pathogens.
Asunto(s)
Vectores Artrópodos , Vesículas Extracelulares , Enfermedades Transmitidas por Vectores , Amebiasis/parasitología , Amebiasis/transmisión , Animales , Vectores Artrópodos/microbiología , Vectores Artrópodos/parasitología , Culicidae/microbiología , Culicidae/parasitología , Vectores de Enfermedades , Exosomas/inmunología , Exosomas/microbiología , Exosomas/parasitología , Vesículas Extracelulares/inmunología , Vesículas Extracelulares/microbiología , Vesículas Extracelulares/parasitología , Filariasis/parasitología , Filariasis/transmisión , Hemípteros/microbiología , Hemípteros/parasitología , Interacciones Huésped-Parásitos/inmunología , Interacciones Huésped-Parásitos/fisiología , Humanos , Inmunomodulación , Leishmaniasis/parasitología , Leishmaniasis/transmisión , Malaria/parasitología , Malaria/transmisión , Psychodidae/microbiología , Psychodidae/parasitología , Tripanosomiasis/parasitología , Tripanosomiasis/transmisión , Enfermedades Transmitidas por Vectores/microbiología , Enfermedades Transmitidas por Vectores/parasitología , Enfermedades Transmitidas por Vectores/transmisión , Virosis/microbiología , Virosis/transmisiónRESUMEN
Recent scientific breakthroughs have significantly expanded our understanding of arthropod vector immunity. Insights in the laboratory have demonstrated how the immune system provides resistance to infection, and in what manner innate defenses protect against a microbial assault. Less understood, however, is the effect of biotic and abiotic factors on microbial-vector interactions and the impact of the immune system on arthropod populations in nature. Furthermore, the influence of genetic plasticity on the immune response against vector-borne pathogens remains mostly elusive. Herein, we discuss evolutionary forces that shape arthropod vector immunity. We focus on resistance, pathogenicity and tolerance to infection. We posit that novel scientific paradigms should emerge when molecular immunologists and evolutionary ecologists work together.
Asunto(s)
Vectores Artrópodos/inmunología , Artrópodos/inmunología , Mamíferos/inmunología , Animales , Evolución Biológica , Ecología , Humanos , Tolerancia Inmunológica , Inmunidad , Transducción de SeñalRESUMEN
Lyme disease, caused by some Borrelia burgdorferi sensu lato, is the most common tick-borne illness in the Northern Hemisphere and the number of cases, and geographic spread, continue to grow. Previously identified B. burgdorferi proteins, lipid immunogens, and live mutants lead the design of canonical vaccines aimed at disrupting infection in the host. Discovery of the mechanism of action of the first vaccine catalyzed the development of new strategies to control Lyme disease that bypassed direct vaccination of the human host. Thus, novel prevention concepts center on proteins produced by B. burgdorferi during tick transit and on tick proteins that mediate feeding and pathogen transmission. A burgeoning area of research is tick immunity as it can unlock mechanistic pathways that could be targeted for disruption. Studies that shed light on the mammalian immune pathways engaged during tick-transmitted B. burgdorferi infection would further development of vaccination strategies against Lyme disease.
Asunto(s)
Borrelia burgdorferi , Ixodes , Enfermedad de Lyme , Garrapatas , Vacunas , Animales , Humanos , Enfermedad de Lyme/prevención & control , VacunaciónRESUMEN
Rickettsial agents are sensed by pattern recognition receptors but lack pathogen-associated molecular patterns commonly observed in facultative intracellular bacteria. Due to these molecular features, the order Rickettsiales can be used to uncover broader principles of bacterial immunity. Here, we used the bacterium Anaplasma phagocytophilum, the agent of human granulocytic anaplasmosis, to reveal a novel microbial surveillance system. Mechanistically, we discovered that upon A. phagocytophilum infection, cytosolic phospholipase A2 cleaves arachidonic acid from phospholipids, which is converted to the eicosanoid prostaglandin E2 (PGE2) via cyclooxygenase 2 (COX2) and the membrane associated prostaglandin E synthase-1 (mPGES-1). PGE2-EP3 receptor signaling leads to activation of the NLRC4 inflammasome and secretion of interleukin (IL)-1ß and IL-18. Importantly, the receptor-interacting serine/threonine-protein kinase 2 (RIPK2) was identified as a major regulator of the immune response against A. phagocytophilum. Accordingly, mice lacking COX2 were more susceptible to A. phagocytophilum, had a defect in IL-18 secretion and exhibited splenomegaly and damage to the splenic architecture. Remarkably, Salmonella-induced NLRC4 inflammasome activation was not affected by either chemical inhibition or genetic ablation of genes associated with PGE2 biosynthesis and signaling. This divergence in immune circuitry was due to reduced levels of the PGE2-EP3 receptor during Salmonella infection when compared to A. phagocytophilum. Collectively, we reveal the existence of a functionally distinct NLRC4 inflammasome illustrated by the rickettsial agent A. phagocytophilum.
Asunto(s)
Anaplasma phagocytophilum/inmunología , Proteínas Reguladoras de la Apoptosis/inmunología , Proteínas de Unión al Calcio/inmunología , Dinoprostona/inmunología , Ehrlichiosis/inmunología , Inflamasomas/inmunología , Subtipo EP3 de Receptores de Prostaglandina E/inmunología , Animales , Modelos Animales de Enfermedad , Ensayo de Inmunoadsorción Enzimática , Immunoblotting , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Reacción en Cadena en Tiempo Real de la PolimerasaRESUMEN
Tick saliva contains a number of effector molecules that inhibit host immunity and facilitate pathogen transmission. How tick proteins regulate immune signaling, however, is incompletely understood. Here, we describe that loop 2 of sialostatin L2, an anti-inflammatory tick protein, binds to annexin A2 and impairs the formation of the NLRC4 inflammasome during infection with the rickettsial agent Anaplasma phagocytophilum Macrophages deficient in annexin A2 secreted significantly smaller amounts of interleukin-1ß (IL-1ß) and IL-18 and had a defect in NLRC4 inflammasome oligomerization and caspase-1 activation. Accordingly, Annexin a2-deficient mice were more susceptible to A. phagocytophilum infection and showed splenomegaly, thrombocytopenia, and monocytopenia. Providing translational support to our findings, better binding of annexin A2 to sialostatin L2 in sera from 21 out of 23 infected patients than in sera from control individuals was also demonstrated. Overall, we establish a unique mode of inflammasome evasion by a pathogen, centered on a blood-feeding arthropod.
Asunto(s)
Anaplasma phagocytophilum/inmunología , Anexina A2/inmunología , Proteínas Reguladoras de la Apoptosis/inmunología , Proteínas de Unión al Calcio/inmunología , Cistatinas/inmunología , Ehrlichiosis/microbiología , Evasión Inmune , Secuencia de Aminoácidos , Anaplasma phagocytophilum/genética , Animales , Anexina A2/química , Anexina A2/genética , Proteínas Reguladoras de la Apoptosis/química , Proteínas Reguladoras de la Apoptosis/genética , Vectores Arácnidos/química , Vectores Arácnidos/genética , Vectores Arácnidos/inmunología , Proteínas de Unión al Calcio/química , Proteínas de Unión al Calcio/genética , Caspasa 1/genética , Caspasa 1/inmunología , Caspasas/genética , Caspasas/inmunología , Caspasas Iniciadoras , Cistatinas/química , Cistatinas/genética , Ehrlichiosis/inmunología , Ehrlichiosis/patología , Escherichia coli/genética , Escherichia coli/metabolismo , Regulación de la Expresión Génica , Humanos , Inflamasomas/genética , Inflamasomas/inmunología , Interleucina-18/genética , Interleucina-18/inmunología , Interleucina-1beta/genética , Interleucina-1beta/inmunología , Ixodes/química , Ixodes/genética , Ixodes/inmunología , Macrófagos/inmunología , Macrófagos/microbiología , Ratones , Modelos Moleculares , Unión Proteica , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/inmunología , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/inmunología , Transducción de SeñalRESUMEN
Saliva from arthropod vectors facilitates blood feeding by altering host inflammation. Whether arthropod saliva counters inflammasome signaling, a protein scaffold that regulates the activity of caspase-1 and cleavage of interleukin-1ß (IL-1ß) and IL-18 into mature molecules, remains elusive. In this study, we provide evidence that a tick salivary protein, sialostatin L2, inhibits inflammasome formation during pathogen infection. We show that sialostatin L2 targets caspase-1 activity during host stimulation with the rickettsial agent Anaplasma phagocytophilum. A. phagocytophilum causes macrophage activation and hemophagocytic syndrome features. The effect of sialostatin L2 in macrophages was not due to direct caspase-1 enzymatic inhibition, and it did not rely on nuclear factor κB or cathepsin L signaling. Reactive oxygen species from NADPH oxidase and the Loop2 domain of sialostatin L2 were important for the regulatory process. Altogether, our data expand the knowledge of immunoregulatory pathways of tick salivary proteins and unveil an important finding in inflammasome biology.
Asunto(s)
Anaplasma phagocytophilum/fisiología , Caspasa 1/metabolismo , Ehrlichiosis/microbiología , Cistatinas Salivales/fisiología , Análisis de Varianza , Animales , Células Cultivadas , Citocinas/metabolismo , Modelos Animales de Enfermedad , Ehrlichiosis/metabolismo , Ehrlichiosis/patología , Inflamasomas/metabolismo , Inflamación/fisiopatología , Macrófagos/metabolismo , Macrófagos/microbiología , Ratones , Ratones Endogámicos C57BL , Especies Reactivas de OxígenoRESUMEN
BACKGROUND: The molecular basis for the focal nature of atherosclerotic lesions is poorly understood. Here, we explored whether disturbed flow patterns activate an innate immune response to form the NLRP3 inflammasome scaffold in vascular endothelial cells via sterol regulatory element binding protein 2 (SREBP2). METHODS AND RESULTS: Oscillatory flow activates SREBP2 and induces NLRP3 inflammasome in endothelial cells. The underlying mechanisms involve SREBP2 transactivating NADPH oxidase 2 and NLRP3. Consistently, SREBP2, NADPH oxidase 2, and NLRP3 levels were elevated in atheroprone areas of mouse aortas, suggesting that the SREBP2-activated NLRP3 inflammasome causes functionally disturbed endothelium with increased inflammation. Mimicking the effect of atheroprone flow, endothelial cell-specific overexpression of the activated form of SREBP2 synergized with hyperlipidemia to increase atherosclerosis in the atheroresistant areas of mouse aortas. CONCLUSIONS: Atheroprone flow induces NLRP3 inflammasome in endothelium through SREBP2 activation. This increased innate immunity in endothelium synergizes with hyperlipidemia to cause topographical distribution of atherosclerotic lesions.
Asunto(s)
Aterosclerosis/inmunología , Proteínas Portadoras/inmunología , Proteína 2 de Unión a Elementos Reguladores de Esteroles/inmunología , Vasculitis/inmunología , Animales , Apolipoproteínas E/genética , Aterosclerosis/metabolismo , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Células Endoteliales/inmunología , Células Endoteliales/metabolismo , Hemodinámica/inmunología , Células Endoteliales de la Vena Umbilical Humana , Humanos , Inmunidad Innata/inmunología , Inflamasomas/inmunología , Inflamasomas/metabolismo , Masculino , Glicoproteínas de Membrana/inmunología , Glicoproteínas de Membrana/metabolismo , Ratones , Ratones Transgénicos , MicroARNs/inmunología , MicroARNs/metabolismo , NADPH Oxidasa 2 , NADPH Oxidasas/inmunología , NADPH Oxidasas/metabolismo , Proteína con Dominio Pirina 3 de la Familia NLR , ARN Interferente Pequeño/genética , Proteína 2 de Unión a Elementos Reguladores de Esteroles/genética , Proteína 2 de Unión a Elementos Reguladores de Esteroles/metabolismo , Estrés Mecánico , Vasculitis/genética , Vasculitis/metabolismoRESUMEN
Ubiquitination (ubiquitylation) is a common protein modification that regulates a multitude of processes within the cell. This modification is typically accomplished through the covalent binding of ubiquitin to a lysine residue onto a target protein and is catalysed by the presence of three enzymes: an activating enzyme (E1), ubiquitin-conjugating enzyme (E2) and ubiquitin-protein ligase (E3). In recent years, ubiquitination has risen as a major signalling regulator of immunity and microbial pathogenesis in the mammalian system. Still, little is known about how ubiquitin relates specifically to vector immunology. Here, we provide a brief overview of ubiquitin biochemistry and describe how ubiquitination regulates immune responses in arthropods of medical relevance. We also discuss scientific gaps in the literature and suggest that, similar to mammals, ubiquitin is a major regulator of immunity in medically important arthropods.
Asunto(s)
Mamíferos/inmunología , Procesamiento Proteico-Postraduccional , Enzimas Activadoras de Ubiquitina/metabolismo , Enzimas Ubiquitina-Conjugadoras/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Animales , Artrópodos/inmunología , Humanos , UbiquitinaciónRESUMEN
Ubiquitination is a posttranslational modification that regulates protein degradation and signaling in eukaryotes. Although it is acknowledged that pathogens exploit ubiquitination to infect mammalian cells, it remains unknown how microbes interact with the ubiquitination machinery in medically relevant arthropods. Here, we show that the ubiquitination machinery is present in the tick Ixodes scapularis and demonstrate that the E3 ubiquitin ligase named x-linked inhibitor of apoptosis protein (XIAP) restricts bacterial colonization of this arthropod vector. We provide evidence that xiap silencing significantly increases tick colonization by the bacterium Anaplasma phagocytophilum, the causative agent of human granulocytic anaplasmosis. We also demonstrate that (i) XIAP polyubiquitination is dependent on the really interesting new gene (RING) catalytic domain, (ii) XIAP polyubiquitination occurs via lysine (K)-63 but not K-48 residues, and (iii) XIAP-dependent K-63 polyubiquitination requires zinc for catalysis. Taken together, our data define a role for ubiquitination during bacterial colonization of disease vectors.
Asunto(s)
Anaplasma phagocytophilum/fisiología , Vectores Arácnidos/enzimología , Ehrlichiosis/microbiología , Ixodes/enzimología , Ubiquitina-Proteína Ligasas/metabolismo , Proteína Inhibidora de la Apoptosis Ligada a X/metabolismo , Animales , Vectores Arácnidos/microbiología , Dominio Catalítico , Humanos , Ixodes/microbiología , Interferencia de ARN , Transducción de Señal , Ubiquitina-Proteína Ligasas/genética , Ubiquitinación , Proteína Inhibidora de la Apoptosis Ligada a X/genéticaRESUMEN
Although genetic manipulation is one of the hallmarks of model organisms, its applicability to non-model species has remained difficult due to our limited understanding of their fundamental biology. For instance, manipulation of a cell line originated from the black-legged tick Ixodes scapularis, an arthropod that serves as a vector for several human pathogens, has yet to be established. Here, we demonstrate the successful genetic modification of the commonly used tick ISE6 line through ectopic expression and clustered regularly interspaced palindromic repeats [(CRISPR)/CRISPR-associated protein 9 (Cas9)] genome editing. We performed ectopic expression using nucleofection and attained CRISPR-Cas9 editing via homology-dependent recombination. Targeting the E3 ubiquitin ligase x-linked inhibitor of apoptosis (xiap) and its substrate p47 led to an alteration in molecular signaling within the immune deficiency network and increased infection of the rickettsial agent Anaplasma phagocytophilum in I. scapularis ISE6 cells. Collectively, our findings complement techniques for the genetic engineering of I. scapularis ticks, which currently limit efficient and scalable molecular genetic screens in vivo.IMPORTANCEGenetic engineering in arachnids has lagged compared to insects, largely because of substantial differences in their biology. This study unveils the implementation of ectopic expression and CRISPR-Cas9 gene editing in a tick cell line. We introduced fluorescently tagged proteins in ISE6 cells and edited its genome via homology-dependent recombination. We ablated the expression of xiap and p47, two signaling molecules present in the immune deficiency (IMD) pathway of Ixodes scapularis. Impairment of the tick IMD pathway, an analogous network of the tumor necrosis factor receptor in mammals, led to enhanced infection of the rickettsial agent Anaplasma phagocytophilum. Altogether, our findings provide a critical technical resource to the scientific community to enable a deeper understanding of biological circuits in the black-legged tick I. scapularis.
Asunto(s)
Anaplasma phagocytophilum , Borrelia burgdorferi , Ixodes , Rickettsia , Animales , Humanos , Borrelia burgdorferi/genética , Anaplasma phagocytophilum/genética , Línea Celular , MamíferosRESUMEN
BACKGROUND: The blacklegged tick, Ixodes scapularis, transmits most vector-borne diseases in the US. It vectors seven pathogens of public health relevance, including the emerging human pathogen Anaplasma phagocytophilum. Nevertheless, it remains critically understudied compared to other arthropod vectors. Ixodes scapularis releases a variety of molecules that assist in the modulation of host responses. Recently, it was found that extracellular vesicles (EVs) carry several of these molecules and may impact microbial transmission to the mammalian host. EV biogenesis has been studied in mammalian systems and is relatively well understood, but the molecular players important for the formation and secretion of EVs in arthropods of public health relevance remain elusive. RabGTPases are among the major molecular players in mammalian EV biogenesis. They influence membrane identity and vesicle budding, uncoating, and motility. METHODS: Using BLAST, an in silico pathway for EV biogenesis in ticks was re-constructed. We identified Rab27 for further study. EVs were collected from ISE6 tick cells after knocking down rab27 to examine its role in tick EV biogenesis. Ixodes scapularis nymphs were injected with small interfering RNAs to knock down rab27 and then fed on naïve and A. phagocytophilum-infected mice to explore the importance of rab27 in tick feeding and bacterial acquisition. RESULTS: Our BLAST analysis identified several of the proteins involved in EV biogenesis in ticks, including Rab27. We show that silencing rab27 in I. scapularis impacts tick fitness. Additionally, ticks acquire less A. phagocytophilum after rab27 silencing. Experiments in the tick ISE6 cell line show that silencing of rab27 causes a distinct range profile of tick EVs, indicating that Rab27 is needed to regulate EV biogenesis. CONCLUSIONS: Rab27 is needed for successful tick feeding and may be important for acquiring A. phagocytophilum during a blood meal. Additionally, silencing rab27 in tick cells results in a shift of extracellular vesicle size. Overall, we have observed that Rab27 plays a key role in tick EV biogenesis and the tripartite interactions among the vector, the mammalian host, and a microbe it encounters.
Asunto(s)
Anaplasma phagocytophilum , Proteínas de Artrópodos , Vesículas Extracelulares , Ixodes , Proteínas rab27 de Unión a GTP , Animales , Humanos , Ratones , Anaplasma phagocytophilum/fisiología , Ixodes/citología , Ixodes/metabolismo , Ixodes/microbiología , Mamíferos , Vesículas Extracelulares/metabolismo , Proteínas rab27 de Unión a GTP/metabolismo , Proteínas de Artrópodos/metabolismoRESUMEN
Uncovering the complexity of systems in non-model organisms is critical for understanding arthropod immunology. Prior efforts have mostly focused on Dipteran insects, which only account for a subset of existing arthropod species in nature. Here we use and develop advanced techniques to describe immune cells (hemocytes) from the clinically relevant tick Ixodes scapularis at a single-cell resolution. We observe molecular alterations in hemocytes upon feeding and infection with either the Lyme disease spirochete Borrelia burgdorferi or the rickettsial agent Anaplasma phagocytophilum. We reveal hemocyte clusters exhibiting defined signatures related to immunity, metabolism, and proliferation. Depletion of phagocytic hemocytes affects hemocytin and astakine levels, two I. scapularis hemocyte markers, impacting blood-feeding, molting behavior, and bacterial acquisition. Mechanistically, astakine alters hemocyte proliferation, whereas hemocytin affects the c-Jun N-terminal kinase (JNK) signaling pathway in I. scapularis. Altogether, we discover a role for tick hemocytes in immunophysiology and provide a valuable resource for comparative biology in arthropods.
Asunto(s)
Anaplasma phagocytophilum , Artrópodos , Borrelia burgdorferi , Ixodes , Enfermedad de Lyme , Animales , Hemocitos , Ixodes/microbiología , Borrelia burgdorferi/fisiologíaRESUMEN
Arthropod-borne pathogens are responsible for hundreds of millions of infections in humans each year. The blacklegged tick, Ixodes scapularis, is the predominant arthropod vector in the United States and is responsible for transmitting several human pathogens, including the Lyme disease spirochete Borrelia burgdorferi and the obligate intracellular rickettsial bacterium Anaplasma phagocytophilum, which causes human granulocytic anaplasmosis. However, tick metabolic response to microbes and whether metabolite allocation occurs upon infection remain unknown. Here we investigated metabolic reprogramming in the tick ectoparasite I. scapularis and determined that the rickettsial bacterium A. phagocytophilum and the spirochete B. burgdorferi induced glycolysis in tick cells. Surprisingly, the endosymbiont Rickettsia buchneri had a minimal effect on bioenergetics. An unbiased metabolomics approach following A. phagocytophilum infection of tick cells showed alterations in carbohydrate, lipid, nucleotide and protein metabolism, including elevated levels of the pleiotropic metabolite ß-aminoisobutyric acid. We manipulated the expression of genes associated with ß-aminoisobutyric acid metabolism in I. scapularis, resulting in feeding impairment, diminished survival and reduced bacterial acquisition post haematophagy. Collectively, we discovered that metabolic reprogramming affects interspecies relationships and fitness in the clinically relevant tick I. scapularis.
RESUMEN
The deer tick transmits nearly half of the known tick-borne pathogens in the United States, and its expanding geographic range increases the risk of human infection. To decrease the abundance of and infection risk from deer ticks, approaches that include vaccines for human use and for animal hosts are desired.