RESUMEN
Anaplasma marginale is an obligate, intracellular, tick-borne bacterial pathogen that causes bovine anaplasmosis, an often severe, production-limiting disease of cattle found worldwide. Methods to control this disease are lacking, in large part due to major knowledge gaps in our understanding of the molecular underpinnings of basic host-pathogen interactions. For example, the surface proteins that serve as adhesins and, thus, likely play a role in pathogen entry into tick cells are largely unknown. To address this knowledge gap, we developed a phage display library and screened 66 A. marginale proteins for their ability to adhere to Dermacentor andersoni tick cells. From this screen, 17 candidate adhesins were identified, including OmpA and multiple members of the Msp1 family, including Msp1b, Mlp3, and Mlp4. We then measured the transcript of ompA and all members of the msp1 gene family through time, and determined that msp1b, mlp2, and mlp4 have increased transcript during tick cell infection, suggesting a possible role in host cell binding or entry. Finally, Msp1a, Msp1b, Mlp3, and OmpA were expressed as recombinant protein. When added to cultured tick cells prior to A. marginale infection, all proteins except the C-terminus of Msp1a reduced A. marginale entry by 2.2- to 4.7-fold. Except OmpA, these adhesins lack orthologs in related pathogens of humans and animals, including Anaplasma phagocytophilum and the Ehrlichia spp., thus limiting their utility in a universal tick transmission-blocking vaccine. However, this work greatly advances efforts toward developing methods to control bovine anaplasmosis and, thus, may help improve global food security.
Asunto(s)
Adhesinas Bacterianas , Anaplasma marginale , Dermacentor , Animales , Anaplasma marginale/genética , Adhesinas Bacterianas/metabolismo , Adhesinas Bacterianas/genética , Dermacentor/microbiología , Bovinos , Adhesión Bacteriana/fisiología , Anaplasmosis/microbiología , Proteínas de la Membrana Bacteriana Externa/metabolismo , Proteínas de la Membrana Bacteriana Externa/genética , Técnicas de Visualización de Superficie Celular , Interacciones Huésped-Patógeno , Enfermedades de los Bovinos/microbiologíaRESUMEN
Anaplasma spp. are obligate intracellular, tick-borne, bacterial pathogens that cause bovine and human anaplasmosis. We lack tools to prevent these diseases in part due to major knowledge gaps in our fundamental understanding of the tick-pathogen interface, including the requirement for and molecules involved in iron transport during tick colonization. We determine that iron is required for the pathogen Anaplasma marginale, which causes bovine anaplasmosis, to replicate in Dermacentor andersoni tick cells. Using bioinformatics and protein modeling, we identified three orthologs of the Gram-negative siderophore-independent iron uptake system, FbpABC. Am069, the A. marginale ortholog of FbpA, lacks predicted iron-binding residues according to the NCBI conserved domain database. However, according to protein modeling, the best structural orthologs of Am069 are iron transport proteins from Cyanobacteria and Campylobacterjejuni. We then determined that all three A. marginale genes are modestly differentially expressed in response to altered host cell iron levels, despite the lack of a Ferric uptake regulator or operon structure. This work is foundational for building a mechanistic understanding of iron uptake, which could lead to interventions to prevent bovine and human anaplasmosis.
Asunto(s)
Anaplasma marginale , Anaplasmosis , Dermacentor , Anaplasma , Anaplasma marginale/genética , Anaplasmosis/microbiología , Animales , Bovinos , Dermacentor/genética , Dermacentor/microbiología , Humanos , HierroRESUMEN
The insect immune deficiency (IMD) pathway is a defense mechanism that senses and responds to Gram-negative bacteria. Ticks lack genes encoding upstream components that initiate the IMD pathway. Despite this deficiency, core signaling molecules are present and functionally restrict tick-borne pathogens. The molecular events preceding activation remain undefined. Here, we show that the unfolded-protein response (UPR) initiates the IMD network. The endoplasmic reticulum (ER) stress receptor IRE1α is phosphorylated in response to tick-borne bacteria but does not splice the mRNA encoding XBP1. Instead, through protein modeling and reciprocal pulldowns, we show that Ixodes IRE1α complexes with TRAF2. Disrupting IRE1α-TRAF2 signaling blocks IMD pathway activation and diminishes the production of reactive oxygen species. Through in vitro, in vivo, and ex vivo techniques, we demonstrate that the UPR-IMD pathway circuitry limits the Lyme disease-causing spirochete Borrelia burgdorferi and the rickettsial agents Anaplasma phagocytophilum and A. marginale (anaplasmosis). Altogether, our study uncovers a novel linkage between the UPR and the IMD pathway in arthropods. IMPORTANCE The ability of an arthropod to harbor and transmit pathogens is termed "vector competency." Many factors influence vector competency, including how arthropod immune processes respond to the microbe. Divergences in innate immunity between arthropods are increasingly being reported. For instance, although ticks lack genes encoding key upstream molecules of the immune deficiency (IMD) pathway, it is still functional and restricts causative agents of Lyme disease (Borrelia burgdorferi) and anaplasmosis (Anaplasma phagocytophilum). How the IMD pathway is activated in ticks without classically defined pathway initiators is not known. Here, we found that a cellular stress response network, the unfolded-protein response (UPR), functions upstream to induce the IMD pathway and restrict transmissible pathogens. Collectively, this explains how the IMD pathway can be activated in the absence of canonical pathway initiators. Given that the UPR is highly conserved, UPR-initiated immunity may be a fundamental principle impacting vector competency across arthropods.
Asunto(s)
Anaplasma phagocytophilum , Anaplasmosis , Artrópodos , Borrelia burgdorferi , Ixodes , Enfermedad de Lyme , Anaplasma phagocytophilum/fisiología , Animales , Endorribonucleasas , Ixodes/genética , Ixodes/microbiología , Proteínas Serina-Treonina Quinasas , Factor 2 Asociado a Receptor de TNFRESUMEN
In the United States, Dermacentor spp. are common vectors of Francisella tularensis subspecies (ssp.), while Ixodes scapularis is not, though the geographic distribution and host range of pathogen and tick overlap. To examine if differences in infection competence at the cellular level underpin these ecological differences, we evaluated the competence of D. andersoni (DAE100) and I. scapularis (ISE6) cell lines to support F. tularensis ssp. novicida (F. novicida) infection. Importantly, D. andersoni is a vector for both F. tularensis spp. tularensis, and F. novicida. We hypothesized F. novicida infection would be more productive in D. andersoni than in I. scapularis cells. Specifically, we determined if there are differences in F. novicida i) invasion, ii) replication, or iii) tick cell viability between DAE100 and ISE6 cells. We further examined the influence of temperature on infection kinetics. Both cell lines were permissive to F. novicida infection; however, there were significantly higher bacterial levels and mortality in DAE100 compared to ISE6 cells. Infection at environmental temperatures prolonged the time bacteria were maintained at high levels and reduced tick cell mortality in both cell lines. Identifying cellular determinants of vector competence is essential in understanding tick-borne disease ecology and designing effective intervention strategies.