ARTICLE https://doi.org/10.1038/s41467-021-23900-8 OPEN Tick extracellular vesicles enable arthropod feeding and promote distinct outcomes of bacterial infection Adela S. Oliva Chávez 1,18, Xiaowei Wang1, Liron Marnin1, Nathan K. Archer 2, Holly L. Hammond1, Erin E. McClure Carroll1,19, Dana K. Shaw 1,20, Brenden G. Tully3, Amanda D. Buskirk4,21, Shelby L. Ford5, L. Rainer Butler1, Preeti Shahi1, Kateryna Morozova6, Cristina C. Clement6,22, Lauren Lawres7, Anya J. O’ Neal 1, Choukri Ben Mamoun 7, Kathleen L. Mason8, Brandi E. Hobbs 1, Glen A. Scoles8,23, Eileen M. Barry 4, Daniel E. Sonenshine9,10, Utpal Pal11, Jesus G. Valenzuela9, Marcelo B. Sztein4,12,13, 1234567890():,; Marcela F. Pasetti4,12, Michael L. Levin5, Michail Kotsyfakis 14, Steven M. Jay 15, Jason F. Huntley3, ✉ Lloyd S. Miller2,16, Laura Santambrogio 17,22 & Joao H. F. Pedra 1 Extracellular vesicles are thought to facilitate pathogen transmission from arthropods to humans and other animals. Here, we reveal that pathogen spreading from arthropods to the mammalian host is multifaceted. Extracellular vesicles from Ixodes scapularis enable tick feeding and promote infection of the mildly virulent rickettsial agent Anaplasma phagocyto- philum through the SNARE proteins Vamp33 and Synaptobrevin 2 and dendritic epidermal T cells. However, extracellular vesicles from the tick Dermacentor andersoni mitigate microbial spreading caused by the lethal pathogen Francisella tularensis. Collectively, we establish that tick extracellular vesicles foster distinct outcomes of bacterial infection and assist in vector feeding by acting on skin immunity. Thus, the biology of arthropods should be taken into consideration when developing strategies to control vector-borne diseases. 1 Department of Microbiology and Immunology, University of Maryland School of Medicine, Baltimore, MD, USA. 2 Department of Dermatology, Johns Hopkins University School of Medicine, Baltimore, MD, USA. 3 Department of Medical Microbiology and Immunology, University of Toledo College of Medicine and Life Sciences, Toledo, OH, USA. 4 Center for Vaccine Development and Global Health, University of Maryland School of Medicine, Baltimore, MD, USA. 5 Centers for Disease Control and Prevention, Atlanta, GA, USA. 6 Department of Pathology, Albert Einstein College of Medicine, Bronx, NY, USA. 7 Section of Infectious Diseases, Department of Internal Medicine, Yale University School of Medicine, New Haven, CT, USA. 8 USDA, ARS, Animal Disease Research Unit, Washington State University, Pullman, WA, USA. 9 Vector Molecular Biology Section, Laboratory of Malaria and Vector Research, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Rockville, MD, USA. 10 Department of Biological Sciences, Old Dominion University, Norfolk, VA, USA. 11 Department of Veterinary Medicine, University of Maryland, College Park, MD, USA. 12 Department of Pediatrics, University of Maryland School of Medicine, Baltimore, MD, USA. 13 Department of Medicine, University of Maryland School of Medicine, Baltimore, MD, USA. 14 Institute of Parasitology, Biology Center of the Czech Academy of Sciences, Ceske Budejovice, Czech Republic. 15 Fischell Department of Bioengineering, University of Maryland, College Park, MD, USA. 16 Immunology, Janssen Research and Development, Spring House, PA, USA. 17 Department of Microbiology and Immunology, Albert Einstein College of Medicine, Bronx, NY, USA. 18Present address: Department of Entomology, Texas A&M University, College Station, TX, USA. 19Present address: Excerpta Medica, Doylestown, PA, USA. 20Present address: Department of Veterinary Microbiology and Pathology, Washington State University, Pullman, WA, USA. 21Present address: Center for Drug Evaluation and Research, Office of Pharmaceutical Quality, Office of Process and Facilities, Division of Microbiology Assessment, Microbiology Assessment Branch III, U.S. Food and Drug Administration, Silver Spring, MD, USA. 22Present address: Department of Radiation Oncology and Physiology and Biophysics, Englander Institute for Precision Medicine, Weill Cornell Medicine, New York, ✉ NY, USA. 23Present address: USDA, ARS, Invasive Insect Biocontrol and Behavior Laboratory, Beltsville, MD, USA. email: [email protected] NATURE COMMUNICATIONS | (2021) 12:3696 | https://doi.org/10.1038/s41467-021-23900-8 | www.nature.com/naturecommunications 1 ARTICLE NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-021-23900-8 rthropod vectors transmit a diverse portfolio of bacterial, shown by transmission electron microscopy (Fig. 1a). In silico viral, and parasitic agents to mammals1. This evolutionary reconstruction of the EV pathway based on the I. scapularis A 36 fi feat is partly due to the secretion of salivary molecules genome identi ed genes associated with exosome biogenesis that disrupt host homeostasis and alter inflammation upon and secretion (Supplementary Data 1). These molecules included blood-feeding2–4. Salivary molecules from arthropod vectors the tetraspanin CD63 and two proteins associated with the redirect host immunity, enable microbial spreading and influence endosomal sorting complex required for transport (ESCRT) disease outcomes in humans and other animals2–4. For instance, machinery: (i) α-1,3/1,6-mannosyltransferase interacting protein saliva from the sandfly Lutzomyia longipalpis enhances trans- X (ALIX) and the (ii) tumor susceptibility gene 101 protein mission of the parasite Leishmania major5. Salivary effectors (TSG101)15–17. Mammalian polyclonal antibodies cross-reacted present in mosquitoes potentiate arbovirus transmission and with markers present in EVs originated from three tick cell lines: augment disease severity in the mammalian host6,7. Tick mole- (i) Amblyomma americanum (i.e., vector of ehrlichiosis37 – AAE2 cules inhibit inflammation and increase transmission of viruses cells); (ii) I. scapularis (i.e., vector of Lyme disease and and bacteria to a variety of animals8–11. Anaplasmosis24 – ISE6 cells); and (iii) D. andersoni (i.e., vector of Although saliva-assisted transmission appears to be universal tularemia38 – DAE100 cells) (Fig. 1b and Supplementary among arthropods that feed on blood2,3,12, the mechanisms that Fig. 1c–e). These EVs presented an average mean size of 173 ± 7, affect interspecific responses reflect unique interactions occurring 137 ± 6, and 183 ± 3 nm, respectively (Supplementary Fig. 1f–h between vectors and their hosts. As an example, the inflammatory and Supplementary Movies 1–3). response caused by the bite of the mosquito Aedes aegypti, which To study the effect of I. scapularis EVs, we silenced the is classically assumed to defend the mammalian host against expression of two tick soluble N-ethylmaleimide-sensitive factor arboviral infection, results in higher Semliki forest and attachment receptor (SNARE) genes (e.g., synaptobrevin 2 and Bunyamwera viral load13. Depletion of neutrophils and inhibition vamp33) through RNA interference (RNAi). Tick studies rely on of inflammasome activity dampens the mosquito’s ability to delivering small interfering RNA (siRNA) to modulate gene enable viral transmission13.Infiltration of neutrophils at the sand expression because complete genetic ablation within ticks is not fly bite site, which is typically judged to be microbicidal, is currently feasible39. No differences in cell viability were observed important for L. major infection and establishment of illness14. between the scrambled (sc) control and silenced (si) treatments Altogether, these studies suggest that the relationship between (Supplementary Fig. 2a). Nanoparticle tracking analysis (NTA) salivary components, host immunity and microbial transmission revealed decreased secretion of tick vesicles in the classical are complex and ill-defined. exosomal range (i.e., 50–150 nm) for both synaptobrevin 2 and Extracellular vesicles (EVs) are a heterogenous population of vamp33 siRNA-treated cells (Fig. 1c, Supplementary Fig. 2b–d, nanovesicles that mediate interspecies communication15–17. and Supplementary Movies 4–7). Exosomes, a subset of EVs that range from approximately 50 to EVs derived from adult I. scapularis salivary glands had an 150 nm in size, are enriched for certain proteins, lipids, nucleic average mean size of 198 ± 4 nm (Fig. 1d and Supplementary acids, and glycoconjugates15–17. Recently, exosomes were shown Movie 8). We then obtained EVs from dissected I. scapularis to facilitate arbovirus transmission to mammals18–21 and two salivary glands cultured ex vivo in a vesicle-free medium to studies examined the molecular cargo of extracellular vesicles analyze their protein content. High-throughput proteomics originated from the evolutionarily distant Asian longhorned tick analysis of their cargo demonstrated a heterogeneous population Haemaphysalis longicornis22,23. The functional plasticity and with proteins typically featured for microvesicles, exosomes, and immunomodulatory potential of arthropod exosomes during exomeres40 (Supplementary Fig. 2e, f, Supplementary Data 2–5, vector transmission remain mostly unknown. To address this and data available via ProteomeXchange, identifier PXD018779). question, we used ticks as arthropod models due to their long- Tick proteins that have an impact on the immune response (e.g., feeding behavior and medical importance24. Herein, we adapted a sialostatins)9 and molecules associated with oxidative stress, protocol to culture tick salivary glands25–27 and developed a metabolism, and cell biology processes were overrepresented salivary