Anaplasma phagocytophilum is an emerging zoonotic pathogen transmitted by Ixodes scapularis that causes human granulocytic anaplasmosis. Here, a high throughput quantitative proteomics approach was used to characterize A. phagocytophilum proteome during rickettsial multiplication and identify proteins involved in infection of the tick vector, I. scapularis. The first step in this research was focused on tick cells infected with A. phagocytophilum and sampled at two time points containing 10-15% and 65-71% infected cells, respectively to identify key bacterial proteins over-represented in high percentage infected cells. The second step was focused on adult female tick guts and salivary glands infected with A. phagocytophilum to compare in vitro results with those occurring during bacterial infection in vivo. The results showed differences in the proteome of A. phagocytophilum in infected ticks with higher impact on protein synthesis and processing than on bacterial replication in tick salivary glands. These results correlated well with the developmental cycle of A. phagocytophilum, in which cells convert from an intracellular reticulated, replicative form to the nondividing infectious dense-core form. The analysis of A. phagocytophilum differentially represented proteins identified stress response (GroEL, HSP70) and surface (MSP4) proteins that were over-represented in high percentage infected tick cells and salivary glands when compared to low percentage infected cells and guts, respectively. The results demonstrated that MSP4, GroEL and HSP70 interact and bind to tick cells, thus playing a role in rickettsia-tick interactions. The most important finding of these studies is the increase in the level of certain bacterial stress response and surface proteins in A. phagocytophilum-infected tick cells and salivary glands with functional implication in tick-pathogen interactions. These results gave a new dimension to the role of these stress response and surface proteins during A. phagocytophilum infection in ticks. Characterization of Anaplasma proteome contributes information on host-pathogen interactions and provides targets for development of novel control strategies for pathogen infection and transmission.

Centro Nacional de Investigaciones Cardiovasculares Melchor Fernández Almagro 3 28029 Madrid Spain

CIRAD UMR CMAEE Site de Duclos Prise d'eau F 97170 Petit Bourg Guadeloupe France; INRA UMR1309 CMAEE F 34398 Montpellier France

Department of Veterinary Pathobiology Center for Veterinary Health Sciences Oklahoma State University Stillwater Oklahoma 74078 United States of America

Institute of Parasitology Biology Centre of the Academy of Sciences of the Czech Republic Branišovská 31 České Budějovice CZ 37005 Czech Republic

Institute of Parasitology Biology Centre of the Academy of Sciences of the Czech Republic Branišovská 31 České Budějovice CZ 37005 Czech Republic; Faculty of Science University of South Bohemia Branišovská 31 České Budějovice CZ 37005 Czech Republic

SaBio Instituto de Investigación en Recursos Cinegéticos IREC CSIC UCLM JCCM Ronda de Toledo s n 13005 Ciudad Real Spain

SaBio Instituto de Investigación en Recursos Cinegéticos IREC CSIC UCLM JCCM Ronda de Toledo s n 13005 Ciudad Real Spain; Center for Infection and Immunity of Lille INSERM U1019 CNRS UMR 8204 Université Lille Nord de France Institut Pasteur de Lille Lille France

SaBio Instituto de Investigación en Recursos Cinegéticos IREC CSIC UCLM JCCM Ronda de Toledo s n 13005 Ciudad Real Spain; Department of Veterinary Pathobiology Center for Veterinary Health Sciences Oklahoma State University Stillwater Oklahoma 74078 United States of America

The Roslin Institute and Royal School of Veterinary Studies University of Edinburgh Easter Bush Midlothian EH25 9RG United Kingdom; The Pirbright Institute Ash Road Pirbright Woking GU24 0NF United Kingdom

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