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Title: The pathogen-occupied vacuoles of anaplasma phagocytophilum and anaplasma marginale interact with the endoplasmic reticulum

item TRUCHAN, HILARY - Virginia Commonwealth University
item HEBERT, KATHRYN - Virginia Commonwealth University
item COCKBURN, CHELSEA - Virginia Commonwealth University
item MAGUNDA, FORGIVEMORE - Washington State University
item Noh, Susan
item CARLYON, JASON - Virginia Commonwealth University

Submitted to: Frontiers in Cellular and Infection Microbiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 2/4/2016
Publication Date: 3/1/2016
Citation: Truchan, H.K., Hebert, K.S., Cockburn, C.L., Magunda, F., Noh, S.M., Carlyon, J.A. 2016. The pathogen-occupied vacuoles of anaplasma phagocytophilum and anaplasma marginale interact with the endoplasmic reticulum. Frontiers in Cellular and Infection Microbiology. doi: 10.3389/fcimb.2016.00022.

Interpretive Summary: Anaplasma phagocytophilum and Anaplasma marginale are closely related tick-transmitted obligate intracellular bacterial pathogens that cause debilitating and potentially fatal diseases. A. phagocytophilum infects neutrophils to cause granulocytic anaplasmosis in humans (HGA), dog, horses and sheep (in the United Kingdom and Europe). HGA is an acute febrile illness that can be accompanied by non-specific symptoms including headache, malaise, myalgia, elevated liver enzymes, leukopenia, and thrombocytopenia. A. marginale, the causative agent of bovine anaplasmosis, is the most prevalent tick borne pathogen of cattle found worldwide. This pathogen is endemic in south eastern and western regions of the U. S and throughout the tropics and causes production limiting disease. It infects erythrocytes, resulting in anemia, weight loss, reduced growth and milk production, and abortion. Following resolution of acute disease, cattle develop life-long persistent infection and serve as a reservoir for ongoing transmission. Currently prevention of both granulocytic and bovine anaplasmosis rely on tick control, either through careful avoidance of ticks in the case of humans, or repeated use of acaricides, which are toxic, expensive and partially effective in cattle. While both of these pathogens cause different diseases and infect different cells in the mammalian host, they share a similar life cycle within the tick. A better understanding of the means by which these pathogens are able to colonize and grow within the tick may lead to improved methods for preventing tick transmission. Upon entrance into the tick, both pathogens are obligated to establish a niche within the cells of the tick midgut. This niche is bound by a protective membrane which protects the pathogen from degradation and allows it to replicate. However, this niche is likely to be nutrient poor. The studies reported in this paper demonstrate that both of these pathogens interact with the endoplasmic reticulum of the host cell. The endoplasmic reticulum is involved in protein and lipid synthesis and may serve as a source of nutrients for both of these pathogens.

Technical Abstract: The genus Anaplasma consists of tick-transmitted obligate intracellular bacteria that invade white or red blood cells to cause debilitating and potentially fatal infections. A. phagocytophilum, a human and veterinary pathogen, infects neutrophils to cause granulocytic anaplasmosis. A. marginale invades bovine erythrocytes. Evidence suggests that both species may also infect endothelial cells in vivo. In mammalian and arthropod host cells, A. phagocytophilum and A. marginale reside in host cell derived pathogen-occupied vacuoles (POVs). While it was recently demonstrated that the A. phagocytophilum-occupied vacuole (ApV) intercepts membrane traffic from the trans-Golgi network, it is unclear if it or the A. marginale-occupied vacuole (AmV) interacts with other secretory organelles. Here, we demonstrate that the ApV and AmV extensively interact with the host endoplasmic reticulum (ER) in endothelial, myeloid, and/or tick cells. ER lumen markers, calreticulin and protein disulfide isomerase, and the ER membrane marker, derlin-1, were pronouncedly recruited to the peripheries of both POVs. ApV association with the ER initiated early and continued throughout the infection cycle. Both the ApV and AmV interacted with the rough ER and smooth ER. However, only rough ER derived vesicles were delivered into the ApV lumen where they localized with intravacuolar bacteria. Transmission electron microscopy identified multiple ER-POV membrane contact sites on the cytosolic faces of both species’ vacuoles that corresponded to areas on the vacuoles’ lumenal faces where intravacuolar Anaplasma organisms closely associated. A. phagocytophilum is known to hijack Rab10, a GTPase that regulates ER dynamics and morphology. Yet, ApV-ER interactions were unhindered in cells in which Rab10 had been knocked down, demonstrating that the GTPase is dispensable for the bacterium to parasitize the ER. These data establish the ApV and AmV as pathogen-host interfaces that directly engage the ER in vertebrate and invertebrate host cells and evidence the conservation of ER parasitism between two Anaplasma species.