The Pathogen-Occupied Vacuoles of Anaplasma phagocytophilum and Anaplasma marginale Interact with the Endoplasmic Reticulum

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 derlin-1-positive 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.

Anaplasma marginale Actively Modulates Vacuolar Maturation during Intracellular Infection of Its Tick Vector, Dermacentor andersoni

“…As reported previously for the AmV in ISE6 cells, ultrastructural evaluation (Fig. 11B) confirmed that rough endoplasmic reticulum (RER) that was devoid of ribosomes on the contact side segmentally covered the outer aspect of the AmVs (29).…”

“…Similar to A. phagocytophilum, the AmVs engaged the ER and the Golgi apparatus early in infection and maintained association throughout the course of infection (29,38,39). The ER and the Golgi apparatus are nutrient-rich compartments of the secretory pathway and likely serve as a rich source of glycoprotein and lipids, which can be degraded to supply amino acids, fatty acids, nitrogen, carbon, and energy to auxotrophic A. marginale (40,41).…”

Anaplasma phagocytophilum MSP4 and HSP70 Proteins Are Involved in Interactions with Host Cells during Pathogen Infection

“…However, less information is available on the bacterial molecules involved in pathogen infection and multiplication (Ge and Rikihisa, 2007; Huang et al, 2010; Lin et al, 2011; Troese et al, 2011; Mastronunzio et al, 2012; Oliva Chávez et al, 2015; Seidman et al, 2015; Villar et al, 2015b; Truchan et al, 2016). Definition of bacterial proteins involved in host-pathogen and vector-pathogen interactions may provide target antigens for the development of vaccines and therapeutics that interfere with pathogen host infection and transmission by ticks (Gomes-Solecki, 2014; de la Fuente and Contreras, 2015).…”

Anaplasma marginale Outer Membrane Protein A Is an Adhesin That Recognizes Sialylated and Fucosylated Glycans and Functionally Depends on an Essential Binding Domain

“…Uninfected and A. marginale (St. Maries strain)-infected RF/6A rhesus monkey choroidal endothelial cells (CRL-1780; American Type Culture Collection, Manassas, VA) and Ixodes scapularis embryonic ISE6 cells were cultured as described previously (8,42,43). Both host cell types infected with A. marginale were originally donated by Ulrike Munderloh (University of Minnesota, Minneapolis, MN).…”

“…Detection of the bacterium colocalizing with the endothelial cell marker von Willebrand factor in tissue sections from an experimentally inoculated calf indicates it is also capable of infecting endothelial cells in vivo and might serve as a reservoir for infection (6). Moreover, endothelial cell lines are useful for studying A. marginale infection in vitro, as they are the only mammalian cell types in which continuous cultivation of these microbes has been achieved (7,8). The immortalized tick cell line ISE6 is susceptible to A. marginale infection and supports its replication, making it a useful model for studying bacteriumtick cell interactions (9)(10)(11).…”