Anaplasma phagocytophilum Asp14 Is an Invasin That Interacts with Mammalian Host Cells via Its C Terminus To Facilitate Infection

Abstract

e Anaplasma phagocytophilum, a member of the family Anaplasmataceae, is the tick-transmitted obligate intracellular bacterium that causes human granulocytic anaplasmosis. The life cycle of A. phagocytophilum is biphasic, transitioning between the noninfectious reticulate cell (RC) and infectious dense-cored (DC) forms. We analyzed the bacterium's DC surface proteome by selective biotinylation of surface proteins, NeutrAvidin affinity purification, and mass spectrometry. Transcriptional profiling of selected outer membrane protein candidates over the course of infection revealed that aph_0248 (designated asp14 [14-kDa A. phagocytophilum surface protein]) expression was upregulated the most during A. phagocytophilum cellular invasion. asp14 transcription was induced during transmission feeding of A. phagocytophilum-infected ticks on mice and was upregulated when the bacterium engaged its receptor, P-selectin glycoprotein ligand 1. Asp14 localized to the A. phagocytophilum surface and was expressed during in vivo infection. Treating DC organisms with Asp14 antiserum or preincubating mammalian host cells with glutathione S-transferase (GST)-Asp14 significantly inhibited infection of host cells. Moreover, preincubating host cells with GST-tagged forms of both Asp14 and outer membrane protein A, another A. phagocytophilum invasin, pronouncedly reduced infection relative to treatment with either protein alone. The Asp14 domain that is sufficient for cellular adherence and invasion lies within the C-terminal 12 to 24 amino acids and is conserved among other Anaplasma and Ehrlichia species. These results identify Asp14 as an A. phagocytophilum surface protein that is critical for infection, delineate its invasion domain, and demonstrate the potential of targeting Asp14 in concert with OmpA for protecting against infection by A. phagocytophilum and other Anaplasmataceae pathogens.

Anaplasma phagocytophilumRab10-dependent parasitism of thetrans-Golgi network is critical for completion of the infection cycle

“…Given these observations for GFP-Rab10, we hypothesized that endogenous Rab10-positive vesicles are delivered into the ApV. Accordingly, we screened infected RF/6A cells with antibodies against endogenous Rab10 and the bacterial outer membrane protein (OMP), Asp14 (14 kDa A. phagocytophilum surface protein) (Kahlon et al ., 2013) or APH0032, which is a bacterial effector that is pronouncedly expressed late (24 to 32 h) during the infection cycle and localizes to the A. phagocytophilum occupied vacuolar membrane (AVM) (Huang et al ., 2010b). In addition to producing a vesicular labelling pattern in the cytosol and at the peripheries of ApVs, endogenous Rab10 signal surrounded individual intravacuolar bacteria in ring-like labelling patterns and partially colocalized with Asp14 signal (Fig.…”

“…Our previous evaluation of the A. phagocytophilum surface proteome identified UMPK as a likely surface protein (Kahlon et al ., 2013). Mining the UMPK tryptic peptides recovered in that study (Kahlon et al ., 2013) revealed that they mapped to the protein’s two predicted surface-exposed regions (Fig. 9B).…”

Subdominant Outer Membrane Antigens in Anaplasma marginale: Conservation, Antigenicity, and Protective Capacity Using Recombinant Protein

“…phagocytophilum OMPs, OmpA and Asp14, shown to mediate entry into host cells in vitro [13, 14]. The level of conservation of these proteins was evaluated using six well-characterized A .…”

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

“…Unbound bacteria were removed and infection was allowed to proceed for 48 h. To determine if recombinant OmpA proteins could antagonize A. marginale infection, RF/6A cells were incubated with GST-AmOmpA, GST-ApOmpA, or GST alone (4 M) for 1 h, after which A. marginale DC organisms were added and incubated with the host cells in the continued presence of recombinant protein for 2 h. Unbound bacteria and proteins were removed and the infection was allowed to proceed for 48 h. Experiments that assessed if antibodies targeting AmOmpA or recombinant OmpA proteins could inhibit A. marginale infection of ISE6 cells were performed identically to those just described, except that A. marginale organisms were incubated with ISE6 cells for 5 h before unbound bacteria were removed, the infection was allowed to proceed for 72 h, and the MOI achieved was approximately 1.7. At the endpoint of each experiment, cells were analyzed by spinning-disk confocal microscopy to determine the percentage of infected cells and number of AmVs per cell (17,20).…”

“…Clues pertaining to the role of A. marginale OmpA (AmOmpA) are provided by recent studies demonstrating the importance of OmpA proteins to cellular invasion by A. phagocytophilum and Ehrlichia chaffeensis, two Anaplasmataceae members that cause potentially fatal infections of humans and animals (17)(18)(19). Indeed, we discovered that A. phagocytophilum OmpA (ApOmpA) is one of a trio of adhesins that cooperatively function to mediate optimal bacterial binding to and invasion of host cells (17,18,20,21). Recombinant ApOmpA binds to host cells, confers adhesiveness and invasiveness to inert beads, and acts as a competitive agonist to inhibit A. phagocytophilum infection in vitro (17,18), confirming that it alone is sufficient to mediate binding and uptake.…”