Identification of Anaplasma marginale Proteins Specifically Upregulated during Colonization of the Tick Vector

Abstract

The transition between infection of the mammalian host and colonization of an arthropod vector is required for the ongoing transmission of a broad array of pathogens, from viruses to protozoa. Understanding how this transition is mediated provides opportunities to disrupt transmission through either chemotherapy or immunization. We used an unbiased proteomic screen to identify Anaplasma marginale proteins specifically upregulated in the tick compared to the mammalian host. Comparative mass spectrometric analysis of proteins separated by two-dimensional gel electrophoresis of uninfected and infected ISE6 cells and infected mammalian cells identified 15 proteins exclusively expressed or upregulated in tick cells. All 15 had originally been annotated as hypothetical proteins. We confirmed quantitative upregulation and expression in situ within the midgut epithelial and salivary gland acinar cells of vector ticks during successful transmission. The results support the hypothesis that A. marginale gene expression is regulated by the specific host environment and, in a broader context, that the core genome evolved in the arthropod vector with differential regulation, allowing adaptation to mammalian hosts. Furthermore, the confirmation of the in situ expression of candidates identified in ISE6 cell lines indicates that this approach may be widely applicable to bacteria in the genera Anaplasma and Ehrlichia, removing a major technical impediment to the identification of new targets for vaccine and chemotherapeutic blocking of transmission.The transition between infection of the mammalian host and colonization of an arthropod vector is required for ongoing transmission of a broad array of pathogens, from viruses to protozoa. Understanding how this transition is mediated provides opportunities to disrupt transmission through either chemotherapy or immunization. Bacteria in the genera Anaplasma and Ehrlichia are obligate intracellular pathogens and effectively invade, survive, and replicate in markedly different cell types in the mammalian host and ixodid ticks, the arthropod vector (4). Impressively, this transition is effected by using a small genome of Ͻ1.5 Mb (2,3,8,9,15). We and others have hypothesized that the bacterial proteome would be specifically molded for each environment, with a core set of proteins expressed universally and subsets specifically up-or downregulated depending on the host/vector environment (6,12,19,26,27). However, there has been only minimal proteomic evidence that supports accepting this hypothesis. The best evidence comes from recent analysis of E. chaffeensis that detected proteins present in either in vitro-infected tick cells or canine macrophages (26); however, unique or upregulated expression of these candidate proteins in the tick cells has not been confirmed. There has been no identification of bacterial proteins specifically upregulated or exclusively expressed during actual colonization in the tick.We addressed this knowledge gap by an unbiased proteomic approach using the St. Mar...

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

“…ISE6 tick cell culture is an acceptable model for studying A. marginale infection of tick cells (9,10). Using this cell line, we discovered that AmOmpA is also important for A. marginale infection of tick cells and that the same AmOmpA 50 -67 domain that is key for the bacterium to optimally invade RF/6A cells is also critical for tick cell infection.…”

“…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).…”

Expression of Anaplasma marginale Ankyrin Repeat-Containing Proteins during Infection of the Mammalian Host and Tick Vector

“…The expression of the three A. marginale ankyrin repeat-bearing proteins in the different host environments was determined using quantitative Western blots (23). Briefly, a large region of each open reading frame was expressed as a recombinant protein and the purified protein used to immunize mice to generate specific monoclonal antibodies for quantitative detection of each protein in infected cells.…”

“…The PCR cycling conditions were 10 cycles of melting at 94°C for 30 s and annealing at 35°C (AM705), 41°C (AM926), or 39°C (AM638) for 30 s with extension at 72°C for 2 min (AM705 and AM638) or 1 min (AM926), followed by 25 cycles of melting at 94°C for 30 s and annealing at 70°C for 30 s and extension at 72°C for 2 min (AM705 and AM638) or 1 min (AM926). Cloning and expression utilized the Gateway expression system (Invitrogen), and the recombinant His-tagged fusion proteins were affinity purified as previously described (23).…”

“…We, and others, have hypothesized that this transition between hosts requires expression of unique proteomes (11,19,23,27). This is supported by proteomic approaches, unbiased as to location or function, which identified both marked upregulation and unique expression of bacterial proteins in the tick vector relative to the mammalian host (23,27). In our recent study using Anaplasma marginale, all 15 proteins shown to be upregulated in tick cells had been originally annotated as hypothetical proteins, consistent with the significant percentage of proteins of unknown function in the genera Anaplasma and Ehrlichia (1-3, 5, 8, 13, 17, 23).…”