Postgenomic Analyses Reveal Development of Infectious Anaplasma phagocytophilum during Transmission from Ticks to Mice

Abstract

Obligate intracellular bacteria of the Rickettsiales order have evolved to colonize both arthropod and mammalian hosts, but few details are known about the bacterial adaptations that occur during transmission from blood-feeding arthropods to mammals. Here we apply proteomics and transcriptome sequencing to Anaplasma phagocytophilum, the agent of human granulocytic anaplasmosis, in Ixodes scapularis tick salivary glands, to detect proteins or genes expressed by the pathogen during transmission feeding by the tick. We detected expression of 139 genes, representing 11% of the open reading frames (ORFs) in the A. phagocytophilum genome. The predominant categories of proteins were ribosomal proteins, cell surface proteins, chaperones, and uncharacterized proteins. There was no evidence of DNA replication enzymes, suggesting that most of the A. phagocytophilum cells were no longer dividing. Instead, protein expression reflected conversion to the extracellular, infectious "dense-core" (DC) form. High expression of a DC-specific marker, APH_1235, further suggested this developmental transition in ticks. We showed that blocking APH_1235 with antibodies reduced A. phagocytophilum infection levels in mammalian cell culture. This work represents a starting point for clarifying essential proteins expressed by A. phagocytophilum during transmission from ticks to mammals and demonstrates that the abundantly expressed, DC-associated APH_1235 protein is important during in vivo infection by A. phagocytophilum.

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

“…Indeed, the increase in asp14 mRNA is coincident with the 4-hour period that A. phagocytophilum requires to efficiently invade HL-60 cells (23)(24)(25). Asp14 is not transcribed in unfed A. phagocytophiluminfected I. scapularis nymphs but is transcriptionally induced during transmission feeding on mice, an observation that is consistent with a recent proteomic profiling study of A. phagocytophilum proteins induced during I. scapularis transmission feeding (46). Thus, while Asp14 is dispensable for A. phagocytophilum survival in ticks, it may be part of the bacterium's armamentarium required for establishing infection in mammals.…”

“…An equally plausible approach would be to focus on late-stage genes, such as APH_0874 and APH_1170, because they may be important for RC-to-DC transition, adhesion, invasion, and/or establishing infection. A precedent for this rationalization is provided by APH_1235, which is a late-stage gene that is pronouncedly upregulated at the DC stage and is critical for propagating infection in HL-60 cell culture (41,46). In summary, this work advances the knowledge of A. phagocytophilum pathogenesis by identifying Asp14 as a novel surface protein that is critical for infection of mammalian host cells and mapping its invasion domain-containing region.…”

“…These studies were performed on mixed populations of DC and RC bacteria (46,47,79). Our study is the first to analyze the A. phagocytophilum DC surface proteome, which facilitated the discovery of the importance of Asp14 to infection.…”

“…To facilitate identification of genes that were upregulated in the infectious DC form compared to the noninfectious RC form, normalized transcript levels for each gene at each time point were calculated as fold changes in expression relative to expression at 16 h, a time point at which the A. phagocytophilum population consists exclusively of RC organisms (26). To monitor asp14 expression during the tick blood meal, A. phagocytophilum-infected I. scapularis nymphs were fed on C3H/HeJ mice, followed by RNA extraction from salivary glands of transmission-fed, unfed, and uninfected control nymphs (46), and RT-qPCR was performed using asp14-specific primers.…”

Anaplasma phagocytophilum Outer Membrane Protein A Interacts with Sialylated Glycoproteins To Promote Infection of Mammalian Host Cells

“…Transmission feeding of A. phagocytophilum-infected I. scapularis nymphs on C3H/HeJ mice and RNA extraction from salivary glands obtained from transmission-fed and uninfected control nymphs were performed as described previously (40). RT-qPCR was performed as described above.…”