Anaplasma phagocytophilum inhibits apoptosis and promotes cytoskeleton rearrangement for infection of tick cells

Abstract

Anaplasma phagocytophilum causes human granulocytic anaplasmosis. Infection with this zoonotic pathogen affects gene expression in both the vertebrate host and the tick vector, Ixodes scapularis. Here, we identified new genes, including spectrin alpha chain or alpha-fodrin (CG8) and voltage-dependent anion-selective channel or mitochondrial porin (T2), that are involved in A. phagocytophilum infection/multiplication and the tick cell response to infection. The pathogen downregulated the expression of CG8 in tick salivary glands and T2 in both the gut and salivary glands to inhibit apoptosis as a mechanism to subvert host cell defenses and increase infection. In the gut, the tick response to infection through CG8 upregulation was used by the pathogen to increase infection due to the cytoskeleton rearrangement that is required for pathogen infection. These results increase our understanding of the role of tick genes during A. phagocytophilum infection and multiplication and demonstrate that the pathogen uses similar strategies to establish infection in both vertebrate and invertebrate hosts.

Fig 1

Gene expression in I. scapularis ISE6 cells and tick developmental stages. The CG2, CG8, CG10, T1, T2, and T3 mRNA levels in ISE6 cells and ticks were determined by real-time RT-PCR. Amplification efficiencies were normalized against the amplification efficiency for tick 16S rRNA, and normalized mRNA levels (average ± SD) are expressed in arbitrary units.

Fig 2

Gene expression in response to A. phagocytophilum and effect of gene knockdown on tick weight and pathogen infection. (A) CG2, CG8, CG10, T1, T2, T3, and subolesin (SUB) mRNA levels in guts, salivary glands, and whole infected and uninfected female ticks were determined by real-time RT-PCR. Amplification efficiencies were normalized against tick 16S rRNA, and mRNA levels (average ± SD) in infected ticks normalized to those in uninfected ticks are expressed in arbitrary units. Normalized C_T values between infected and uninfected tick cells and ticks were compared by Student's t test (*, P < 0.05; n = 12 to 20). (B) Female ticks were injected with dsRNA and fed on a sheep. Tick weight was determined after completion of feeding and compared between ticks injected with test dsRNA and control ticks injected with the unrelated Rs86 dsRNA by Student's t test (*, P < 0.05, **, P < 0.01; n = 10). (C) A. phagocytophilum infection levels were characterized in ISE6 tick cells after RNAi. Normalized C_T values were compared between test and negative-control tick cells incubated with the unrelated Rs86 dsRNA by Student's t test (*, P < 0.05; n = 10). (D) A. phagocytophilum infection levels were characterized in female I. scapularis tick guts and salivary glands after RNAi. Normalized C_T values were compared between test and negative-control ticks injected with the unrelated Rs86 dsRNA by Student's t test (*, P < 0.05; **, P < 0.01; n = 12 to 20). Fold differences in expression were determined with respect to expression in the controls incubated with the unrelated Rs86 dsRNA.

Fig 3

Analysis of CG8 and T2 protein sequences. (A) Phylogenetic analysis of CG8 protein sequences. (B) Phylogenetic analysis of T2 protein sequences. The evolutionary histories were inferred using the neighbor-joining method. Tick sequences are marked with dots. (C) Amino acid sequence alignment of tick T2 ortholog sequences in I. scapularis (GenBank accession no. XP_002408065), R. microplus (GenBank accession no. ADT82652), and A. variegatum (GenBank accession no. DAA34069). Asterisks denote amino acids conserved among all sequences analyzed.

Fig 4

T2 and CG8 protein levels decrease in A. phagocytophilum-infected ISE6 tick cells. (A) Flow cytometry profile histogram showing in cells from early and late infection (infected cells [IC]) and uninfected cells (UC) the isotype control (IgG IC, green; IgG UC, yellow), T2 (T2 IC, purple; T2 UC, orange), and CG8 (CG8 IC, red; CG8 UC, blue) MFI peaks visualized by use of an FITC-conjugated secondary antibody and CellQuest Pro software. (B) Ratio of MFI for infected cells to MFI for uninfected cells for MSP4, subolesin (SUB), T2, and CG8 in cells from early and late infection and uninfected cells. Positive and negative values denote higher and lower protein levels in infected cells, respectively, with respect to the levels in the uninfected controls. MFI was calculated as the MFI of the test labeled sample minus the MFI of the isotype control. (C) Representative images of immunofluorescence analysis of uninfected and A. phagocytophilum-infected ISE6 tick cells. Tick cells were stained with rabbit anti-tick proteins antibodies (green, FITC). (a and b) Bright-field image of infected cells (a) and preimmune control serum-treated infected cells (b), which gave results to similar those for uninfected cells; (c and d) uninfected (c) and infected (d) cells stained with antisubolesin antibodies; (e and f) uninfected (e) and infected (f) cells stained with anti-MSP4 antibodies; (g and h) uninfected (g) and infected (h) cells stained with anti-T2 antibodies; (i and j) uninfected (i) and infected (j) cells stained with anti-CG8 antibodies. Bars, 10 μm (a, b) and 20 μm (c to j).

Fig 5

Role of T2 in mitochondrially induced apoptosis. (A) CASP9 levels (OD₄₅₀ values; average ± SD) were compared between treatments by Student's t test (*, P < 0.05; **, P < 0.002; n = 4). (B) Hexokinase activity was characterized in uninfected and A. phagocytophilum-infected ISE6 tick cells, and levels (in mU/ml; average ± SD) were compared between treatments by Student's t test (*, P < 0.0001; n = 6). (C) Mitochondrially induced apoptosis pathway showing the effect of A. phagocytophilum infection on reducing T2 levels, which results in the inhibition of tick cell apoptosis.