Tissue-Specific Signatures in the Transcriptional Response to Anaplasma phagocytophilum Infection of Ixodes scapularis and Ixodes ricinus Tick Cell Lines

Abstract

Anaplasma phagocytophilum are transmitted by Ixodes spp. ticks and have become one of the most common and relevant tick-borne pathogens due to their impact on human and animal health. Recent results have increased our understanding of the molecular interactions between Ixodes scapularis and A. phagocytophilum through the demonstration of tissue-specific molecular pathways that ensure pathogen infection, development and transmission by ticks. However, little is known about the Ixodes ricinus genes and proteins involved in the response to A. phagocytophilum infection. The tick species I. scapularis and I. ricinus are evolutionarily closely related and therefore similar responses are expected in A. phagocytophilum-infected cells. However, differences may exist between I. scapularis ISE6 and I. ricinus IRE/CTVM20 tick cells associated with tissue-specific signatures of these cell lines. To address this hypothesis, the transcriptional response to A. phagocytophilum infection was characterized by RNA sequencing and compared between I. scapularis ISE6 and I. ricinus IRE/CTVM20 tick cell lines. The transcriptional response to infection of I. scapularis ISE6 cells resembled that of tick hemocytes while the response in I. ricinus IRE/CTVM20 cells was more closely related to that reported previously in infected tick midguts. The inhibition of cell apoptosis by A. phagocytophilum appears to be a key adaptation mechanism to facilitate infection of both vertebrate and tick cells and was used to investigate further the tissue-specific response of tick cell lines to pathogen infection. The results supported a role for the intrinsic pathway in the inhibition of cell apoptosis by A. phagocytophilum infection of I. scapularis ISE6 cells. In contrast, the results in I. ricinus IRE/CTVM20 cells were similar to those obtained in tick midguts and suggested a role for the JAK/STAT pathway in the inhibition of apoptosis in tick cells infected with A. phagocytophilum. Nevertheless, tick cell lines were derived from embryonated eggs and may contain various cell populations with different morphology and behavior that could affect transcriptional response to infection. These results suggested tissue-specific signatures in I. scapularis ISE6 and I. ricinus IRE/CTVM20 tick cell line response to A. phagocytophilum infection that support their use as models for the study of tick-pathogen interactions.

Keywords: anaplasma; apoptosis; rickettsia; tick; tick cell line; transcriptomics.

Figure 1

Validation of RNAseq results. (A,B) The expression of selected differentially expressed genes was characterized by real-time RT-PCR using total RNA extracted from infected and uninfected ISE6 and IRE/CTVM20 tick cells. The mRNA levels were normalized against tick 16S rRNA and cyclophilin using the genNorm method. Normalized Ct-values were represented as average + S.D. and compared between infected and uninfected tick cells by Student's t-test with unequal variance (^*P < 0.05; N = 5 biological replicates). (C) Comparison between RNAseq and real-time RT-PCR results showing a good correlation between both methods.

Figure 2

Biological processes affected by A. phagocytophilum infection of I. ricinus IRE/CTVM20 and I. scapularis ISE6 cells. (A) Differentially expressed genes were functionally annotated and grouped according to the BP of the encoded proteins. The number of genes on each BP is shown. (B) Percentage of upregulated genes in each BP. (C) Percentage of downregulated genes in each BP. UR, upregulated genes; DR, downregulated genes.

Figure 3

Molecular function of differentially expressed genes in response to A. phagocytophilum infection of I. ricinus IRE/CTVM20 and I. scapularis ISE6 cells. (A) Differentially expressed genes were functionally annotated and grouped according to the MF of the encoded proteins. The number of genes on each MF is shown. (B) Percentage of upregulated genes in each MF. (C) Percentage of downregulated genes in each MF. UR, upregulated genes; DR, downregulated genes.

Figure 4

GO normalized data for differentially expressed genes in response to A. phagocytophilum infection of I. ricinus IRE/CTVM20 and I. scapularis ISE6 cells. (A) BP for upregulated genes. (B) BP for downregulated genes. (C) MF for upregulated genes. (D) MF for downregulated genes. For all BP and MF categories in both upregulated and downregulated genes, values were compared between tick cell lines by Chi² test and resulted in significant differences at p < 0.01 for all except for MF of downregulated genes in which differences were not significant between both tick cell lines.

Figure 5

Analysis of most differentially expressed genes in response to A. phagocytophilum infection of I. ricinus IRE/CTVM20 and I. scapularis ISE6 cells. (A) Proteins encoded by the 10 most upregulated genes in I. ricinus IRE/CTVM20 cells. (B) Proteins encoded by the 4 most upregulated genes in I. scapularis ISE6 cells. (C) Proteins encoded by the 10 most downregulated genes in I. ricinus IRE/CTVM20 cells. (D) Proteins encoded by the 10 most downregulated genes in I. scapularis ISE6 cells. The InterPro motifs obtained using DAVID were used to evaluate the fold enrichment of differentially expressed genes in the corresponding tick cell line (p ≤ 0.05).

Figure 6

Transcriptional profile of genes differentially expressed in both A. phagocytophilum-infected I. ricinus IRE/CTVM20 and I. scapularis ISE6 cells and in comparison with infected tick samples. Differential expression (p ≤ 0.05; q ≤ 0.05) is shown for I. scapularis ISE6 cells, I. ricinus IRE/CTVM20 cells, and I. scapularis nymphs and adult midguts and salivary glands. Data for tick samples was obtained from Ayllón et al. (2015a).

Figure 7

Tissue-specific signatures in the inhibition of tick cell apoptosis by A. phagocytophilum. (A) Tissue-specific response in the inhibition of tick cell apoptosis by A. phagocytophilum. The annotation of JAK/STAT pathway genes and the effect of A. phagocytophilum infection on tick tissue response were obtained from previous reports (Ayllón et al., ; Villar et al., 2015a). (B) Schematic representation of the effect of A. phagocytophilum on the inhibition of the JAK/STAT pathway to establish infection in I. ricinus IRE/CTVM20 cells. In infected I. ricinus IRE/CTVM20 cells, transcriptomics results reported here showed upregulation of JAKs resulting in activation of the JAK/STAT pathway. Additionally, downregulation of SOCS enhances the effect of induced cytoprotective factors such as Bcl-2IP, which in turn reduce CASP expression to inhibit apoptosis.