Anaplasma phagocytophilum Inhibits Apoptosis and Promotes Cytoskeleton Rearrangement for Infection of Tick Cells

Abstract

dAnaplasma 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.T icks are ectoparasites of animals and humans and are considered to be the most important arthropod vector of pathogens in some regions (1). Ixodes scapularis Say (Acari: Ixodidae) is an important vector of pathogens that infect and cause disease in humans and domestic animals in the United States. Anaplasma phagocytophilum (Rickettsiales: Anaplasmataceae), the focus of this study, is the causative agent of human, canine, and equine granulocytic anaplasmosis and tick-borne fever of ruminants (2, 3).A. phagocytophilum is an intracellular bacterium that infects vertebrate host neutrophils, where it multiplies within a parasitophorous vacuole, thus evading host defenses while inhibiting apoptosis and promoting cytoskeleton rearrangement for infection and multiplication (4-8). Tick-A. phagocytophilum interactions are not as well characterized as those between pathogen and vertebrate hosts (4). While A. phagocytophilum has been shown to infect I. scapularis gut cells (9) and salivary glands (10), the developmental cycle of this pathogen has not been described in ticks. Tick proteins such as Salp16, subolesin, antifreeze glycoprotein IAFGP, and alpha1-3-fucosyltransferease were differentially regulated and required for A. phagocytophilum infection of I. scapularis (10-20). Activation of heat shock proteins and other stress response proteins in ticks and cultured tick cells in response to A. phagocytophilum infection was also characterized by proteomics and transcriptomics analyses (21).The overall goal of our research is to characterize molecular interactions at the vector-pathogen interface and develop vaccines for the control of tick infestations and pathogen infection/transmission. Our hypothesis is that tick genes differentially expressed in response to pathogen infection would include those involved in pathogen infection, multiplication, and transmission, as well as in the tick protective response to infection. In this researc...

Infection of Ixodes spp. tick cells with different Anaplasma phagocytophilum isolates induces the inhibition of apoptotic cell death

“…Nevertheless, results showed differences between both A. phagocytophilum isolates and tick cell lines. For example, as previously reported for the NY18 isolate in ISE6 cells (Ayllón et al, 2013) phagocytophilum isolates (Fig. 4B).…”

“…Functional characterization using RNA interference (RNAi) demonstrated that porin knockdown significantly increases colonization of I. scapularis ticks by A. phagocytophilum but does not affect tick feeding, thus showing how bacterial inhibition of porin expression increases tick vectorial capacity for this pathogen (authors' unpublished results). These results demonstrate how A. phagocytophilum has evolved to use similar strategies to establish infection in both vertebrate and invertebrate hosts (Ayllón et al, 2013).…”

“…Previous results suggested that mitochondrial Porin is involved in inhibition of the intrinsic apoptosis pathway by A. phagocytophilum infection of tick cells (Ayllón et al, 2013). The intrinsic apoptosis pathway is induced by Bcl-2 proteins through Porin resulting in Cytochrome c release and activation of CASP9, and is inhibited by HK and inhibitors of apoptosis (IAP) (Fig.…”

“…The presence of A. phagocytophilum DNA in the blood samples was confirmed by real-time PCR (Ayllón et al, 2013). White blood cells (WBC) were harvested from the blood samples one week after collection as follows.…”

“…Different studies have shown that A. phagocytophilum infection upregulates the expression of anti-apoptotic blc-2 genes, blocks cell surface fatty acid synthase (FAS) clustering during spontaneous neutrophil apoptosis and inhibits cleavage of pro-Caspase 8 and Caspase 8 activation as well as secretion Anaplasma translocated substrate-1 (Ats-1) preventing mitochondrial response to apoptotic signals (Niu et al, 2010;Rikihisa, 2010). Inhibition of apoptosis has also been observed in the I. scapularis cell line ISE6 in which A. phagocytophilum infection downregulates mitochondrial porin inducing mitochondrial dysfunction and thereby inhibition of the intrinsic apoptosis pathway to subvert host cell defenses and increase infection (Ayllón et al, 2013). Functional characterization using RNA interference (RNAi) demonstrated that porin knockdown significantly increases colonization of I. scapularis ticks by A. phagocytophilum but does not affect tick feeding, thus showing how bacterial inhibition of porin expression increases tick vectorial capacity for this pathogen (authors' unpublished results).…”