Anaplasma phagocytophilum is an obligate intracellular rickettsial pathogen vectored primarily by Ixodes spp. and causes human granulocytic anaplasmosis (HGA), equine, and canine granulocytic anaplasmosis, and tick-borne fever (TBF) (de la Fuente et al., 2008). In the vertebrate host, A. phagocytophilum infects neutrophils where the pathogen multiplies within a parasitophorous vacuole or morula (Ayllón et al., 2015a; Severo et al., 2015). In the absence of transovarial passage, ticks must acquire infection in each generation during a bloodmeal. A. phagocytophilum initially infects tick midgut cells and then subsequently develops in the salivary glands for transmission to susceptible hosts during tick feeding. Bacteria from the B. burgdorferi sensu lato complex are transmitted by Ixodid ticks and cause various symptoms associated with Lyme disease (Radolf et al., 2012). B. burgdorferi s.l. are acquired by larvae or nymphs from an infected host as they are not transovarially transmitted (Rollend et al., 2013). In the tick, spirochetes colonize the midgut and then traverse into the hemocoel and migrate to salivary glands for transmission during tick feeding (Pal et al., 2004; Ramamoorthi et al., 2005; Zhang L. et al., 2011; Coumou et al., 2016).

Ticks transmit a range of viruses that are of significant public and veterinary health concern (Table 1). It is estimated that these viruses spend over 95% of their life cycle within the tick vector. Tick-borne encephalitis virus (TBEV) causes neurological disease in humans, whereas louping ill virus (LIV) causes neurological disease in sheep (Labuda and Nuttall, 2003). Ixodid ticks transmit these viruses to particular host species through a bite (Doherty and Reid, 1971; Mansfield et al., 2016). Crimean-Congo hemorrhagic fever virus (CCHFV) is transmitted to humans by the bite of infected ticks (Hyalomma spp. are the most competent vectors) or by direct contact with blood or tissues of viremic patients or animals, causing a disease characterized by fever, headache, myalgia, and hemorrhagic manifestations (Papa, 2010). If the appropriate receptors are present in the tick, following a blood meal TBEV and CCHFV enter vector host cells by endocytosis (Labuda and Nuttall, 2003; Simon et al., 2009; Garrison et al., 2013; Shtanko et al., 2014; Suda et al., 2016). These viruses replicate in the lining of the tick midgut where they disseminate to the hemolymph and subsequently infect different tissues reaching the highest titers in the salivary glands and reproductive organs to exit the cell via exocytosis (Dickson and Turell, 1992).

Babesia spp. are tick-borne Apicomplexan protozoans which invade vertebrate host erythrocytes, where all hemoparasite phases occur (Yokoyama et al., 2006; Chauvin et al., 2009; Florin-Christensen and Schnittger, 2009). Babesia bovis and Babesia bigemina, transmitted mainly by Rhipicephalus microplus and Rhipicephalus annulatus, are considered the most important species for their great economic impact on the cattle industry. Humans are accidental hosts, but human babesiosis caused by Babesia microti is now considered an emerging zoonosis as cases are increasing yearly (Schnittger et al., 2012). Ticks become infected with Babesia parasites when ingesting blood cells containing piroplasms, which develop into male and female gametes in the tick midgut (Uilenberg, 2006). The zygotes then multiply and invade numerous tick organs including the ovaries, which results in transovarial passage for some species such as B. bovis and B. bigemina but not B. microti (Uilenberg, 2006). When ticks attach to a new host, the sporozoites mature and the parasites are transmitted with tick saliva and infect red blood cells (Uilenberg, 2006).

Tick-pathogen protein-protein interactions play a crucial role during pathogen infection, persistence and transmission. The analysis of A. phagocytophilum proteins differentially represented during infection in ticks demonstrated that heat shock protein 70 (HSP70) and major surface protein 4 (MSP4) interact and bind to tick cells, thus playing a role in tick-pathogen interactions (Villar et al., 2015b). The type IV secretion system (T4SS) was proposed to be involved in the secretion of HSP70 and the MSP4 interaction with tick cells may induce the secretion of vesicles at the phagocytic cup to aid in adhesin secretion for rickettsial infection of tick cells (Villar et al., 2015b). Recent results have advanced our understanding of the molecular factors that are involved in the acquisition, persistence and transmission of B. burgdorferi in ticks (Rosa et al., 2005; Kung et al., 2013). An important protein involved in spirochete colonization of the tick midgut is the outer surface protein A (OspA), which binds to the tick receptor for OspA (TROSPA) (Pal et al., 2004). An I. scapularis dystroglycan like protein (ISDLP) as well as a tick receptor for the B. burgdorferi protein BBE31 (TRE31) help spirochetes traverse from the tick midgut into the hemocoel (Zhang L. et al., 2011; Coumou et al., 2016). B. burgdorferi outer surface protein C (OspC), produced when bacteria leave the tick midgut, binds to tick salivary protein 15 (Salp15) (Ramamoorthi et al., 2005), providing protection against mammalian antibody/complement-mediated immune response during bacterial transmission (Garg et al., 2006; Schuijt et al., 2011a). The TROSPA homolog in the B. bigemina vectors, R. microplus, and R. annulatus was proposed to be a putative receptor for Babesia ligands based on the decrease in infection after RNAi and vaccination experiments targeting this protein (Antunes et al., 2012; Merino et al., 2013). Flaviviruses and CCHFV enter vertebrate and vector host cells by attachment of viral envelope proteins to host receptors, which activates the actin-dependent clathrin-mediated endocytic pathway (Labuda and Nuttall, 2003; Simon et al., 2009; Garrison et al., 2013).

Intracellular bacteria induce cytoskeletal rearrangement to establish infection (Ireton, 2013). In I. scapularis, A. phagocytophilum remodels tick cytoskeleton by altering the ratio between monomeric globular G actin and filamentous F actin to facilitate infection through selective regulation of gene transcription in association with the RNA polymerase II and the TATA-binding protein (Sultana et al., 2010). In I. scapularis midgut cells, the up-regulation of Spectrin alpha chain or Alpha-fodrin in response to infection results in cytoskeleton remodeling that is used by A. phagocytophilum to facilitate infection (Ayllón et al., 2013, Figure 2A). Although not functionally characterized, a proteomics analysis in I. ricinus tick salivary glands showed the under-representation of cytoskeleton proteins in response to Borrelia infection, suggesting that some Borrelia strains promote a cytoskeleton rearrangement in ticks (Cotté et al., 2014, Figure 2B).

Apoptosis is an intrinsic immune defense mechanism in response to microbial infection that results in reduction of infected cells, but several pathogens have developed different strategies to inhibit cell apoptosis in order to enhance their infection, replication and survival (Ashida et al., 2011). Infection of tick salivary glands with A. phagocytophilum results in inhibition of the intrinsic apoptosis pathway through porin down-regulation, favoring bacterial infection (Ayllón et al., 2015a). Tick cells respond to infection via activation of the extrinsic apoptosis pathway, which limits A. phagocytophilum infection and promotes tick survival (Ayllón et al., 2015a). In tick midguts, A. phagocytophilum infection results in activation of the Janus kinase/signal transducers and activators of transcription (JAK/STAT) pathway, which inhibits apoptosis and promotes pathogen infection (Ayllón et al., 2015a). The ISE6 cultured cells, derived from embryonic I. scapularis, have provided a model for tick hemocyte responses to pathogen infection. In this cell line, A. phagocytophilum infection promotes protein misfolding in the endoplasmic reticulum (ER), counteracting the tick cell response to infection. However, tick cells respond by activating protein targeting and degradation, which reduces ER stress and apoptosis, thus favoring A. phagocytophilum infection (Villar et al., 2015a). Additionally, A. phagocytophilum may benefit from the tick cells ability to limit pathogen infection through phosphoenolpyruvate carboxykinase (PEPCK) inhibition that results in lower glucose metabolism and the reduction in the availability of essential metabolites for bacterial growth, which leads to the inhibition of cell apoptosis that increases infection in tick cells (Villar et al., 2015a). These results show that the inhibition of tick cell apoptosis is a physiologically relevant mechanism used by A. phagocytophilum to facilitate infection and multiplication in both tick and vertebrate host cells (de la Fuente et al., 2016, Figure 4). Infection of I. ricinus cells with flaviviruses leads to the differential expression of a large number of genes involved in a variety of cellular functions, including up-regulation of genes such as cytochrome c associated with cellular stress and apoptosis (Mansfield et al., 2017). However, the lack of detection of caspase genes, and the up-regulation of genes that inhibit apoptosis (including hsp70) suggest that flavivirus infection inhibits tick cell apoptosis in order to promote cell survival during infection as previously shown for A. phagocytophilum (Ayllón et al., 2015a; Alberdi et al., 2016).

Tick vector competence is influenced by the ability of transmitted pathogens to evade tick innate immune response (Hajdušek et al., 2013). Several humoral and cell-mediated immune response pathways are involved in tick innate immunity, and play a role in defense to Anaplasma, Borrelia, flavivirus, and Babesia infection or are manipulated by pathogens to facilitate infection (Turell, 2007; Hajdušek et al., 2013; Mansfield et al., 2017, Figure 2). With respect to the tick innate immune response, A. phagocytophilum subverts tick RNAi by mechanisms other than reduction of Tudor staphylococcal nuclease (Tudor-SN) levels to preserve a protein that is important for tick feeding (Ayllón et al., 2015b). In contrast, Subolesin (SUB), also involved in tick innate immune response for limiting pathogen infection (Naranjo et al., 2013; de la Fuente and Contreras, 2015), is not manipulated by A. phagocytophilum. SUB has been shown to be required for tick feeding and reproduction and for pathogen infection, and therefore the preservation of this protein is important for both tick and pathogen survival (de la Fuente and Contreras, 2015). In I. scapularis, the x-linked inhibitor of apoptosis protein (XIAP) interacts with the E2 conjugating enzyme Bendless affecting positive and negative regulators of the immune deficiency (IMD) pathway resulting in protection against infection by A. phagocytophilum (Severo et al., 2013).

After molting, tick nymphs attach and start feeding, displaying an altered midgut transcriptome when infected with B. burgdorferi (Rudenko et al., 2005). Some of the genes affected by infection include innate immune factors (defensin and thioredoxin peroxidase) that possibly limit tick Borrelia infection. Tick salivary protein 20 (Salp20) belongs to a protein family with complement-inhibitory activity that blocks the host alternative complement pathway and assists in Borrelia transmission (Hourcade et al., 2016). Tick salivary lectin pathway inhibitor (TSLPI) inhibits the human lectin complement pathway by interfering with the mannose binding lectin activity and enables transmission of Borrelia by protecting it from complement-mediated killing (Schuijt et al., 2011b; Wagemakers et al., 2016). Recently, Smith et al. (2016) showed that I. scapularis respond to interferon gamma acquired in the blood meal when parasitizing on B. burgdorferi-infected mice, leading to the up-regulation of the Rho-like GTPase and induction of antimicrobial peptides to inhibit pathogen infection.

Preliminary studies focusing on transcriptomic changes induced by TBEV infection of I. scapularis and I. ricinus cells have revealed the role of particular proteins within tick innate immune pathways that act to control infection (Weisheit et al., 2015). A similar approach has identified this response in tick cells infected with LIV and TBEV, with a range of transcripts being up and down-regulated (Weisheit et al., 2015; Mansfield et al., 2017). Flavivirus infection also induced transcripts associated with activation of innate immune pathways in tick cells, including JAK/STAT and Mitogen-activated protein kinase (MAPK) pathways (Mansfield et al., 2017), with additional up-regulation of genes with host resistance functions, including FK506 binding protein (FKBP) and the antiviral helicase Slh1 (Mansfield et al., 2017, Figure 2C). CCHFV is capable of evading the tick innate immune response. Following intracoelomic CCHFV inoculation, virus titers in male and female ticks are the same and infection rates and titers in salivary glands, ovaries, and testes increase upon blood feeding (Dickson and Turell, 1992). Therefore, viral replication in tissues associated with possible CCHFV transmission in infected ticks may be stimulated by attachment and feeding on susceptible hosts. This might reduce the stress induced by viral replication while ticks are waiting to find a vertebrate host, but increase the potential for viral transmission once the host is infested (Turell, 2007).

Using different methodologies, some molecules have been identified as being implicated in tick-Babesia interactions (Hajdušek et al., 2013). Genes involved in immunity, stress, and defense responses showed up-regulation in response to B. bovis infection (Heekin et al., 2012), while genes encoding for calreticulin, kunitz-type serine protease inhibitors and microplusin which exhibits antimicrobial activity, were differentially expressed in B. bovis/B. bigemina infected Rhipicephalus ticks (Rachinsky et al., 2007; Antunes et al., 2012; Heekin et al., 2013; Lu et al., 2016). Tick SUB (Almazán et al., 2005) was shown to be up-regulated in B. microti inoculated intrahemocoelically into Rhipicephalus haemaphysaloides (Lu et al., 2016) and B. bigemina-infected R. microplus (Merino et al., 2013) (Figure 2D). The putative role of SUB in B. bigemina infection in ticks was supported by showing a decrease in pathogen levels in ticks fed on cattle immunized with recombinant SUB (Merino et al., 2013).

Intracellular pathogens manipulate the transcriptional programs of their host cells via epigenetic mechanisms, leading to stress, and inflammatory responses (Gómez-Díaz et al., 2012). Recently, A. phagocytophilum was shown to manipulate tick cell epigenetics to increase the levels of the histone modifying enzymes (HMEs), histone acetyltransferases (HATs; 300/CBP), and histone deacetylases (HDACs and Sirtuins) resulting in the inhibition of cell apoptosis to facilitate pathogen infection and multiplication (Cabezas-Cruz et al., 2016). The results of this study suggested that a compensatory mechanism might exist by which A. phagocytophilum differentially manipulates tick HMEs to regulate transcription and apoptosis in a tissue-specific manner to facilitate infection but preserving tick fitness to guarantee survival of both pathogens and ticks (Cabezas-Cruz et al., 2016). As previously discussed (Cabezas-Cruz et al., 2016), the mechanisms by which A. phagocytophilum affects tick cell epigenetics is unknown but effector proteins such as AnkA, secreted through T4SS or other secretion mechanisms probably control it (Garcia-Garcia et al., 2009a,b; Rennoll-Bankert et al., 2015). It has been previously demonstrated that A. phagocytophilum AnkA recruits host histone deacetylase 1 (HDAC1) and modifies neutrophils gene expression (Garcia-Garcia et al., 2009a,b; Rennoll-Bankert et al., 2015). Interestingly, the homolog of HDAC1 in I. scapularis was overrepresented upon A. phagocytophilum infection in tick salivary glands (Cabezas-Cruz et al., 2016). It remains to be tested whether A. phagocytophilum AnkA plays the same role in ticks as in vertebrate neutrophils.

The characterization of I. scapularis-A. phagocytophilum molecular interactions revealed complex responses by both ticks and pathogens that were necessary for maintenance of tick health while ensuring robust vector capacity (Ayllón et al., 2015a; Villar et al., 2015a; Gulia-Nuss et al., 2016; de la Fuente et al., 2016). Several lines of evidence suggest that tick-pathogen associations evolved to form “intimate epigenetic relationships” that have the potential to increase tick fitness (Cabezas-Cruz et al., 2017). At the tick-pathogen interface, A. phagocytophilum induces an antifreeze glycoprotein (IAFGP) and heat shock proteins (HSPs) to increase tick survival and feeding fitness (Neelakanta et al., 2010; Busby et al., 2012). Neelakanta et al. (2010) demonstrated that I. scapularis ticks infected with A. phagocytophilum show enhanced fitness against freezing injury due to the induced expression of IAFGP. They further showed that improved survival of infected ticks correlated with higher bacterial infection, therefore providing a direct link between pathogen infection and tick fitness in unfavorable ecological conditions. The fact that Borrelia and TBEV-infected ticks choose a higher questing height suggests that these pathogens help ticks to survive under dry conditions. In agreement with this hypothesis, I. ricinus infected by B. burgdorferi move less toward a humid environment and their survival is higher in highly desiccating conditions (Hermann and Gern, 2010; Herrmann and Gern, 2012). The tick histamine release factor (tHRF), up-regulated in B. burgdorferi-infected I. scapularis during feeding, facilitates tick engorgement and B. burgdorferi infection by increasing the blood flow to the tick-bite site and modulating vascular permeability (Dai et al., 2010).

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.