The Apicomplexan parasites represent a major class of pathogens with a wide host range. They have emerged as one of the most successful intracellular parasites, which efficiently modulate the host for their survival benefits. In this review, we focus on the potential sabotage mechanisms adopted by the five well-studied pathogens of human and veterinary importance: Plasmodium falciparum (malaria), Babesia bovis (babesiosis), Theileria annulata (theileriosis), Toxoplasma gondii (toxoplasmosis), and Cryptosporidium parvum (cryptosporidiosis). These parasites are morphologically similar; however, variations exist in the context of host range, mode of infection, invasion, and replication inside the host (Table 1).

Beginning with transmission, P. falciparum, T. annulata, and B. bovis are vector borne; however, C. parvum and T. gondii do not require a vector and the host is infected by oocyst-ingestion (Table 1). T. annulata solely infects animals impacting their health and causing huge economic loss, whereas other parasites have broader host preference range. P. falciparum and T. gondii infections affect human health and cause mortality worldwide. On the other hand, B. bovis and C. parvum are comparatively less pathogenic with fewer reported cases of mortality and morbidity.

In this review, we epitomize the major blueprint of the pathways targeted by these parasites to sabotage the host defense mechanism for their survival and consequent disease progression.

Plasmodium falciparum is considered the most lethal among the Plasmodium species, as it accounts for serious illness and high mortality (1–5). Two hundred fourteen million new cases of malaria are reported worldwide with a 35% mortality rate reported for children below 5 years of age (6).

Malaria transmission cycle starts with the female Anopheles feeding on a mammalian host. Thereafter, Plasmodium smartly exploits host cell machinery in numerous ways discussed hereafter to complete its life cycle (7–9). The sporozoites harbored in the salivary gland enter the host blood stream and passes on to the hepatic sinusoid (10–12). The presence of antihistamines and immunomodulators in the salivary gland secretion protects Plasmodium from the initial host immune response (10, 11, 13). The endothelial cell lining the liver sinusoid, guarded by kupffer cells (liver macrophages) prevents sporozoite entry into the hepatocytes (12, 14, 15). The circumsporozoite protein (CSP) of the parasite interacts with LRP-1 (low-density lipoprotein receptor-related protein) present on the kupffer cells thereby upregulating cAMP. Thereafter, cAMP mediates EPAC (exchange protein activated by cAMP) inhibition of reactive oxygen species (ROS) production ultimately suppressing the macrophage defense (15–17) (Figure 1). Simultaneously, the expression of TNFα, IL-6, and monocyte chemoattractant protein-1 (MCP-1) is downregulated and there is an increased production of anti-inflammatory IL-10 cytokine (15). The sporozoite also downregulates expression of kupffer cells MHC-1 and IL-12 to overturn their antigen presenting ability and ease infiltration of sporozoites into hepatocytes (15, 17). All these events result in the successful invasion.

Furthermore, the role of calcium (Ca²⁺) in activating various parasite proteins involved in the process of invasion, egress, motility, and cell cycle regulation has been observed (18–21). In Plasmodium, endoplasmic reticulum and acidocalcisomes are the major Ca²⁺ reservoirs which are also observed in Toxoplasma. The activation of protein kinase G (PKG) by an unknown parasitic signal during invasion or egress releases Ca²⁺ from the parasite endoplasmic reticulum mediated by cyclic guanosine monophosphate (cGMP) (20). Furthermore, phosphoinositide phospholipase C (PI-PLC) is activated by cGMP-dependent PKG which results in hydrolysis of phosphatidylinositol 4, 5-bisphosphate (PIP2) to diacylglycerol (DAG) and inositol 1,4,5-trisphosphate (IP3). The translocation of IP3 on to the ER surface causes efflux of Ca²⁺ to the cytoplasm by the formation of IP3-Ca²⁺ channel (20, 22) (Figure 1). The increase in the cytoplasmic Ca²⁺ levels activates various calcium-dependent proteases and kinases, like calcium-dependent protein kinases (PfCDPK), double C2 domain protein (PfDOC2) which induce the secretion of microneme and rhoptry proteins for cell adherence and invasion (20, 23, 24). The hepatocyte invasion of the sporozoites occurs via Ca²⁺-mediated activation and secretion of microneme proteins, CSP and thrombospondin-related adhesion protein (TRAP) such as Trap-like protein (TLP) (12, 15, 25) (Figure 1). The CSP secreted to the apex in association with actin covers the surface of the sporozoites and its glycosyl phosphatidyl inositol (GPI) anchored C terminus helps in the invasion of sporozoites (12, 26, 27). PfTRAP (TLP) protein interaction with actomyosin motor complex helps in gliding movement of the parasite (15, 25, 28).

The transcellular migration by sporozoites is mediated by the secretion of perforin protein SPECT or perforin like protein1 (PfPLP1), which is also demonstrated to be important in cell traversal, to perforate the hepatocytes (12, 29). Hepatocytic growth factor (HGF) is released by the perforated hepatocyte (30, 31), which activates c-MET receptor tyrosine kinase (c-MET RTK) on them resulting in the activation of tyrosine residues at the cytoplasmic domain of the c-MET receptor (32, 33). This recruits phosphoinositide 3-kinase (PI3-K) which phosphorylates and sequesters proapoptotic proteins of the BCL-2 family (Bad, Bim, PUMA) through AKT (32–35). AKT, which activates anti-apoptotic proteins (BCl-2, BCL-XL, A1), inhibits Bax on the outer mitochondrial membrane and hinders the permeabilization of the mitochondrial membrane and the subsequent release of proapoptotic signaling molecule such as cytochrome-c (Cyt-c) and eventually blocks apoptosis (Figure 2) (12, 27, 32, 33).

Once the parasite has already invaded the hepatocyte, host cell apoptosis block is independent of the PI3-K pathway. It seems that the direct intervention of parasite proteins is necessary for modulating the host survival signal (36). One such example is hypoxia mediated by host-dependent HIF-α through AMPK activation which promotes proliferation and parasite survival in the liver (37). The presence of the autophagy marker Atg8 on P. falciparum might suggest the parasite’s involving degradative functions, but it instead majorly contributes toward biogenic process (38, 39). After the exoerythrocytic merogony, they trigger apoptosis, although this does not seem to occur by activating the caspase-dependent pathway nor via the expression of phosphatidylserine (36, 40). However, a serine-repeated antigen (SERA), a cysteine protease identified in Plasmodium berghei, is upregulated and is reported to be playing a role in parasite-induced cell death, parasitophorous vacuole (PV) disruption and merosome formation at the time of exoerythrocytic merozoite egress (12). It suggests that the parasite secretory proteins are mediators of host cell apoptosis in the late liver stage. Among seven calcium-dependent protein kinases (CDPK1–7) known in Plasmodium, inhibition of PfCDPK5 leads to schizont stage arrest (20).

Erythrocyte invasion of Plasmodium occurs in two stages. First, the interaction of merozoite with the erythrocyte causes host cytoskeletal distortion with the help of high Ca²⁺ level, increasing the contact area between the two favoring merozoite entry and the alignment of its apical pole (14, 19, 20). The role of calcineurin (CnA, CnB) has been implicated in merozoite attachment to erythrocytes, which when knocked down results in impaired invasion (20, 41). Furthermore, DOC2 activation induces microneme secretion of erythrocyte binding antigen (EBA175) and AMA1 (microneme apical antigen1), which are involved in the attachment (20, 42) (Figure 1). At the second stage of invasion, AMA1, in association with RON2 (rhoptry neck protein), binds to the erythrocyte ligand resulting in the formation of tight junction via TRAP (43). It has also been observed that the localization of formin at the apical pole nucleates the parasite F-actin with its FH2 domain and helps in parasite motility (44). The forward propulsion of the actin-myosin filament helps in the invasion of merozoite and encapsulation into the PV in the host cytoplasm (14, 45–47). Plasmodium invasion entails increased erythrocyte membrane permeability in order to gain nutrients from the extracellular fluid for its survival (45, 48) and further, utilizes the NF-kB-dependent pathway to inhibit host cell apoptosis (49). Following the invasion, the parasite secretes proteins essential for survival, cell adhesion, and pathogenicity. These are transported from the cytosol to the plasma membrane through vesicular transport. The interplay of protein export elements (PEXEL) and Plasmodium translocon of exported proteins (PTEX), cause the exported proteins to be transported to the plasma membrane of the RBC (50–52).

Cell cytolysis and infecting new cells are an essential strategy associated with disease progression in Plasmodium. Parasite egress is a tightly regulated proteolytic activity of parasite proteases PfSUB1 and dipeptidyl peptidase 3 (DPAP3) in response to increased Ca²⁺ level in the cytosol of the parasite. The PfSUB1, an exoneme secreted protease acts on the PV and the plasma membrane of RBC causing cell rupture and egress (53, 54). Millholland et al. 2013 showed the role of host calpains in cytolysis. Krebs cycle intermediates, formed during replication, act through host GPCR, diffused from host plasma membrane to the PV, also help in cytolysis. The ligand activation of GPCR signal through Gαq activates PKC via PLC activation. PKC phosphorylates and liberates adducin (a protein that maintains cytoskeletal integrity) resulting in host cell cytoskeletal deformation, which in turn opens the TRPC6 cation channel on the plasma membrane of RBC. The influx of Ca²⁺ from the extracellular fluid activates CaMK (Ca²⁺/calmodulin dependent kinase) via calcium-dependent calmodulin, which phosphorylates host cytoskeletal substrates also resulting in a rapid influx of Ca²⁺. This Ca²⁺ activates and releases host calpain, which causes lysis and dissolution of host cytoskeleton facilitating parasite release (55, 56). The modulation of host survival signaling by Plasmodium allows them to successfully establish a specific environment where they can proliferate and differentiate leading to pathogenesis.

Despite substantial progress in the malaria research, restraining the disease still remains a challenge. P falciparum parasites owing to their multiple forms/stages, antigenic polymorphisms and AT-rich genome have further impended the problem. Current chemotherapy is based on using artemisinin and artemisinin-based combination therapies (ACTs), however, reports of drug resistance have already emerged. An effective vaccine should be the ultimate goal for long-term control of the disease. To date only the RTS, S/AS01 vaccine, targeting the CSP 178 of P. falciparum has reached phase three trials, but has not shown much efficacy (57). There are still many gaps in the understanding of the invasion process of P. falciparum, such as which molecules signal the release of Ca²⁺, which leads to adherence and invasion by activating many pathways. These pathways playing role in Ca²⁺ release can be targeted for identifying the novel antigens for developing future vaccines and therapeutics.

Babesiosis is a hemolytic disease prevalent in tropical and subtropical parts of the world with a broad host range. B. microti and B. divergens infect humans and have emerged as a public health concern predominantly in the United States and Europe, respectively (58). Human cases of babesiosis have surfaced recently in Asian countries, including India and Korea (59). B. bovis and B. bigemina both infect cattle but, however, higher morbidity and mortality are associated with B. bovis resulting in a huge economic loss (60). There are very limited studies investigating the parasite and its host interactions as compared to the other apicomplexan parasites. Owing to the striking similarity between Babesia and Plasmodium (61), the disease pathogenesis is considered to be similar to malaria during infection in cattle (60).

Babesia multiplies in the host erythrocyte in a similar fashion to Plasmodium, and they are transovarially transmitted in the vector except for B. microti (Table 1) (62). The life cycle of the parasite begins with tick feeding on host blood and simultaneously releasing sporozoites into the host bloodstream (63). Invasion occurs in a similar fashion as observed for Plasmodium, Toxoplasma, and Cryptosporidium via gliding mechanism using microneme and rhoptry secretions (61, 64). Parasite loosely attaches to the surface of the host RBC by its surface GPI anchored proteins and within the apical secretory organelles. Variant erythrocyte surface antigen 1 (VESA1), a heterodimeric protein of Babesia is known to play role in cytoadherence to the host erythrocyte surface (62, 65). After entry of the sporozoites into the red blood cells, they divide by binary fission and produce merozoites. Erythrocyte lysis further allows each merozoite to invade a new RBC and successive merogonies follow (62, 64, 66, 67). Merozoites interact with the RBC surface receptors and play a major role in invasion.

In Babesia, the role of Ca²⁺ has been primarily described in invasion and egress mechanism of the parasite, however, the modulation of the host signaling pathways are not thoroughly understood. In B. bovis, Ca²⁺-dependent protein kinase inhibitor showed growth limiting effects (68), though, in B. divergens, it impacted egress of the merozoites from erythrocytes (69, 70). Since there is no PV formation in Babesia, less Ca²⁺ is released during egress as compared to P. falciparum parasites (Table 2) (70, 71).

Transovarial transmissions in tick vector and straight entry of sporozoites into erythrocytes are some key features, which make Babesia parasites distinct from Plasmodium or Theileria parasites. Few studies to understand the mechanism of disease pathogenesis during Babesia infections have been published. Also, the mechanism of entry and transmission of the parasites are poorly defined. It will be important to investigate the parasite invasion and evasion strategies along with parasite vector interactions for identifying key genes that might play an important role in immune evasion or disease pathogenesis.

Theileria annulata and T. parva cause tropical theileriosis and east coast fever, respectively, in ruminants predominantly in cattle causing enormous economic loss to the livestock industry (106, 107). Tick vector transmits the parasite upon feeding on animal through the saliva (108–110). After entering the blood stream, it infects WBC of different lineage, T. parva infects B cells and T cells whereas T. annulata infects B cells and cells of monocyte lineage. The sporozoites, i.e., the infective stage of the parasite passively invade the host cell by zippering mechanism, which is unlike other apicomplexan discussed in which a tight continuous junction is formed between the host cell surface and the parasite sporozoites (108). In addition, the role of MHC class I molecule (Figure 1) (111), intrasporozoite Ca²⁺ and protein kinases of host and parasite and the G-protein linked signaling has been shown in invasion (75, 112). After entry into the host cell, parasite rhoptries and its microsphere discharges dissolve the enveloping PV membrane (108), and move to the host cell cytoplasm rather than to PV in comparison to other apicomplexan parasites and provides it with an advantage of escaping lysosomal degradation (Table 1). Additional advantage of staying in the host cytoplasm allows the parasite to modulate several signaling pathways, such as TGF-β, JNK, PI3-K, NF-κB, src kinase, and casein kinase 2 (CK2) (80, 100, 113–115).

Theileria transforms their host cell into a cell with a cancerous phenotype by modulating several host cell kinases and activating transcription factors (116). Several studies have been done to identify parasite protein instigating epigenetic changes that may lead to successful transformation. T. annulata protein, TaPIN (secretory prolyl isomerase Pin) has been reported to promote transformation by degrading FBW7, a host ubiquitin ligase via stabilizing c-JUN (117). p104 and TaSP are surface proteins which have been reported to be phosphorylated in a host cell cycle-dependent manner and might be involved in transformation (118). Two more proteins are TashAT group of protein, which contains AT hook DNA-binding motif and nuclear localization signal and found to be localized in host nucleus (119). SuAT1, a parasite gene, contains AT hook DNA-binding polypeptide and predicted signal peptide, PEST motifs and nuclear localization signals, which may interact with the host cell and play a role in transformation (120). Studies to identify epigenetics changes are scarce, only one study has shown the role of oncomiR mir155 in repressing DET1 protein (involve in c-Jun ubiquitination) and stabilizing c-Jun (121). The parasite schizont hijacks the host mitotic assembly resulting in its clonal expansion (122–124).

Theileria transformed cells can be reversed, unlike tumor cells upon treatment with BW720c (122). The transformation occurs by modulating several signaling pathways which ultimately inhibits apoptosis, increases proliferation, and encourages metastatis (116). NF-κB is constitutively expressed in Theileria infected cells, which in turn upregulates many anti-apoptotic proteins, such as c-FLIP, IAPs, Bcl-2, and Bcl-XL, and induces Gadd45β that blocks the pro-JNKK2-mediated apoptotic JNK pathway. NF-κB is activated by recruitment and phosphorylation of IKK signalosome α and β subunits, which further phosphorylates inhibitory κB (iκB) setting NF-κB free to translocate to the nucleus (125). Infected cells release a plethora of cytokines and growth factors that activate TGF-β receptor (I and II) and TNF-α receptor (126). TGF-β2 activates smad2/3 and subsequently smad4, which over-expresses COX-2 resulting in increased levels of prostaglandins and downregulates PKIG, a potent inhibitor of PKA pathway. Simultaneously, TGF-β2 is accounted for parasite motility and invasiveness by activating Rho–ROCK kinase and recruiting an adaptor protein growth factor receptor-bound protein 2 (Grb2) to TGF-RII receptor. The signaling descends by Grb2, activating downstream PI3-K/AKT and JNK pathway (80, 127). Activator protein 1 (AP1), a JNK activated transcription factor drives B cell integration cluster (BIC) transcription upregulating miRNA 155 which inhibits DET1 resulting in accumulation of c-Jun and increased proliferation (89, 121, 128–131). B-1 a bovine analog of MMP9 (ECM degrading proteinase) containing AP1 binding sites also helps in detachment and metastasis of the infected cell to other organs (89, 132, 133).

Theileria modulates the host PI3-K/AKT pathway to be regulated by granulocyte-macrophage colony-stimulating factor (GM-CSF), depending on an autocrine loop and, hence, sharing a major role in cell proliferation (134, 135). Phosphorylation of the AKT protein by class I PI3-K facilitates the release of Rb bound E2F transcription factor, activating MDM2 (E3 protein ubiquitin ligase and negative regulator of p53 tumor suppressor gene) directly or through mammalian target of rapamycin (mTOR). AKT-mediated inhibition of several proapoptotic genes (bad, foxo) and GSK-3β help the infected cells to combat the stress-induced mitochondrial-mediated apoptosis and to maintain elevated c-Myc levels, respectively (Table 2; Figure 2) (136). Hypoxia-inducing factor (HIF-1α) is activated by mTOR pathway and by constitutive NF-κB and AP1 production (137). Increased levels of ROS during infection and HIF-1 expression induce the Warburg effect allowing the parasite to establish uncontrolled proliferation (96, 103, 138, 139). PI3-K/AKT pathway, therefore, plays a very important role in survival and proliferation as well as in metastasis of Theileria. PTEN, an inhibitor of the PI3-K pathway and activator of p53 are suppressed majorly by NF-κB and CK2 (82, 140, 141). CK2 also dampens TNF/Fas-mediated apoptosis and accelerates iκB degradation augmenting NF-κB activation. Activation of JAK/STAT pathway via GM-CSF autocrine loop enhances c-myc expression, whereas phosphorylation by CK2 stabilizes this potent oncogene primarily upregulated in Theileria infected cells (Figure 2) (82, 115).

The proliferation of infected cells is followed by evasion and metastasis, which involves cytoskeleton alteration. TNF-α binding to its receptor recruits TNF receptor-associated factor 2 (TRAF2), which may either activates NF-κB or a mitogen-activated protein kinase, MAP4K4 (134). MAP4K4 bifurcates into JNK activation and ERM (ezrin/radixin/moesin) cytoskeletal protein phosphorylation, helping in actin dynamics through Rho kinase. ERM may also be activated through src kinases (72). Interfering with autophagic mechanisms also augments survival of the parasite within the cell. Dampening peptide presentation by CD4⁺ and CD8⁺ infected cells is a way opted by the parasite to increase its chances of survival enhancing the establishment of infection (94). In contrast to other apicomplexan parasites, Theileria usually does not egress and remains attached to the host mitotic assembly. Although under unfavorable circumstances the schizont ruptures releasing merozoites, which invade RBCs, forming piroplasm, which is taken up by the tick and the cycle resumes. Theileria parasites have thereby evolved a wide range of strategies to help them survive and proliferate inside the host cells.

Theileria parasites are considered as the smartest among the apicomplexan group for their ability to manipulate the host cells. However, the parasite proteins and molecular mechanisms behind the host cell manipulation are still not clear. Very few proteins are identified till now, which are involved in the host–parasite interaction. The presence of a heterogeneous population of T. annulata parasite strains in the field is making it difficult for a currently used attenuated vaccine (schizont stage) to be effective against T. annulata parasites. Resistance against burpaquone, a drug currently being used for the treatment of theileriosis, and acaricide, used for controlling the tick vector, has hampered the control of the disease. In this post genomic era with the availability of advanced genomic and proteomic tools, better studies are needed to dissect the pathways modulated by Theileria in detail and select new targets for the development of second-generation drugs and vaccines for disease control.

A substantial amount of research has been done to gain insights into pathways by which these parasites modulate and undermine the host defense, yet gaps in knowledge still prevail and many questions remain unanswered. In this review, we have attempted to include all the major work carried out in this field. Advancement in gene editing technologies and whole genome sequencing of these pathogens lead us to better understand the manipulation strategies used by the parasites. Emerging problems of either drug resistance or unavailability of an effective vaccine against some of the parasites make the precise comprehension of the sabotage techniques employed by the parasites a primary requisite in order to curb the morbidity rate.

As discussed above, it is apparent that few of the host defense pathways targeted by these parasites to survive and proliferate in the host cell are common among the mentioned pathogens. Host cell invasion by Plasmodium, Babesia, Toxoplasma, and Cryptosporidium occur via gliding movement, whereas in Theileria an overall different process known as zippering takes place. Plasmodium, Toxoplasma, and Cryptosporidium reside and replicate inside the PV in the host, however, Theileria and Babesia survives in the host cytoplasm. Theileria clearly takes an advantage of staying in the host cytoplasm by modulating numerous pathways, though any such information about Babesia has to be still investigated. Several pathways are modulated by majority of these parasites such as host cytoskeleton remodeling, Ca²⁺⁺ modulated signaling pathways, and apoptotic pathways which helps in their survival. In the review, we have discussed the cross talks happening between the parasite and the host and observed that the multifaceted nature of the parasite gives them an upper hand over the host.

Future studies focusing on (i)

Exploring the parasite proteins and their role in host–parasite interface interaction will provide in-depth understanding of the invasion process. These targets can be further utilized to develop vaccine or drugs.

Therefore, targeting the common pathways playing crucial role in all parasites survival and dissemination may be a good approach to understand disease pathogenesis and controlling the disease.

In order to deal with these cunning pathogens, we need all the necessary information to be able to target important molecules for a vaccine or drug development. However, a considerable amount of research and thorough screening of presently available literature is still required to better understand how these parasites exploit their hosts for their own survival. Apicomplexan parasites infecting human beings such as Plasmodium is hugely funded and globally studied; however, so is not the case when it comes to parasites such as Babesia and Theileria, which are of veterinary importance. Therefore, for more in-depth understanding of these pathogens, tenacious research is expected which would only be possible through the combined efforts of researchers and support from funding agencies on a global range.

All authors mentioned have made a significant effort and contributed intellectually to the work and approved it for publication.

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.