The complete proteome-based phylogenetic analysis of Dictyostelium, Acanthamoeba, and Entamoeba shows that animals and fungi are close to amoebozoa group (Song et al., 2005). Nevertheless, amoebozoa are distinct from early diverging unicellular eukaryotes, Leishmania, Trypanosoma, Plasmodium, Giardia, and plant as well. The more remarkable similarities despite their early divergence in amoebozoa and metazoan proteins translate into a generally higher degree of functional conservation between them. The universal domain architectures aid in delineating and organizing the proteins in their families and participating in particular cellular pathways in lower eukaryotes.

The conserved sequence-based search identifies CRIB/PBD/GBD containing proteins; ten in Dictyostelium discoideum, fourteen in Acanthamoeba castellani, nine in Entamoeba histolytica, only one protein in Trypanosoma cruzi, and Giardia lamblia. In humans, twenty-seven CRIB containing effector proteins have been identified and reported in earlier studies (Pirone et al., 2001) (Figure 1). The available functional, biophysical and biochemical characterization has been explored in detail to classify the identified CRIB containing effector proteins. Apart from the CRIB motif, the conserved structural features in identified proteins of Amoebozoa fall into PAK or PAK like kinase and actin-associated or actin assembly protein. In Acanthamoeba and Entamoeba, very few proteins have been studied earlier, on the other hand, a lot of literature is available for Dictyostelium proteins. However, in other parasite protozoans (Giardia and Trypanosoma), the single identified CRIB domain protein does not show any similarities with the conserved domains of Cdc42/Rac effector. The identified CRIB containing protein families indicated here are PAK/PAK related kinase and actin assembly protein families found conventionally during evolution, while Ser/Thr and cytosolic Tyrosine kinase, adaptor family proteins present in humans are non-conventional.

Dictyostelium discoideum, a soil-dwelling social amoeba, is a unicellular eukaryote that forms multicellular structure fruiting bodies under limiting nutrition conditions (Gaudet et al., 2008). The D. discoideum genome (∼34 Mb) entirely encrypts about 10,300 proteins, including numerous protein families; some are involved in fundamental processes like post-translational modification, secondary metabolism, and signal transduction belong to cellular activities like cell adhesion and cytoskeleton control (Kuspa and Loomis, 2006; Loomis, 2006). Numerous Dictyostelium proteins are more similar to human orthologs than yeast, probably due to higher evolutionary changes along the fungal lineage. The small, simple genome and complex transcriptome made it an easy-going prototypical organism to dissect the signaling pathways and their elements with typical relationships throughout the metazoans (Li and Purugganan, 2011; Bozzaro, 2019).

In this study, ten CRIB domain (or PBD/GBD) proteins were found fit functionally to be Cdc42/Rac effector proteins. Conserved sequence and structural features group them into p21-activated kinase (six protein), WASP family (three protein) and a novel gelsolin-related protein (Figure 2). However, a genomic study mentions that eight PAKs (PAKa-h) are present in D. discoideum (Arasada et al., 2006), with no structural and functional shreds of evidence support. In our study we found that PAKe (Q54B33) and PAKh (Q556S2) have a kinase domain but lack a consensus CRIB domain and other accessory domains present in human homologues. Thus, these two proteins may potentially be candidates of some other subfamily of Ser/Thr kinase that is a matter of further investigation or may be pseudo PAK like E. histolytica PAK1 (Labruyere et al., 2000; Labruyere et al., 2003). The novel gelsolin homolog identified here is encoded by the gnrC gene, categorically a putative actin-binding protein (Q551I6), which suggests its regulation by a small gtpase. This is the unique protein in Dictyostelium with two CRIB domains present consecutively at its N-terminal. However, no study reports any details regarding the Rac protein, which activates it.

Out of six PAK family members identified in the search, PAKa, PAKb/MIHCK, PAKc, and PAKd have some similar structural domains of human Group-I PAKs. PAKf (Q869T7) and PAKg (Q556S2) possess the conserved CRIB and kinase domain; typical structural features of PAK homologs of metazoans and yeast but need to be experimentally validated. One member has identical features to human WASP protein, while, the other two (WASP like-B (Q7KWP7) and WASP like-C (Q54QH4)) have comparable domain characteristics to human N-WASP derived as WASP-related protein (Figure 2).

In Dictyostelium, PAK and WASP are the CRIB-containing effectors of Rho family GTPases, which regulate chemotaxis, phagocytosis, and cytokinesis (Wilkins and Insall, 2001; Park et al., 2004). No clear homologues of Rho and Cdc42 are present, though 15 members of Rac have been reported in the Rho GTPase family (Rivero et al., 2001; Rivero and Somesh, 2002). RacB has been proposed as a functional equivalent to Cdc42 (Rivero et al., 2001). Additionally, a component of SCAR (suppressor of cAMP-receptor)/WAVE complex, four IQGAPs, ten formins, two PCH (full form) family, several lipid kinases/phospholipase and NADPH oxidase components are also present which represent CRIB-independent effectors of Rho family (Vlahou and Rivero, 2006). Experimental evidence remarkably suggested that a CRIB motif is present in coronin protein, which interacts with GDP bound gtpase (Swaminathan et al., 2014; Swaminathan et al., 2015). In general, coronin activates the actin nucleation factor Arp2/3 complex and IQGAPs (Shina and Noegel, 2008). The presence of the CRIB motif varied amongst lower and higher eukaryotes’ coronin proteins. Nevertheless, the sequence comparison of the conserved CRIB motif and the coronin CRIB region indicated moderate similarity while displaying Cdc42/Rac binding. Reasonable similarity can possibly explain that the region not annotated as CRIB in protein domain databases can contribute to the protein-protein interaction mechanism.

WASP family consist of one WASP, two WASP related and one WASP like (SCAR) subfamilies proteins, which are the positive regulators of Arp2/3 complex in actin polymerization (Seastone et al., 2001; Myers et al., 2005). WASP controls Arp2/3 complex spatially and temporally in D. discoideum via interaction of Rac with the CRIB motif. WASP, an actin nucleation-promoting factor, also functions as a controller of cellular localization of Rac, contributing to the maintenance of front-rear polarity (Amato et al., 2019). WASP-A encoded by wasA gene has a WH1 (WASP-homology 1) domain that interacts with poly-Proline helices (Myers et al., 2005). RacC works as a connection between WASP activation and chemo-attractant stimulation in the signaling pathway regulating F-actin assembly during chemotaxis (Han et al., 2006). WASP-B regulates F-actin polymerization through attenuation that is important for regulating the dynamics of pseudopod extension and retraction (Chung et al., 2013). However, SCAR subfamily proteins possess a C-terminal VCA domain akin to human WASP and N-WASP but lack an extended N-terminal WH1 region and GBD domains (Symons et al., 1996). The human homolog of SCAR is called WAVE. SCAR/WAVE is a multi-protein complex with PIR121, Nap125, Abi2, and HSp300 components, each encoded by a single gene present in the Dictyostelium genome (Blagg et al., 2003). WASP compensates for the loss of function of SCAR/WAVE proteins in Dictyostelium (Veltman et al., 2012).

PAK family has six proteins positioned in two different clades, classified in two separate classes; PAKa-d and PAKf/g, based on phylogenetic analysis of the catalytic kinase domain. The consequence of such distinction is not clear in Dictyostelium to humans who also have two PAK groups (Arasada et al., 2006). The class I PAKs in Dictyostelium functionally established to be involved in cell polarity, actin-myosin assemble and phagocytosis (Lee et al., 2004; Yang et al., 2013; Garcia et al., 2014; Phillips and Gomer, 2014). The six PAK isoforms share high sequence identity ∼50–70% in the catalytic kinase domain and PBD regions, while the rarely display any homology outside these regions. PAKa and PAKb, both of which have CRIB/AI domains that have been linked to myosin II regulation (de la Roche and Cote, 2001).

Dictyostelium PAKa possesses a potential poly-Proline tract for SH3 domain interaction, a highly acidic N-terminal domain followed by a CRIB and a C-terminal kinase domain. The CRIB/PBD domain preferentially interacts with DdRac1B and HsCdc42 and then translocate to myosin II filament to regulate myosin heavy chain kinases (MHCKs). PAKa inhibits MHCK-B, C, and D to stabilize myosin II assembly in response to upstream cAMP response. Despite dynamic subcellular localization, PAKa co-localizes with myosin in all the cell movement process (de la Roche and Cote, 2001). PAKa was also identified at the cytokinesis cleavage furrow, and localized to rear end of the polarized migrating cell, and posterior cortex during chemotaxis (Chung and Firtel, 1999; Muller-Taubenberger et al., 2002). The signaling cascades regulated by PAKa are also dependent on coronin (Swaminathan et al., 2015).

Dictyostelium PAKb consists of an N-terminal Proline-rich region, followed by a consensus PBD, a Glutamine and Asparagine residues rich linker, and a C-terminal kinase domain for catalysis. DdPAKb, previously termed as myosin I heavy chain kinase (MIHCK), was identified (Lee et al., 1996) through its phosphorylation and regulation of a single-headed myosin I (DdMyoD) (de la Roche and Cote, 2001). PAKb phosphorylates TEDS (Thr, Glu, Asp, or Ser residue) rule site located in the MyoD motor (de la Roche and Cote, 2001). Dictyostelium Rac1a/b/c, RacA (a RhoBTB protein), RacB, RacC and RacF1 activate PAKb to regulate myosin driven motility on actin. PAKb localizes in the cytosol and enriches at the leading edge of the cells during migration, macropinocytosis, and phagocytosis, sites that show prevalence of myosin I as well (de la Roche et al., 2005). Thus PAKb plays pivotal role in myosin I activation during these events. However, loss of function mutants’ have impaired functions dependent on myosin I (Wu et al., 1996). In contrast, constitutively active PAKb mutant increases the rate of myosin I dependent processes such as pinocytosis/phagocytosis and disrupts cytokinesis. The constitutively active and C-terminal truncated active PAKb shows localization at rear-end of the migrating cell and cleavage-furrow during cell division (Yang et al., 2013). The notable fact here is the opposite localization of PAKa and PAKb in migrating cells, one at the rear end and the other at the posterior end, respectively, suggests that the two proteins do not have an overlapping function. However, both PAKa and PAKb function synergistically during phagocytosis and not pinocytosis.

The structural features of PAKc include a PH domain followed by a CRIB and a kinase domain, with a C-terminal extension G_ßγ binding domain. PAKc PH domain, related to the fungal Cla4p-like PAKs, is alone responsible for the cytosolic localization (Phillips and Gomer, 2014). The PH plus CRIB domain exhibits weak membrane localization in response to chemo-attractant stimulation (Lee et al., 2004). PAKc is activated rapidly and transiently in response to chemo-attractant stimulation that enriches it at the plasma membrane. PAKc functions to inhibit lateral pseudopodia to restrict pseudopod formation to the plasma membrane facing the chemo-attractant source (Lee et al., 2004). PAKc CRIB domain preferentially binds to RacC GTP-bound form. The highly conserved Arginine 34 is required for inositol binding in the PH domain. The CRIB domain is strongly similar to human PAK1 consisting of overlapping CRIB and AID (auto-inhibitory domain). The C-terminal G_ßγ binding domain shows strong conservation of the C-terminal yeast Ste20, required for transient localization to the Dictyostelium plasma membrane (Lee et al., 2004).

PAKd contains an N-terminal CH domain and additional C1 domain upstream of the CRIB domain. PAKd was implicated in F-actin aggregation during developmental processes, and actin polymerization in response to stimulation by a chemo attractant (Phillips and Gomer, 2014). Upon cell starvation, PAKd moves to cellular extensions, suggesting its presence in the Golgi apparatus (Garcia et al., 2014). PAKd kinase activity is regulated through the binding of CRIB domain to activated Cdc42/Rac molecules.

Experimental records strongly confirm that Dictyostelium coronins (Coronin A and B) have a Rac activated CRIB motif (Uetrecht and Bear, 2006). None of the previous studies on CRIB containing proteins included coronin family under Cdc42/Rac effector proteins (Clemen et al., 2008). In D. discoideum CoroninA, residues 117–133 harbor the CRIB motif highly homologous to CRIB motifs of other conventional effector proteins. Structural characterization indicates that half of the CRIB motif lies on the solvent accessible face, while the other half is embedded inside. Coronin prefers the GDP form of GTPases for binding to the CRIB motif, which is interestingly exceptional to all other CRIB containing proteins. Analysis of coronin lacking mutants reveals its role in cell motility, phagocytosis and cytokinesis (Vinet et al., 2014). Coronin functions as a Rho protein GDP dissociation inhibitor (RhoGDI) that interacts with Rac GTPases in their inactive GDP bound form, thus preventing their availability to PAKs. It also interacts with PAKs (PAKa) directly to regulate their activity (Swaminathan et al., 2015).

Acanthamoeba is the solitary free-living soil amoeba that diverged earlier than other amoebozoans (Shabardina et al., 2018; Corsaro, 2020). The genome contains ∼15,455 coding genes and comparative genomic studies from other metazoans established many putative protein families who play an interpreting role of cytoskeleton machinery and signaling related to cell motility and cytokinesis (Clarke et al., 2013). The putative proteins involved in cytoskeleton regulation through small GTPases in downstream pathways are not explored thoroughly (Mullins and Pollard, 1999).

In silico search for the conserved CRIB domain retrieved fourteen proteins in Acanthamoeba castellanii (Figure 3). The only characterized protein in this repertoire is a Myosin I heavy chain kinase (MIHCK), belongs to the p21 activated kinase family (Brzeska et al., 1997). Including MIHCK (Q93107), seven proteins (L8GCL9, L8GPX2, L8GVV0, L8GW48, L8H707, L8HHB6, and L8HET0) have kinase domains homologous to human PAKs along with some variant domains. L8GPX2 shares a domain with myosin I heavy chain kinase, while, L8H707 and L8GW48 have substantial similarities with PAK proteins. L8HHB6 (39%) and L8GCL9 (42%) are potential Ser/Thr kinases that show moderate similarity with human PAK6 and can be categorized under the PAK family after experimental validation. L8HET0 kinase domain shows 24% similarity with HsMRCKβ but no other domains so it could be probable PAK family member. L8GVV0 shows 30% similarity with HsPAK3 can also be prospective PAK family member subjected to experimental backing. Two proteins (L8H2P8 and L8H6L6) with WH1 domain and strong homology with WASP proteins could be potential WASP proteins in Acanthamoeba. Apart from these two, another protein (L8H3X7) possesses WASP like domain characteristics/homology with WASP related proteins of metazoans. Two proteins (L8GKX1 and L8GYC7) composed of unique domains along with the CRIB domain, which does not have homology with any conventional/non-conventional proteins of higher eukaryotes, can be classified as p21 rho-binding domain-containing protein. We also found one protein (L8H695) with Leucine-rich repeats along with the CRIB domain, and have classified it as a unique group. Lastly, L8GFZ9 was with similar structural features to the coronin family, but the functional role and interactive GTPase molecule are yet to be explored. The extensive repertoire of CRIB containing proteins present in Acanthamoeba indicates and supports the ancient origin of the CRIB motif and its linkage to various effector proteins of Rho mediated signaling (see Supplementary Table S1). However, the relation with the Rho family of protein and detailed functional study will help link these proteins with cellular processes and their subcellular localization.

Acanthamoeba MIHCK, member of the PAK family, phosphorylates a heavy chain of myosin IC and activates it. MIHCK, from Dictyostelium, was validated as PAKb, which performs a similar function to phosphorylate and activate myosin I. AcMIHCK has a homologous region to human PAK1, PBD (Residue 93–149), which includes the CRIB motif (Residue 93–100) and as IS domain, but lacks the kinase inhibitory (KI) domain region. It also has a putative calmodulin-binding region at its N-terminal, before PBD (Brzeska et al., 2001). The typical C-terminal Ser/Thr kinase domain has the characteristic Ser-627 phosphorylation site. The region between PBD and catalytic domain (residue 158–449) is highly Proline-rich, including multiple PXXP motifs (class I) that provide potential binding to SH3 domains (Brzeska et al., 1996). Acanthamoeba MIHCK mechanism of regulation is quite similar to mammalian PAK1. Acanhtamoeba MIHCK is fully phosphorylated (Brzeska et al., 1990a; Brzeska et al., 1999), while Dictyostelium MIHCK is partially activated in vitro by autophosphorylation in the presence of Rac and lipids (Lee et al., 1998). Human PAK1 also requires Rac or lipids for autophosphorylation (Manser et al., 1994). Calcium-dependent calmodulin inhibits phospholipids’ stimulation in Acanthamoeba MIHCK (Brzeska et al., 1992) and Dictyostelium MIHCK/PAKb (Lee et al., 1998) but is not required for mammalian PAKs. Also, the lipid that activates mammalian PAK1 (Bokoch et al., 1998) differs from those that activate Acanthamoeba and Dictyostelium proteins (Brzeska et al., 1990a; Brzeska et al., 1990b).

Entamoeba histolytica is a primitive unicellular eukaryote and amitrochondrian protozoan parasite, which causes dysentery and liver abscess. Amoebic pathogenicity is selected coincidently in the lumen of the intestine because the parasite uses the same methods to kill bacteria or cause disease by damaging the host cells (Bosch and Siderovski, 2015). Amoebic phagocytosis and its mechanism show similarities with the action of macrophages during the phagocytosis of bacteria and unwanted cells, which supports the idea of coincidental selection (Ghosh and Samuelson, 1997; Labruyere et al., 2019). The parasite uses anterior pseudopods and posterior uroids to move inside the human intestine. The host complement system, lectin ConA (multivalent), and anti-amoeba antibodies target the invading amoebic cell, initiating the formation of cap by rearward recruitment of surface receptors and increasing the local receptor-ligand concentration (Calderon, 1980). The defense mechanism of E. histolytica against host immune response includes surface receptor capping in the uroids and membrane shedding (Espinosa-Cantellano and Martinez-Palomo, 1994). E. histolytica cytoskeleton functions actively in the capping process (Espinosa-Cantellano and Martinez-Palomo, 1994; Rath and Gourinath, 2020). The invasion and survival inside the host tissue are maintained through the phagocytosis of RBC, lumen cells and surrounding cells. It has been demonstrated the role of cytoskeleton assembly in the Entamoeba and a human macrophage (Marchat et al., 2020). Phagocytosis is a dynamic and regulated process that involved a varsity of proteins ranging from actin-binding protein, motor protein, small GTPases, kinases and phosphatases (Godbold and Mann, 1998; Marquay Markiewicz et al., 2011; Anwar and Gourinath, 2016; Gautam et al., 2017; Agarwal et al., 2019; Gautam et al., 2019). ∼20 Rho family GTPases and numerous downstream signaling effectors are present in these single-celled trophozoites that coordinate actin dynamics in pathogenesis-related processes. Hence, cell migration and chemotaxis, followed by adherence to the epithelium in the host intestine, and host cell killing and phagocytosis are all regulated by Rho family signaling toolkit (Bosch and Siderovski, 2013).

The domain-based search retrieved a total of nine CRIB domain-containing proteins (Figure 4). The homology with metazoans proteins classifies them under conventional effector proteins of Rac/Cdc42. Six (PAK2-7) and one pseudo-PAK (PAK1) belong to the p21-activated kinase with their typical PBD and kinase domain architecture. However, an earlier study shows that one isoform of PAK lacks CRIB/PBD domain in N-terminal but has C-terminal kinase domain homologous to yeast ste20 (Gangopadhyay et al., 1997). Earlier kinome study (Anamika et al., 2008) predicted 17 homologous PAKs in the E. histolytica genome. Only with the six homologues match the typical PAK features, and thus, it is now confirmed that Entamoeba possesses only six PAK isoforms, including one pseudo-PAK. Interestingly, out of six, three PAKs (PAK2, PAK3 and PAK5) have additional PH domains (Figure 4). Three proteins (C4M2L6, C4LZJ6, C4M0R3) have a domain that is homologous to C-terminal of human WASP, but no experimental studies have so far been performed to characterize its structure and function (Figure 4). The Arp2/3 complex nucleates new actin filaments, when activated by nucleation promoting factors like WASP or SCAR. No available research yet describes any WASP or SCAR protein in Entamoeba, hence, the actin nucleation activity may be regulated by other unidentified proteins. Exploring the predicted putative WASP here might reveal its role in actin nucleation and regulation of actin cytoskeleton. However, CARMIL protein binds Arp2/3 complex with an exact mechanism used by WASP via its acidic motif. CARMIL homologues have been discovered through proteomic analysis of the phagosome (Okada et al., 2005; Clark et al., 2007; Tolstrup et al., 2007) and they further provide essential clues for understanding actin nucleation. A recent in silico study on actin-binding proteins (Rath and Gourinath, 2020) from our lab shows that E. histolytica harbors three WASH (Wiskoit-Aldrich syndrome protein and SCAR homolog) proteins C4MBT4, C4LTV1 and C4M2Y0. Additionally, E. histolytica encodes six formin genes that accelerate actin filament assembly in eukaryotic cells, however, no IQGAPs are reported yet. Three proteins (C4M137, C4M943, C4M5U0), which have similar structural features to the coronin protein family, are also present in Entamoeba (see Supplementary Table S1) but the CRIB motif is not yet defined it them like Dictyostelium. Out of the seven PAK family members, the detailed account of experimental evidence is available only for five; PAK6 and PAK7 have not been characterized yet (see Supplementary Table S1).

Entamoeba PAK (PAK1) is a pseudo-PAK because it lacks the consensus CRIB motif (or PBD); still, it interacts with EhRac1 via the N-terminal region, suggesting that it is a regulatory domain necessary for the maintenance of cell polarity (Labruyere et al., 2003). Migrating trophozoites display PAK1 at their leading edge, where it functions in amoeboid cellular polarity and motility, along with human red blood cell phagocytosis (Labruyere et al., 2000). The protein shares 33% identity with rat KPAK and yeast STE20 (Gangopadhyay et al., 1997). The Proline-rich N-terminal domain can potentially bind to SH3 domains of adapters like Nck (non-catalytic region of Tyrosine kinase adapter protein) or PIX (PAK Interacting eXchange factor) (Galisteo et al., 1996; Manser et al., 1998). Constitutive EhPAK expression alters the new adhesion site formation in E. histolytica (Labruyere et al., 2003).

PAK2 PBD selectively binds activated EhRacA during receptor capping and collagen matrix invasion (Arias-Romero et al., 2006). PAK2 probably controls cell movement, surface receptor capping and cytokinesis (Arias-Romero et al., 2006). The biochemical studies conducted on the C-terminal kinase domain of PAK2 described its activity towards myelin basic protein. Interestingly, the PAK and EhRacA complex homology model showed the specific interaction in PAK2 residues Met-121 and His-123 with RacA Tyr-40; and PAK2 residue Phe-145 with RacA Asp-63, Arg-66, Leu-67 and Leu-70 (Arias-Romero et al., 2006). A detailed investigation to elucidate the critical residues influencing the binding energy would guide the rationale development of small molecules that inhibit such interaction. The possible interaction of other GTPases with PAK2 can also be investigated experimentally.

Un-stimulated cells show cytoplasmic PAK3 distribution, while capping protein induction relocates it to the caps (Dutta et al., 2007). PAK3 undergoes autophosphorylation and phosphorylates histone H1 in vivo, and in vitro studies displays kinase activity in the absence of small GTPases (Dutta et al., 2007). Maximum enzymatic activity is achieved after the autophosphorylation of a critical residue present in the activation-loop of many protein kinases. It is yet to be established whether an increase in activity or change in localization is observed upon gtpase binding. PAK3 sequential feature reports a PH domain (residues 2–82), a PBD/CRIB domain (residues 84–141) and a kinase domain (residue 142–447) at its C-terminal. PAK3 shares 40% identity (50% similarity) with Dictyostelium PAK (PAKc). All the typically conserved XI subdomains from Ser/Thr kinases are visible in its kinase domain. The only variation observed, is the replacement of the conserved Leucine by Tyrosine in subdomain VII.

PAK4 and PAK5 are highly specific effectors of EhRacC (Bosch and Siderovski, 2015). EhRacC^Q65LGTP and EhPAK4-PBD reveal a deviation of PBD α-helix in an otherwise conserved Rho/effector interface. The side chains of EhPAK4 PBD residues line up the EhRacC binding interface. The similar residues are well conserved in EhPAK5, hinting at a common interaction surface for the same Rho gtpase. Asp-17 of EhPAK4 (Glu-108 in EhPAK5) forms a salt bridge with Arg-30 of EhRacC, while Phe-21 of EhPAK4 (Tyr-112 of EhPAK5) contributes towards a hydrophobic RacC interface (Bosch and Siderovski, 2015). Further experiments need to be conducted to for RacC effectors PAK4 and PAK5 to elucidate their biological functions. We still lack the knowledge that correlates the autoinhibition modes of E. histolytica PAK isoforms with mammalian group-I and group-II PAKs. Here, we also acknowledge the unresolved question of the signaling specificity between Rho family gtpase and PAKs.

Entamoeba has three coronins; two belonging to short tail (Coronin1: C4M943, Coronin 2: C4M137) are 70% identical to each other, and one long tail (Coronin 3: C4M5U0). The predicted function of long coronin is crucial in the amoeboid migration and pseudopod regulation (Tolstrup et al., 2007).

The genus Giardia comprises several species that inhabit intestinal tracts of vertebrates (fish, amphibians, reptiles, birds, rodents). It is one of the most pervasive intestinal pathogens that infects a wide range of mammals; for example, human and agricultural livestock such as cattle and sheep. However, Giardia lamblia (alternatively referred to as Giardia intestinalis and Giardia duodenalis) infects and causes giardiasis in humans, suggesting a zoonotic transmission (Ryan and Caccio, 2013). The life cycle is simple, involving two morphogenetic stages; 1) cyst form, which is environmentally resistant and infectious, and 2) vegetative trophozoite stage, which colonizes the small intestine and becomes invasive to cause disease. During encystation, the parasite relays the signals to produce, transport, and secrete the cyst wall protein (CWP). It has been demonstrated that flagella and disk structures modulate motility and host intestinal epithelial cell attachment (Di Genova and Tonelli, 2016). The molecular mechanism behind the regulation of these processes remains abstract. The sole Rho family gtpase, GlRac, regulates endomembrane organization and CWP trafficking (Krtkova et al., 2016). Subcellular localization studies indicate the association of GlRac with endoplasmic reticulum and Golgi apparatus like encystation-specific vesicles (ESV).

The CRIB domain search identifies only one protein (C6LUS0), which has the CRIB and the kinase domains homologous with other PAKs (see Supplementary Table S1). The earlier studies indicated that Giardia lamblia has only one Rac in the complete Rho family, so the identified protein in our search can be the prospective effector protein (Krtkova et al., 2016). The interaction and co-localization studies on proposed protein with known Rac will provide thoughtful insights on the signaling mechanism of cytoskeleton assembly in the parasite.

Trypanosoma cruzi causes an encumbering severe illness in humans, known as the Chagas disease, which affects millions of people globally (de Souza et al., 2010). The various species of Trypanosoma belongs to kinetoplastid protozoans in an evolutionary context (Gupta et al., 2020). The complex life cycle involves four developmental stages: 1) epimastigotes; 2) metacyclic trypomastigotes; 3) amastigotes; and 4) bloodstream trypomastigotes (Zuma et al., 2021). Trypomastigotes and extracellular amastigotes are the only infective forms that are able to invade almost any nucleated host cell (Ferri and Edreira, 2021). During the invasion, bidirectional signaling pathways are triggered in both the parasite and the host cell. The CRIB domain search identifies only one protein (Q4DHX7) in Trypanosoma cruzi (see Supplementary Table S1) which intrigued us to carryout in depth literature search for cytoskeletal regulation signaling pathways. However, we cannot categorize this CRIB domain-containing protein in any existing families of effector protein because it doesn’t any depict homologous features to other eukaryotic proteins. Further investigation is required to know more about the protein function and relation to the CRIB domain-containing protein family from other lower eukaryotes.

Interestingly, few proteins have been found that regulate cytoskeletal pathways but are only related to the invasion of the extracellular amastigote form of T. Cruzi. Extracellular amastigote engulfed by mammalian cells via phagocytic cup based on actin-dependent cytoskeleton changes (Mortara et al., 2005). Extracellular amastigotes secretes proteins like P21, mevalonate kinase (MVK) and specific-surface protein 4 (Ssp4), which mediate host cell signaling during phagocytosis. P21 is a 21 kDa secretory protein (da Silva et al., 2009), related to ERK and PI3K signaling pathways during phagocytosis and cytoskeleton remodelling (Rodrigues et al., 2012; Teixeira et al., 2015). The recombinant version of P21 (rP21) interacts with the CXCR4 chemokine receptor, inducing actin assembly to drive phagocytosis and modulate the PI3K-dependent expression of an actin-related gene (da Silva et al., 2009; Rodrigues et al., 2012). TcMVK is involved in protein glycosylation and cytoskeletal assembly through activation of p38/ERK, FAK (focal adhesion kinase) components and PAK signaling trails (Ferreira et al., 2016). Amastigote form invades the host cell through Ssp-4, another secretory molecule predicted to function as Rac1/WAVE2 and Cdc42/N-WASP signaling mediators (Florentino et al., 2018). TcSsp4 is majorly a surface GPI-anchored glycoprotein whose expression doesn’t correlate with infection, but glycosylation of protein is linked with host cell invasion. Highly infective strain’s amastigotes secrete a differentially glycosylated Ssp4 that recruits Galectin-3 (Gal3) to mediate host cell surface and parasite interaction (Florentino et al., 2018). Recent studies on actin-binding proteins from kinetoplastids, proposed a protein (Q4DEX0) as coronin because it has homologous WD repeat and coronin domains characteristic to amoebozoa and higher eukaryotes (Gupta et al., 2020).

Leishmania donovani also belongs to kinetoplastids, a unicellular protozoan parasite causing a fatal disease, visceral leishmaniasis, in humans. In vertebrates, it is present as invasive promastigote and amastigote, which cause infection. The best survival strategy used by promastigotes during establishment of infection in macrophages is to inhibit the fusion of phagosome and endosome (Desjardins and Descoteaux, 1997). Lipophosphoglycan (LPG), a significant surface glycoconjugate of promastigote, is crucial for intracellular survival (Handman et al., 1986). The pathogen uses the human host macrophage cell cytoskeletal assembly to sustain and prevent phagosome maturation (Scianimanico et al., 1999; Lodge and Descoteaux, 2008). However, recent report on the actin-binding protein repertoire is classifying the presence of various proteins in the pathogen itself (Gupta et al., 2020). Earlier information reveals that L. donovani recruits human Cdc42 and Rac1 to form an F-actin coat around its phagosome as protective measure from macrophage killing (Lodge and Descoteaux, 2005; Lerm et al., 2006).

The domain search retrieves one protein (Q4QEZ0), which has conserved Ser/Thr kinase domain homologous to PAK catalytic domain but no CRIB domain was present in it (see Supplementary Table S1). A coronin homolog protein (E9BGF4) is reported recently in the genome-based studies (Gupta et al., 2020). The functional characterization to link it with CRIB containing effector proteins is yet to be established.

Human is placed as the top ranking evolutionary evolved organism, but it is a host for several pathogens. The understanding of molecular mechanisms of host and pathogen proteins helps to prevent infections and diseases. The cellular signaling process is complex in this system due to presence of multi-layered system of tissues and organs. However, cytoskeleton signaling and its regulation are somehow similar in most of the cells and different in some cells at the same time. Investigations on complexity in regulating cytoskeleton dynamics, mediated by Rho family GTPases is fundamental to processes like motility (Murali and Rajalingam, 2014), adhesion, differentiation and development. All the twenty-seven proteins, which have been identified in the CRIB domain-based search, are effector proteins of Rac/Cdc42. The brief information of CRIB containing effectors is accounted in this study to understand the link and origin of CRIB motif and association with protein in social amoebas (Kumar et al., 2009), intestinal pathogens and kinetoplastids. In the human proteome, nine different CRIB containing protein families are present (Figure 5, see Supplementary Table S2). Additionally, six coronin isoforms are also identified in human; some of them have shown the presence of CRIB motif via experimental evidence while others need to be explored (see Supplementary Table S2).