Insights Into Peptidyl-Prolyl Cis-Trans Isomerases From Clinically Important Protozoans: From Structure to Potential Biotechnological Applications

1. Introduction

Parasitic protozoa are responsible for a significant number of diseases worldwide, leading to an estimated 1.1 million combined deaths annually [1]. Owing to their relevance in public health, numerous parasite genomes have been partially or entirely described to better understand the genes and proteins associated with these organisms [2].

Peptidyl-prolyl cis/trans isomerases (PPIases) are specialized enzymes that catalyze conformational changes in proteins and are highly conserved in all living organisms, including archaea, bacteria, protists, and eukaryotes. They participate in various biological processes, such as protein folding, protein trafficking, cell signaling, and immune response, and they can act as virulence factors [3,4,5].

The overall domain architecture of human PPIases (hPPIases) shows remarkable similarity to that in other organisms. hPPIase orthologs have been found in clinically important protozoan parasites, such as Trichomonas, Entamoeba, Giardia, Trypanosoma, Leishmania, Plasmodium, and Toxoplasma, emphasizing their importance in the survival, development, and pathogenicity of these organisms [6]. However, much remains unknown about the role of PPIases in parasites.

PPIases facilitate the interconversion of the cis- and trans-isomers of the N-terminal bond preceding a proline residue (X-Pro) in nascent proteins (Figure 1A). The trans conformation is generally more favorable for most amino acids (aa), except for proline, where the difference in free energy between the cis and trans conformations is small. Furthermore, most cis prolines are exposed on the protein surface [7]. Although the exact mechanism of action of PPIases has not been determined, several theories, including substrate desolvation, substrate autocatalysis, preferential transition-state binding, and nucleophilic catalysis, have been proposed. The proper conformation of these bonds in a protein is crucial since different functions can rely on the distinction between the cis and trans states [5].

The superfamily of PPIases includes four families of nonhomologous proteins: cyclophilins (CyPs), FK506-binding proteins (FKBPs), parvulins (Pars), and protein phosphatase 2A phosphatase activators (PTPAs). Each family can be distinguished by specific inhibitors (Figure 1C). For instance, CyPs and FKBPs are inhibited by immunosuppressive drugs such as cyclosporin A (CsA) and FK506 (tacrolimus), respectively. FKBPs can also be inhibited by rapamycin (sirolimus). Pars, on the other hand, are inhibited by the natural compound juglone (5-hydroxy-1,4-naphthalenedione). However, specific PTPA inhibitors have not yet been identified [4,8,9,10]. PPIases are characterized by a catalytic domain located in the central region of the protein that is responsible for the PPIase activity. Additionally, these enzymes have isoforms with sequence extensions in either the N- or C-terminal region or in both regions, and these isoforms are associated with their specific cellular functions.

The discovery that mammalian tissues contain CyPs with a high affinity for CsA marked the beginning of PPIase research [3,11]. Human CyPs (hCyPs) have been extensively studied. The structure of CyPs is a closed eight-stranded antiparallel ꞵ-barrel structure with two α-helixes enclosing the barrel from either side (Figure 1B). These proteins share a common domain of ~109 aa, known as the cyclophilin-like domain (CLD) [12]. Four hCyPs have been identified based on their localization to specific organelles: hCyP19 is linked to the nuclear spliceome, CyPB is found in the endoplasmic reticulum (ER), CyPC is present in the membrane and has a signal peptide (SP) for the ER, and CyPD is associated with the mitochondria because an SP directs it to this organelle [12,13]. Furthermore, hCyPs share high sequence homology within their own family, and their active region contains highly conserved aa residues, such as R55, F60, M61, Q63, A101, F113, W121, L122, and H126 [14].

FKBPs contain an FKBP domain that is responsible for PPIase catalytic activity. hFKBP12 is a well-studied reference protein in this family. Structurally, FKBPs comprise a five-stranded β-sheet wrapped around one short α-helix, forming a β-barrel similar to that of CyPs (Figure 1B) [15,16,17]. These proteins are regarded as divergent due to their lack of universally conserved aa residues, which underlies their unique and varied nature [17].

Pars, the third type of enzyme with PPIase activity, are structurally unrelated to CyPs and FKBPs. The term “parvulins” is derived from the Latin word “parvulus”, meaning “very small”, and refers to the low molecular weight of these proteins [18,19,20]. hPar (Pin1) is a well-characterized nuclear protein, and its tertiary structure contains four β-strands, one α-helix and a loop. Pin1 consists of two domains: 1) the N-terminal WW domain, which is a protein‒protein interaction motif related to its cellular localization through recognition of proline-rich peptide motifs (PRMs) and phosphorylated phosphate Ser/Thr-Pro sites [21], and 2) the C-terminal catalytic domain (PpiC), which promotes the isomerization of the Ser-Pro or Thr-Pro bonds [22,23] (Figure 1B).

In addition to the extensive investigations of hPPIases, additional research is needed to understand the role of PPIases in parasites. This work provides an overview of the current information on PPIases reported in databases (VEuPathDB and UniProt) of clinically relevant protozoan parasites, such as Trichomonas vaginalis, Entamoeba histolytica, Giardia intestinalis, Trypanosoma cruzi, Trypanosoma brucei, Leishmania major, Leishmania donovani, Plasmodium falciparum, Plasmodium vivax, and Toxoplasma gondii. We focus on the structural characteristics, localization, and functions of these proteins. Similarly, we analyze several biotechnological aspects: we have collected important information about the expression processes, purification, and activity of recombinantly produced protozoan PPIases, and we discuss the possible biotechnological applications of these proteins in assisted protein refolding.

2. Parasite PPIases: Disease, Genome Database, and Structural Characteristics

2.1. Anaerobic or Microaerophilic Protozoan Parasites

2.1.1. Trichomonas vaginalis

Trichomoniasis is a sexually transmitted disease caused by the protist parasite T. vaginalis. Infected people can present a variety of symptoms, but most are asymptomatic. Trichomonal infection has been linked to other sexually transmitted diseases, such as HIV, and is associated with HIV transmission [24].

The genome of T. vaginalis strain G3 encodes 13 CyPs, 9 FKBPs, and 3 Pars, as reported in the TrichDB database (https://trichdb.org/trichdb/app/) (Release 63 03May2023) [25] (Table 1). In this review, we used the nomenclature for PPIases reported by Galat (2003) [13], in which PPIases are named with a prefix of two letters indicating the species followed by a number for the calculated molecular weight in kDa.

The PPIases from T. vaginalis present sequence variations within each family, with few conserved regions among them. Phylogenetic analysis revealed that the CyPs were grouped into clades with low bootstrap values, suggesting that they do not share a common origin. In contrast, the FKBPs and Pars formed distinct clades with higher bootstrap values, indicating a common evolutionary origin for these proteins. However, TvCyP44 was found to share a clade with the FKBPs, which might indicate that it is more closely related to FKBPs than to CyPs, despite containing a CLD (Supplementary Figure S1).

Most CyPs from T. vaginalis retain the aa residues essential for catalytic activity within the highly conserved CLD identified in TvCyP19 (referred to as TvCyP1 by Hsu et al., 2014) [26]: H⁶², R⁶³, F⁶⁸, M⁶⁹, Q⁷¹, G⁸⁰, A¹⁰⁹, N¹¹⁰, A¹¹¹, Q¹¹⁹, F¹²¹, W¹²⁹, L¹³⁰, and H¹³⁴. Exceptions to this pattern were observed in TvCyP21, TvCyP37, and TvCyP44 (Supplementary Figure S2).

The structures of only two of the CyPs of T. vaginalis have been solved: TvCyP19 and TvCyP19.9 (referred to as TvCyP1 and TvCyP2, respectively, by Hsu et al., 2014; 2020) [26,27]. Both proteins present the typical structure, characterized by a β-barrel composed of eight antiparallel β-strands and two α-helices. TvCyP19 mainly consists of the CLD domain, whereas TvC19.9, in addition to the catalytic domain, has a longer N-terminal segment. An important difference between the two CyPs is that TvCyP19 is a dimer, whereas TvCyP19.9 is a monomer [28,29].

We analyzed the sequence identities of CyPs from T. vaginalis, taking the sequence of TvCyP19 as a reference (Supplementary Table S1). Low-molecular-weight CyPs (TvCyP14, TvCyP18, TvCyP19.2, TvCyP19.8, TvCyP20, TvCyP21, TvCyP22, and TvCyP23) have sequence identities of approximately 40%-70% (Table S1) with respect to TvCyP19. The eight small TvCyPs are characterized by having only the CLD in their sequence, similar to TvCyP19 and TvCyP19.9 (Figure 2A,B). Moreover, the high-molecular-weight CyPs (TvCyP37, TvCyP44, and TvCyP63) have low sequence identity to TvCyP19 (Supplementary Table S1). These TvCyPs exhibit a unique domain in addition to the conserved CLD. For example, TvCyP37 contains the SF-CC1 domain and the RRM motif at its C-terminus (Figure 2C). The SF-CC1 domain is characteristic of RNA splicing factors and is marked by Arg- and Ser-rich sequences, typically followed by RNA recognition domains; this feature is also found in TvCyP37 [30]. Similarly, the presence of an RNA splicing factor has been noted in CyPs from other organisms, such as humans and Arabidopsis thaliana [31], as along with the RNA recognition motif (RRM), which is involved in nucleic acid and/or protein recognition. The structural versatility of RRM interactions contributes to the diverse biological functions of RRM-containing proteins [32].

While TvCyP44 has an N-terminal U-box domain, a specialized type of RING finger that differs from other RING fingers in the lack of metal binding sites [33]. This domain has an estimated length of 67 aa (Figure 2D) and has been identified in ubiquitin ligase-like proteins in Saccharomyces cerevisiae that serve as scaffolds for proteins during ubiquitination, which is associated with protein degradation pathways [34,35]. TvCyP63 contains a WD40 domain near the N-terminus, spanning aa 32-349 (Figure 2E). This WD40 domain is also present in other large CyPs, such as PfCyP87, PvCyP83, and TgCyP86 from P. falciparum, P. vivax, and T. gondii, respectively. The WD40 domains function in anchoring to other proteins or in DNA binding. These domains are present in a wide variety of proteins with diverse functions, including chaperone proteins, but have no catalytic activity [36].

Compared to CyPs, T. vaginalis FKBPs presented lower sequence identities (less than 24%) when TvFKBP-12 was used as a reference (Supplementary Table S1). This low similarity is attributed to the absence of consensus sequences in FKBPs. Each of the nine TvFKBPs has a FKBP domain with a sequence length in the range of ~84 to 100 aa near the N-terminus (Figure 2F-H). Six of the FKBPs (TvFKBP-12, TvFKBP-15.1, TvFKBP-15.2, TvFKBP-19, TvFKBP-20, and TvFKBP-33) range from 12 to 33 kDa and consist of a single FKBP domain that spans most of the protein sequence (Figure 2F). In contrast, three of the FKBPs (TvFKBP-30, TvFKBP-32, and TvFKBP-63) include another domain in addition to the catalytic domain. TvFKBP-30 and TvFKBP-32 contain three tetratricopeptide repeat (TPR) motifs near the C-terminus (Figure 2G). TPRs are structural motifs that usually comprise approximately 34 aa and mediate protein–protein interactions and the assembly of multiprotein complexes [37]. TvFKBP-63 contains an MukB domain (Figure 2H), which is found in Mukb proteins and is associated with chromatin remodeling [38]. No other reports of FKBPs containing MukB domains were found.

Two of the three T. vaginalis Par proteins, TvPar17.8 and TvPar17.9, have similar molecular weights and 41.6% identity (Supplementary Table S1). Both proteins have a PpiC domain close to the C-terminus, which covers most of the protein, ~100 aa. In addition to this domain, the three TvPar proteins possess a WW domain of ~30 aa near the N-terminus (Figure 2I-J).

The third TvPar, with a molecular weight of ~102 kDa (TvPar102), contains the PpiC domain, which spans ~100 aa near the N-terminus. In addition, the WW domain near the N-terminus contains a suppressor of forked (Suf) domain (Figure 2J), which is commonly associated with mRNA formation and polyadenylation in organisms, such as Drosophila melanogaster [39]. Understanding the role of the Suf domain in TvPars could provide valuable insights.

TvPar102 is the largest Par identified among these clinically important parasites and is significantly larger than the typical (~13 kDa) Pars. Its distinct C-terminal end, which consists of a series of α-helixes, sets it apart from other proteins in this group. However, its function remains unknown.

2.1.2. Entamoeba histolytica

Amoebiasis is a parasitic disease caused by protozoan parasites belonging to the genus Entamoeba, among which E. histolytica is the most pathogenic species. This infection is transmitted through oral-fecal contamination, often via the consumption of food or water contaminated with parasite cysts, which migrate from the small intestine to the large intestine, where they divide by binary fission and are eliminated in the feces. Amoeboid forms may migrate out of the intestine and invade other organs of the body. This disease causes dysentery and various intestinal problems, affecting approximately 500 million people worldwide and causing more than one hundred thousand deaths per year [44].

The E. histolytica HM1-IMSS strain reference genome in the AmoebaDB database [25] (https://amoebadb.org/amoeba/app/) (Release 63 03May2023) contains six genes encoding CyPs, five genes encoding FKBPs, and two genes encoding Pars (Table 2).

Alignment of the EhCyP18 CLD with CLDs of CyPs from other organisms revealed high sequence conservation of this domain, in addition to the conservation of important aa residues in the active site (Supplementary Figure S3). Unlike the EhCyPs, the five FKBPs in E. histolytica are poorly conserved, which is unsurprising since FKBPs are known to be highly divergent. The lowest sequence identities among the E. histolytica PPIases were found between its two Pars, EhPar13 and EhPar13.25, which share 20% identity, indicating significant divergence between these proteins.

The CyPs found in E. histolytica have molecular weights between 10 and 40 kDa, and the CLD domain is consistently located at the N-terminus. Interestingly, EhCyP10 is the smallest known CyP among parasites and contains a CLD. EhCyP21 and EhCyP22 contain an N-terminal sequence that allows them to localize to the ER. Notably, in addition to the CLD domain, CyPs EhCyP10 and EhCyP40 also contain an RRM domain of ~77-78 aa at the C-terminus.

The five FKBPs of E. histolytica are globular proteins with molecular weights ranging from 18 to 46 kDa. Interestingly, in addition to the PPIase-FKBP domain, four of the FKBPs (EhFKBP-29, EhFKBP-35, EhFKBP-43, and EhFKBP-46) possess the TPR domain, which is related to protein‒protein interactions. In contrast, EhFKBP-18 contains only the FKBP-like PPIase domain and a signal sequence at the N-terminus with unknown function. E. histolytica Pars contain only the PpiC-like PPIase domain; both Pars are small in size, which is common in the Pars known thus far [47]

2.1.3. Giardia intestinalis

Giardiasis, caused by the protozoan parasite Giardia intestinalis (also known as G. duodenalis or G. lamblia), is a disease of global concern, affecting both developed and undeveloped nations. While asymptomatic cases are common, various intestinal and extraintestinal symptoms and postinfection problems have been recorded [48]. Giardiasis was designated a neglected disease by the WHO in 2004, highlighting its public health significance [49]. G. intestinalis is a complex species comprising eight genetically related groups (assemblages A to H). Assemblages A and B, which are responsible for infecting humans, are subject to debate regarding their potential classification as different Giardia species due to significant genetic differences. Further research is required to resolve this question [50,51,52].

In this review, we focused on PPIases from the G. intestinalis genome reported in the GiardiaDB database (https://giardiadb.org/giardiadb/app) (Release 63 03May2023) [25], specifically from reference isolates WB (assemblage A), DH (subassemblage AII), and GS (assemblage B), which are all responsible for human infections [50,53,54]. These G. intestinalis isolates contain two CyPs (molecular weight < 25 kDa) and six FKBPs (molecular weight < 39 kDa), and no Pars were reported, in contrast to parasites such as T. vaginalis, Trypanosoma, Leishmania, or Toxoplasma, which have more than 20 PPIases with molecular weights < 100 kDa.

The numbers of CyPs and FKBPs in the Giardia assemblage A and B genomes are relatively consistent between the isolates. Some PPIases are the same in both assemblages (Table 3), indicating potential similarities and a common ancestor (Figure 3A). In the DH isolate (subassemblage AII), the two GiCyPs and five GiFKBPs showed high sequence identity (>94.24%) compared to that of the WB isolate. However, differences in molecular weight were found for only one GiCyP and one GiFKBP (Table 3).

All G. intestinalis CyPs share a common CLD domain of ~89 aa, spanning 159 residues (Supplementary Table S2) (Figure 3C). The GiCyP18 proteins in the WB and GS isolates share 99.4% identity (Supplementary Table S3) and contain only a CLD domain (Supplementary Table S2) (Figure 3C). The GiCyP21 proteins in the WB and GS isolates share 94.2% identity (Supplementary Table S3) and have some characteristics in common with hCyPB [12]. These proteins contain an SP of 13 residues, two nonstructural regions at both ends of the CLD domain, and an additional 9 residues at the C-terminus but lack the C-terminal ER sequence found in human hCyPB (Table S2). In the DH isolate, GiCyP25 contains regions resembling those in hCyPB and a transmembrane helix (TMH) segment (Supplementary Table S2) (Figure 3C).

We also explored 3D GiCyP models using the AlphaFold database in UniProt (Release 2023_01). Despite the lack of crystallized GiCyPs to date, our analysis revealed that all the GiCyPs share a secondary and tertiary structure similar to that of hCyPA, as previously described [4,14]. Specifically, the secondary structure comprises eight β-strands, two α-helixes, two small helixes, and twelve regions of random-coil turns, which are also widely reported in other species (Figure 3C).

In the 3D structures, we observed that the central domain consists of eight β-strands flanked by an α-helix at each end of the barrel. These regions are connected by random-coil turn structures that are exposed on the protein surface. To determine the active site residues, we referenced the protein domain families in the Conserved Domains Database (CDD) [55] (Figure 3B). Notably, these active site residues coincide with the residues involved in CsA binding, including W123 in GiCyP1 (referred to as GiCyP19 in this review) (Table 3). GiCyP19 was the first CyP characterized from the G. intestinalis WB strain by Yu et al., 2002 [56]. It should be noted that although the sequence of this gene had not yet been deposited in a database at the time of its publication, through BLAST analysis we found an identity of around 98% of the sequence of the original publication with that of GiCyP19 found in the Giardia spp. genome.

Giardia FKBPs (Table 3) share similarities in size and aa sequence among isolates (Tables S2 and S3) (Figure 3D). The molecular weights of the proteins ranged from ~12 to 39 kDa, and their isoelectric points varied significantly from highly acidic at 4.6 to highly alkaline at 9.5 (Supplementary Table S2). Similar variations in size and charge have been observed in other organisms [13,57]. Among the Giardia FKBPs, only GiFKBP-12 has been crystallized (PDB: 2LGO) [58]. Its FKBP domain is located at the C-terminus and consists of 88 to 97 aa, forming six antiparallel β-strands and one α-helix. It contains conserved residues responsible for inhibitor binding (Figure 3D) [59]. Other Giardia FKBPs possess additional regions along with the FKBP domain (Table 2). Some have a small disordered region of approximately 21 aa at the N-terminus (GiFKBP-12), while others have an SP of 15 or 18 aa (GiFKBP-24, GiFKBP-28, GiFKBP-29). Only one FKBP (GiFKBP-38) contain a TPR region (Supplementary Table S2) formed by six antiparallel α-helixes (Figure 3E).

2.2. Trypanosomatid Parasites

2.2.1. Trypanosoma cruzi and Trypanosoma brucei gambiense

T. cruzi and T. brucei gambiense are protozoan parasites that cause significant health impacts through trypanosomiasis. T. cruzi causes Chagas disease (also known as American trypanosomiasis), which is transmitted to humans and other mammals by Triatominae insects. T. brucei gambiense and T. brucei rhodesiense cause sleeping sickness (also known as African trypanosomiasis), which is transmitted by the tsetse fly [66]. According to the WHO, between 6 and 7 million people worldwide, in Latin America, are infected by T. cruzi, and T. brucei gambiense accounts for more than 95% of the reported cases of African trypanosomiasis [67].

The CyPs in the T. cruzi CL Brener Esmeraldo-like genome have been reported in the TriTrypDB database (https://tritrypdb.org/tritrypdb/app/) (Release 62 09 Mar 2023) [25] and include 15 paralogs [68]. We analyzed the 3D structure of these CyPs using the predicted structures generated by AlphaFold in the UniProt database (Release 2023_01). All these CyPs share the highly conserved CLD domain, an eight-stranded antiparallel ꞵ-barrel structure accompanied by two α-helixes and show some differences in regions outside the CLD domain. TcCyP19 consists predominantly of the CLD domain, and the other fourteen are isoforms (Table 4). For instance, TcCyP21 and TcCyP24 have SPs with lengths of 26 and 25 aa, respectively. Notably, TcCyP21 is the only T. cruzi CyP with a reported crystal structure (PDB:1XO7) [58], and its PDB ID is linked to a different UniProt ID (Q4DPB9) due to its origin in the T. cruzi CL Brener non-Esmeraldo-like genome. However, alignment of the two protein sequences revealed 97.9% identity.

Furthermore, TcCyP22, TcCyP26, TcCyP30, and TcCyP42 contain an elongated region in the N-terminus, while TcCyP20 has a small elongated region in the C-terminus. Additionally, TcCyP25, TcCyP28, TcCyP29, TcCyP35.3, and TcCyP35 (previously named TcCyP34 by Potenza et al., 2006) [69] exhibit elongated regions in both the N- and C-terminal segments. Moreover, TcCyP38 (previously named TcCyP40) [69] contains the TPR motif. Finally, TcCyP103 (previously named TcCyP110) [69] is the largest CyP (103 kDa) and contains an array of structures adjacent to the CLD domain, which were identified as a disordered region with both basic and acidic residues.

Table 4. Peptidyl-prolyl cis-trans isomerase repertoire from Trypanosoma cruzi¹.

Parasite	UniProt	TriTrypDB	NCBI	PDB	PPIase name	Localization3	Function3	References
T. cruzi CL Brener	Q4E4L9	TcCLB.506925.300 (CYPA)	XP_821578.1		TcCyP192	Extracellular space	Promotes ROS production in host cells	[68,76,77,78,80]
	Q4DC03	TcCLB.507009.100	XP_811912.1		TcCyP20
	Q4DNC9	TcCLB.507521.70	XP_815879.1	1XO7	TcCyP212			[68,78]
	Q4DI85	TcCLB.504035.70	XP_814080.1		TcCyP222	Mitochondria	Cell death regulation	[68,79]
	Q4CXV1	TcCLB.506413.80	XP_806960.1		TcCyP24
	Q4DFL3	TcCLB.508323.94	XP_813175.1		TcCyP25			[68,78]
	Q4D4K3	TcCLB.503885.40	XP_809302.1		TcCyP26
	Q4CX88	TcCLB.509499.10	XP_806737.1		TcCyP282			[68,78]
	Q4DQI8	TcCLB.505807.10	XP_816616.1		TcCyP29
	Q4DNS3	TcCLB.511589.50	XP_816007.1		TcCyP30	Membrane		[43,68]
	Q4DM35	TcCLB.511577.40 (CYP35)	XP_815421.1		TcCyP35 (TcCyP34)2			[68,78]
	Q4DVC9	TcCLB.511217.120	XP_818332.1		TcCyP35.3 (TcCyP35)			[68]
	Q4E4G0	TcCLB.506885.400 (CYP40)	XP_821542.1		TcCyP38 (TcCyP40)2			[43,68]
	Q4DG41	TcCLB.510761.44	XP_813344.1		TcCyP42	Membrane		[43,68]
	Q4D1M5	TcCLB.504215.10	XP_808273.1		TcCyP103 TcCyP110			[68]
T. cruzi Y	Q09734	TcYC6_0113560	CAA49346.1	1JVW	TcFKBP222 (TcMIP)	Extracellular space	Host cell entry/invasion	[69,70]
T. cruzi CL Brener	Q4D5W5	TcCLB.508169.69	XP_809772.1		TcFKBP12
	Q4DFL5	TcCLB.508323.84	XP_813174.1		TcFKBP12.2
	Q4D7F5	TcCLB.511731.89	XP_810317.1		TcFKBP35
	Q4CZN2	TcCLB.511353.10	XP_807578.1		TcFKBP52
	Q4CYE6	TcCLB.507629.39	XP_807152.1		TcFKBP93
T. cruzi CL Brener	Q4D8F7	TcCLB.508567.70 (Pin1)	XP_810661.1		TcPar12.62 (TcPin1)	Cytosol		[72,81]
	Q4D394	TcCLB.506697.50	XP_808848.1		TcPar132 (TcPar14)			[73]
	Q4D9J4	TcCLB.506857.60 (Par45)	XP_811046.1		TcPar452	Nucleus		[73]

¹In parenthesis, PPIasa names previously reported. ²More information is in Table 11. ³The localization and function of PPIases were taken from the references cited or from the UniProt database, which were predicted by the Gene Ontology Consortium [43]. Spaces in blank: not reported data.

Table 4. Peptidyl-prolyl cis-trans isomerase repertoire from Trypanosoma cruzi¹.

Parasite	UniProt	TriTrypDB	NCBI	PDB	PPIase name	Localization3	Function3	References
T. cruzi CL Brener	Q4E4L9	TcCLB.506925.300 (CYPA)	XP_821578.1		TcCyP192	Extracellular space	Promotes ROS production in host cells	[68,76,77,78,80]
	Q4DC03	TcCLB.507009.100	XP_811912.1		TcCyP20
	Q4DNC9	TcCLB.507521.70	XP_815879.1	1XO7	TcCyP212			[68,78]
	Q4DI85	TcCLB.504035.70	XP_814080.1		TcCyP222	Mitochondria	Cell death regulation	[68,79]
	Q4CXV1	TcCLB.506413.80	XP_806960.1		TcCyP24
	Q4DFL3	TcCLB.508323.94	XP_813175.1		TcCyP25			[68,78]
	Q4D4K3	TcCLB.503885.40	XP_809302.1		TcCyP26
	Q4CX88	TcCLB.509499.10	XP_806737.1		TcCyP282			[68,78]
	Q4DQI8	TcCLB.505807.10	XP_816616.1		TcCyP29
	Q4DNS3	TcCLB.511589.50	XP_816007.1		TcCyP30	Membrane		[43,68]
	Q4DM35	TcCLB.511577.40 (CYP35)	XP_815421.1		TcCyP35 (TcCyP34)2			[68,78]
	Q4DVC9	TcCLB.511217.120	XP_818332.1		TcCyP35.3 (TcCyP35)			[68]
	Q4E4G0	TcCLB.506885.400 (CYP40)	XP_821542.1		TcCyP38 (TcCyP40)2			[43,68]
	Q4DG41	TcCLB.510761.44	XP_813344.1		TcCyP42	Membrane		[43,68]
	Q4D1M5	TcCLB.504215.10	XP_808273.1		TcCyP103 TcCyP110			[68]
T. cruzi Y	Q09734	TcYC6_0113560	CAA49346.1	1JVW	TcFKBP222 (TcMIP)	Extracellular space	Host cell entry/invasion	[69,70]
T. cruzi CL Brener	Q4D5W5	TcCLB.508169.69	XP_809772.1		TcFKBP12
	Q4DFL5	TcCLB.508323.84	XP_813174.1		TcFKBP12.2
	Q4D7F5	TcCLB.511731.89	XP_810317.1		TcFKBP35
	Q4CZN2	TcCLB.511353.10	XP_807578.1		TcFKBP52
	Q4CYE6	TcCLB.507629.39	XP_807152.1		TcFKBP93
T. cruzi CL Brener	Q4D8F7	TcCLB.508567.70 (Pin1)	XP_810661.1		TcPar12.62 (TcPin1)	Cytosol		[72,81]
	Q4D394	TcCLB.506697.50	XP_808848.1		TcPar132 (TcPar14)			[73]
	Q4D9J4	TcCLB.506857.60 (Par45)	XP_811046.1		TcPar452	Nucleus		[73]

¹In parenthesis, PPIasa names previously reported. ²More information is in Table 11. ³The localization and function of PPIases were taken from the references cited or from the UniProt database, which were predicted by the Gene Ontology Consortium [43]. Spaces in blank: not reported data.

The TriTrypDB database includes 6 T. cruzi FKBP genes [68] (Table 4). Structural analysis revealed that these FKBPs range in length from 12 to 93 kDa and share similarities in the catalytic domain. Two FKBPs, TcFKBP-12 and TcFKBP-12.2, consist mostly of the FKBP domain. The four remaining proteins contain an FKBP domain plus other motifs. In the case of TcFKBP-22 (also known as TcMIP, the Microphage Infectivity Potentiator by Moro et al., 1995) [70] contains a 29 aa SP. TcFKBP-22 is from the T. cruzi strain Y and is the only T. cruzi FKBP that has been characterized and crystallized (PDB: 1JVW) [Berman et al., 2000 71]. TcFKBP-35 has extensive elongation in its C-terminus, and TcFKBP-52 and TcFKBP-93 have elongated regions in their N-terminus that have been identified as coiled coils. These coiled coils are involved in various biological functions as molecular spacers within proteins, influencing the architecture of organelles such as centrioles and the Golgi apparatus and facilitating the binding of transport vesicles to the Golgi apparatus [72].

Finally, T. cruzi contains three Pars [68] (Table 4) ranging from 12 to 45 kDa, all of which possess the PpiC domain. TcPar12.6 (previously named TcPin1 by Erben et al., 2007 [73], and a homolog of hPin1) consists entirely of the PpiC domain and lacks the protein‒protein interaction-related WW domain at the N-terminus. In addition, TcPar13 (previously named TcPin14 by Erben et al., 2010 [74], and a homolog of hPar14) has an elongated region in its N-terminus, which is described as disordered, and lacks the WW domain. Unlike TcPar13, hParv14 lacks the N-terminal WW domain but has an unstructured N-terminal extension that is essential for its nuclear localization and DNA binding. The third Par, TcPar45, has an elongated N-terminal segment and contains a forkhead-associated domain (FHA) instead of a WW domain; FHA domains play a role in recognizing phosphopeptides related to biological processes [75].

The genome of the T. brucei gambiense strain DAL972 is closely related to the T. brucei genome, which suggests that the DAL972 genome is an effective scaffold for any T. brucei genome sequence [76]. The T. brucei gambiense strain DAL972 genome in TriTrypDB (Release 62 09 Mar 2023) [25] encodes 19 members of the CyP family (four more than T. cruzi), 6 members of the FKBP family, and 3 members of the Pars family [77] (Table 5).

We analyzed the 3D models of these PPIases and observed similarities with those in T. cruzi. The TbgCyPs exhibit elongated N- or C-terminal segments or both (TbgCyP21.1, TbgCyP21.4, TbgCyP25.55, TbgCyP27.1, TbgCyP27.4, TbgCyP29, TbgCyP30, TbgCyP33, TbgCyP43, TbgCyP46, and TbgCyP100). TbgCyP38, like TcCyP38, contains a TPR motif. Furthermore, four CyPs have SP domains (TbgCyP21.2, TbgCyP24, TbgCyP25.56, and TbgCyP58). T. cruzi does not contain a 58 kDa CyP, unlike T. brucei gambiense. TbgCyP58 also contains an RRM motif, which is involved in nucleic acid and/or protein recognition. T. brucei gambiense has three CyPs (TbgCyP19, TbgCyP20.3, and TbgCyP20.5) that consist predominantly of the CLD domain, whereas T. cruzi has only one CyP with this domain.

We found that the T. brucei gambiense FKBPs also contain elongated N- and C-terminal regions (TbgFKBP12.3 and TbgFKBP36), disordered regions and a coiled coil (TbgFKBP92), and an SP (TbgFKBP21). Additionally, TbgFKBP48 contains a disordered region paired with coiled coils and a TPR motif. Only TbgFKBP12 consists almost exclusively of the FKBP domain.

The T. brucei gambiense Pars are also homologous to T. cruzi Pars. TbgPar12 and TbgPar13 share more than 68% sequence identity with TcPar12.6 and TcPar13, respectively. TbgPar12 consists mainly of the PpiC domain, while TbgPar13 contains an elongated N-terminal segment that is reported to be disordered. Moreover, TbgPar42 shares 60% sequence identity with TcPar45 and contains both a PpiC domain and an FHA domain.

Table 5. Peptidyl-prolyl cis-trans isomerase repertoire from Trypanosoma brucei gambiense¹.

UniProt	TriTrypDB	NCBI	PPIase name2	Localization4	Function4	References
							D0A5M6	Tbg972.11.920 (CYPA)	XP_011779241.1	TbgCyP19(TbgCyPA)	Cytoplasm, flagellum, and extracellular space		[82,83]
C9ZYX4	Tbg972.9.6990	XP_011776889.1	TbgCyP20.3
D0A8E1	Tbg972.11.10610	XP_011780206.1	TbgCyP20.5
C9ZIV0	Tbg.972.2.170	XP_011771617.1	TbgCyP21.1
C9ZT99	Tbg972.7.5450	XP_011774914.1	TbgCyP21.2	Extracellular space		[83]
C9ZWH7	Tbg972.8.7100	XP_011776042.1	TbgCyP21.4
C9ZRQ0	Tbg972.7.160	XP_011774319.1	TbgCyP24
C9ZSQ5	Tbg972.7.3760	XP_011774720.1	TbgCyP25.55
C9ZNS2	Tbg972.5.1880	XP_011773337.1	TbgCyP25.56
C9ZWA7	Tbg972.8.6340	XP_011775972.1	TbgCyP27.1
C9ZXF5	Tbg972.9.1740	XP_011776370.1	TbgCyP27.4
C9ZQE6	Tbg972.6.1040	XP_011773911.1	TbgCyP29
C9ZVY5	Tbg972.8.5140	XP_011775850.1	TbgCyP30
C9ZUX8	Tbg972.8.1650	XP_011775493.1	TbgCyP33
C9ZYI8	Tbg972.9.5630	XP_011776753.1	TbgCyP38	Extracellular space		[83]
C9ZZI1	Tbg972.9.9060	XP_011777096.1	TbgCyP43	Membrane		[43]
C9ZIB2	Tbg972.1.930	XP_011771345.1	TbgCyP46
C9ZPQ4	Tbg972.5.5220	XP_011773669.1	TbgCyP58	Nucleus		[43]
C9ZZU0	Tbg972.10.15980	XP_011778762.1	TbgCyP100
D0A2I5	Tbg972.10.5640	XP_011777743.1	TbgFKBP12
C9ZSQ4	Tbg972.7.3750	XP_011774719.1	TbgFKBP12.33(TbgFKBP12)	Flagellar pocket	Motility and cytokinesis	[84]
D0A0P0	Tbg972.10.19020 (MIP)	XP_011779062.1	TbgFKBP21
D0A0P1	Tbg972.10.19030	XP_011779063.1	TbgFKBP36
D0A0V5	Tbg972.10.19710	XP_011779127.1	TbgFKBP48	Extracellular space		[83]
D0A6H9	Tbg972.11.3980	XP_011779544.1	TbgFKBP92
C9ZUI9	Tbg972.8.300 (Pin1)	XP_011775354.1	TbgPar123(TbgPin1)	Cytoplasm		[85]
C9ZKX9	Tbg972.3.3260	XP_011772278.1	TbgPar13(TbgPar14)			[85]
C9ZRL7	Tbg972.7.2770 (Par45)	XP_011774600.1	TbgPar423	Nucleus	Cell growth	[85]

¹Isolate: Trypanosoma brucei gambiense DAL972. ² In parenthesis, PPIase names previously reported. ³More information is in Table 11. ⁴The localization and function of PPIases were taken from the references cited or from the UniProt database, which were predicted by the Gene Ontology Consortium [43]. Spaces in blank: not reported data.

Table 5. Peptidyl-prolyl cis-trans isomerase repertoire from Trypanosoma brucei gambiense¹.

UniProt	TriTrypDB	NCBI	PPIase name2	Localization4	Function4	References
							D0A5M6	Tbg972.11.920 (CYPA)	XP_011779241.1	TbgCyP19(TbgCyPA)	Cytoplasm, flagellum, and extracellular space		[82,83]
C9ZYX4	Tbg972.9.6990	XP_011776889.1	TbgCyP20.3
D0A8E1	Tbg972.11.10610	XP_011780206.1	TbgCyP20.5
C9ZIV0	Tbg.972.2.170	XP_011771617.1	TbgCyP21.1
C9ZT99	Tbg972.7.5450	XP_011774914.1	TbgCyP21.2	Extracellular space		[83]
C9ZWH7	Tbg972.8.7100	XP_011776042.1	TbgCyP21.4
C9ZRQ0	Tbg972.7.160	XP_011774319.1	TbgCyP24
C9ZSQ5	Tbg972.7.3760	XP_011774720.1	TbgCyP25.55
C9ZNS2	Tbg972.5.1880	XP_011773337.1	TbgCyP25.56
C9ZWA7	Tbg972.8.6340	XP_011775972.1	TbgCyP27.1
C9ZXF5	Tbg972.9.1740	XP_011776370.1	TbgCyP27.4
C9ZQE6	Tbg972.6.1040	XP_011773911.1	TbgCyP29
C9ZVY5	Tbg972.8.5140	XP_011775850.1	TbgCyP30
C9ZUX8	Tbg972.8.1650	XP_011775493.1	TbgCyP33
C9ZYI8	Tbg972.9.5630	XP_011776753.1	TbgCyP38	Extracellular space		[83]
C9ZZI1	Tbg972.9.9060	XP_011777096.1	TbgCyP43	Membrane		[43]
C9ZIB2	Tbg972.1.930	XP_011771345.1	TbgCyP46
C9ZPQ4	Tbg972.5.5220	XP_011773669.1	TbgCyP58	Nucleus		[43]
C9ZZU0	Tbg972.10.15980	XP_011778762.1	TbgCyP100
D0A2I5	Tbg972.10.5640	XP_011777743.1	TbgFKBP12
C9ZSQ4	Tbg972.7.3750	XP_011774719.1	TbgFKBP12.33(TbgFKBP12)	Flagellar pocket	Motility and cytokinesis	[84]
D0A0P0	Tbg972.10.19020 (MIP)	XP_011779062.1	TbgFKBP21
D0A0P1	Tbg972.10.19030	XP_011779063.1	TbgFKBP36
D0A0V5	Tbg972.10.19710	XP_011779127.1	TbgFKBP48	Extracellular space		[83]
D0A6H9	Tbg972.11.3980	XP_011779544.1	TbgFKBP92
C9ZUI9	Tbg972.8.300 (Pin1)	XP_011775354.1	TbgPar123(TbgPin1)	Cytoplasm		[85]
C9ZKX9	Tbg972.3.3260	XP_011772278.1	TbgPar13(TbgPar14)			[85]
C9ZRL7	Tbg972.7.2770 (Par45)	XP_011774600.1	TbgPar423	Nucleus	Cell growth	[85]

¹Isolate: Trypanosoma brucei gambiense DAL972. ² In parenthesis, PPIase names previously reported. ³More information is in Table 11. ⁴The localization and function of PPIases were taken from the references cited or from the UniProt database, which were predicted by the Gene Ontology Consortium [43]. Spaces in blank: not reported data.

2.2.2. Leishmania major and Leishmania donovani

Leishmaniasis is caused by protozoan parasites in the Leishmania genus, which are transmitted via infected sandfly mosquito bites to humans and other animals. Between 700,000 and one million new cases of leishmaniasis are estimated to occur annually worldwide [87]. L. major and L. donovani are linked to cutaneous and visceral leishmaniasis, respectively, in Asia, Africa, and parts of Europe [88].

The TriTrypDB database (https://tritrypdb.org/tritrypdb/app/) (Release 63 03May23) [25] contains at least 24 PPIase genes from the L. major isolate Friendlin reference genome [89] and the same number from the L. donovani BPK282A1 reference genome [90]. Both species contain 17 CyPs, 5 FKBPs, and 2 Pars (Table 6 and Table 7). Despite their high overall sequence identity and similar molecular weights, LmPar47 and LdPar17 exhibit only 34% sequence identity, mainly due to differences in molecular weight (Supplementary Table S4). Interestingly, the gene encoding LdCyP108, which is categorized as a conserved hypothetical protein in the Leishmania database (TriTrypDB), was confirmed to be a CyP through further verification via the UniProt and NCBI databases. The number of PPIases in Leishmania is comparable to that in other trypanosomatids, such as T. cruzi and T. brucei (Table 4 and Table 5).

The first PPIases discovered in L. major and L. donovani were CyPs identified during studies involving Cyclosporin A (CsA) [91,92]. Leishmania CyPs vary in size from 19 to 108 kDa, and many of them have additional N-terminal or C-terminal extensions or both alongside the CLD domain [93]. Three Leishmania CyPs stand out in particular: LmCyP24.6 (also known as LmaCyP5), which features a PLD (prokaryotic lipid attachment domain); LmCyP38 (also known as LmaCyP40), which is distinguished by two additional TPR domains; and LdCyP38.4 (also known as LdCyP40), which contains a TPR domain at its C-terminus.

The crystal structure of LdCyP20 (PDB: 2HAQ) from L. donovani closely resembles that of other CyPs, with- an eight-stranded β-barrel and two α-helixes, albeit with minor differences from hCyPA [94]. LmCyP32 from L. major (PDB: 2HQJ) shares the same secondary structure with slight variations in the loops. Additionally, L. major has two more crystallized CyPs, LmCyP25 (PDB: 7AIH) and LmCyP29 (PDB: 7AM2), both of which are part of a large subunit of the L. major mitoribosome [95].

The FKBPs identified in both L. major and L. donovani display notable similarities. These proteins share the FKBP PPIase domain and fall within the 11.8 to 48 kDa range. Interestingly, only LmFKBP48 and LdFKBP47 contain an additional TPR domain at the C-terminus. Among Leishmania Pars, LmPar13, LdPar12, and LdPar17 are characterized by a PpiC PPIase domain and similar molecular weights. In the case of LmPar47, an FHA domain is present alongside the PpiC domain.

2.3. Apicomplexan Parasites

2.3.1. Plasmodium falciparum and Plasmodium vivax

The Plasmodium genus causes malaria, which is transmitted by infected female Anopheles mosquitoes. P. falciparum and P. vivax are the most prominent species that cause malaria in humans due to their characteristics and impact on public health. P. falciparum is the most prevalent pathogen in Africa, and P. vivax is the dominant parasite in most countries outside of sub-Saharan Africa. According to the latest report, there were 247 million cases of malaria in 2021 [102].

The reference genome of the P. falciparum 3D7 isolate is widely used in malaria research. According to the PlasmoDB database (https://plasmodb.org/plasmo/app) (Release 63 03May2023) [25], this genome contains 13 genes encoding PPIases: 11 CyPs and 2 FKBPs. To date, no Pars have been reported (Table 8). The new reference genome for P. vivax was obtained from the P01 isolate [103]. According to PlasmoDB (Release 63 03May2023), this genome contains the same number of PPIases as P. falciparum. Thus, this family is conserved between species even though the P. vivax genome presents almost twice the genetic diversity of P. falciparum [104,105].

The P. falciparum CyPs have molecular weights ranging from 19 to 87 kDa. They contain a conserved CLD domain with a typical eight-strand β-barrel and two α-helixes. Most of the PfCyPs contain a CLD region of ~142-165 aa, but the largest CLD region is 209 aa (PfCyP81) (Table 8). PfCyP19 and PfCyP18.6 (also known as PfCyP19C) consist only of the CLD domain, and the rest are CyP isoforms. For example, PfCyP22 contains an SP, and PfCyP23 has a coiled coil near the N-terminus. PfCyP25 has N-terminal extensions, whereas PfCyP26 and PfCyP53 have C-terminal extensions, and PfCyP32 and PfCyP72 have extensions in both the N- and C-terminal regions. PfCyP87 contains a WD40 repeat, and PfCyP81 contains a region related to the SYF2 family (Figure 4A,B). This analysis was similar to that reported by Marín-Menéndez & Bell (2011) [106], with some differences in the length of regions or domains. Additionally, two crystallized structures of the CyP catalytic domain have been reported for P. falciparum: PfCyP19 (PDB: 1QNG) and PfCyP87 (PDB: 2FU0) [107] (Table 8).

To date, no P. vivax CyPs have been characterized (Table 9). Approximately half the CyP sequences of the two Plasmodium species exhibited ≥80% identity. However, PvCyP29, PvCyP52, PvCyP65, PvCyP71, and PvCyP87 have below-average identity (Supplementary Table S5). PvCyP19 consists predominantly of the CLD domain. Outside this domain, the P. vivax homolog CyPs show similarities to PfCyP. For example, PvCyP21 contains an SP, and PvCyP83 contains a WD40 repeat. The CyPs with elongated segments in the N-terminus, C-terminus, or both are PvCyP18, PvCyP23, PvCyP26, PvCyP29, PvCyP32, and PvCyP52. Intriguingly, P. vivax has two CyPs with distinct molecular weights, PvCyP29 and PvCyP65, instead of PfCyP25 and PfCyP81. PvCyP65 also contains a region belonging to the SYF2 family, as suggested for PfCyP81.

P. falciparum and P. vivax possess only two FKBPs, in contrast to most other parasites mentioned, which typically have at least five FKBPs. Some of the Plasmodium FKBPs have 60% identity (PfFKBP25.6 and PvFKBP25) and 80% identity (PfFKBP35 and PvFKBP34) (Supplementary Table S5). The conserved FK506 binding domains of three Plasmodium FKBPs have been crystallized: PfFKBP35 (PDB: 2OFN) [107], PvFKBP25 (PDB: 4JYS) [109], and PvFKBP34 (PDB: 2KI3) [110] (Table 8 and Table 9). This domain shares many of its secondary structures, comprising a six-stranded β-sheet and a short α-helix, with an additional β-strand at the N-terminus. In addition to this domain, PfFKBP25 and PvFKBP25 contain extensions at the N-terminus. PvFKBP25 is considered an atypical FKBP that lacks catalytic activity and does not have the conserved active site in typical FKBPs [109,111]. PfFKBP35 and PvFKBP34 have three and one TPR domains, respectively. These domains control the dimeric form of PfFKBP35, while the FKBP domain remains a monomer in solution [112].

Table 9. Peptidyl-prolyl cis-trans isomerase repertoire from Plasmodium vivax^1,2.

UniProt	PlasmoDB	NCBI	PDB	PPIase name	Localization	References
							A0A1G4HCW7	PVP01_0916900 (CYP19C)	XP_001615280.1		PvCyP18.5
A0A1G4HBM6	PVP01_0818200 (CYP19A)	XP_001614493.1		PvCyP19
A0A1G4HCM3	PVP01_0916400	XP_001615276.1		PvCyP21
A0A1G4HDR7	PVP01_1005100 (CYP23)	XP_001613671.1		PvCyP23
A0A1G4H2Q1	PVP01_1301700 (CYP26)	XP_001616500.1		PvCyP26
A0A1G4GR33	PVP01_0115700 (CYP24)	XP_001608574.1		PvCyP29
A0A1G4H4X8	PVP01_1434000 (CYP32)	XP_001617250.1		PvCyP32
A0A1G4HIV6	PVP01_1325800	CAG9475874.1		PvCyP52
A0A1G4HAY2	PVP01_0729200	XP_001614845.1		PvCyP71
A0A1G4GR20	PVP01_0117200 (CYP81)	CAG9485095.1		PvCyP65	Nucleus	[43]
A0A1G4HEA6	PVP01_1023800 (CYP87)	XP_001613274.1		PvCyP83
A0A1G4H4D0	PVP01_1414200	XP_001617060.1	4JYS	PvFKBP253		[108]
A0A565A3M9	PVP01_1464500	XP_001613999.1	2KI3	PvFKBP343(PvFKBP35)		[109,121]

¹Isolate: Plasmodium vivax P01. ² In parenthesis, PPIAse names previously reported. ³ More information is in Table 11. ⁴The localization and function of PPIases reported in the references cited or predicted by the Gene Ontology Consortium [43] in the UniProt database. Spaces in blank: no reported data.

Table 9. Peptidyl-prolyl cis-trans isomerase repertoire from Plasmodium vivax^1,2.

UniProt	PlasmoDB	NCBI	PDB	PPIase name	Localization	References
							A0A1G4HCW7	PVP01_0916900 (CYP19C)	XP_001615280.1		PvCyP18.5
A0A1G4HBM6	PVP01_0818200 (CYP19A)	XP_001614493.1		PvCyP19
A0A1G4HCM3	PVP01_0916400	XP_001615276.1		PvCyP21
A0A1G4HDR7	PVP01_1005100 (CYP23)	XP_001613671.1		PvCyP23
A0A1G4H2Q1	PVP01_1301700 (CYP26)	XP_001616500.1		PvCyP26
A0A1G4GR33	PVP01_0115700 (CYP24)	XP_001608574.1		PvCyP29
A0A1G4H4X8	PVP01_1434000 (CYP32)	XP_001617250.1		PvCyP32
A0A1G4HIV6	PVP01_1325800	CAG9475874.1		PvCyP52
A0A1G4HAY2	PVP01_0729200	XP_001614845.1		PvCyP71
A0A1G4GR20	PVP01_0117200 (CYP81)	CAG9485095.1		PvCyP65	Nucleus	[43]
A0A1G4HEA6	PVP01_1023800 (CYP87)	XP_001613274.1		PvCyP83
A0A1G4H4D0	PVP01_1414200	XP_001617060.1	4JYS	PvFKBP253		[108]
A0A565A3M9	PVP01_1464500	XP_001613999.1	2KI3	PvFKBP343(PvFKBP35)		[109,121]

¹Isolate: Plasmodium vivax P01. ² In parenthesis, PPIAse names previously reported. ³ More information is in Table 11. ⁴The localization and function of PPIases reported in the references cited or predicted by the Gene Ontology Consortium [43] in the UniProt database. Spaces in blank: no reported data.

2.3.2. Toxoplasma gondii

Toxoplasmosis is caused by the parasite Toxoplasma gondii in warm-blooded animals, including humans. Most infected individuals with strong immune systems do not show symptoms and do not require treatment. However, pregnant women and immunocompromised individuals need to be cautious, as toxoplasmosis can cause severe health problems [123]. The T. gondii ME49 isolate is type II, which is considered the priority type due to having the closest association with human disease [124]. According to the ToxoDB database (https://toxodb.org/toxo/app) (Release 63 03May23) [25], the T. gondii ME49 genome contains 20 genes encoding PPIases, 13 of which are CyPs, 4 are FKBPs, 2 are Pars, and 1 is a dual PPIase (FKBP-CyP) (Table 10).

The size of CyPs in T. gondii ranges from 18 to 86 kDa. Two crystallized CyP proteins, TgCyP64 (PDB: 3BKP) [58] and TgCyP69 (PDB: 3BO7) [58], exhibit conserved secondary structures within the CLD domain, featuring eight β-strands and two α-helices. TgCyP18 mainly consists of the CLD domain, and the rest of the CyPs are isoforms. TgCyP20 contains an SP, and TgCyP21, TgCyP23, TgCyP26, TgCyP32, TgCyP35, and TgCyP38 have a TMH domain and extra extensions at the N- or C-terminus or both in addition to the CLD domain. TgCyP38 initially appeared to be CyP20 due to its molecular weight, as previously reported [126]. However, a closer analysis revealed that the ~20 kDa region corresponds to the CLD. This finding suggested that this protein undergoes a specific posttranslational modification process, distinct from glycosylation, to remove its N-terminal extension. Additionally, TgCyP38 also has a transmembrane domain that appears to play an important role in its folding, assembly, and function [124].

Moreover, TgCyP66.2 has a coiled coil structure and is associated with the 2YF2 family due to sequence similarities, while TgCyP66.25 contains an RRM. Additionally, TgCyP69 contains a U-box domain. TgCyP86, the largest CyP in this organism (Table 10), contains a WD40 repeat, similar to the largest CyPs in other parasites, such as P. falciparum and P. vivax.

T. gondii FKBPs range in size from 38 to 67 kDa and share similarities at the structural level, primarily due to their FKBP domain. Notably, none of these FKBPs have TPRs, unlike those in other parasites. An intriguing PPIase in T. gondii is TgFCBP57, which is classified as a dual-family PPIase because it possesses both FKBP and CyP domains at the N- and C-termini, respectively, linked by TPRs [127] (Table 10). Figure 5 shows that the PPIase domains of other Apicomplexa parasites, such as P. falciparum and P. vivax, are highly conserved, even for this dual PPIase, showing very similar three-dimensional structures. Although an RNAi study showed that PPIases are essential for T. gondii growth [127], the specific function(s) of the FKBPs have yet to be determined.

Two members of the Parv family are present: TgPar13 and TgPar96 (Table 10). TgPar13 comprises an entire PpiC domain. TgPar96 is the second largest Parv after TvPar102. This Par is 912 aa long and contains a PpiC domain within the last 146 aa, along with an FHA domain. However, the sequences outside the catalytic domain have not been well characterized. These structural and sequence resemblances among parasite PPIases demonstrate their shared evolutionary history.

3. Localization and Functions of PPIases in Parasites

The localization and functions of PPIases in clinical protozoan parasites are not yet well understood. In this section, we provide a broad outline of the importance of these PPIase isoform-specific extensions, which can provide valuable insights into the precise mechanisms by which PPIases regulate essential cellular processes, making them attractive targets for further research and potential therapeutic interventions.

T. vaginalis contains only two characterized CyPs, TvCyP19 and TvCyP19.9 (known as TvCyP1 and TvCyP2, respectively). TvCyP19 localizes to the cytoplasm and to hydrogenosomes. It interacts with the transcription factor Myb1, participating in the translocation of the transcription factor to the nucleus [26]. TvCyP19.9 is present in ER membranes and can associate with TvCyP19. Thus, both CyPs could be involved in a putative trafficking pathway [27]. Almost all the PPIases of T. vaginalis (except TvFKBP-63) have UniProt annotations indicating their subcellular locations (Table 1). Most of the PPIases are predicted to be present in the cytoplasm and a few in the ER (TvFKBP-15.1 and TvFKBP-15.2) or nucleus (TvCyP14, TvCyP37, TvCyP44, TvCyP63, and TvPar102) (Table 1).

Limited information is available regarding the functions and subcellular location of PPIases in E. histolytica. However, predictions made by UniProt suggest subcellular locations for these proteins. EhCyP18, EhCyP20, EhCyP21, and EhCyP22 may be located in the cytoplasm, EhCyP40 in the ER, EhPar13 in the nucleus, and EhPar13.2 in the cytosol and nucleus. The locations of the remaining E. histolytica PPIases could not be predicted (Table 2).

There is little information on the subcellular localization of G. intestinalis PPIases. Only certain Giardia PPIases, such as GiCyP21 (DH isolate), exhibit membrane localization, and according to UniProt annotations, GiCyP18, GiCyP21, GiFKBP-13, GiFKBP-24, and GiFKBP-38 of the WB isolate have a cytoplasmic presence [43] (Table 3). Only three reports are available on Giardia PPIase localization and functions. In 2017, Ma’ayeh et al. [63] identified PPIases in the Giardia secretome from the isolates WB and GS: GiCyP18 and GiFKBP-38 were found in both, GiCyP21 in WB and GiFKBP-12 in GS. Interaction with the host cells resulted in the secretion of five PPIases from both isolates: GiCyP18, GiCyP21, GiFKBP-12, GiFKBP-24, and GiFKBP-38 (Table 3). Moreover, these authors proposed that non-SP secreted proteins, such as GiCyP18 (in WB and GS isolates), which lacks SP, might be released via vesicles. Additionally, GiCyP18 (in the WB isolate), a highly expressed secreted protein [131], has been suggested to play a role in triggering macrophage pyroptosis via TLR4 signaling [64]. However, the detailed role of PPIases in giardiasis remains to be explored.

Among trypanosomatids, T. cruzi has been the focus of the most PPIase research. TcCyP19 is the most studied CyP and shares 71.9% identity with hCyPA [77]. TcCyP19 is the main CyP expressed and secreted by T. cruzi [69,81]. This protein is expressed in all stages of T. cruzi [78]. Furthermore, TcCyP19, released by the epimastigote form, inhibits insect antimicrobial peptides, increasing parasite survival [132]. Furthermore, this resistance is related to the mechanism of benznidazole resistance [133]. TcCyP19 is also involved in the modulation of ROS production during infection, promoting T. cruzi proliferation [78]. Recently, TcCyP19 has been identified as a promising target of treatment for this disease [134] and seems to be a promising biomarker for evaluating trypanocidal therapies and diagnosing the disease [135].

Moreover, TcCyP21, a low-abundance protein with an SP (Figure 6A), was identified in a membrane-enriched fraction [136]. TcCyP22, a homolog of mammalian CyPD, localizes to the mitochondria in all three stages of the T. cruzi life cycle and is involved in parasite cell death under oxidative stress. In addition, TcCyP21, TcCyP22, TcCyP24, and TcCyP25 are predicted to localize to the mitochondria [80].

Furthermore, the UniProt annotations for TcCyP30 and TcCyP42, two of the four CyPs with elongated regions in their N-terminus, indicate that these CyPs exhibit cell membrane localization due to their transmembrane regions. Mitochondrial TcCyP22 is another CyP with this extended region. Thus, these elongations are not random or disordered regions; rather, they could represent specific localization signals that have not been previously reported.

TcFKBP-22 (TcMIP) is the only T. cruzi FKBP whose function and localization have been previously described. TcFKBP-22 possesses an SP (Figure 6B) and is secreted by trypomastigotes, playing an important role in the process of host cell entry and invasion [70,137]. Additionally, information on two of the three Pars has been reported (Figure 6C). TcPar12.6 is cytosolic and nonessential for cell proliferation, although the protein is present in all parasite stages [82]. TcPar45 is more nuclear than cytosolic [74] due to its phosphopeptide recognition module (FHA domain). This Par might be involved in a wide variety of cellular processes, such as signal transduction, protein transport, transcription, protein degradation, and DNA repair [75].

In this review, we infer that the functional PPIases in T. brucei brucei are closely related to those in the T. brucei gambiense strain (>98.7% identity) and likely present the same location and function in T. brucei gambiense strain DAL972. T. brucei gambiense CyP19 (TbgCyP19), a hCyPA homolog, is secreted and localized to the cytosol and flagellum [82,84]. Due to this diversity of location, this gene product is suggested to have a role in the Trypanosoma survival strategy. TbgCyP21.2 and TbgCyP38 are other secreted CyPs that might be part of protein complexes, considering that TbgCyP38 possesses a predicted TPR motif. Additionally, CyPs lacking an SP could be secreted through microvesicles as an alternative secretory pathway [84]. However, only two of these CyPs have UniProt annotations indicating their subcellular localization. TbgCyP43, which has an elongation in its N-terminus, is predicted to localize to the parasite membrane, and TbgCyP58 is predicted to localize to the nucleus due to the presence of an SP and a structural motif for RNA recognition (RRM).

Among the T. brucei FKBP proteins, only TbgFKBP-12.3 has been characterized. It is associated with the cytoskeleton and located in the flagellar pocket, mainly in the bloodstream form of the parasite. TbgFKBP-12.3 contributes to cytokinesis in the bloodstream form and to motility in the procyclic form [85]. Interestingly, only TbgFKBP-48 was identified as a secreted protein [84]. TbgFKBP-48 is the one containing a predicted TPR, which, like that of TbgCyP38 mentioned above, suggests a potential role in the assembly of protein complexes. Among Pars, only the localization of TbgPar12 (TbgPin1) and TbgPar42 has been previously described [86]. TbgPar12 is localized to the cytosol, and TbPar42 is localized to the nucleus, similar to their T. cruzi homologs TcPar12.6 and TcPar45, respectively. Furthermore, the association of TbPar42 with cell growth suggests that its function might resemble that of its counterpart Parv in T. cruzi.

Experimental evidence regarding the localization and function of PPIases in other trypanosomatids, such as Leishmania, remains limited. In the case of L. major, the UniProt database predicts that the majority of the PPIases are localized to the cytoplasm (Table 6). Moreover, the discovery that the two crystallized CyPs (LmCyP25 and LmCyP29) of L. major are components of the mitoribosome [95] suggests that these CyPs are involved in the cis-trans isomerization of newly synthesized peptides. This mechanism is similar to that of the E. coli trigger factor PPIase, which catalyzes the cis-trans isomerization of RNAse T1 at the 50S ribosomal subunit [138]. Moreover, the Vibrio cholerae trigger factor has a similar interaction with the 50S ribosomal subunit, suggesting its involvement in the cis-trans isomerization of novel peptides [139].

Among L. donovani CyPs, LdCyP20.4 (also referred to as LdCyP) is a noncytolytic CyP [92] that is released into the cytoplasm and is localized in the ER of the parasite under stress conditions. This translocation pattern suggests a regulatory role during transformation in L. donovani [101]. In addition, LdCyP20.4 has a chaperone function that contributes to the disaggregation of adenosine kinase (AK) aggregates in vitro and prevents AK aggregation in vivo [98,140]. Moreover, according to Yau et al., 2010 [93], LdCyP19 (CyP2 identified by MS) and LdCyP38 (also known as LmCyP40) might be implicated in Leishmania growth or differentiation. Interestingly, LdCyP38 was identified as a phosphoprotein in amastigotes, and LdFKBP-47 was identified as a phosphoprotein in both stages [140,142]. This finding suggested that this posttranslational modification can regulate protein activity, location, and interactions in a stage-specific manner. Nonetheless, further research is needed to validate the precise roles of these PPIases.

Among P. falciparum CyPs, PfCyP22 and PfCyP19 may be cytosolic CyPs [117]. Additionally, PfCyP22 is localized to the membrane [119], which is consistent with the UniProt annotation. P. falciparum is the second parasite, following T. vaginalis, with the most UniProt annotations for subcellular CyP location. CyPs are found in the cytoplasm (PfCyP26 and PfCyP32), mitochondria (PfCyP32), and nucleus (PfCyP18.6, PfCyP23, PfCyP53, PfCyP72, PfCyP81, and PfCyP87). Among the P. vivax CyPs, only PvCyP65 is suggested to localize to the nucleus (Table 8 and Table 9). The prevalence of the prediction of nuclear localization for these CyPs is intriguing. Notably, PfKBP-35 is the only FKBP with a nuclear prediction in UniProt, which is supported by the data of Kumar et al. (2005) [115], suggesting a role in parasite nucleosome interactions [143].

The localization of T. gondii PPIases is similar. However, studies on the localization and functions of these CyPs are rare. For example, TgCyp18-induced nitric oxide production plays a critical role in inhibiting parasite replication and triggering bradyzoite development [128]. TgCyP20 is a secreted protein that interacts with cysteine‒cysteine chemokine receptor 5 (CCR5) and triggers IL-12 production [129]. Interestingly, its PPIase activity is not necessary for the CCR5 interaction but is required for IL-12 induction [144]. T. gondii is known to employ a sophisticated strategy of manipulating pro- and anti-inflammatory host cell signaling to promote parasite growth and dissemination while preserving host survival. Furthermore, the UniProt database suggests that TgCyP66.21 and TgCyP66.25 are localized to the nucleus, while TgCyP38 and TgFKBP-38 are found in the membrane. Nonetheless, additional research is required to explore other functions of T. gondii PPIases.

Although some advances have been made in understanding the localization and functions of PPIases in parasites, much remains unknown. Furthermore, understanding the significance of isoform-specific extensions of PPIases might provide valuable insights into the precise mechanisms of these proteins in parasite biology and pathogenesis, making them attractive targets for further investigation and potential therapeutic interventions.

4. Recombinant Expression and Purification of PPIases from Clinically Important Protists

Producing recombinant proteins from protist parasites is often difficult because of the challenges of both the expression of the protein in the model organism itself and the heterologous expression of recombinant proteins with enzymatic activity [145]. This difficulty arises from the uniqueness of protist protein sequences, as well as the intrinsic complexity of certain proteins [146].

The lack of information about PPIase proteins in many protists has underscored the importance of their recombinant production for molecular and biochemical characterization. Researchers have successfully generated recombinant PPIases with full enzymatic activity through heterologous expression. Various expression vectors, both commercial and modified, can include tags, such as His, GST, or the solubility tag SUMO (Table 11). E. coli is the preferred expression platform; multiple strains have been used, including Rosetta, JM109, XL-Blue, and BL21 (DE3), the last of which is the most widely used. Most of the purification methods used involve affinity and ion-exchange chromatography, e.g., IMAC, IEX, and GST/GSH Sepharose (Table 11).

Table 11. Recombinant PPIases from protozoan parasites expressed in E. coli^1,2.

Parasite	PPIasa	UniProt	kDa	pI	Expression system		Purification	Catalytic Efficiency3	Inhibition		References
					Strain	Vector		kcat/Km.	Inhibitor	IC50 nM4
T. cruzi	TcCyP19	Q9U664	18.9	8.4	M15, XL1Blue	pQE30	IMAC		CsA H-7-94 F-7-62 MeVal-4	14.42-18.4 12.54 13.3 15.25	[66,77,79,81]
	TcCyP21	Q4DPB9	21.1	9.1	BL21 pLysS	pET14	IMAC		CsA H-7-94 F-7-62 MeVal-4	28.74 23.64 25.15 30.04	[69,79]
	TcCyP25	Q9NAT5	25.6	8.5	Origami	pRSETA	IMAC		CsA H-7-94 F-7-62 MeVal-4	31.7 17.2 17.8 29.98	[69,79]
	TcCyP28	O76990	28.4	9.7	BL21 RIL	pET41b	IMAC		CsA H-7-94 F-7-62 MeVal-4	13.05 9.16 10.06 13.52	[69,79]
	TcCyP34	K2NAL4	33.4	9.0	BL21(DE3)	pRSETA	IMAC		CsA H-7-94 F-7-62 MeVal-4	>2005	[69,79]
	TcCyP38 (TcCyP40)	Q6V7K6	38.4	5.7	M15	pQE30	IMAC		CsA H-7-94 F-7-62 MeVal-4	>2005	[69]
T. brucei	TbgCyP19 (TbCypA)	D0A5M6	18.7	8.3	M15	pQE30	IMAC				[83]
T. vaginalis	TvCyP19 (TvCyP1)	A2DT06	19	7.7	BL21	pET32a	IMAC, IEX, AC	7.1 M-1s-1 4.0 M-1s-1	CsA	7.5	[26,28]
	TvCyP19.9 (TvCyP2)	A2DLL4	20	9.1	BL21	pET, pGEX2t pET29b	IMAC, IEX, AC	4.5 M-1s-1			[27,29]
L. major	LmaCyP19	O02614	19	7.7	M15	pQE30, pREP4 pET14b, pTYB1 pGEX4T-3	IMAC, HIC, AC	1.5x106 M-1s-1 2.6x106 M-1s-1	CsA	Ki=0.53	[96,97]
	LmaCyP38 (LmaCyp40)	E9AFV2	38.4	5.6	BL21	pGEX-5X-Strep	AC				[93]
L. donovani	LdCyP20.4 (LdCyP)	Q9U9R3	17.7	6.9	BL21 pLysS	pET3a, pQE32	IMAC				[92,94,99,100]
T. gondii	TgCyP18	A0A125YZ79	18.3	6.9	BL21	pET28a	IMAC, AC, SEC, RPC	1.0x104 M-1s-1			[130]
	TgCyP20	S8F7V1	19.6	6.0			AC				[126,129]
	TgCyP23	A0A125YL73	22.9	7.0	BL21	pET28a	IMAC, SEC	3.8x106 M-1s-1			[130]
P. falciparum	PfCyP19 (PfCyP19A)	Q76NN7	19	8.2	BL21	pET-3a, pET22b+	IMAC	6.3x106 M-1s-1 1.2x107 M-1s-1	CsA CsC CsD Rapamycin FK506	10 581 238 >5000 >10000	[106,113,114,116,117]
	PfCyP22 (PfCyP19B)	Q8IIK8	22	7.1	BL21	pET22b+	IMAC	2.3x106 M-1s-1 5.7x106 M-1s-1	CsA	10	[106,116,117,118]
	PfCyP18.6 (PfCyP19C)	Q8IIK3	18.6	5.9	BL21	pET22b+	IMAC				[106,114]
	PfCyP23	Q8I3I0	23.2	5.3	BL21	pET22b+	IMAC				[106,114]
	PfCyP25 (PfCyP24)	Q8I6S4	24.9	6.7	BL21	pET22b+	IMAC				[106,109,114,120]
	PfCyP26	Q8I621	26.4	8.5	BL21	pET22b+	IMAC				[106,114]
	PfCyP32	Q8I5Q4	32.3	9.8	Rosseta	pET22b+	IMAC				[106,114]
	PfCyP53 (PfCyP52)	Q8ILM0	52.7	7.0	BL21	pET22b+	IMAC				[106,114]
E. histolytica	EhCyP18 (EhCyP)	O15729	18.1		XL1Blue	pTrcHis A	IMAC		CsA	10	[46]
G. intestinalis	GiCyP19 (GiCyP1)		19		BL21	pGEX 4T-1	AC		CsA	500	[56]
	GiCyP18	A8BC67	18	8.4	BL21	pColdI	IMAC				[64]
T. cruzi	TcFKBP22 (TcMIP)	Q09734	22.1	6.8	XL1 Blue	pGEX-2T	AC	0.745 M-1s-1	FK506	410	[70,71]
T. gondii	TgFCBP57	Q4VKI5	57.2	5.5	BL21(DE3)	pET15b	IMAC		FK506 CsA	70 750	[127]
P. falciparum	PfFKBP35	Q8I4V8	34.8	5.4	BL21, TB1	pMALc2X, pSUMO	IMAC, SEC, AC	1.7x104 M-1s-1 1x105 M-1s-1	FK506 Rapamcyin D44	320,260 480 125	[108,112,115,121]
P. vivax	PvFKBP25	A0A1G4H4D0	25.2	9.5	BL21(DE3)	pNIC28-Bsa4	IMAC, SEC				[109]
	PvFKBP34 (PvFKBP35)	A0A565A3M9	34	6.1	BL21(DE3)	pSUMO	IMAC, SEC	1x105 M-1s-1	FK506 D44	160 125	[109,121]
G. intestinalis	GiFKBP12	Q8I6M8	12	9.2	BL21(DE3)-R3-RARE	AVA0421	IMAC, SEC				[59]
T. cruzi	TcPar12.6 (TcPin1)	Q4D8F7/ Q4DKA4	12.6	7.7	JM109	pQE30	IMAC	3.97x105 M-1s-1 1.54x104 M-1s-1			[73,82]
	TcPar13 (TcPar14)	Q4D394/ Q4E641	13.3	9.4	BL21(DE3)-CodonPlus RIL	pET-22b+	IMAC, SEC	0.194 M-1s-1			[74]
	TcPar45	Q4D9J4/ Q4DH56	45.5	8.7	BL21(DE3)-CodonPlus RIL	PET28a	IMAC, SEC	7.1x103 M-1s-1			[74]
T. brucei	TbgPar12 (TbPin1)	C9ZUI9	12.5	6		pET28b	SEC				[86]
	TbPar42	C9ZRL7	41.7	7.1		pET28b	SEC				[86]
L. major	LmaPar13 (LmPIN1)	Q4QII4	12.6	7.2	BL21		IMAC, SEC				[93,98]

¹Experimental data reported in the references cited. ² In parenthesis, PPIAse names previously reported. ³Catalytic Efficiency determined by Kofron assay. ⁴Values for IC₅₀. Ki only for LmaCyP19. ⁵ >200 nm for the four inhibitors. Spaces in blank: no reported data. Affinity chromatography (AC). Immobilized metal affinity chromatography (IMAC). Size exclusion chromatography (SEC). Hydrophobic Interaction Chromatography (HIC). Reverse phase chromatography (RPC). Ion exchange chromatography (IEX). Cyclosporin A (CsA), Cyclosporin C (CsC), Cyclosporin D (CsD). pI= Isoelectric point. IC₅₀= inhibitory concentration at 50%. K_i= inhibitory constant.

Table 11. Recombinant PPIases from protozoan parasites expressed in E. coli^1,2.

Parasite	PPIasa	UniProt	kDa	pI	Expression system		Purification	Catalytic Efficiency3	Inhibition		References
					Strain	Vector		kcat/Km.	Inhibitor	IC50 nM4
T. cruzi	TcCyP19	Q9U664	18.9	8.4	M15, XL1Blue	pQE30	IMAC		CsA H-7-94 F-7-62 MeVal-4	14.42-18.4 12.54 13.3 15.25	[66,77,79,81]
	TcCyP21	Q4DPB9	21.1	9.1	BL21 pLysS	pET14	IMAC		CsA H-7-94 F-7-62 MeVal-4	28.74 23.64 25.15 30.04	[69,79]
	TcCyP25	Q9NAT5	25.6	8.5	Origami	pRSETA	IMAC		CsA H-7-94 F-7-62 MeVal-4	31.7 17.2 17.8 29.98	[69,79]
	TcCyP28	O76990	28.4	9.7	BL21 RIL	pET41b	IMAC		CsA H-7-94 F-7-62 MeVal-4	13.05 9.16 10.06 13.52	[69,79]
	TcCyP34	K2NAL4	33.4	9.0	BL21(DE3)	pRSETA	IMAC		CsA H-7-94 F-7-62 MeVal-4	>2005	[69,79]
	TcCyP38 (TcCyP40)	Q6V7K6	38.4	5.7	M15	pQE30	IMAC		CsA H-7-94 F-7-62 MeVal-4	>2005	[69]
T. brucei	TbgCyP19 (TbCypA)	D0A5M6	18.7	8.3	M15	pQE30	IMAC				[83]
T. vaginalis	TvCyP19 (TvCyP1)	A2DT06	19	7.7	BL21	pET32a	IMAC, IEX, AC	7.1 M-1s-1 4.0 M-1s-1	CsA	7.5	[26,28]
	TvCyP19.9 (TvCyP2)	A2DLL4	20	9.1	BL21	pET, pGEX2t pET29b	IMAC, IEX, AC	4.5 M-1s-1			[27,29]
L. major	LmaCyP19	O02614	19	7.7	M15	pQE30, pREP4 pET14b, pTYB1 pGEX4T-3	IMAC, HIC, AC	1.5x106 M-1s-1 2.6x106 M-1s-1	CsA	Ki=0.53	[96,97]
	LmaCyP38 (LmaCyp40)	E9AFV2	38.4	5.6	BL21	pGEX-5X-Strep	AC				[93]
L. donovani	LdCyP20.4 (LdCyP)	Q9U9R3	17.7	6.9	BL21 pLysS	pET3a, pQE32	IMAC				[92,94,99,100]
T. gondii	TgCyP18	A0A125YZ79	18.3	6.9	BL21	pET28a	IMAC, AC, SEC, RPC	1.0x104 M-1s-1			[130]
	TgCyP20	S8F7V1	19.6	6.0			AC				[126,129]
	TgCyP23	A0A125YL73	22.9	7.0	BL21	pET28a	IMAC, SEC	3.8x106 M-1s-1			[130]
P. falciparum	PfCyP19 (PfCyP19A)	Q76NN7	19	8.2	BL21	pET-3a, pET22b+	IMAC	6.3x106 M-1s-1 1.2x107 M-1s-1	CsA CsC CsD Rapamycin FK506	10 581 238 >5000 >10000	[106,113,114,116,117]
	PfCyP22 (PfCyP19B)	Q8IIK8	22	7.1	BL21	pET22b+	IMAC	2.3x106 M-1s-1 5.7x106 M-1s-1	CsA	10	[106,116,117,118]
	PfCyP18.6 (PfCyP19C)	Q8IIK3	18.6	5.9	BL21	pET22b+	IMAC				[106,114]
	PfCyP23	Q8I3I0	23.2	5.3	BL21	pET22b+	IMAC				[106,114]
	PfCyP25 (PfCyP24)	Q8I6S4	24.9	6.7	BL21	pET22b+	IMAC				[106,109,114,120]
	PfCyP26	Q8I621	26.4	8.5	BL21	pET22b+	IMAC				[106,114]
	PfCyP32	Q8I5Q4	32.3	9.8	Rosseta	pET22b+	IMAC				[106,114]
	PfCyP53 (PfCyP52)	Q8ILM0	52.7	7.0	BL21	pET22b+	IMAC				[106,114]
E. histolytica	EhCyP18 (EhCyP)	O15729	18.1		XL1Blue	pTrcHis A	IMAC		CsA	10	[46]
G. intestinalis	GiCyP19 (GiCyP1)		19		BL21	pGEX 4T-1	AC		CsA	500	[56]
	GiCyP18	A8BC67	18	8.4	BL21	pColdI	IMAC				[64]
T. cruzi	TcFKBP22 (TcMIP)	Q09734	22.1	6.8	XL1 Blue	pGEX-2T	AC	0.745 M-1s-1	FK506	410	[70,71]
T. gondii	TgFCBP57	Q4VKI5	57.2	5.5	BL21(DE3)	pET15b	IMAC		FK506 CsA	70 750	[127]
P. falciparum	PfFKBP35	Q8I4V8	34.8	5.4	BL21, TB1	pMALc2X, pSUMO	IMAC, SEC, AC	1.7x104 M-1s-1 1x105 M-1s-1	FK506 Rapamcyin D44	320,260 480 125	[108,112,115,121]
P. vivax	PvFKBP25	A0A1G4H4D0	25.2	9.5	BL21(DE3)	pNIC28-Bsa4	IMAC, SEC				[109]
	PvFKBP34 (PvFKBP35)	A0A565A3M9	34	6.1	BL21(DE3)	pSUMO	IMAC, SEC	1x105 M-1s-1	FK506 D44	160 125	[109,121]
G. intestinalis	GiFKBP12	Q8I6M8	12	9.2	BL21(DE3)-R3-RARE	AVA0421	IMAC, SEC				[59]
T. cruzi	TcPar12.6 (TcPin1)	Q4D8F7/ Q4DKA4	12.6	7.7	JM109	pQE30	IMAC	3.97x105 M-1s-1 1.54x104 M-1s-1			[73,82]
	TcPar13 (TcPar14)	Q4D394/ Q4E641	13.3	9.4	BL21(DE3)-CodonPlus RIL	pET-22b+	IMAC, SEC	0.194 M-1s-1			[74]
	TcPar45	Q4D9J4/ Q4DH56	45.5	8.7	BL21(DE3)-CodonPlus RIL	PET28a	IMAC, SEC	7.1x103 M-1s-1			[74]
T. brucei	TbgPar12 (TbPin1)	C9ZUI9	12.5	6		pET28b	SEC				[86]
	TbPar42	C9ZRL7	41.7	7.1		pET28b	SEC				[86]
L. major	LmaPar13 (LmPIN1)	Q4QII4	12.6	7.2	BL21		IMAC, SEC				[93,98]

¹Experimental data reported in the references cited. ² In parenthesis, PPIAse names previously reported. ³Catalytic Efficiency determined by Kofron assay. ⁴Values for IC₅₀. Ki only for LmaCyP19. ⁵ >200 nm for the four inhibitors. Spaces in blank: no reported data. Affinity chromatography (AC). Immobilized metal affinity chromatography (IMAC). Size exclusion chromatography (SEC). Hydrophobic Interaction Chromatography (HIC). Reverse phase chromatography (RPC). Ion exchange chromatography (IEX). Cyclosporin A (CsA), Cyclosporin C (CsC), Cyclosporin D (CsD). pI= Isoelectric point. IC₅₀= inhibitory concentration at 50%. K_i= inhibitory constant.

Among the clinically important parasites, PPIases from E. histolytica, G. intestinalis, and P. vivax have undergone the least heterologous production and study. In contrast, more than 50% of recombinant PPIases produced are from T. cruzi and P. falciparum (Table 11). These recombinant PPIases (rPPIases) from parasites have been produced for four primary purposes: structural analysis, biological characterization, antibody production for further studies, and research on their potential as therapeutic targets.

The primary focus of rPPIase production has been on CyPs, with less on FKBPs and Pars. Only five FKBPs have been produced: G. intestinalis (GiFKBP-12), T. cruzi (TcFKBP-22), P. falciparum (PfFKBP-35), P. vivax (PvFKBP34) and T. gondii (TgFCBP-57). Only six Pars have been produced from trypanosomatids, T. cruzi (TcPar12.6, TcPar14, and TcPar45), T. brucei (TbgPar12 and TbPar42), and L. major (LmPar13). The scant production of recombinant FKBPs can be attributed to the sequence complexity of some of these proteins. Furthermore, both FKBPs and Pars are relatively new discoveries compared to CyPs, which is another reason they are only beginning to be studied in protist parasites (Table 11).

Notably, most of the protozoan rPPIases have been obtained in the soluble fraction, except for TgCyP18, which was purified from inclusion bodies but was not used in activity assays [129]. These soluble proteins have molecular weights between 18 and 30 kDa (Table 11). Difficulties in obtaining large recombinant proteins were evident with certain CyPs from P. falciparum, such as PfCyP32, PfCyP72, PfCyP81, and PfCyP87, which cannot be cloned or expressed in E. coli. Consequently, only the CLD was expressed, except for PfCyP81, which could not be produced at all.

Interestingly, many rPPIases from parasites were expressed in the soluble fraction, in contrast to the general challenges faced in obtaining recombinant proteins from protist parasites. Typically, it is estimated that only 30-50% of parasite proteins are heterologously expressed, and an even smaller fraction of those proteins are successfully purified [106].

5. Assays of the Activity of PPIases from Clinically Important Protists

Most of the activity assays conducted for the recombinant PPIases discussed in this review are based on a spectrophotometric assay proposed by Fischer (1984) [147] and modified by Kofron et al. (1991) [148]. The Kofron assay is commonly used to evaluate the cis-trans isomerization of the chromogenic peptide N-suc-APPF-pNA by PPIases via a chymotrypsin-coupled method. Additionally, modifications have been made to the chromogenic substrate sequence to assess the enzymatic affinity of PPIases. For example, succinyl-Ala-Leu-Pro-Phe-p-nitroanilide has been widely used for analyzing FKBP PPIase activity [149]. In the case of Pars from T. brucei, activity was evaluated using the phosphorylated peptide SSYFSG[p]TPLEDDSD, as Pars are known to have activity on phosphorylated peptides [86]. A protease-free variant of the Kofron assay has also been used to evaluate PPIase activity. For instance, in the case of T. cruzi Pars, a succinyl-Ala-Glu-Pro-Phe-p-nitroanilide substrate was used that includes a negatively charged glutamyl instead of a positively charged alanine, modifying the classical substrate of the assay [74,82].

Several CyPs, such as PfCyP19 and PfCyP22 from P. falciparum, LmCyP19 from L. major and TgCyP23 from T. gondii, have demonstrated high levels of activity, comparable to those of hCyPA (Kcat/Km = 4.9 × 10⁶ M^-1s^-1) [116,130]. Moreover, among the recombinant CyPs from protist parasites, TvCyP19 and TvCyP19.9 from T. vaginalis exhibited the lowest PPIase activity. Their activity is lower than that of hCyPA [26,27,130], which could be attributed to differing substrate affinities. These data suggest that these TvCyPs might exhibit different activities on other substrates (Table 11). Moreover, it is important to note that some CyPs showed no PPIase activity, such as several P. falciparum recombinant CyPs (PfCyP18.6, PfCyP23, PfCyP25, PfCyP26, PfCyP32 CLD and PfCyP25 CLD); however, the activities of these proteins were evaluated using two different methods: the classical Kofron assay and the RNAsa T refolding. One possible reason for the lack of enzymatic activity might be the absence of H¹²⁶ in the catalytic site, an aa residue considered crucial for binding to CsA in hCyP18. Notably, some hCyPs lack PPIase activity while still retaining their chaperone role. Hence, it is plausible that both functions are not universally associated with all PPIases [114].

In terms of FKBP activity, only three out of the six recombinant protist FKBPs produced thus far have been evaluated. Among these, the P. falciparum and P. vivax FKBPs, PfFKBP-35 and PvFKBP-34, exhibited similar activities, both of which were greater than the activity of T. cruzi TcFKBP-22 (Table 11). In contrast, PvFKBP-25 showed no PPIase activity, which could be attributed to mutations in the active site, similar to those in T. gondii CyPs. These data suggest that this FKBP in P. falciparum might differ from the others [109]. Notably, protist FKBPs generally exhibit lower activity than CyPs. This difference was also observed for hFKBP, which has an activity 25 times lower than that of hCyP. This significant difference in activity could be due to the varying affinities of FKBPs for the substrate used in the Kofron assay [150].

Only Pars from T. cruzi were analyzed with two different substrates. Specifically, rTcPar14 and rTcPar45 demonstrated very high affinities for the succinyl-Ala-Arg-Pro-Phe-NH-Np substrate, while their affinities were minimal or negligible for substrates lacking arginine immediately preceding proline [74]. Conversely, TcPar12.6 exhibited a greater affinity for the Ala-Glu-Pro-Phe-p-nitroanilide substrate than for the other substrates tested [73].

These data are significant because they highlight the importance of considering cases where recombinant PPIases exhibit little or no activity. These proteins might possess distinct functions or higher activity levels with different substrates. Therefore, identifying specific substrates for parasite PPIases represents a vital area of research that deserves further development.

6. Inhibition Assays of rPPIases from Protozoan Parasites

The importance of PPIase inhibitors has been highlighted since the discovery of CyPs that bind CsA, a molecule with immunosuppressive activity [11]. The antiparasitic activity of CsA is more strongly associated with calcineurin inhibition than with PPIase inhibition [151]. To fully understand the mechanism by which CsA inhibits infection, it is necessary to identify the parasite CsA receptor [130]. An important part of parasite rPPIase studies is identifying whether known inhibitors (CsA, FK506, and rapamycin) or new inhibitors inhibit a wide variety of biological processes in which PPIases are involved.

For example, the recombinant CyPs that showed high sensitivity to CsA were TvCyP19 and TvCyP19.9 from T. vaginalis; EhCyP18 from E. histolytica; PfCyP19; PfCyP22 from P. falciparum; and TgCyP23 from T. gondii in the nanomolar range (IC₅₀= 0.6-10 nM), comparable to the inhibition of hCyPA (6.6 nM) [116]. Intermediate sensitivity to CsA (IC₅₀ 13-31 nM) was observed for GiCyP19 from G. intestinalis and for TcCyP19, TcCyP21, TcCyP25, and TcCyP28 from T. cruzi. However, the lowest sensitivity to CsA (IC₅₀ of 160 nM) was determined for T. cruzi TcCyP35.1 and TcCyP38, and for Leishmania LmCyP38 and LdCyP20.4 (Table 11). These values are comparable to those of hCyP40 [152]. In contrast, the dual PPIase TgFCBP-57 from T gondii required high concentrations of CsA for inhibition (Table 11).

Moreover, competitive inhibition constant (K_i) of PPIases has been reported only for LmCyP19 (K_i =5.2 nM) from L. major and PfCyP19 from P. falciparum (K_i =3.3-14.4 nM), which showed similar affinities for CsA [97,116]. These values are also comparable to the K_i value of a mammalian CyP (K_i=3 nM) [153]. Interestingly, not all recombinant CyPs have detectable enzymatic activity: The P. falciparum CyPs, which have molecular weights between 18.6 and 53 kDa, do not (Table 11). Moreover, despite their strong in vitro inhibitory effects on P. falciparum CyPs (Table 11), the CsA derivatives did not exhibit significant antimalarial activity in in vivo tests, unlike CsA [116]. Among the nine recombinant CyPs, only one exhibited high sensitivity to CsA derivatives (Table 11). In addition, no immunosuppressive CsA derivatives demonstrated IC₅₀ values comparable to those of CsA in inhibition assays using recombinant T. cruzi CyPs, except for TcCyP35.1 and TcCyP38, which required high concentrations of inhibitors [79]. Variations in affinities between PPIases and their inhibitors, such as TgCyP18.4 and TgCyP23, have been attributed to changes in crucial binding site residues. These alterations influence the affinities of these PPIases for CsA [130].

The inhibition assays with FK506 and the four FKBs showed IC₅₀ values in the nanomolar range (70-410 nM) (Table 11). Other inhibitors tested on FKBPs, such as L-685-818, rapamycin, and D44, also had IC₅₀ values in the nanomolar range (Table 11). Notably the double inhibition of TgFCBP-57 from T. gondii by CsA and FK506 identified this protein as an FCBP, a protein with both CLD and FKBP domains. It is the only FCBP from protozoa that has been recombinantly produced thus far [127]. These inhibitors also reduced parasitic infection or growth [69,153].

Interestingly, the inhibitor D44 selectively targeted PfFKBP-35 through its PPIase activity and inhibited P. falciparum growth [154]. Notably, this PPIase can inhibit calcineurin independently of the presence of the inhibitor [127]. There are no reports on inhibition assays of protozoan Pars, although the inhibitor juglone inhibits this type of PPIase [8]. Since a unique feature of Par is binding to phosphopeptides by means of a positively charged surface, inhibitors of this protein may require negatively charged substituents [155].

Finding specific and relevant inhibitors for PPIases is challenging due to several factors, including the superficiality of their binding sites. This characteristic makes it difficult to create small-molecule inhibitors that can bind to enzymes with high affinity. Additionally, PPIases generally have structurally conserved binding sites across different families, further complicating the search for inhibitors [155]. Finally, inhibiting PPIase activity may not always affect parasite infection, as evidenced in some studies.

7. Protozoan PPIases Biotechnological Applications

The important roles of protozoan parasite PPIases in processes such as protein folding, sexual differentiation, virulence, and immunomodulation make these proteins potential drug targets. Many PPIases play important roles in the parasite as virulence factors or are important in the life cycle of the parasite.

These PPIases have the potential to be used as inhibitors of viral infections in the future. For example, TgCyP18 from T. gondii was found to be an inhibitor of HIV-1 cell fusion and cell-free virus infection. This protein binds to the human immunodeficiency virus (HIV) coreceptor CCR5 and inhibits viral fusion and infection of T cells and macrophages. Importantly, such findings may lead to new anti-HIV drugs [156,157]. Moreover, TgCyP18 has potential for use as a vaccine antigen: It has been tested in combination with vehicle and the adjuvant BCG in a vaccine against T. gondii. This vaccine proved to be highly immunogenic and to have good protection efficacy against T. gondii infection in BALB/c mice [158,159].

In addition to their potential biomedical applications, PPIases have other potential biotechnological uses. These enzymes have also been used to assist in the in vitro refolding of denatured proteins. One example is the refolding of human creatine kinase, a protein with many prolines in its sequence. This enzyme was denatured in 6 M urea and refolded in the absence or presence of human PPIase. The results showed that PPIase accelerated the slow phase of refolding, and the enzyme became active at the end of the refolding process. This highlighted the cis-trans isomerization of its prolines as the critical step in the refolding of human creatine kinase [160]. However, no research has been done on refolding assisted by parasite PPIases, which would be interesting to analyze, given the potential of parasite PPIases to assist in the refolding of proteins with many prolines.

8. Conclusions

PPIases are found in large numbers in most clinically important protozoans. However, these enzymes have not been fully studied, possibly because many of them have complex structures. Notably, PPIases play important roles as chaperones, participating in various parasite functions. Thus, several PPIases are considered virulence factors, suggesting that they are potential targets for therapeutic inhibition and vaccine antigens against parasitic infections. Therefore, the recombinant production of protozoan PPIases is an important and necessary tool to expand the biological and biotechnological information on these enzymes and illuminate their potential as therapeutic targets. Interestingly, PPIases from different parasites are often recombinantly produced in a soluble form and with catalytic activity. These characteristics endow these proteins with great potential for use in different biotechnological applications.