|
 
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| REVIEW ARTICLE |
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| Year : 2015 | Volume
: 8
| Issue : 1 | Page : 14-37 |
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Heat shock proteins and parasitic diseases: Part II. Protozoa
Sherif M Abaza MD
Department of Parasitology, Faculty of Medicine, Suez Canal University, Ismailia, Egypt
| Date of Submission | 22-Feb-2015 |
| Date of Acceptance | 17-Apr-2015 |
| Date of Web Publication | 24-Aug-2015 |
Correspondence Address:
Sherif M Abaza
Department of Parasitology, Faculty of Medicine, Suez Canal University, Ismailia
Egypt
 Source of Support: None, Conflict of Interest: None  | Check |
DOI: 10.4103/1687-7942.163407
List of contents
1. Plasmodium spp.
1.1. Introduction
1.2. Historical background
1.3. Applications
2. Leishmania spp.
2.1. Introduction
2.2. Applications
3. Trypanosoma spp.
3.1. African trypanosomiasis
3.1.1. Applications
3.2. American trypanosomiasis
3.2.1. Introduction
3.2.2. Applications
4. Toxoplasma gondii
4.1. Applications
5. Cryptosporidium spp.
5.1. Applications
6. Other protozoa
6.1. Babesia spp.
6.2. Microsporidium spp.
6.3. Giardia lamblia
6.4. Eimeria spp.
6.5. Trichomonas vaginalis
6.6. Entamoeba histolytica
6.7. Free living amoeba
6.8. Cyclospora cayetanensis
6.9. Blastocystis spp.
6.10. Theileria spp.
Concluding Remarks
References
Abbreviations
ASS: African sleeping sickness; BiP: Binding protein; BS: Bloodstream forms; CL: Cutaneous leishmaniasis; COWP: Cryptosporidium oocyst wall protein; CTL: Cytotoxic T-lymphocyte; Cy: Cytosolic; ER: Endoplasmic reticulum; GA: Geldanamycin; GP60: Glycoprotein 60; HSP: Heat shock protein; ITS: Internal transcribed spacer; KMP-11: Kinetoplastid membrane protein-11 gene; MAb: Monoclonal antibody; MCL: Mucocutaneous leishmaniasis; MHC: Major histocompatibility complexe; Mit: Mitochondrial; NO: Nitric oxide; PS: Procyclic forms; PTEX: Plasmodium translocon of exported proteins; sHSP: small heat shock protein; SSU: Small subunit; TL: Tegumentary leishmaniasis; TLR: Toll like receptor; VL: Visceral leishmaniasis; VSG: Variant surface glycoprotein. Keywords: apoptosis, drug target, heat shock protein, immune response, parasite survival, protective immunity, protozoa, serodiagnostic marker, vaccine candidate, virulence
How to cite this article:
Abaza SM. Heat shock proteins and parasitic diseases: Part II. Protozoa. Parasitol United J 2015;8:14-37 |
| 1. Plasmodium species | |  |
1.1. Introduction
It is apparent that heat shock proteins (HSPs) play an important role in the survival of Plasmodium spp. against temperature changes associated with its passage from the cold-blooded mosquito vector to the warm-blooded human host. Added to that, there is increased temperature during febrile episodes in malaria. Moreover, it was found that P. falciparum has a proteome with a majority of asparagine (Asn) residues in its amino acid repeats [1] . The presence of Asn-rich sequences in P. falciparum proteins leads to increased aggregate formation that is enhanced under stress conditions with increased temperature. Therefore, P. falciparum has to survive conditions that increase Asn repeat-rich protein aggregation [2] . It was also reported that the capacity of P. falciparum to grow and thrive inside the host bloodstream depends on its ability to export ~5% of its encoded genome (200-300 proteins) into the host cell cytosol [3] . These exported proteins (EXPs) play an important role in virulence by promoting cell rigidity and adhesion of infected cells [4] . To gain access to the host cell cytosol, EXPs must cross the parasite plasma membrane and the parasitophorous vacuole membrane. Transfer across the parasite envelope additionally requires a Plasmodium translocon of exported proteins (PTEX). Discovery of this element was a major advance as it permitted forecasting of the exported proteome of Plasmodium spp. In their studies, Australian investigators identified a translocon that comprised HSP101, PTEX150, and exported protein 2 (EXP2) with two additional accessory proteins PTEX88 and thioredoxin 2 (TRX2). They suggested that this translocon might offer new strategy for development of new drug targets [5] .
At least six P. falciparum HSP70 homologs have been identified across the cytosolic (Cy), endoplasmic reticulum (ER), and mitochondria (Mit) [3] . Of these, only PfHSP70-1 received widespread research attention due to its potential use as a vaccine candidate. A review article discussed its Cy localization, the identical sequence of PfHSP70-1 and PfHSP70-x, and their ability to move intranuclearly when exposed to stress. The authors also proved that PfHSP70-2 originates from ER and interacts with more P. falciparum proteins compared with PfHSP70-1 and PfHSP70-3 (Mit HSP70 homolog) [6] . In another review article also published in 2007, Indian scientists comparatively analyzed structures, complexes, substrate client proteins, and functions of Plasmodium HSPs. Among 92 chaperones encoded by the Plasmodium genomic structure, they discussed the possible applications of HSP90 as a drug target, HSP70 as a vaccine candidate, and HSP40 as the best representative HSP [7] . In 2009, Chiang and his colleagues presented 5 events for the essential functions of HSP70 and HSP40 in P. falciparum life cycle: 1) HSP40 interacts with HSP70 through a conserved bundle known as J domain which enhances HSP70 ATPase activity, 2) HSP70-HSP40 pairs transport across membranes and are essential for parasite's membrane integrity, 3) The formed J domain is required for 'knob' formation to bind parasitized red blood cells (RBCs) to the vascular endothelium, 4) both HSPs are necessary to offset cellular stresses encountered during the P. falciparum life cycle, 5) molecular chaperones of both HSPs help retaining newly synthesized polypeptides in soluble conformations and facilitate protein folding; a function which enables P. falciparum to exhibit protein synthesis when the trophozoite stage starts to initiate several rounds of intracellular division. Based on these events, P. falciparum viability should be sensitive to HSP70-HSP40 inhibition [8] .
Several review articles were published during 2010-2014. Shonhai [9] analyzed and discussed Plasmodium HSP90, HSP70/HSP40 pairing, and small HSPs (sHSPs) structural and functional features, and their potentiality and importance for parasite growth, development, and virulence. He also proposed mechanisms of action of drug targets that inhibit HSPs [9] . Another team from South Africa reviewed also the potential uses of Plasmodium HSPs (90, 70, and 40) as new drug targets against malaria [10] . Rug and Maier [11] reviewed the potential roles of HSP40 in Plasmodium spp. In 2013, a third team from South Africa reviewed Plasmodium HSP70-J protein potentials as a novel antimalarial drug target [12] . Indian reviewers analyzed HSP90 functions in Plasmodium spp. growth and development during febrile malarial attacks and claimed the development of seven antimalarial drugs as well as 30 anticancer therapies under development using research for HSP90 inhibitors [13] . In 2014, three review articles were published. In the first one from Australia, the reviewers discussed PfHSP70-PfHSP40 partnerships. They mentioned that, despite poor and extensive characterization of PfHSP40 and PfHSP70, both interact together and share in several functions, including protein homoeostasis, cytoprotection, and protein trafficking across the parasitophorous vacuole as well as into the infected RBCs [14] . In the second review conducted in Singapore, the reviewers claimed the identification of more than ten cochaperones for HSP90 in the P. falciparum genome of which only five (PfHop, Pfp23, PfAha1, PfPP5, and PfFKBP35) were experimentally proved to interact with HSP90. They also discussed the regulatory roles of PfHSP90's cochaperons as well as their potential influence in the development of new antimalarial drugs targeting PfHSP90 [15] . The last review article, which was also published by Australian reviewers, dealt with exported parasite proteins and their relation to virulence and in generating a new trafficking system in the infected RBCs [16] .
On the other hand, Pavithra et al. [17] carried out a system-level analysis of chaperone networks in P. falciparum, which allowed the scientists to: 1) Predict presumed functions of several hypothetical proteins in P. falciparum, 2) Draw attention to some chaperones that have not been characterized yet, 3) Highlight chaperone systems that are missing in the malarial parasite, 4) Provide a possible basis for the antimalarial activity of drugs affecting the chaperone system; and finally, 5) Determine the functional significance of chaperones during the different developmental stages of the parasite.
They concluded that understanding P. falciparum biology is essential for rational development of antimalarial drugs [17] . Furthermore, new observations regarding P. falciparum biology in vivo were reported. On the basis of the comparison of gene expression profiles of different isolates, parasites were categorized into three clusters of which cluster 1 represented starvation response and cluster 3 represented environmental stress response [18] . In another article, investigators analyzed the transcript levels of 103 chaperones from different P. falciparum clinical isolates. They found that most of the chaperones are highly overexpressed and display a specific pattern. Their results suggested overexpression of Mit and apicoplast chaperones in cluster 1 category, with overexpression of Cy chaperones in cluster 2 category. Cluster 3 consisted of two subclusters, one of which overexpressed Cy chaperones. They also found HSP70 overexpression in cluster 2, whereas HSP90 showed maximum upregulation in cluster 2 and in a specific subpopulation in cluster 3. They concluded that parasites of cluster 3 (representing environmental stress response) are further subclustered on the basis of hsp90 gene expression, and that HSPs gene expression in different P. falciparum clinical isolates is important in understanding host-parasite interactions and, subsequently, treatment of severe malaria [19] .
1.2. Historical background
Ardeshir et al. [20] recognized a 75 kDa surface protein (P75) localized in P. falciparum merozoite. Its sequence proved to be homologous with that of HSP70, suggesting its essential role in P. falciparum life cycle. The investigators also suggested the conservative character of the p75 gene as they found its identical size in nine P. falciparum strains [20] . One year later, another genomic DNA clone isolated from P. falciparum was described to encode a 72 kDa protein whose sequence proved homologous to HSP78 (glucose-regulated protein of rat and hamster) [21] . Indian investigators tested serum samples of patients cured of P. falciparum malaria for detection of antischizont and anti-HSP70 antibodies, and found increased levels of schizont antibodies that significantly inhibited merozoite invasion in vitro. Although they established increased levels of anti-HSP70, no correlation between anti-HSP70 and inhibition of merozoite invasion was detected [22] . In the same year, the same investigators evaluated the human immune response to P. falciparum HSP70. They found that serum samples from more than 50% of patients cured of the disease (with no parasitemia) had no detectable antibodies against PfHSP70, and exhibited antibody response to total schizont antigens. Therefore, they concluded that HSP70 was not a suitable vaccine candidate [23] . Similar results were obtained when Brazilian investigators investigated IgG levels against recombinant protein derived from PfHSP70 in individuals living in areas with unstable and hypoendemic malarial transmission [24] .
In 1992, Sharma [25] identified five HSP genes from P. falciparum, and he discussed their biological roles as stress proteins to protect the parasite from various stresses encountered in the host. In addition, he attributed their antigenicity to the nonhomologous sequences in the C-terminal region [25] . In 1993, American scientists detected expression of proteins with similar sequence to HSP70 in the hepatic stages of malarial parasites. They concluded their potential role in stimulation of immune responses against infected hepatocytes [26] . Later, it was found that the parasitic growth rates among five different P. falciparum isolates were not affected after increasing the temperature to 39°C for 30 min, with marked significant HSP70 expression. The investigators emphasized that the P. falciparum hsp70 gene responded to heat stress by expression of PfHSP70 to protect the parasite from being killed during malarial fever [27] . Moreover, it was shown that monoclonal antibodies (mAbs) generated by mice immunized with P. berghei-infected RBCs were reactive with HSP70 expressed in hepatic and erythrocytic stages. In contrast to previous results, the investigators demonstrated HSP70 expression in malarial sporozoites [28] .
1.3. Applications
1. Life cycle development: PfHSP90 was shown to have an essential role in parasite development within the erythrocytic stages during the frequent malarial febrile episodes [29] . In another Indian study using the HSP90 inhibitor [geldanamycin (GA)], the investigators observed the significant role of HSP90 in parasite development and growth [30] . In addition, PfHSP40 was found to share in the regulation of transport of exported proteins across several membranes to target RBCs [31] . HSP40 members are divided into four distinct classes on the basis of their encoded domains. Type IV HSP40 protein (PfL2550w), which was named P. falciparum gametocyte erythrocyte Cy protein (PfGECO), was found to have a role in sexual differentiation. Using northern blotting, the investigators demonstrated that PfGECO was transcribed from asexual stages to stage III gametocytes, and it was predominantly expressed in stage I-IV gametocytes. The investigators concluded that gametocytes sustain many exported proteins from the asexual blood stages to facilitate the changes in shape that occur during their five stages of development and/or to provide protection throughout it. Therefore, type IV HSP40 genes do have an essential role during gametocyte sequestration [32] . By using the molecular genetics approach, investigators showed that Plasmodium HSP20 is critical for fast sporozoite locomotion in vitro and for efficient natural malarial transmission in vivo. Based on their observational studies, gliding sporozoites motility was critical for 1) their entry and dispersion into the mosquito salivary glands, 2) intradermal migration from the mosquito to blood of the host, 3) penetration of the endothelial barrier of liver sinusoid and successful invasion and migration through the liver parenchyma. The investigators postulated that HSP20 directly contributes to cell-substrate adhesion by physical interaction with transmembrane parasite invasins. According to their explanation, HSP20 modulates the motor complex that generates the traction forces during sporozoite locomotion, and it regulates the turnover of actin polymerization and competes with actomyosin interaction, or both [33] .
2. Parasite invasion and virulence: HSP70 was identified as one of the five major proteins responsible for regulation of parasite actin polymerization, which provides the mechanism for localized actin filament growth and movement of the parasite into the host cell [34] . In another study, the investigators claimed that initial exposure of the parasites to HSPs provided better survival and improved infectivity. Accordingly, their conclusion was that febrile episodes promoted intraerythrocytic development of the parasite [30] . In 2012, other investigators discovered the ability of Cy HSP110 to prevent Asn repeat-rich protein aggregation in vitro and in vivo. They concluded that HSP110 is vital for proteostasis of P. falciparum proteome, allowing the propagation of these repeats within the parasite proteome, and for parasite growth and survival during its brief exposure to febrile temperatures [2] . Recently, after investigating a conserved Plasmodium protein (named plasmoDJ1) as regards its response to stress and its role in parasite growth and development, it was found that the expression of this particular protein in all intraerythrocytic stages and ookinetes increased upon heat stress as well as oxidative stress due to H 2 O 2 and artemisinin treatment. The study showed that plasmoDJ1 not only reduced H 2 O 2 and significantly decreased protease activities but also attenuated virulence and reduced oocyst production upon plasmoDJ1 gene knockout [35] .
3. Elicitation of immune response: γ δ T cells collected from experimental P. yoelii-infected mice were capable of proliferation in vitro in response to PfHSP60 and PfHSP70, suggesting the immunological role of HSPs in cellular immune response [36] . HSP65 was found to be strongly expressed in splenic cells of mice infected with a nonlethal strain of P. yoelii and slightly expressed in mice infected with a lethal strain. The investigators suggested the important role of HSP65 in protection against malarial infection in mice [37] . Two years later, the same investigators extended their studies to confirm the crucial role of HSP65 in the prevention of macrophage apoptosis in P. yoelii-infected mice, and CD4 + T cells were proven to be essential for HSP65 expression [38] . In another study conducted by the same investigators in Thailand on malaria patients, IgG, IgM, and IgA significantly increased against HSP90, whereas only IgM became significantly elevated in response to HSP70 and IgA in response to HSP65. The investigators concluded that in malarial infection the antigenic potential of HSP90 is higher than that of HSP70 and HSP65 [39] . In Korea, investigators attempted to characterize the molecular properties of HSP70 of P. vivax. They obtained isolates from Korea, Myanmar, Thailand, and Indonesia and amplified the gene encoding PvHSP70. They tested it against patients' sera infected with P. vivax and P. falciparum as well as in patients with other parasitic diseases and in healthy controls. The results strongly suggested high antigenicity of this HSP and the investigators recommended further studies to evaluate the biological significance of immune responses to PvHSP70 [40] .
4. Diagnosis: As a diagnostic marker, IgG mAbs were raised against PfHSP70 conjugate and used in comparison with malarial antigen conjugate and polyclonal IgG antibodies against P. falciparum malarial conjugate in the diagnosis of P. falciparum malaria. Using a rapid agglutination test, results showed 90% sensitivity and 80% specificity. Similar sensitivity and specificity were obtained with polyclonal Abs, while malarial antigen conjugates gave lower results [41] . In Portugal, investigators succeeded in developing a rapid, simple, and sensitive immunoassay to detect malarial antigens in infected blood cultures. The assay utilized labeled recombinant PfHSP70 bound to gold nanoparticles functionalized with anti-HSP70 mAbs. The immunoassay succeeded in detecting malarial antigen at a 3% parasitemia level [42] .
5. Drug Targets: Indian investigators analyzed the P. falciparum genome and found a high degree of sequence identity between PfHSP90 and yeast HSP90. This result allowed them to derive a 3D structure for PfHsp90 by homology modeling. Accordingly, they claimed that understanding the chemical structure of PfHSP90 would greatly help in the development of new drug targets against malaria [43] . A novel class of antimalarial agents was identified by American scientists in 2009. They evaluated the efficacy of nine pyrimidinone amides (HSP70 modulators) to inhibit the replication of P. falciparum in parasitized RBCs. Their results showed that the compounds altered the ATPase activity of purified PfHSP70 [8] . In addition, intraperitoneal administration of 17-(allylamino)-17-demethoxygeldanamycin (HSP90 inhibitor) to P. berghei-infected mice inhibited parasite growth [44] . The obtained results suggested the possibility of chaperone-targeted therapy for the treatment of a variety of protozoan infections. In another study, 'harman' was identified as an alkaloid extracted from leaves of Guiera senegalensis (herbs found in West and Central Africa). In their studies, Canadian investigators demonstrated that 'harmine' (harman derivative) inhibited P. falciparum HSP90 by competition for the N-terminal ATP-binding domain. They concluded that harmine derivatives displayed preferential binding for PfHSP90 orthologs and provided an ATP-binding domain that confers species specificity [45] . Two years later, the same team of scientists confirmed the specific affinity of harmine to PfHSP90 and unexpectantly discovered a related compound 'harmalol' that showed higher affinity for the human HSP90 ATP-binding pocket better than harmine. The investigators extended their studies to show that harmine had potential synergistic effects on chloroquine and artemisinin in vitro as well as in vivo in P. berghei-infected mice [46] . In the same year, American scientists showed the ability of PfHSP110 for proteostasis of the P. falciparum proteome, which motivated the search for an inhibitor to hamper the proteostasis of the parasite proteome [2] . Very recently, similar results were obtained and the investigators demonstrated the essential and universal roles of PfHSP101 in exporting proteins; they provided strong evidence for PTEX function in protein translocation into the host cell [47] . In addition, Elsworth et al. [48] discussed the functions of PTEX and the possibility of its use as a prime antimalarial drug target. The investigators showed that the major virulence factor PfEMP1 was significantly reduced in PTEX knockdown parasites, and that modest PTEX knockdown had a strong effect on erythrocytic stages both in vitro and in vivo [48] . Finally, a purine analog HSP90 inhibitor (PU-H71) proved to be active against PfHSP90. In addition, the investigators found that PU-H71 had high affinity to the PfHSP90 ATP-binding domain and inhibited ATPase activity. PU-H71 also synergized chloroquine to reduce the parasite load and improve survival rates in P. berghei experimentally infected mice. Therefore, they concluded that PU-H71 exhibits potent antimalarial activity both in vitro and in vivo [49] .
6. Vaccination: In three experiments, American scientists used gene encoding P. yoelii HSP60 for immunization of mice against challenge infection. In the first experiment, 40% protection was observed, whereas instead of protection there was delay in the onset of parasitemia in the second experiment. In the third experiment, there was neither reduction of erythrocytic stages nor development of exoerythrocytic stages. The investigators recommended further evaluation of the use of HSP60 in the development of malarial vaccines [50] . The results obtained from a study conducted in Sweden emphasized the potential role of HSP70 as an adjuvant in a DNA vaccine candidate against P. falciparum [51] . Furthermore, Spanish investigators were able to identify four proteins characterized by high immunogenic blood stage antigens using proteomic analysis of P. yoelii antigens. These were two ER proteins (disulfide isomerase and HSP70), a digestive protease plasmepsin, and a ribosome-associated protein. They claimed that isolation of parasitic antigens using IgG from sera of malaria-protected individuals could be a novel strategy for malaria vaccine development [52] .
| 2. Leishmania species | | |
2.1. Introduction
It was reported that HSP70 and HSP83 were abundant in Leishmania promastigotes and remained constitutively expressed in amastigotes to protect the organism against temperature change from 22 to 28°C in the sandfly to 33-37°C in the mammalian host [53] . In addition to HSP70 and HSP83, the same investigators characterized sHSPs with lower degree of conservation that shared in the protection of L. mexicana amastigotes against heat stress [54] .
2.2. Applications
1. Virulence and parasite growth: The role of L. major HSP100 was studied and the investigators found that loss of HSP100 chiefly affected the initial phase of the infection, i.e. development from promastigotes to amastigotes. They also found that the impact of HSP100 is stage specific, which indicated its protective role during the establishment of the parasite within the mammalian host [55] . L. donovani HSP100 was also found to be clustered, with the majority of its molecules localized close to the cytoplasmic membrane [56] . Furthermore, a gene encoding HSP100 (expressed exclusively in amastigotes) was established as a virulence factor by a reverse genetic approach. Gene replacement mutants (lacking this gene) were found avirulent in BALB/c mice, and the virulence was recovered on spontaneous clonal divergence within the mutant population leading to emergence of virulent parasites. In their study, the investigators identified and characterized another virulence gene encoding a 46-kDa protein whose overexpression restored infectivity to the mutant L. major strains [57].
2. Immune response elicitation: L. braziliensis genes encoding HSP70 and HSP83 were cloned, identified, and expressed as recombinant rLbHSP83 and rLbHSP70, and the leukocyte responses and anti-IgG antibody titers in the sera of infected patients with different clinical forms of disease were compared. The investigators found that the differential cytokine elicitation patterns of LbHSP83a and LbHSP83b indicated their potential importance in the development of improved immunotherapeutic or immunoprophylactic targets [58] . Proteins of L. chagasi promastigotes were separated into 67 fractions and tested for their ability to stimulate proliferation of peripheral blood mononuclear cells. The investigators suggested that the 69-kDa protein fraction (with similar sequence identity to HSP70) was able to elicit human T-cell immune responses, an important step toward vaccine development [59] . In addition, both L. infantum HSP70 and HSP83 were proved to be potent mitogens for murine splenocytes and able to induce proliferation on B-cell populations purified from BALB/c spleen [60] . Similar results were obtained in a study conducted in Spain, which revealed that the immune response generated during cutaneous leishmaniasis (CL) and mucocutaneous leishmaniasis (MCL) was elicited specifically by the parasitic histone and HSP70 [61] .
3. Diagnosis: There was much controversy regarding the potential use of HSP70 as a serodiagnostic marker in leishmaniasis. The first description and characterization of L. donovani HSP70 was published in 1990. The investigators demonstrated the presence of antibodies against the carboxy-terminal region of L. donovani HSP70 in more than 50% of visceral leishmaniasis (VL) patients and in all samples of Chagas disease. The investigators suggested at that time its importance during the growth of both parasites [62] . Two years later, recombinant L. donovani HSP70 and HSP90 showed strong humoral immune response, which was evident by strong reaction with antibodies in ELIZA when tested against sera from L. donovani-infected patients, but not with sera from Chagas disease patients. The investigators considered HSP70 as a specific biomarker for VL serodiagnosis [63] . Similar results were obtained when sera from patients with CL, malaria, schistosomiasis, and Chagas disease did not recognize the recombinant L. donovani HSP7O using western blots. The investigators suggested that in VL, an immunodominant anti-HSP7O response was raised against a species-specific epitope in the carboxy-terminal region of HSP70 [64] . However, in another study conducted in India, the investigators identified 20 clones that were reactive with pooled sera from Kala azar-infected patients. The clone with the largest cDNA insert and expressing HSP70 was tested for its suitability in serological assays, but the results revealed that it was unreliable for serodiagnosis of Kala azar [65] . One year later, Spanish investigators reported that recombinant L. infantum HSP70 (rLiHSP70) was not reliable as a disease-specific marker for serodiagnosis as it was recognized by sera from patients with Chagas disease [66] . In 2003, another team from Spain isolated the single-copy gene encoding HSP70 of L. braziliensis (663 amino acids) from the genomic DNA library. They showed that its main antigenic determinant was located in the carboxy-terminal end. Using this fragment for specific serodiagnosis of CL and MCL, their results showed 70% sensitivity and 100% specificity [67] . In Iran, the L. infantum hsp70 gene segment was amplified, cloned, and evaluated for VL serodiagnosis. The ELISA results showed that HSP70 was recognized by 81.1% (30/37) of VL patients and 6.3% (5/63) of controls [68] . From L. infantum cDNA and genomic library, the investigators selected five gene members to evaluate their reactivity against a panel of Leishmania spp.-infected canine and human sera. One of these loci was a gene encoding a protein homolog to members of the Cy HSP70 family. Although it cross-reacted with antibodies from a certain proportion of dogs with other infectious diseases, some serum samples from patients with CL, and serum samples from patients with Chagas disease, its optical density (OD) values were low. However, it showed moderate reactivity with sera from most VL patients. The investigators concluded that none of these selected gene members was efficient for diagnosing all canine or human VL cases [69] . Moreover, in an attempt to identify L. donovani protein antigen with high specificity and sensitivity, and in the same time the test becomes negative on patient recovery, Indian investigators succeeded to identify an immunoreactive BHUP1 antigen from L. donovani HSP70. The antigen proved to be 90% specific among healthy individuals living in the endemic region, and 54% of the cured VL patients turned negative when tested with 1-year follow-up. Thus, the investigators considered L. donovani-specific HSP70 (BHUP1) protein as a potential diagnostic and prognostic marker in VL. They also recommended BHUP1 for a rapid immunochromatographic test [70] . Recently, Brazilian investigators identified specific B-cell epitopes from calpain-like cysteine peptidase, thiol-dependent reductase 1, and HSP70 of L. infantum using in-silico analysis. The detected antigens were suggested to be used as diagnostic biomarkers in VL [71] .
As regards HSP83, research showed that recombinant L. infantum HSP83 (rLiHSP83) elicited strong humoral immune response in 90% of sera from dogs with VL. The results suggested its use in serodiagnostic assays for canine leishmaniasis [72] . The specificity of the antibody response in patients with diffuse CL was evaluated using three immunodominant leishmanial antigens [HSP70, HSP83, and Leishmania eukaryotic initiation factor 4A (LeIF)]. The results revealed an antibody specificity pattern that was predominantly restricted to HSP83. The investigators considered Leishmania HSP83 as an important antigen in the diagnosis of infections caused by L. amazonensis [73] . Recently, rLiHSP83 was evaluated with ELISA for serodiagnosis of CL, MCL, and VL as well as in samples from other diseases such as Chagas disease, toxoplasmosis, and malaria. It was also evaluated in treated patients during a follow-up to test anti-rHSP83 antigen antibody titers. Evaluation of rLiHSP83 in comparison with L. major-like total promastigote antigen showed significantly higher sensitivity and 100% specificity of rLiHSP83. The investigators also did not detect a significant decrease in antibody levels after treatment. Therefore, they concluded that rLiHSP83 is a good biomarker for serodiagnosis of leishmaniasis [74] . Finally, using sandwich ELISA and western blotting, it was concluded that combined L. donovani HSP70 and HSP83 are good serodiagnostic biomarkers in VL. The investigators attributed their results to the induction of strong cell-mediated and humoral immune responses during leishmaniasis [75] .
For tegumentary leishmaniasis (TL), the recombinant L. major hsp60 gene was cloned, sequenced, and screened against sera from patients with TL. The results revealed that rLmHSP60 elicited humoral immune response suggesting its use as a potential antigen for TL serodiagnosis [76] . In another study, recombinant L. infantum (rLiHSP83) was evaluated in comparison with L. major-like total antigen using ELISA. Both antigens were tested against sera from patients with CL and MCL and sera from Chagas disease patients as well as sera from normal blood bank donors. Both antigens showed similar results except for the absence of cross-reactivity that occurred with sera from patients with Chagas disease on use of rLiHSP83. Therefore, the investigators considered it to be a good antigen for routine use in serodiagnosis of TL [77] . On the other hand, the restriction fragment length polymorphism (RFLP) protocol was designed using HSP70 to diagnose and identify Leishmania spp. causing TL in skin scrapings with a sensitivity and specificity of 95 and 100%, respectively [78] . Recently, another group of investigators compared six L. infantum-L. chagasi recombinant proteins, including HSP70 and soluble L. infantum-L. chagasi antigen (SLA), for TL serodiagnosis. All recombinant proteins were recognized by sera from TL patients, and rHSP70 showed the best performance (sensitivity and specificity), which was superior to SLA in the diagnosis of CL. The investigators concluded that rHSP70 and/or in combination with different antigens should be considered a good biomarker in serodiagnosis of TL, especially in endemic areas [79] . In another recent study conducted in Brazil, the investigators examined the ability of L. braziliensis rHSP83.1 in TL serodiagnosis against samples from TL and VL patients and from dogs infected with canine VL. Their results showed the potential use of rHSP83.1 in TL serodiagnosis [80] .
On the other hand, the Leishmania sHSPs family includes the most widespread but most poorly conserved collection of HSPs. In an attempt to characterize L. amazonensis HSP20, investigators utilized the complete bioinformatics of the L. major genome database to identify a protein entry. They found that the 17.5 kDa predicted protein that had 155 amino acids was the only sHSP family member found in all Leishmania spp. On the basis of these data, L. amazonensis HSP20 was expressed and used as a serodiagnostic marker to determine the presence of specific IgG in the sera of Leishmania-infected animals and human patients with VL. Results revealed that HSP20 was recognized by all dog sera, but with limited and moderate antigenicity by 30 and 62% of the assayed human sera and sera from experimentally infected hamsters, respectively. Accordingly, it was concluded that the production of anti-HSP20 antibodies coincided with the onset of disease symptoms. The investigators suggested the use of HSP20 as a useful serodiagnostic marker for active leishmaniasis [81] .
4. Species identification: Leishmania HSP70 genes were found to represent adequate targets for sensitive typing of neotropical Leishmania spp. in host tissues. They encode for several major antigens that may be used for probing of the genetic variability of molecules possibly engaged in immunopathology. In addition, they present a lower rate of genetic variation compared with other markers [e.g. glycoprotein 63 (gp63) or rDNA internal transcribed spacer (ITS)] [82] . On the other hand, a species-specific segment in the L. donovani genome employing the carboxy-terminal region of the rHSP70 was amplified. In their study, the PCR assay identified L. donovani in hepatic and splenic infected tissues, in aspirates from lesions of post-Kala azar dermal leishmaniasis, and in bone marrow aspirates from VL patients [83] .
Due to the multiple Leishmania species in some endemic areas such as Brazil, the investigators analyzed the hsp70 gene sequence in several Leishmania species to identify specific restriction enzymes that could be used for PCR-RFLP-based identification. They suggested that the new identification protocol would correct the limitations of the gold standard technique (multilocus enzyme electrophoresis) used for routine Leishmania typing [84] . In addition, the HSP70 PCR-RFLP assay utilizing BccI as the restriction enzyme could differentiate between L. panamensis and L. guyanensis (species of the subgenus Viannia with very high genomic similarity), which are considered potential causes of CL and MCL. The new restriction enzyme overcame the failure of HaeIII (as restriction enzyme) because it showed the same pattern for both species [85] . In the same year, the same investigators published another work presenting a single PCR-RFLP method based on analysis of 51 HSP70 sequences. The assay showed different patterns for 11 species; L. infantum, L. donovani, L. tropica, L. aethiopica, L. major, L. lainsoni, L. naiffi, L. braziliensis, L. peruviana, L. guyanensis, and L. panamensis by applying two subsequent digests. However, only three species (L. mexicana, L. amazonensis, and L. garnhami) failed to generate species-specific patterns [86] . Two years later, they improved the sensitivity and specificity of the technique using alternative PCR primers and RFLPs, and succeeded in testing it on 114 globally representative Leishmania strains and on various other parasites [87] . Later, another team from Brazil analyzed the Leishmania hsp70 gene to select primers ranging from 234 to 384 bp for use as restriction enzyme for the PCR-RFLP protocol. The investigators tested the ability of these primers to distinguish between Leishmania spp. (70 strains, including reference strains and strains circulating in Brazil) in comparison with other PCR-based assays for CL molecular diagnosis using a panel of clinical samples from several endemic areas in Brazil. The results showed that the 234-bp region was promising, given its utility for detecting and identifying Leishmania spp. in clinical samples. Their ITS1 PCR-RFLP yielded similar results to those of 234-bp HSP70 PCR-RFLP. The investigators concluded that the newly developed assay could detect Leishmania spp. in clinical samples and discriminate between all of the species circulating in Brazil, thus rendering this assay particularly useful in endemic areas [88] .
On the basis of multilocus sequence typing, a recent method for differentiation of Leishmania spp., Chinese investigators used five enzyme-encoding genes and two conserved genes; one of them is HSP70. Results demonstrated that Chinese Leishmania isolates were of two types: clade A1 and clade A2. The first was most closely related to L. donovani strains from India and Africa, whereas the second was found to be related to the European strains of L. infantum. The investigators reported that the study provided a basis for further understanding of the phylogeny and evolutionary history of the Chinese Leishmania spp. [89] . From Belgium, scientists presented a completely validated and globally applicable standardized protocol for the use of HSP70 sequences in Leishmania typing without parasite culture. The method is especially suited for use in clinics in nonendemic regions, which have relatively few cases/year. The described method was developed in the framework of a European consortium of tropical infectious disease clinics called 'LeishMan' (http://www.leishman.eu) to characterize Leishmania spp. using a standardized PCR assay. The investigators recommended extension of their strategy to identify additional strains with associated clinical information, and to establish a global database of L. parasites [90] . Finally, Thailand investigators used PCR amplification and sequence analysis of the L. siamensis hsp70 gene to identify species of infected Sergentomyia (vectors of autochthonous VL). The latter was considered an emerging disease in Thailand caused by L. siamensis resulting in VL and disseminated dermal leishmaniasis. The investigators found that the captured Sergentomyia gemmea was a potential vector of L. siamensis in Thailand [91] .
5. Drug therapy and resistance: It was reported that L. infantum HSP70 is encoded by two genes. Investigators studied the effects of either absence or mutation of one of these genes on promastigotes. Major alteration in the growth of promastigotes and frequent aberrant forms were observed. Extending the study by re-expressing HSP70-II in mutant promastigotes restored growth rate, recovery of normal morphology, and increased macrophage interactions. It was suggested that the role played by HSP70-II expression in Leishmania virulence recommends the use of this gene as a new drug target in leishmaniasis [92] . More recently, two small acidic proteins (Lbp23A and Lbp23B) were identified as cochaperones for LbHSP90. Both recombinant proteins shared similar structures and interacted with LbHSP90 and inhibited its ATPase activity with variable efficiencies, but Lbp23A was more stable than Lbp23B [93] . For HSPs application in drug resistance, the investigators analyzed eight gene expressions in ten resistant and four sensitive clinical isolates of L. donovani using quantitative real-time PCR in an attempt to distinguish antimony-resistant and antimony-sensitive strains. They found significant expression of four of the evaluated genes; HSP83 was included in antimony-resistant strains [94] .
6. Vaccination: HSP70 was considered one of several potential immunogenic antigens. L. major HSP70 showed potential importance in stimulating humoral immune responses [95] , and HSP70 from L. donovani was identified through proteomic analysis inducing Th1 type immune response in cured and/or endemic VL [96],[97],[98] . The efficacy of a DNA vaccine with purified P4 nuclease protein along with the adjuvants HSP70 or interleukin (IL)-12 in immunizing BALB/c mice against L. amazonensis and L. major was evaluated. The results showed that P4/IL-12 produced complete protection against infection with L. amazonensis but not against L. major, whereas P4/HSP70 produced complete protection against L. major and partial protection against L. amazonensis. The investigators concluded that both P4 and HSP70 are vaccine candidates that could potentially be useful in DNA-based vaccines for both L. amazonensis and L. major [99] . The immunogenicity of three L. infantum antigens; HSP70, paraflagellar rod protein-2, and kinetoplastida membrane protein-11 (KMP-11), was evaluated in experimentally infected dogs. The investigators found that these recombinant antigens induced increased production of interferon γ (IFN-γ) and tumor necrosis factor α (TNF-α), and they concluded their importance as vaccine candidates for control of canine VL [100] .
Sachdeva and his colleagues [101] presented the first study in which the L. donovani hsp70 gene was used as an adjuvant to a polytope DNA vaccine. The investigators evaluated three vaccine formulations in a mouse model; gp63 DNA vaccine (encoding the region of gp63 gene of L. donovani), polytope DNA vaccine and polytope DNA vaccine fused to L. donovani HSP70 as genetic adjuvant to improve T cell responses as it has both cytokine and chaperone functions. The latter vaccine resulted in significant reduction in the number of amastigotes in the liver and spleen after four weeks of challenge infection. The investigators assumed that the HSP70-fused polytope antigen was internalized by receptor-mediated endocytosis, thus constituting the antigenic HSP-associated peptides [via their cell surface major histocompatibility complex (MHC) class I] presented to CD8 + T cells, consequently improving antigen presenting function. They concluded its efficiency as a preventive strategy for VL as it enhanced the cytolytic activity of splenocytes isolated from vaccinated BALB/c mice and induced strong Th1 responses [101] . Finally, in an Indian study conducted recently, investigators developed recombinant L. donovani HSP70 (rLdHSP70) to evaluate its potentiality to stimulate immune responses in lymphocytes of cured Leishmania-infected hamsters and in the peripheral blood mononuclear cells of cured VL patients. They used rLdHSP70 either individually or in combination with recombinant L. donovani proteins such as elongation factor-2, protein disulfide isomerase, and triose phosphate isomerase (TPI). Results showed that all vaccinated hamsters had enhanced proliferative response compared with control animals. In addition, they noted (1) remarkable nitric oxide (NO) production, (2) significant delayed-type hypersensitivity response, (3) significant upregulation of Th1 type of immune response (high levels of IFN-γ and IL-12), and (4) IgG and IgG1 levels that proportionally increased with very low L. donovani loads in vaccinated animals. Therefore, the investigators postulated that vaccines based on a combination of LdHSP70 with Th1-stimulatory proteins could be an essential strategies for vaccine development against VL [102] .
HSP100 was also reported as an efficient vaccine candidate. It was reported that CL produced self-limiting infection in C57/BL/6 mice and progressive and systemic infection in BALB/c mice. In their experimental studies, German investigators vaccinated both types of mice with hsp100 gene of mutated L. major wild strain. Their results encouraged the idea of generating a safe attenuated vaccine using targeted replacement of single genes required for parasite pathogenicity like the hsp100 gene [103] . On the other hand, combined use of HSP70 and HSP83 as vaccine administered alone or with one of two adjuvants (MPLA and ALD) in L. donovani-infected BALB/c mice was evaluated. The used measures for evaluation included hepatic parasite load and IgG2a, IFN-γ, and IL-2 levels. The three vaccines gave efficient results, but use of adjuvants (especially MPLA) significantly raised the IgG2a, IFN-γ, and IL-2 levels [104] .
| 3. Trypanosoma species | | |
3.1. African trypanosomiasis
3.1.1. Applications
1. Parasite survival: T. brucei HSP60 (63.7 kDa) was found to be expressed by both bloodstream (BS) and procyclic (PC) forms. It has 51.3 and 94.5% amino acid identity to the mammalian homologs P1 protein and T. cruzi HSP60, respectively [105] . Furthermore, Mit HSP60 and HSP70 demonstrated elevated expression in T. brucei stumpy forms, and they were both markers of morphological transformation from stumpy to PC forms [106] .
2. Immune response elicitation: T. congolense 69 kDa protein is conserved among all developmental stages of African trypanosomes. It was analyzed and proved to have 45-65% identity with HSP70s, and it was responsible for the major immunogenicity in trypanosomes that cause African sleeping sickness (ASS) [107] . In 2000, a study conducted in Belgium compared between the effects of anti-T. brucei HSP60, anti-invariant surface gp70, and anti-variant surface glycoprotein (VSG) in humoral immune responses during ASS in BALB/c mice. The results revealed that HSP60 was specifically recognized during the entire course of infection. Immunoglobulins G2a and G2b (induced mainly in a T-cell-independent manner) were detected during the first peak of parasitemia, whereas G1 and G3 (due to a specific T-cell-mediated response) were observed at the end of the infection. The investigators were convinced that HSP60 causes a significant host humoral immune response of an autoimmune character. They concluded that, during the course of the disease, several switch factors and B-cell activation pathways can determine the humoral immune response, which might mean that these responses are the outcome of mixed antiparasite and antihost immune reactions [108] . The same investigators conducted another study using T. brucei-purified flagellar antigens to generate polyclonal antisera in rats that recognized several proteins other than VSG. Screening of the T. brucei proteome form cDNA library, ~67% of the selected specific cDNA inserts encoded trypanosome HSP60 [109] .
3. Pathogenesis: The role of the hsp70.1 gene in ASS caused by T. congolense was investigated, and the parameters assessed were survival time, levels of parasitemia, anemia, and course of infection. Results revealed that susceptible A/J mice developed only moderate anemia, whereas HSP70.1-deficient C57BL/6 J and resistant wild-type C57BL/6 J mice controlled parasitemia, but developed severe anemia in the late stage of infection [110] .
4. Diagnosis: In 2002, investigators from Kenya reported that T. congolense 69 kDa protein was homologous to mammalian immunoglobulin-binding protein (BiP) and used the latter in an indirect ELISA technique for diagnosis of bovine ASS. Results showed limited sensitivity, and further evaluation was recommended [111] . Later, anti-BiP mAbs were used against sera from T. congolense experimentally infected cattle. The HSP70/BiP-based inhibition ELISA assay showed good sensitivity, with an improved sensitivity in secondary infections [112] . In addition, aiming to find out a diagnostic marker for ASS caused by T. brucei, German scientists selected several proteins including HSP70, histone H2B, histone H3, phosphoglycerate kinase (PGKC), rhodesain, and others for analysis against patient sera. They found that the majority of sera reacted with HPS70, but recommended its use as a component of a multiplex diagnostic assay containing several immunogenic proteins to ASS [113] . On the other hand, the T. congolense Mit hsp70 gene (MTP) was cloned and Japanese investigators determined its entire nucleic acid sequence. They found that the specific antigenic epitope was located in its C-terminal region. No significant differences were detected in transcription and translation levels between BS and PC forms, which indicated that it is similarly expressed in both forms and hence could be used as a diagnostic marker. They examined the MTP antigenic epitope against specific anti-MTP antibody production in hosts with primary infection, and detected MTP-specific antibodies produced in sera from infected mice. This detection was stable irrespective of the route of infection; intraperitoneal or subcutaneous. The investigators recommended the use of specific anti-MTP antibodies against sera from naturally infected animals [114] .
5. Drug therapy: The main function of HSP90, also known as HSP83 or HSP86 because of the variable molecular weight among different orthologs, is to limit protein aggregation during thermal stress [115] . The HSP90 inhibitor (17AAG) was used as a drug target against T. evansi, which causes surra in domestic animals. Targeting HSP90 for the treatment of a variety of human and animal protozoan infections was suggested [44] . Later, HSP90 inhibitors [GA, and GA analogs (17AAG and 17DMAG), radicicol and novobiocin] were investigated as drug targets. The results revealed that 17AAG and 17DMAG were the most effective against T. brucei, as the former caused severe morphological abnormalities and the latter cured infected mice [116] . Another recent study conducted by collaborative teams from the USA, UK, and Canada published a detailed structural and chemical characterization of T. brucei HSP83 as a potential drug target against ASS. The investigators could identify a benzamide derivative compound capable of interacting with TbHSP83 with inhibition of parasitic growth in vitro [117] .
Besides the previous HSPs applications in ASS, T. brucei possesses a large HSP40 complement that consists mostly of type III HSP40. In their work, Louw et al. [118] expressed and purified a novel HSP40 that potentially functions as a novel cochaperone of HSP70 in T. brucei.
3.2. American trypanosomiasis
3.2.1. Introduction
It was reported that different protective functions attributed to HSPs in heart injury patients included the repair of ion channels, the restoration of redox balance, interaction with NO-induced protection, inhibition of proinflammatory cytokines, and prevention of activation of the apoptosis pathway [119] . This was supported by the detection of a significant number of HSPs (40 spots for 13 different HSPs) in the myocardium of chronic Chagas cardiomyopathy [120] . Brazilian investigators identified 12 spots (HSP70 isoforms) and claimed that HSP70 increased in response to ischemic stress. In addition, there was increase in myocardial HSP60 production, which was previously reported to be associated with the development of chronic heart failure [121] .
3.3. Applications
1. Parasite survival: Enhanced synthesis of HSP60, HSP70, and HSP90 was detected in T. cruzi-infected mice exposed to increased temperature (39°C) using antibody immunoprecipitation analysis. The investigators also analyzed the histopathological changes in the cardiac and skeletal muscles of infected mice maintained at room temperature or at 36°C. It was found that transfer of mice from room temperature to 36°C led to decreased numbers of circulating parasites with consequently increased survival rate and reduction in parasitemia [122] .
2. Autoimmunity and pathogenesis: T. cruzi HSP70 was first recognized in 1990, when it was investigated as a specific target of humoral autoimmunity with a role in the pathogenesis of Chagas disease. The results strongly argued against its role in the pathogenesis of Chagas disease [123] . In 1993, Spanish scientists investigated its immunological response in infected sera compared with controls. There was reactivity in both groups but it was significantly higher in patient sera. However, mapping of T. cruzi HSP70 epitopes could identify the particular immunogenic patterns of Chagas disease pathogenesis [124] .
3. Diagnosis: T. cruzi ER HSP7O was found to encode a 78-kDa glucose-regulated protein (grp78). It was cloned and molecularly characterized to identify the immunogenicity of T. cruzi antigens in Chagas disease. The investigators analyzed other members of the HSP70 family in their studies to clarify the importance of HSP70 in the parasite's biology as well as host immune response. They found that grp78 is the most immunogenic protein as it reacted strongly with sera from infected mice [125] . Similar results were obtained in a study conducted in Brazil, when the best sensitivity and specificity were obtained using grp78 (ER T. cruzi HSP70) in comparison with Cy HSP70 and Mit HSP70. In addition, the investigators used grp78 with T. cruzi flagellar calcium-BiP, and the diagnostic sensitivity increased from 90 to 97% but increased leishmanial reactivity only from 3 to 8%. The investigators also concluded that none of the three HSP70 antigens was effective in distinguishing cured and uncured treated patients, which may be considered indicative of effective treatment [126] . Later, the IgG subclass antibody responses against three different T. cruzi antigens; crude antigen, KMP-11 gene, and HSP70 as well as against T. rangeli HSP70 were analyzed. The results showed the ability of T. cruzi KMP-11 and T. rangeli HSP70 to act as a marker for Chagas disease (healthy or infected, acute or chronic) [127] .
4. Species differentiation: In a study conducted recently in Cuba, investigators succeeded in using the hsp70 gene to differentiate between T. cruzi and T. rangeli strains in single and mixed infections using PCR-RFLP [128] .
5. Drug targets: Recently, it was found that activation of CD8 + cytotoxic T lymphocyte (CTL) response is one of the targets for development of immunotherapy against Chagas disease. In this study, 30 peptides were selected, synthesized, and tested for human leukocyte antigen (HLA-A*02:01) binding. Four HSP70 immunodominant epitopes (210-218, 255-263, 316-324, and 345-353) were recognized, two of which (210-218 and 316-324) were also recognized by CTL of HLA-A*02:01 from Chagas patients [129] .
6. Vaccination: HSP70 could act as an adjuvant with T. cruzi KMP11 to immunize mice against Chagas disease. Its combination with the vaccine leads to MHC class I processing pathway and elicits CD8 + response [130] . Its use in combination with T. cruzi paraflagellar rod proteins (PFR2 and PFR3) produced activation of PFR2 antigen-specific CTLs manifested by increase in IL-12 and IFN-γ and decrease in the percentage of cells expressing IL-4. The investigators concluded that PFR2-HSP70 provided a protective response in T. cruzi-infected mice [131] . Moreover, truncated TcHSP70 was shown to induce human dendritic cell (DC) maturation in patients with Chagas disease. This was manifested by increase in the expression levels of CD83, CD86, IL-12, TNF-α, and IL-6 [132] . Finally, plasmids encoding PFR2-HSP70 and PFR3-HSP70 were used to immunize transgenic mice, and the immunodominant CD8 + T-cell epitopes for both proteins were recognized by CTLs from sera of patients. Results showed that some peptides had high binding affinity to the HLA-A*02:01 molecule, and T cells from chronic patients showed proinflammatory cytokine production (IFN-γ, TNF-α, and IL-6) [133] . Interestingly, the chaperone or ATPase domains of TcHSP70 was used as an adjuvant in DNA vaccine with Entamoeba histolytica surface collagen-BiP peroxiredoxin to induce effective immune response and to protect hamsters against amoebic liver abscess development [134] .
7. Apoptosis: T. cruzi HSP65 plays an essential role in preventing macrophage apoptosis and contributes to host resistance against Chagas disease. Its expression mechanism was investigated, and results revealed that macrophages of resistant C57BL/6 and DBA/2 infected mice showed strong HSP65 expression compared with that of susceptible BALB/c mice. In addition, CD4 + T cells were responsible for the induction of HSP65 expression in macrophages [135] .
| 4. Toxoplasma gondii | | |
4.1. Applications
1. Antigen presentation and autoreactivity: The results obtained from a study conducted in Japan revealed that heatshock cognate protein (HSC71) had a potential role in presenting T. gondii antigen to CD4 + CTL. The investigators infected P36 cell culture with T. gondii and used specific anti-HLA-DR mAbs to show CD4 + CTL lytic activity to the T. gondii-infected cells. Using flow cytometric analysis, HSC71 was found to be expressed on the cell surface of infected and uninfected P36 cells [136] . On the other hand, because of the homologous sequence between TgHSP70 and mouse mHSP70 and human hHSP70, another group of Japanese investigators speculated that it is possible to induce autoreactive immune responses in T. gondii-infected hosts. They were able to demonstrate the production of anti-TgHSP70 antibody that cross-reacted with self mHSP70 and showed that B-1a cells are responsible for this autoreactivity in T. gondii-infected BALB/c (resistant strain) and B6 mice (susceptible strain) [137] .
2. Anaphylactic shock reaction: The expression of TgHSP70 was found to increase rapidly just before the death of infected host cells, indicating its role as a warning signal during acute toxoplasmosis [138] . It was also shown that TgHSP70 caused deterioration of the host defense through downregulation of NO release [139] , production of anti-HSP70 autoantibodies [140] , and activation of B cells and DCs [141],[142] . In Japan, scientists observed that the mechanism of lethal anaphylactic reaction induced by TgHSP70 injection in T. gondii-infected mice was IFN-γ dependent and through platelet-activating factor, not by an IgE-dependent pathway [143] . In 2010, Japanese investigators evaluated the use of T. gondii hsp70 gene vaccination in mice against TgHSP70-induced anaphylactic reaction in toxoplasmosis. They found that the vaccine targeted peripheral DCs, and produced prolonged survival rates in immunized infected mice. Therefore, they concluded its ability to induce protective immunity against toxoplasmosis [144] .
3. Protective immunity: It was shown that mice immunized with Toxoplasma cell homogenates acquired protective immunity only against infection with a low-virulence strain but not against a highly virulent strain [145] . One year later, the same team of investigators reported that HSP65 expressed in peritoneal exudate cells of Toxoplasma-infected mice was capable of generating HSP65 production and expression on the macrophage surface, thus developing effective immunity. However, it was found that HSP65 expression depends on strain virulence; it increased in a relatively avirulent strain and was not expressed in a more virulent strain [146] . Contradicting to these results, Australian investigators observed HSP65 expression on host macrophage surface in all strains in vivo and in vitro. On the other hand, HSP70 was only detected in virulent strains in vivo, and poorly expressed in avirulent strains. It was neither detected in vitro nor in infection of immunocompromised mice, and was poorly expressed in avirulent strains [147] . Furthermore, Japanese investigators extended their studies to demonstrate that HSP65 expression within and on macrophage surfaces markedly increased in the presence of γ δ T cells. Mice depleted of these T cells died in the early stages of infection, whereas mice depleted of α β T cells survived but infection could not be controlled in its late stages. The investigators attributed these results to the increase in the number of T cells bearing the γ δ receptor with thymic (Thy-1 + and Thy-1 - ) phenotypes in the peritoneal cavity and spleen [148] . Similar results were obtained in the same year using low-virulent and high-virulent T. gondii strains [149] . One year later, the same investigators demonstrated that γ δ T cells induced HSP65 expression through secretion of IFN-γ and TNF-α and NO production [150] . They added that extra-Thy rather than intra-Thy γ δ T cells have crucial roles in HSP65 expression in toxoplasmosis [151] . Later, another Japanese team found that natural killer T cells were responsible for suppression of protective immunity against toxoplasmosis through generation of IL4, which interfered with γ δ T cells [152] . Besides HSP65, HSP90 was first identified as an antigen with high ability to elicit IgG production during chronic infection in comparison with the acute stage [153] .
Moreover, TgHSP70 was found to be responsible for inhibition of inducible NO synthase expression and NO production. The investigators observed this effect only in mice infected with virulent strains; i.e. virulent T. gondii is protected from host immune responses by producing HSP70. They attributed their results to the genetic structures of the gene encoding HSP70 in virulent and avirulent strains, which may have important implications for the synthesis and stability of this protein [154] . Another study was conducted to evaluate the effects of toll-like receptors (TLR4, TLR2), and myeloid differentiation primary response gene 88 (myd88) in T. gondii-infected mice (wild-type) and mice deficient of these factors. The investigators observed high mortality rate and high levels of T. gondii load in different organs in myd88-deficient infected mice. Meanwhile, high levels of anti-mouse HSP70 autoantibody and anti-T. gondii HSP70 antibody production were detected in the sera of myd88-deficient mice [155] . Another group of investigators compared between T. gondii HSP70 and lipopolysaccharide (LPS) effects on spleen B and T cells and measured the proliferative responses in vitro. They found that HSP70 induced prominent B-cell proliferation in infected and uninfected mice, which was not significantly inhibited by polymyxin B (a potent inhibitor of LPS). They also found that both HSP70-induced and LPS-induced proliferative responses of spleen cells required TLR4 as a receptor, whereas MyD88 was involved only in LPS-induced proliferative response [141] .
4. Virulence and pathogenesis: German scientists characterized the bradyzoite specifically expressed gene (hsp30, formally known as bag1) believed to be responsible for tachyzoite-bradyzoite transformation (an important step in the pathogenesis of toxoplasmosis) [156] . In another study, investigators analyzed the roles of IFN-γ and TgHSP70 in bradyzoites/tachyzoites inter-conversion by measuring the mortality rates of wild type and IFN-γ knockout in T. gondii-infected mice. They found that IFN-γ is essential but not sufficient for protective immunity against toxoplasmosis in the wild type mice [138] . Moreover, it was found that the 82-kDa protein secreted by extracellular tachyzoites reacted specifically with mAb (Tg485). Screening mAb with T. gondii cDNA expression library, followed by amplification and sequencing, revealed significant homology to HSP90 of other Apicomplexan species [157] . Furthermore, another study conducted in Australia showed a relationship between T. gondii virulence and HSP70 expression [154] . A recent study from Argentina showed that the absence of palmitoylation of TgHSP20, which is synthesized as a mature protein in the Cy, causes its accumulation in the inner membrane complex and interference with host cell invasion by T. gondii [158] .
Regarding the identification and characterization of T. gondii sHSPs, five of them were first described by the Argentinian group: HSP20, HSP21, HSP28, HSP29, and HSP30. They are located in different cell compartments and share the homologous α-crystallin domain. They are expressed in both tachyzoites and bradyzoites, with the exception of HSP28 (predominant in tachyzoites) and HSP30 (only in bradyzoites). In their study, the researchers found that 1) Cy HSP21 shares the same subcellular localization as HSP30, 2) HSP28 is only mitochondrial and present in both T. gondii stages (tachyzoite and bradyzoite), 3) HSP29 is a membrane-associated protein in both stages, with its role to regulate membrane fluidity and preserve membrane integrity during thermal fluctuations, and 4) HSP20 is shown in T. gondii apical region of both stages. The investigators concluded that sHSPs localized to the apical surface or in the membrane are of particular interest in view of their potential role in host cell invasion [159] . Later, the same investigators showed that HSP20 co-localized with the motor complex at the outer surface of the inner membrane complex. This unique subcellular localization indicated its role in motility [160] .
5. Diagnosis: The potential value of serum IgG anti-TgHSP70.1 was evaluated as a diagnostic biomarker of ocular toxoplasmosis in a case-control study conducted in France. The study included three groups (laboratory-confirmed and clinically suspected cases as well as healthy blood donors). Significant IgG increased levels were detected in the first two groups compared with the third group [161] . Recombinant T. gondii antigens were evaluated for use in the diagnosis of toxoplasmosis. The investigators compared three proteins; HSP20, surface antigen (SAG1), and dense granule (GRA7) in human samples infected with toxoplasmosis using IgG ELISA. They found that the combination of two or three recombinant antigens improved the sensitivity values, whereas none of them reacted against seronegative IgG and IgM human sera. Therefore, they concluded its possible use as a diagnostic biomarker in toxoplasmosis [162] .
6. Drug target: The use of GA (HSP90 inhibitor) significantly disturbed the intracellular growth of T. gondii [157] . Using fluorescence microscopy, investigators found increased levels of HSP90 in tachyzoites Cy and in the nucleus and Cy of mature bradyzoites. These findings suggested the potential role of HSP90 in tachyzoiteͻbradyzoite transformation, which was confirmed using GA [163] .
7. Vaccination: Mice immunized with recombinant TgHSP30/bag1 enhanced protective immunity against toxoplasmosis, whereas recombinant TgHSP70 inhibited it [139] . One year later, the same Japanese investigators published another study to localize the region of HSP70 involved in inhibition of host protective immunity and deterioration of toxoplasmosis. They utilized its full length, its NH 2 and carboxy terminal regions, and they measured IgG levels and counted cyst numbers in the brain tissue. They found that mice immunized by full length or carboxy-terminal region showed deleterious effects and attributed these results to 2 reasons; 1) activation of immunosuppression by down-regulating NO release of macrophages, 2) HSP70-full length and its carboxy-terminal region may function as a source of autoimmunity [164] . Similar results were obtained as the investigators observed deteriorating effects on host immune responses on intraperitoneal injection of TgHSP70. They found increased production of IL4 and IL10 (Th2 cytokines) as well as reduced NO production from peritoneal macrophages indicating the role of T. gondii HSP70 in immunosuppression [140] .
In 2003, three genes were used to vaccinate mice against toxoplasmosis: HSP70 (virulent tachyzoite-specific gene), HSP30 (a bradyzoite-specific gene), and SAG1 (a tachyzoite-specific gene). Results revealed that the hsp70 gene was the most effective, showing the lowest parasite burden in various organs of experimental T. gondii-infected mice, and it lasted for more than 3 months [165] . In 2012, Wei [166] constructed a polyvalent vaccine using the hsp70 gene to immunize BALB/c mice. He evaluated the vaccine effects by assessment of CD4 + , CD8 + , immunoglobulins (IgG, IgM, and IgA), and cytokines (IFN-γ, TNF, IL-2, IL-4, and IL-12). Results showed strong and significant cellular and humoral immune responses in the supernatant of cultured spleen cells and in sera of immunized mice [166] . Investigators in Argentina raised antibodies against TgHSP20 in rabbits and then incubated T. gondii tachyzoites in rabbit serum. Reduced parasite invasion (57%) and tachyzoite gliding activity (49%) were elicited. In addition, the rabbit sera reduced Neospora caninum invasion of host cells [167] .
8. Apoptosis: HSP65 of infected macrophages was shown to prevent apoptosis by T. gondii, contributing to the protective immunity against T. gondii low-virulence strains. When mice were susceptible to high-virulent strains, no HSP65 expression was obtained and macrophages showed marked apoptosis. The investigators claimed that mice infected with high-virulent strains might have mechanism(s) that interfere with HSP65 expression [168] .
| 5. Cryptosporidium species | | |
5.1. Applications
1. Viability: In a study on C. parvum oocyst viability, German scientists used HSP70 as a marker. The induction ratio of HSP70 mRNA production in oocysts subjected to heat stress was assessed. Results showed its slight increase in viable oocysts that were heat shocked at 45°C for 20 min, indicating that HSP70 mRNA induction ratio varied according to oocyst viability [169] .
2. Host-parasite relationship: Another HSP70 application was attempted to understand host-parasite relationship in cryptosporidiosis. Results of the multi-locus genetic characterization of small subunit (SSU) rRNA, actin and HSP70 genes of 15 Cryptosporidium spp. revealed that 1) host adaptation is a general phenomenon in cryptosporidiosis, 2) specific genotypes infect specific groups of animals, 3) C. parvum bovine genotype and C. meleagridis that infected rodents and mammals, respectively, were able to infect humans [170] .
3. Diagnosis: The utility of HSP70 was first reported in the diagnosis of cryptosporidiosis when the investigators used two pairs of PCR primers for oocyst detection in drinking water supplies to avoid waterborne outbreaks in the USA. One of the used primers recognized all Cryptosporidium spp., whereas the other targeted C. parvum HSP70 [171] . In 2001, Australian investigators suggested the use of HSP70 to monitor water sources as it is a sensitive biomarker for detection of viable oocysts even at low concentration levels [172] . In 2009, a nanotechnology technique was developed to diagnose C. parvum in clinical samples using oligonucleotide-functionalized gold nanoparticles-targeted HSP70 mRNA from C. parvum oocysts. The nanoparticle probes were complementary to two adjacent target regions on the HSP70 mRNA from C. parvum and did not cross-react with other organisms. The investigators concluded that the ability of nanoparticles to identify HSP70 from C. parvum oocysts will promote research for the use of nanotechnology in the diagnosis of parasitic diseases [173] .
4. Species identification, genotyping and phylogenetic studies: The first work using HSP70 in discrimination between Cryptosporidium species/genotypes was published in 2000. The investigators used genes encoding 18S ribosomal DNA, HSP70 and acetyl coenzyme A synthetase in molecular characterization of Cryptosporidium species in HIV patients. They identified 4 genotypes; C. parvum human and cattle genotypes, C. felis and C. meleagridis [174] . In the same year, HSP70 nucleotide sequences and phylogenetic analysis of Cryptosporidium species were identified. The investigators sequenced ~1,950 bp of the hsp70 genes from 40 Cryptosporidium samples isolated from human and various animals (C. parvum, C. baileyi, C. felis, C. meleagridis, C. muris, C. serpentis, C. wrairi, and unknown species from a desert monitor). They concluded that hsp70 gene offered advantages over SSU rRNA for phylogenetic studies of Cryptosporidium species; 1) higher heterogeneity in hsp70 gene sequences to be better target for genotyping, 2) the arms of mutations (deletion and insertions) are limited in hsp70 gene sequences [175] . One year later, PCR analysis of C. parvum hsp70 gene on 10 isolates was performed and they found its ability to detect C. parvum oocysts genotypes 1 and 2 (5 samples each) [176] . In addition, human samples with clinical cryptosporidiosis from UK were subjected to genomic analysis at hsp70 gene. The results revealed that 149 samples were C. parvum either genotype 1 (~49%) or 2 (~46%), and the investigators concluded the importance of HSP70 in screening and monitoring genotype distribution of cryptosporidiosis for the epidemiological studies [177] .
Analysis of DNA sequences of nine genes belonging to HNJ-1 (a Japanese reference human strain) showed high identity to the reported genotype 2 sequence. Of these genes, hsp70, α-tubulin and β-tubulin, 18S rRNA, and Cryptosporidium oocyst wall protein (COWP) were included [178] . Gene sequence analysis of the 18S rRNA and hsp70 gene of Cryptosporidium from infected cows, sheep, and goats showed that C. parvum was the only species infecting cows. Hence, it was concluded that cows were the possible source of maintaining cryptosporidiosis in farms in Spain [179] .
Phylogenetic analyses of three loci, including hsp70, confirmed that C. macropodum, a species isolated from kangaroos, was genetically different from other known species (C. parvum, C. hominis, C. suis, and C. canis). The investigators considered it a new species, although its oocysts are morphologically identical to the other species [180] . In another study, genes encoding HSP70 and gp60 were used to genotype symptomatic Cryptosporidium samples. The Belgian investigators found C. hominis and C. parvum in 54 and 46% of their samples, respectively. They also observed that the gp60 gene sequencing revealed the predominance of IbA10G2 (a C. hominis subgenotype) and IIaA15G2R1 (a C. parvum subgenotype) in the isolated samples [181] . Using DNA sequence analysis of the 18S rRNA, actin, and hsp70 gene, the investigators could identify C. andersoni and C. ryanae as the dominant species affecting cattle in China [182] .
Several studies utilizing HSP70 in the molecular characterization of Cryptosporidium spp. were published during the last few years (2010-2014). Using 18S rRNA, actin, COWP, HSP70, and gp60, the investigators identified five isolates from cockatiel birds; three of them were new genotypes of C. meleagridis. The latter was reported as a significant cause of cryptosporidiosis in man [183] . To improve PCR methods used for molecular species identification in cryptosporidiosis, Canadian investigators used the immunomagnetic separation (IMS) technique, by which fragments of the genes encoding Cryptosporidium 18S rRNA, HSP70, and COWP were amplified to determine the optimal genes used in molecular identification. Using primers directed against HSP70 they were able to diagnose 60 and 58 cases by IMS-PCR and PCR alone, respectively, in comparison with using 18S rRNA (43 and 42 cases) and gene encoding COWP (56 and 45 cases) [184] . In China, investigators isolated a new strain from a calf and they used SSU rRNA and hsp70 gene sequences to identify it as a new isolate; C. serpentis [185] . Using the same markers, the investigators characterized a new Cryptosporidium genotype in seals [186] . Another new Cryptosporidium genotype (C. viatorum n. sp.) was identified in ten diarrheic British patients coming from India during 2007-2010. Sequences of C. viatorum isolates at HSP70, actin, and SSR RNA loci were not identical to those of C. parvum or C. hominis [187] . Moreover, using genetic identification based on the 18S ribosomal RNA and HSP70, investigators succeeded in assigning four genotypes to infected pigs in Canada; pig genotype II (61%), C. suis (36%), C. parvum (2%), and Cryptosporidium mouse genotype (1%) [188] . On the basis of sequence analyses of four genes encoding 18S rRNA, HSP70, actin, and gp60, other investigators found that two Cryptosporidium isolates belonged to C. hominis, subtype IdA21, which is a rare subtype in China [189] . Further, using genetic analysis of partial 18S rRNA, HSP70, COWP, and actin genes, Chinese investigators claimed that samples isolated from giant pandas were different from those identified from Cryptosporidium spp. and genotypes. Accordingly, they considered these isolates as a new genotype [190] . Similar data were obtained on using the same gene sequences to characterize Cryptosporidium isolates from domestic mice in China (C. tyzzeri) [191] and domestic pigs in the Czech Republic (C. scrofarum) [192] . In Central Vietnam, investigators assigned only one genotype of C. avian and two genotypes of C. suis to all infected ostriches [193] and pigs [194] , respectively. HSP70 was also used with 18S rDNA to diagnose cryptosporidiosis in seals, although immunofluorescence microscopy failed to detect Cryptosporidium oocysts in their fecal samples [195] . HSP70 was also used for phylogenetic analysis of seven samples isolated from Mongolian diarrheic patients that were identified as C. parvum bovine genotype [196] .
Apart from C. felis of cats and C. baileyi of birds, the genomic analysis of other Cryptosporidium spp. affecting man revealed that the hsp90 gene had a conserved sequence identity. On the basis of these data, investigators used it in species identification and were able to identify and differentiate between other species (C. hominis, C. parvum, C. meleagridis, C. canis, C. muris, C. suis, and C. andersoni) [197] .
5. Vaccination: C. andersoni HSP70 was cloned and its recombinant protein was identified (rCaHSP70). In their experimental studies on BALB/c and C57BL/6 mice, the investigators showed that purified rCaHSP70 proved to be highly antigenic and may be used as an immunodiagnostic marker, whereas its constructed plasmid could be used as a candidate vaccine [198] .
| 6. Other protozoa | | |
6.1. Babesia More Details species
B. divergens HSP70 is a highly conserved cytoplasmic protein identified from five isolates from different hosts (human and bovine). The investigators detected HSP70 as an early antigen during acute human Babesiosis More Details, a result that encouraged them to use it as a candidate vaccine [199] . Later, American scientists cloned and expressed B. microti HSP70 and evaluated its use in mice immunization. They found that 30% of the mice survived the challenge infection versus none of the controls [200] . Furthermore, when used in phylogenetic analysis of Babesia spp. and Theileria spp., HSP70 genes of different isolates showed nucleotide sequences with more variety than those of 18S rDNA. Accordingly, species of Babesia and Theileria were classified into 3 groups; A) Babesia infecting dogs such as B. divergens, B. odocoilei, B. bovis, B. caballi, and B. ovis, B) Theileria spp. and C) B. microti and B. rodhaini [201] .
Because expression levels of HSP70 from B. gibsoni and B. microti showed a high degree of conservation, investigators were encouraged to compare between the immunoprotective properties of recombinant BgHSP70 and BmHSP70. Mice immunized with both recombinant proteins elicited high antibody levels and significant reductions in peripheral parasitemia. Accordingly, the use of HSP70 as a candidate vaccine was recommended [202] . Besides, HSP70 was also used in the diagnosis of B. canis vogeli in dogs using multiplex quantitative real-time PCR [203] . On the other hand, using quantitative real-time reverse transcription-PCR, Japanese investigators examined the copy number of B. gibsoni HSP70 transcripts in the strains that showed resistance to diminazene aceturate (DA). Results showed significantly lower levels of HSP70 transcripts in the DA-resistant strains in comparison with the wild-type B. gibsoni. Hence, it was suggested that HSP70 transcript levels were reduced because of parasite exposure to low concentrations of DA. Further studies were recommended to explain the relation between HSP70 and the mechanism of DA resistance in babesiosis [204] . B. gibsoni HSP70 was also found to be a highly immunogenic protein expressed on the surface of exoerythrocytic stages in canine babesiosis [205] . In addition to HSP70, investigators attempted to identify potentially protective antigens from B. bovis by analyzing memory CD4 + T lymphocyte recognition of fractionated merozoite antigens. They detected a 20-kDa protein (Bbo20) with a B lymphocyte epitope that was conserved in different strains and showed 86.4% identity with another protein identified in B. bigemina (Bbg20). Blast analysis of amino acid sequences of both 20 kDa proteins showed significant similarity with HSP20 [206] .
6.2. Microsporidium species
Genes of mitochondrion-related chaperon 60 and Mit HSP70 were found to be coded in the nuclear DNA. These include chaperonin 60 (Cpn60) in E. histolytica, T. vaginalis, and G. lamblia and Mit HSP70 in T. vaginalis and three microsporidians, Nosema locustae, Vairimorpha necatrix, and Encephalitozoon cunuculi [207] . In a study conducted in Japan, Mit HSP70 was used in phylogenetic analysis, when the investigators sequenced genes encoding Mit HSP70 from G. lamblia, E. histolytica, and Encephalitozoon hellem. Their results demonstrated that these protozoa are not primitively amitochodrial, but they lost their mitochondria during their evolutionary history [208] . Although microsporidia have no identifiable mitochondria organelle, Mit HSP70 was eventually expressed from an ancient Mit organelle called mitosome. This discovery placed microsporidia in amitochondriates with E. histolytica, G. lamblia, and T. vaginalis [209] . Expression levels of microsporidial HSP70 were shown in infected insects using affinity chromatography and immunoblotting [210] . Using transmission immunoelectron microscopy, Encephalitozoon cuniculi HSP70 was found to be localized in the spore internal structures [211] .
6.3. Giardia lamblia
The first article that reported the 57 kDa antigen as Giardia HSP was published in 1992. The investigators reported that children with acute infection had specific IgG and IgA response to that antigen [212] . One year later, they published another article evaluating IgG and IgA responses to the 57 kDa antigen in Giardia-infected children with persistent diarrhea and severe malnourishment using SDS-PAGE. Results revealed the absence of IgA response to Giardia HSP antigen in chronic giardiasis, suggesting that specific IgA production was an essential factor for determining parasite clearance [213] . In 2009, Belgian investigators sequenced the genes encoding β-giardin, triose phosphate isomerase (TPI), and glutamate dehydrogenase (GDH) to genotype Giardia isolated from symptomatic patients. Assemblage B was detected in majority of samples (74.4%), however, using a novel species-specific PCR based on tpi gene, mixed infections with both assemblage A and B were detected in 32.4% [181] .HSP70 was found to be essential in excystation of Giardia cysts. In their studies to assess the importance of phosphorylated proteins during excystation, investigators found two phosphoproteins; one of them was HSP70 [214] . In an attempt to determine the infectivity and virulence of a Giardia isolate, mRNA expression was evaluated by quantitative measurements of two biomarkers; HSPs and variant-specific protein (VSP). Using reverse transcription-PCR, investigators measured HSPs in cysts and VSP in trophozoites to assess trophozoite attachment to a cell monolayer (Caco-2 cell culture). They found direct linear correlation between the used biomarkers [215] . In addition, evaluating three kits for DNA extraction, investigators found that use of combined markers (genes encoding HSP and β-giardin) gave better results in the detection and genotyping of G. lamblia isolates than using each marker alone. However, when used alone, the HSP marker gave a higher limit of detection and genotyping compared with the β-giardin marker [216] .
4. Eimeria species
HSP90 was found to be essential for proper cell functions in coccidian protozoa. Molecular analysis of HSP90 from E. tenella and E. acervulina showed high identity and similar nucleotide sequences. However, transcripts detected in all developmental stages of E. tenella disappeared in oocysts undergoing sporulation and in fully sporulated oocysts in E. acervulina. The researchers suggested that regulation systems in messenger RNA underwent different expressions in the two species [217] . Investigating the importance of HSP90 in the intracellular growth of E. tenella, investigators showed increase in EtHSP90 expression in the parasitophorous vacuole membrane, indicating its importance for parasite intracellular growth. Moreover, when specific antibodies and GA were used to attenuate EtHSP90 functions, the parasite was unable to invade and grow in the host cell [218] . Recently, the effect of diclazuril as anticoccidial therapy on HSP90 expression of E. tenella merozoites was attempted. The results showed that the drug directly reduced HSP70 in second-generation merozoites. The investigators concluded that inhibition of HSP90 production might be a promising target for new therapy of E. tenella infection [219] .
On the other hand, HSP70 expression was related to E. tenella virulent strains. Quantitative analysis of HSP70 showed significant gradual decrease in sporozoites and the expressed HSP70 in the excysted sporozoites would be involved in the parasite pathogenicity [220] . Using anti-HSP70 MAbs with immunogold labelling, Spanish investigators succeeded to localize and analyze HSP70 in E. tenella oocysts. They also performed ultrastructural study to visualize the synaptonemal complexes (SCs) to evaluate its chromosomal behavior in presence and absence of HSP70 expression. Meanwhile, inhibition of HSP70 synthesis was inhibited by 3 different doses of quercetin. The investigators found that with increasing the quercetin dose, the number of immunogold particles decreased which means reduction in the HSP70 synthesis. When the dose increased, they found defects in SCs formation followed by sporulation failure (in the last high quercetin dose). The investigators concluded the importance of HSP70 in the sporulation of E. tenella oocysts [221] . HSP70 was also used as an adjuvant in vaccinating chicken against avian coccidiosis. The investigators used the parasite antigen (Ag) in the presence and absence of Ag-loaded DCs or Ag-pulsed DC-derived exosomes (HSP70, CD80 and flotillin, MHC I and II). The results revealed that chicken immunized in the presence of Ag-loaded DCs or Ag-pulsed DC-derived exosomes showed better results in the form of reduction in 1) fecal oocyst shedding, 2) intestinal lesions, and 3) mortality rate, while their body weight increased [222] .
6.5. Trichomonas vaginalis
Using immunoprecipitation, American investigators tested the antigenicity of T. vaginalis HSPs against sera from infected patients and controls. While the majority of HSPs were immunoprecipitated by sera from infected patients as well as controls, only the 38-kDa HSP was immunoprecipitated by sera from infected females, indicating its specificity in trichomoniasis [223] . PCR-RFLP employing the heat-inducible Cy HSP70 was utilized in subtyping T. vaginalis isolates by American scientists. That probe was used as it simultaneously measures the polymorphisms present at multiple gene loci in the Cy hsp70 gene family. They found 10 distinct RFLP pattern subtypes in 36 isolates that did not correlate with metronidazole resistance [224] . Using RFLP-PCR with the same probe, Egyptian investigators reported no relation between T. vaginalis subtypes and presence of T. vaginalis virus. The investigators detected six distinct RFLP pattern subtypes in 13 symptomatic and seven asymptomatic T. vaginalis isolates [225] . On the other hand, a study conducted in 2009 revealed a high degree of diversity among T. vaginalis clinical isolates. As reported, 105 different HSP70 RFLP patterns were detected in 129 isolates; 85 isolates produced unique patterns and 20 additional patterns were shared among 44 isolates. The American investigators found identical patterns in isolates from different geographic locations, confirming the theory of clonal propagation proposed for T. vaginalis isolates. The high genetic diversity among T. vaginalis isolates was attributed to the variable clinical manifestations in trichomoniasis [226] .
6.6. Entamoeba histolytica
The first report dealing with E. histolytica HSPs published in 1992 constructed a cDNA library from E. histolytica trophozoites. Proteins from this library were screened against serum IgG from a patient with invasive amebiasis. Most of the immunopositive proteins were ~70% identical to the human HSP70. When the investigators screened them again with 12 serum samples from infected patients, only three samples expressed HSP70 [227] .
6.7. Free living amoeba
Canadian investigators reported Mit HSP60 expression on abundant organelles within Mastigamoeba balamuthi [228] . Another study confirmed that Cy HSP70 was essential in the proliferation and cytotoxicity of Naegleria fowleri [229] . In addition, HSP70 was detected in Acanthamoeba (nine strains) and in free-living freshwater amoebae (15 strains, belonging to genus Amoeba and Trichamoeba) [230] .
6.8. Cyclospora cayetanensis
A nested PCR method employing the hsp70 gene was developed to diagnose C. cayetanensis infections. This technique was tested in 16 isolates from three different endemic regions: Nepal, Mexico, and Peru. The results revealed that the hsp70 gene could be considered a useful genetic marker for rapid detection of C. cayetanensis [231] .
6.9. Blastocystis species
In Japan, determination of nucleotide sequences encoding Cy HSP70 with other factors (SSU rRNA, translation elongation factor 2 and the noncatalytic 'B' subunit of vacuolar ATPase) enabled the investigators to suggest that Blastocystis (stramenopiles) is the closest relative of alveolates [232] .
6.10. Theileria species
Sera from Theileria-infected sheep were used to screen clones of the T. lestoquardi expression library to identify a specific clone that could be used as a diagnostic marker. The investigators found a reacting clone with high identity to HSP70, which proved to be ~94% sensitive and ~90% specific in diagnosis using ELISA, especially in epidemiological surveys [233] . In addition, HSP70 was used as an adjuvant with the major T. sergenti surface protein (p33), and the results indicated that HSP70-p33 is a promising candidate vaccine against theileriosis [234] .
| Concluding remarks | | |
- HSPs play essential roles in vector-transmitted protozoa such as Plasmodium, Leishmania, Trypanosome, and Babesia. They are responsible for their survival against temperature changes and the febrile conditions in malaria, leishmaniasis, trypanosomiasis, and toxoplasmosis. They are also responsible for their growth and virulence, as well as for their ability to elicit host immune response. Thus, their potential uses as serodiagnostic markers, drug targets, and vaccine candidates have been interesting topics for studies conducted in the last two decades.
- In malignant malaria, the P. falciparum proteome contains Asn-rich sequences, which lead to increased aggregate formation that is enhanced under stress conditions during febrile attacks, another threat by P. falciparum when HSPs have to counteract. Besides, HSPs have to help the organism export ~5% of its encoded genome into the host passing the plasma membrane and the parasitophorous vacuole membrane. PTEX is a translocon that helps in the trafficking across the membranes, and HSPs were identified as constitutents of these PTEXs.
- There is a partnership between HSP40 and HSP70 controlling the events in the life cycle of P. falciparum, and its viability is sensitive to HSP70-HSP40 inhibition. Small HSPs (HSP20) also have potential roles in parasite growth and development, as well as in virulence. Cy HSP110 showed its ability to prevent Asn repeat-rich protein aggregation in vitro and in vivo. P. falciparum HSP70 was found to be a serodiagnostic marker and vaccine candidate.
- HSP100 has a potential role in Leishmania parasite growth and virulence, whereas HSP20 was suggested to be a useful serodiagnostic marker for active leishmaniasis. HSP70 and HSP83 proved to be highly immunogenic and both have prospective importance in the development of improved immunotherapeutic and/or immunoprophylactic targets. Both HSPs were able to elicit human T-cell immune responses and to induce B-cell proliferation. Therefore, both were considered serodiagnostic markers in VL and TL. The gene encoding HSP70 represented a good target for species identification and genotyping, and it was suggested as a new drug target and an adjuvant with a polytope DNA vaccine in leishmaniasis. Furthermore, the use of recombinant HSP70 proved to exhibit protective immunity to all vaccinated mice.
- In African trypanosomiasis, HSP60 was expressed by both BS and PC forms indicating its responsibility for their survival. It also caused a significant host humoral immune response during the entire course of the disease. HSP70 was suggested as a serodiagnostic marker in ASS, whereas the Mit hsp70 gene was found to have a specific antigenic epitope located in its C-terminal region. Investigating new drug targets in ASS revealed drugs that inhibit HSP90 (e.g. GA and GA analogs) and that interact with HSP83 (e.g. benzamide derivative), leading to inhibition of parasite growth.
- In American trypanosomiasis, mapping of HSP70 epitopes identified the immunogenic motifs in the pathogenesis of Chagas disease. Thus, it was used as a marker for this disease (healthy or infected, acute or chronic). The gene encoding the hsp70 gene was used to differentiate between T. cruzi and T. rangeli strains in single and mixed infections. It was also used as a drug target and/or adjuvant in a plasmide vaccine candidate to immunize mice against challenge infection. Interestingly, its ATPase domains were used as adjuvants in a DNA vaccine with E. histolytica-BiP to protect hamsters against amoebic liver abscess. On the other hand, HSP65 has an essential role in preventing macrophage apoptosis contributing to host resistance against infection.
- In toxoplasmosis, a homologous sequence between HSP70s of T. gondii, mouse and human was detected, and the possibility of TgHSP70 to induce autoreactive immune responses in infected hosts with subsequent occurrence of lethal anaphylactic shock reaction was reported. The hsp70 gene was useful for immunizing mice against anaphylactic reaction, whereas its use in a polyvalent vaccine showed strong and significant results in vaccination. There was also a relationship between virulence and HSP70 expression, and its potential use as a diagnostic biomarker of ocular toxoplasmosis was reported.
- Besides, HSP65 expression by macrophages was reported to develop effective immunity, but its expression depends on strain virulence. Therefore, HSP65 was found to prevent apoptosis of infected macrophages by T. gondii low-virulence strains. HSP90 has the ability to elicit IgG production during chronic toxoplasmosis, and HSP90 inhibitors showed significant results for use as drug targets. The role of sHSPs in toxoplasmosis requires further evaluation. They are localized in the apical surface, indicating their role in motility, and they are expressed in both tachyzoites and bradyzoites. Reduction of both parasite invasion and tachyzoites gliding activity was observed on incubation of antibodies against HSP20 with T. gondii tachyzoites.
- In cryptosporidiosis, HSP70 was the only HSP that proved to have several applications; 1) understanding host-parasite relationship, 2) assessment of oocyst viability, 3) detection of oocysts in drinking water supplies, 4) species identification and genotyping (phylogenetic analysis), and 5) immunization of mice against challenge infection.
- Studies on HSP70s constituted the majority of research published on protozoa. It was essential in excystation of Giardia cysts, and in proliferation and cytotoxicity of N. fowleri. The gene encoding HSP70 was used in phylogenetic analysis of G. lamblia, T. vaginalis, and species of Babesia, Theileria, Blastocystis, and Encephalitozoon. It was also used in the diagnosis of C. cayetanensis, Theileria, and in the diagnosis of babesiosis in dogs. In addition, HSP70 proved to be useful as a vaccine candidate in babesiosis, and as an adjuvant against avian coccidiosis.
| Acknowledgements | | |
Conflicts of interest
There are no conflicts of interest.
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