Transcript

Toxoplasma gondii and Heat Shock Proteins

Aykut Özgür1, Lütfi Tutar2, Kübra Açıkalın Çoşkun1, Esen Tutar3 and Yusuf Tutar*,4

1Gaziosmanpaşa University, Faculty of Natural Sciences and Engineering, Department of Bioengineering, Tokat, Turkey

2Kahramanmaraş Sütçü İmam University, Faculty of Science and Letters, Biology Department, Avşar Campus, Kahramanmaraş, Turkey

3Kahramanmaraş Sütçü İmam University, ÜSKİM Research Center, Central Laboratory, Avşar Campus, Kahramanmaraş, Turkey

4Cumhuriyet University, Faculty of Pharmacy, Department of Biochemistry, Sivas Turkey

Toxoplasma gondii is a well-described intracellular parasitic protozoan which is carried by humans, warm blooded animals and birds. In the world, around a third of people are infected by T. gondii as a result of toxoplasmosis. In humans, the life cycle of T. gondii has two stages: tachyzoite stage and bradyzoite stage. Tachyzoites form causes acute infection and tachyzoites transforms to bradyzoites form, this slow growing non-pathogen form is transmissible to the other organisms [1, 2]. A number of studies have reported that, Heat shock proteins (Hsps) expression is increased with viral, bacterial, or parasitic infections, fungi, and inflammation. Therefore, Hsps are associated with infection diseases especially toxoplasmosis [3, 4]. We isolated Hsp40, Hsp70 and Hsp100 from infective T. gondii RH strain, and biochemical characterization and bioinformatics analysis of these proteins were performed. Our results showed that Hsp40, Hsp70 and Hsp100 prevent protein aggregation and induce refolding. Consequently, these Hsps may play essential roles in the mechanisms of bradyzoite development and life cycles of T. gondii. In this chapter, we will mainly focus on potential roles of heat shock proteins to T. gondii survival in the host organism using experimental and computational data.

Keywords Toxoplasma gondii; Heat shock proteins

1. Why protein folding?

Proteins are probably the most important class of macromolecules for every living cell. They play significant role in a variety of functions including catalyzing biochemical reactions, cell signaling, cellular transport, acid-base balance and mechanical support. Proteins must have correct three-dimensional structure in order to perform these functions properly [3,5]. In cell, all proteins are synthesized as linear amino acid chain. During or after biosynthesis, small proteins fold spontaneously in contrast folding mechanism of large proteins is a complex processes [3]. Under some conditions (molecular crowding in the cytosol, cellular stresses and the milieu) newly synthesized proteins may not fold correctly and proper folded proteins can lose their native conformation rapidly. Thus, the biological activities of proteins are decreased, and they have a tendency for aggregation. The accumulation of misfolded proteins leads to several diseases such as Alzheimer’s, Parkinson’s, prion and Huntington diseases [3,5-8].

2. Protein Folders; Heat Shock Proteins

Heat shock proteins (Hsps) are highly conserved proteins which are expressed in response to cellular and environmental stresses. Hsps are participated in formation of proper protein conformation, prevention of protein aggregation and elimination misfolded proteins. Hsps are localized in different compartments of cell (cytosol, endoplasmic reticulum and mitochondria), and they are named according to their molecular weight: small Hsps (<30 kDa), Hsp40, Hsp60, Hsp70, Hsp90 and Hsp100. Overexpression of Hsps can be triggered by exposure to high temperature, infections, inflammation, drugs, malignancy, oxidative stresses, growth factors and fever. Therefore, expression level of Hsps is associated with metabolic diseases, cancer, viral, bacterial and parasitic infections [3,4,9,10].

3. Toxoplasma gondii and Toxoplasmosis

Toxoplasma gondii is a well-described ubiquitous obligate intracellular parasite which is a member of phylum Apicomplexa. The first discovery of the Toxoplasma gondii was from the rodent Ctenodactylus gundi by Nicole and Manceaux (1908). Researchers initially thought that organism was Leishmania however later they noticed that it was a new organism and they named it as Toxoplasma gondii (Ctenodactylus gundi) [1,11,12]. T. gondii can cause infection, known as toxoplasmosis. In the world, one out of every three people is affected by toxoplasmosis. Human may be infected with T. gondii by two ways: congenital and postnatal. Congenital transmission occurs from infected mother to the fetus through the placenta by tachyzoite form of the parasite. Postnatal transmission occurs by consumption of infected animal meat and unwashed vegetables which are contaminated by cat feces. In this case, toxoplasmosis infection progresses rapidly in immune compromised human (for example HIV patients) as a result of encephalitis and

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systemic disease occur. In the early stage of pregnancy T. gondii cause abortion; however in the last of stage of pregnancy the organism damages brain, eye and other tissues [13-17]. In humans and mammalians T. gondii follows an asexual replication cycle and proliferative forms of the parasite (tachyzoites and bradyzoites) are available in the body while oocysts form in the cat feces. Tachyzoites are rapidly-growing form of parasite, and they are monitored in the acute phase of toxoplasmosis. Tachyzoites enter the cell with phagocytosis. In the advancing period of the toxoplasmosis, tachyzoites are converted into slow dividing dormant bradyzoites forms. The tachyzoite–bradyzoite conversion triggers principal infection and subsequently recurrence of the disease. Bradyzoites can remain latent in the tissues, and cysts form in nervous and muscle tissues. In the case of suppression of the immune system, cysts open and conversion of bradyzoites to tachyzoites occurs readily. Life cycle of parasite begins with cat ingestion of a rodent infected with the parasite. Number of parasite increases in the digestive system of the cat and disperses to the environment through cat feces and the parasite transmits to humans orally. Furthermore, recent studies have indicated a correlation between toxoplasmosis and neurodegenerative diseases (prion, Alzheimer’s diseases, schizophrenia etc.) and cancer. The formation of bradyzoites is associated with alterations in the environmental conditions and in cellular stress levels. It is known that all of these factors induce the expression of Hsps during conversion from tachyzoites to bradyzoites, and also inhibition of Hsps suppresses bradyzoite development [14,18-21].

4. Structure and Function of Protein Folder Family

4.1. Hsp100

Hsp100 is a large conserved chaperone protein which is a member of Clp/Hsp100 family of AAA+-ATPases. Expression level of Hsp100 is very low in unstressed cells, but increases under cellular stresses. Hsp100 forms six-membered ring complexes and it functions in disaggregating insoluble protein aggregates and separation of large protein aggregates into smaller pieces. Hsp100 contains highly conserved nucleotide binding domains (NBDs) and peptide binding sites (Fig.1). This large protein family identified with five conserved motifs: Walker A, Walker B, sensor1, sensor2 and arginine fingers. Generally, Hsp100 has two nucleotides NBDs (NBD1 and NBD2) which have been shown to bind and hydrolyze ATP. Walker A (also known as Walker loop or P-loop) and Walker B are conserved motifs which are widely found in part of nucleotide binding domains of proteins. According to the amino acid sequences of Hsp100 family, GXXXXCLT/S (X donate any amino acid) and hhhhDE (h donate any hydrophobic amino acid) are conserved Walker A and Walker B motifs respectively. Walker A interacts with the phosphate groups of the ATP. Walker B contains conserved acidic residues and it functions in coordination of ATP hydrolysis. In these residues, aspartic acid contacts with Mg+2 ions, and glutamic acid is essential for ATP hydrolysis. Sensor 1 motif has characteristic conserved threonine residue which interacts with the γ-phosphate of ATP via hydrogen bond. Sensor 2 motif is occurred in conserved GAR sequences, and also generates α-helix structure across the top of the ATP-binding site. Moreover, Sensor 2 is involved in an interaction with conserved arginine residue for interacting with γ-phosphate of ATP [22-27].

Fig. 1 Conserved domains of Hsp100 [24]

Hsp100 and Hsp70/Hsp40 chaperone system establish cooperation, and the chaperone activity is occurred via ATP hydrolysis. Potential mechanism of protein disaggregation may be explained by crowbar effect of Hsp100. In this model, large protein aggregates interacts first with Hsp100, and then with Hsp70 and Hsp40. Protein aggregates enter into tunnel like hollow spherical structure of Hsp100, and then composed smaller protein aggregates gain a proper three dimensional structure through Hsp70 and Hsp40 complex function located at the end of the tunnel [3].

4.2. Hsp40

Hsp40, also known as co-chaperone, is a chaperon protein family which has more than 100 members. Hsp40 is mainly involved in protection of proteins from irreversible protein aggregation. In this process, Hsp40 works together with Hsp70. There are three types of Hsp40 according to their structural differences including Type I, Type II and Type III (Fig. 2). Type I consist of J domain, glycine-phenyl alanine rich region (G/F), zinc finger like region (ZFLR) and C-

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terminal domain II, Type II possess J domain, glycine-phenyl alanine rich region (G/F), glycine-methionine rich region (G/M) and C-terminal domain I-II; however, Type III contains only J-domain [28,29].

Fig. 2 Conserved domains of different types of Hsp40 (Redrawn from reference 29)

J-domain characterizes universally conserved histidine-proline-aspartic acid (HPD) motif. J-domain interacts with Hsp70 ATPase domain, and ATPase activity of Hsp70 is stimulated with this interaction. V-shaped ZFLR domains have characteristic four repeated Cys-X-X-Cys-X-Gly-X-Gly (X is any amino acid) motif which is involved in substrate binding and protein-protein interaction. G/F region (approximately 40% glycine and 15% phenylalanine) provides hydrophobic region for binding substrate. Additionally, G/F region is involved in modulating the conformation of substrates. Briefly, Hsp40 may provide bound protein substrates to Hsp70 [3,30,31].

4.3. Hsp60

Hsp60 is abundant protein which is particularly found in mitochondria and cytoplasm. Hsp60 is responsible for protein folding, transport and maintenance of mitochondrial proteins, and DNA metabolism. In E. coli, GroEL protein is structurally and functionally homolog to Hsp60. In order to solve protein folding mechanisms; GroEL has been recognized as a model for elucidating the role of Hsp60. Structurally, GroEL is composed of back-to-back heptameric rings, and each subunit of heptameric rings is comprised of three domains: the apical, the equatorial and the intermediate domain [32,33]. In protein folding processes, Hsp60 works coordinately with other Hsps, and requires hydrolyze energy of ATP. Hsp60 acts different as a chaperone than that of other Hsps. 50 kDa substrate proteins can be encapsulated in barrel shaped Hsp60. If the capacity is exceeded, Hsp10 closes the barrel [3,34].

4.4. Hsp90

Hsp90 is highly conserved chaperon protein which is widely found in endoplasmic reticulum, cytosol and mitochondria. Hsp90 is one of the most expressed cellular proteins and plays important role in protein folding and refolding, cell signaling, regulation of steroid hormone receptors and kinases, cell cycle control and transcriptional regulation. Hsp90 protein contains three functional domains, N-terminal domain (NTD), middle domain (MD) and C-terminal domain (CTD) (Fig. 3). NTD has an ATP binding site and shows ATPase activity. Hsp90 has highly a flexible and dynamic structure, and large conformational changes are occurred with binding and hydrolyzing of ATP. CTD is important for Hsp90 dimerization. MEEVD motif of CTD provides binding site for some co-chaperones such as Hsp organizing protein (Hop), FK506-binding protein (FKBP52) and protein phosphatase 5 (PP5). Furthermore, MD also has binding sites for client proteins and co-chaperones [35-37].

Fig. 3 Conserved domains of Hsp90 [37]

Hsp90 is similar to the structure of Hsp70; however, their functions don’t show similarities. Hsp90 does not act on nascent protein folding; indeed it acts in the late stages of the folding. To perform functions, Hsp90 must collaborate with Hsp70 and co-chaperones, for example Hop. According to the proposed model, Hsp70-Hsp40 complex interacts with misfolded proteins, and Hop is involved in this complex. Then this complex interacts with Hsp90. Hereby, Hop is an important actor in mediating the association of Hsp70 and Hsp90. Thus, irreversible protein aggregation is mostly

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suppressed by participation of Hsp90 in this process. However, mechanism of Hsp90 chaperone action is not fully understood yet [3,38].

4.5. Hsp70

Hsp70 is a well-known conserved chaperone protein which is expressed in different cellular compartments. Hsp70 is an important part of the cell's machinery for folding of newly synthesized polypeptides, refolding and solubilization of misfolded proteins, and protection of cells from cellular stresses. Also, it is involved in prevention of apoptosis and translocation of proteins across membranes. In protein folding, Hsp70 collaborates with other chaperone proteins especially Hsp40 and Hsp100 (This argument is explained under Hsp100 section). Hsp70 has three functional domains: 44 kDa ATPase domain, 25 kDa substrate binding domain and 18 kDa C-terminal domain. U-shaped ATPase domain has four subdomains (IA, IB, IIA and IIB) and shows ATPase activity. The hydrolysis of ATP triggers the binding of Hsp70 to peptide substrate. Therefore, Hsp70 shows high affinity for substrate at ADP state [39-42]. Substrate binding domain has lid and protein-binding groove. Substrate binding domain provides hydrophobic regions for substrate protein. Energy from ATPase activity of Hsp70 helps movement of the lid domain. The lid is open when Hsp70 is ATP bound. In this case, nascent proteins bind in this region. On the other hand, the lid is closed when Hsp70 are ADP bound and hydrolyzed ATP, and substrate proteins are bound to the substrate binding domain to allow a hydrophobic environment for proper folding [3,43].

4.6. Small Hsps

Small Hsps (sHsps) are group of chaperon proteins which have molecular weights lower than 40 kDa. In contrast to other Hsp families, sHsps are not conserved proteins among organisms. However, their increased expression level upon stresses is the most similar property with other Hsp family. They have been identified in different parts of cell. All members of this protein family contain a 100 amino acid α-crystallin domain. Unlikely, sHsps are not used in protein folding as other Hsps. sHsps are interacted with partially folded proteins. sHsps may stabilize these unfolded substrate proteins, and they prevent aggregation. sHsps do not have an ATP binding site; therefore their functions are independent from ATP. However, other ATP-dependent chaperone proteins coordinate sHsps activities. Moreover, sHsps are also involved in RNA stabilization and cytoskeleton interaction [3,44-47].

5. Importance of Hsps in Life Cycle of T. gondii

Numerous studies have been suggested important roles for Hsps in the life cycle of intracellular parasites. Both parasite and host cell benefit from the Hsps to defend against harmful effects during interactions. Hsps are dominant antigen for many pathogens, and they are generated as immune response in infections [48,49]. In order to improve vaccine and treatment methods for toxoplasmosis, the functions of Hsps must be known in the life cycle of T. gondii. Available information indicates that the expression level of the host and T. gondii-derived Hsps (T.g. Hsp70) are significant parameters in toxoplasmosis. Generally, increased expression level of Hsps is associated with bradyzoite development and conversion from bradyzoites to tachyzoites. Among Hsp family, Hsp70 is the most important chaperone protein which plays protective roles for T. gondii and host cell. T.g. Hsp70 is highly expressed just before death of the host. T.g. Hsp70 stimulates down-regulation of nitric oxide (NO) release from peritoneal macrophages, production of anti-Hsp70 antibody by B1 cells, inhibition of cytokine binding and NFκB (nuclear factor kappa-light-chain-enhancer of activated B cells) in host cell. Furthermore, Hsp70 mediates activation of B and dendritic cells [50-53]. Also, apoptosis is involved in the interaction between the T. gondii and the host. In toxoplasmosis, T lymphocytes and leukocytes induce apoptosis; in contrast, T. gondii inhibits the apoptotic program of the host cell. T. gondii interacts with caspase cascade, and expression of anti-apoptotic molecules is increased in infected host cell. Therefore, the intracellular environment is facilitated for the development of T. gondii. Hsp family directly interferes not only with apoptosis related molecules, but also regulates transcription of anti-apoptotic molecules such as Bcl-2 protein family. Especially, Hsp27 and Hsp70 have important roles for modulating of infected host cell. These Hsps contribute to degradation of apoptosis-regulatory proteins and inhibit key effectors of the apoptotic pathway [54-56]. Hsp40-Gok1, Hsp70-Ayk1 and Hsp100-Ipek1 were isolated from T. gondii RH strain using recombinant DNA technology and their functional and structural properties were investigated with in silico analyses and Flourescence, nanoDSC, FTIR, ATP hydrolysis, luciferase folding and luciferase aggregation experiments by our research group. Denaturation curves calculated by fluorescence and nanoDSC signals showed similar thermodynamic stability with homolog Hsps. FTIR experiments indicated significant alterations in Hsps secondary structure upon ligand binding. Hsps activity determination for substrate protein folding with denatured luciferase showed similar trends with Hsp homologs. Additionally, ATP hydrolyses experiments with a regenerative system showed an increase in the rate if Ayk1 coordinates with Gok1 as expected. In summary, Gok1, Ayk1 and Ipek1 involve in protein refolding processes in T. gondii. Moreover, expression level of Hsps increase with development of bradyzoites forms from tachyzoites forms [57].

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Fig. 4 Modelling of Hsp100-Ipek1

In our study, isolated Hsp40-Gok1, Hsp70-Ayk1 and Hsp100-Ipek1 models are created with SWISS-MODEL (Fig 4-6) (http://swissmodel.expasy.org/). According to models, their secondary structures showed similarities with Hsps homolog, and conserved domains of Hsp40, Hsp70 and Hsp100 were determined in Hsp40-Gok1, Hsp70-Ayk1 and Hsp100-Ipek1.

Fig. 5 Modelling of Hsp70-Ayk1 Prevention of substrate protein aggregation with isolated Gok1, Ayk1, and Ipek1 was also performed. Chemically denatured substrate protein luciferase was treated with Gok1, Ayk1, and Ipek1 alone with or without nucleotides. Then different combinations of the isolated Hsps with or without nucleotides were tested. Combination of Ayk1-Gok1-Ipek1 in the presence of ATP but not ADP showed increased folding activity as determined by luminometric method. Therefore, Ayk1-Gok1-Ipek1 complex help T. gondii to prevent protein aggregation and may help the parasite survival under stress conditions including but not limited to host invasion.

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Fig. 6 Modelling of Hsp40-Gok1

6. Evolutionary Conservation of Hsps

6.1. Phylogenetic analysis

Hsp protein families are evolutionary conserved across all of three domains of life to a certain degree. We aimed to shed light on evolutionary conservation and phylogeny with giving examples for each Hsp families in this part of the chapter. We performed a bioinformatics approach to show phylogeny and conservation of Hsps from different organisms including Toxoplasma gondii ME49 strain. The genome database (http://toxodb.org) was used to determine sequence homologues to members of the HSP families, and to obtain deposited protein sequences of Hsp20, Hsp60, and Hsp90 of Toxoplasma gondii ME49 strain. Also, our sequenced DNAs of Hsp40-Gok1, Hsp70-Ayk1, and Hsp100-Ipek1 (from RH strain) validated and used for Hsp40, Hsp70, and Hsp100 protein sequences respectively for determination of homologues Hsp proteins from different organisms. Protein sequences were scanned with protein blast (blastp) for the occurrence of patterns stored in non-redundant protein sequences (nr) database of NCBI (http://blast.ncbi.nlm.nih.gov). For phylogenetic analysis, selected Hsp sequences were used. Selected Hsp protein sequences were aligned using the ClustalW algorithm and the data set was used to build a phylogenetic tree for each Hsp family with the MEGA5 software [58]. The trees were made using the neighbor-joining algorithm with Poisson-corrected amino acid distances. The reliability of clustering patterns in the tree was tested by bootstrapping (1000 replicates). NCBI taxonomy browser (http://www.ncbi.nlm.nih.gov/Taxonomy) took as a reference for to determine hierarchical taxonomical ranks of analyzed species. The trees show different organisms on tree nodes branched on the basis of their Hsp20, Hsp40, Hsp60, Hsp 70, Hsp90, and Hsp100 proteins (Fig. 7-12). In Figure 7, Hsp40s (DnaJs) of T. gondii ME49 and Neospora caninum Liverpool, Sarcocystidae family members of apicomplexans, highly related to each other with the bootstrap value of 100%, this is consistent with the situation that both species taxonomically members of same family. Also other apicomplexans (2 Plasmodium species, Babesia bovis, Toxoplasma gondii, Neospora caninum, and 2 Cryptosporidium species) take place in the same branch and supported by lower bootstrap values, and diverged branch from main tree. A kinotoplastid (Trypasonoma cruzi) diverged from the branch including bilateria animals that human (Homo sapiens), African clawed frog (Xenopus leavis), and Masrupenaeus japonicus by a high bootstrap value of 95%. The node for ameobozoa (Acanthamoeba castellanii and Dictyostelium fasciculatum) is supported by a higher boostrap value i.e. 92%. Furthermore ameobozoa node (Acanthamoeba castellanii and Dictyostelium fasciculatum) diverged with boostrap value of %97 from the node including two distantly related species, Albugo laibachii and Oxyricha trifallax. This gives an idea about evolutionary conservation of DnaJ (Hsp40) proteins with taxonomically distant species.

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Fig. 7 Phylogenetic tree of Hsp40 (DnaJ) proteins with bootstrap support values on the nodes. Phylogenetic tree of Hsp70s from different alveolata (Fig. 8) showed that species under same taxonomical rank fall into same node with very higher bootstrap values and different species of Plasmodium formed separate monophyletic group in 100% of the bootstraps. However, all apicomplexans were not formed a monophyletic group which indicated their Hsp70 homology to a certain degree.

Fig. 8 Phylogenetic tree of Hsp70 proteins with bootstrap support values on the nodes.

T. gondii ME49 makes totally diverged branch from the main tree on the basis of Clp/Hsp100 proteins since other 17 proteins from the domain Bacteria but T. gondii take place in domain Eukaryota in Fig.9. In Clp/Hsp100 tree bacterial species formed a monophyletic group and also species in the same family i.e. Escherichia coli o157:H7 to Pectobacterium carotovorum subsp. carotovorum WPP14 (top to bottom of tree) fall into same node by a high bootstrap value of 100% (Fig. 9).

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Fig. 9 Phylogenetic tree of Clp/Hsp100 proteins with bootstrap support values on the nodes. In the Phylogenetic tree of sHsp proteins (Fig. 10) node for basidiomycetes of kingdom Fungi (Pholiota nameko, Auricularia delicate, and Paxillus involutus) is supported by very high bootstrap value i.e 100% while the node for peach (Prunus persica) diverged from a very low bootstrap value from basidiomycetes. Apicomplexans (Neospora caninum, Toxoplasma gondii, 2 species of Babesia, and 3 species of Plasmodium) node is supported by very high bootstrap value i.e 98%. Except for Dictyotelium fasciculatum (ameobozoan Eukaryote) rest of the other species belongs to Bacteria and supported by lower bootstrap values. sHsp phylogenetic tree was consistent with the taxonomical positions of basidiomycetes and apicomplexans but not for the rest. Moreover, position of the species basidiomycetes and peach is supported by very low bootstrap value in Fig.10 but it is consistent with the phylogenetic position of Fungi and Viridiplantae kingdoms.

Fig. 10 Phylogenetic tree of small heat shock proteins (Hsp20) with bootstrap support values on the nodes. In Hsp60 phylogenetic tree in figure 11, organism divided in two main branches. Flowering plants (Prunus dulcius, Zea mays, and Arabidopsis thaliana) is formed a monophyletic group and supported by very high bootstrap value (100%) and the moss Physcomitrella patens subsp. patens separated from flowering plants with 99% bootstrap value, and also green algae (Osterococcus lucimarinus, Volvox carterii, and Ulva pertusa) separated from former mentioned plants with 93% bootstrap value showing a consistency with their taxonomical positions. While apicomplexans (Theileria orientalis, Babesia bovis, Toxoplasma gondii, and 3 Plasmodium species) fall into second main branch are supported by 100% bootstrap value, node for the rest is supported by lower bootstrap values.

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Fig. 11 Phylogenetic tree of Hsp60 with bootstrap support values on the nodes. Bootstrap consensus tree of different organisms on the basis of Hsp90 proteins showed two main branches (Fig. 12); in the second main branch green algae and flowering plants (Viridiplantae Kingdom) formed a monophyletic group with 100% bootstrap value while rest of the alveolata species are supported by very low bootstrap values in the first main branch. Furthermore, Hsp90s of dinoflagellates (Karlodinium veneficum and Amphidinium carterae) are supported well by 100% bootstrap, but nested in the taxonomically distant apicomplexans group.

Fig. 12 Phylogenetic tree of Hsp90 with bootstrap support values on the nodes. Except for Clp/Hsp100 tree, all phylogenetic trees clearly showed the Hsps of T. gondii ME49 and Neospora caninum Liverpool highly related to each other with a very high bootstrap value of 100% because of two species taxonomically members of Sarcocystidae family of apicomplexans. Furthermore, T. gondii nested the same branches with apicomplexans in all phylogenetic trees which shows the evolutionary conservation of Hsp proteins and their homology to a certain degree. Then again, plant Hsps were clustered together in accordance with their taxonomical hierarchy but on the higher taxonomical ranks (Fig. 11-12). Phylogenetic tree of Clp/Hsp100 proteins (Fig. 9) separated bacterial species and eukaryotic T. gondii, but not resolved taxonomical relation of bacteria. In Figure 7, Hsp40s (DnaJs) of bilateria animals clustered together with a high bootstrap value and this branch diverged from other distantly related species. Hereby, the Hsp proteins may not be a good marker to resolve lower taxonomical ranks (i.e. family, genus, and species) but their evolutionary conservation is clear on the higher taxonomical levels, and they are highly conserved at least in the same kingdom.

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7. Current Prospects and Future Plans

Hsps are universally conserved proteins among organisms as analyzed in the previous section. Currently several studies target different Hsps to develop drugs for a variety of diseases including systemic and neurodegenerative diseases. Hsps are hydrophobic proteins and interact with almost all protein types whether in the refolding process or in the biochemical pathways involved. This feature makes Hsps perfect candidate for drug-vaccine design and delivery. Several examples of Hsp-based applications-treatments may be found in the literature. An example to these efforts is Hsp90. This highly expressed protein in human has been used for cancer drug development. ATP inhibitors of Hsp90 are widely used in cancer treatments. T. gondii affects one out of three people in the world and the organism probably employs its Hsps at different stages of the host infections. Therefore, our lab isolated three key Hsp proteins from virulent RH strain and biochemically characterized these proteins. We will submit the sequences to Pubmed database soon. And our lab is currently working on determination of functional and structural differences between human and T. gondii Hsps for developing a T. gondii specific drug.

Acknowledgements This work was funded by TUBITAK (110T928) project and through a seed grant from the Turkish National Academy of Sciences (TUBA GEBIP 2008-29).

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Microbial pathogens and strategies for combating them: science, technology and education (A. Méndez-Vilas, Ed.)

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