RESUMEN
Leishmaniasis is a parasitic disease of medical importance widely distributed around the world. Several methods are available for diagnosis but molecular approaches are highly recommended. To improve the sensitivity of an existing hsp20 gene based-PCR protocol to detect Leishmania parasites, primers were redesigned to amplify a shorter fragment using a new PCR variant (PCR-hsp20S). In this study, we aimed at characterizing the performance of the new method on cutaneous clinical samples and compare it with the former PCR-hsp20. The analytical sensitivity of the PCR-hsp20S was evaluated using DNA dilutions (100-0.1 pg) from Leishmania donovani and resulted in the detection of 10 fg of parasitic DNA, the equivalent to 0.05 parasite genome. For the diagnostic evaluation, a panel of 127 human clinical samples was used to calculate the parameters of sensitivity, specificity, accuracy, and positive and negative predictive values of the PCR-hsp20S. Diagnostic sensitivity was 94% (CI, 89.1-99.7%) and the specificity of 100% (CI, 98.6-100%). The same panel was also evaluated with the PCR-hsp20 to calculate the agreement between both molecular assays and to compare their performances. While both hsp20-based PCRs showed a good agreement coefficient (kappa index = 0.6), the performance of the novel variant, PCR-hsp20S, was significantly higher in terms of sensitivity (P = 0.0001) allowing the accurate detection of a higher number of Leishmania-positive clinical samples. We endorse the use of the PCR-hsp20S over the former protocol for the detection of Leishmania parasites from cutaneous clinical samples. In addition, as an improved sensitivity was achieved with the new method merely through the amplification of a shorter gene fragment, this investigation constitutes an experimental proof of this concept.
Asunto(s)
Proteínas del Choque Térmico HSP20/genética , Leishmania donovani/aislamiento & purificación , Leishmaniasis/parasitología , Técnicas de Diagnóstico Molecular/métodos , Reacción en Cadena de la Polimerasa/métodos , Animales , Cartilla de ADN/química , ADN Protozoario/genética , Humanos , Leishmania donovani/genética , Proteínas Protozoarias/genética , Sensibilidad y Especificidad , Piel/parasitologíaRESUMEN
In the present study, we evaluated the transcriptional response of four stress-related genes in three Oenococcus oeni strains after acclimation at two different temperatures. Gene expression was analyzed at time zero and after 48 h acclimation at 18 and 21 °C. After the acclimation period cells were inoculated into sterile Pinot noir wine and MLF was followed for 25 days to investigate if different acclimation temperatures could influence cell survival and MLF performance. L-malic acid consumption, population survival, and transcriptional behavior were different upon the acclimation temperature. rmlB and hsp20 genes presented a considerable increase in their expression level when strains were acclimated at 18 °C particularly in the psychrotrophic strains UNQOe19 and UNQOe4 isolated from Patagonian Pinot noir wine in comparison with the control strain (ATCC 27310). The increase in rmlB and hsp20 expression could account for the better survival of these strains in Pinot noir in comparison with the control strain. In addition, Patagonian populations acclimated at 18 °C were able to consume a higher percentage of L-malic acid in comparison with cells acclimated at 21 °C. Our results suggest that gene expression analysis of cells acclimated at sub-optimal temperatures could benefit the selection of psychrotrophic strains aimed as starter cultures.
Asunto(s)
Adaptación Biológica , Frío , Perfilación de la Expresión Génica , Oenococcus/genética , Oenococcus/efectos de la radiación , Estrés Fisiológico , Vino/microbiología , Argentina , Chile , Proteínas del Choque Térmico HSP20/genética , Hidroliasas/genética , Malatos/metabolismo , Viabilidad Microbiana/efectos de la radiaciónRESUMEN
Small heat shock proteins (HSPs) are molecular chaperones with ATP-independent properties. They are involved in a variety of physiological and stress processes. In this study, the full-length HSP 20 (HSP20) from Pinctada martensii, designated as PmHSP20, was obtained from hemocytes using rapid amplification of cDNA ends technology. The PmHSP20 cDNA was 952 bp in length, containing an open reading frame of 534 bp that encoded 177-amino acid residues, with an isoelectric point of 5.86 and molecular weight of 20.24 kDa. The sequence of this deduced polypeptide contained typical structure and function domains conserved in the HSP20 family, providing evidence that PmHSP20 belongs to the HSP20 family. The PmHSP20 mRNA expression levels were detected in various tissues of P. martensii and in hemocytes after challenges with the bacteria Vibrio harveyi and lipopolysaccharide (LPS) using quantitative real-time polymerase chain reaction amplification. The results indicated that PmHSP20 is constitutively expressed in all tissues tested and might be involved in the immune response. The upregulation of PmHSP20 after V. harveyi and LPS challenge suggests that PmHSP20 plays an important role in anti-bacterial immunity. Studies on PmHSP20 are a valuable resource to further explore the immune system in pearl oysters and might enhance our knowledge of molluscan innate immunity.
Asunto(s)
Proteínas del Choque Térmico HSP20/genética , Pinctada/genética , Animales , Proteínas del Choque Térmico HSP20/química , Proteínas del Choque Térmico HSP20/metabolismo , Hemocitos/metabolismo , Hemocitos/microbiología , Pinctada/metabolismo , Dominios Proteicos , Estrés Fisiológico , Regulación hacia Arriba , Vibrio/patogenicidadRESUMEN
In Azotobacter vinelandii, a cyst-forming bacterium, the alternative sigma factor RpoS is essential to the formation of cysts resistant to desiccation and to synthesis of the cyst-specific lipids, alkylresorcinols. In this study, we carried out a proteome analysis of vegetative cells and cysts of A. vinelandii strain AEIV and its rpoS mutant derivative AErpoS. This analysis allowed us to identify a small heat-shock protein, Hsp20, as one of the most abundant proteins of cysts regulated by RpoS. Inactivation of hsp20 did not affect the synthesis of alkylresorcinols or the formation of cysts with WT morphology; however, the cysts formed by the hsp20 mutant strain were unable to resist desiccation. We also demonstrated that expression of hsp20 from an RpoS-independent promoter in the AErpoS mutant strain is not enough to restore the phenotype of resistance to desiccation. These results indicate that Hsp20 is essential for the resistance to desiccation of A. vinelandii cysts, probably by preventing the aggregation of proteins caused by the lack of water. To our knowledge, this is the first report of a small heat-shock protein that is essential for desiccation resistance in bacteria.
Asunto(s)
Azotobacter vinelandii/genética , Azotobacter vinelandii/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Proteínas del Choque Térmico HSP20/genética , Proteínas del Choque Térmico HSP20/metabolismo , Factor sigma/genética , Factor sigma/metabolismo , Secuencia de Bases , Desecación , Silenciador del Gen , Proteínas del Choque Térmico HSP20/química , Metabolismo de los Lípidos , Datos de Secuencia Molecular , Mutación , Regiones Promotoras Genéticas , Proteoma , Proteómica , Procesamiento Postranscripcional del ARN , Transcripción GenéticaRESUMEN
OBJECTIVES: Explore a new target for molecular diagnosis of Leishmania. MATERIALS AND METHODS: We evaluated the utility of the gene that encodes the heat shock protein 20-kDa (Hsp20) for detecting Leishmania by polymerase chain reaction (PCR). PCR was normalized and analytical parameters were determined, as well as the validity and diagnostic accuracy, and concordance with the PCR - 18S. PCR-Hsp20 with DNA was obtained from a group of clinical samples from different sources. RESULTS: The analytical parameters were adequate. The sensitivity obtained was 86% and the specificity was 100%. The concordance with the reference method was good (κ = 0.731), which supports its potential use for diagnosis. The possibility of subsequent identification of the species by sequencing the amplified product gives an additional advantage. CONCLUSIONS: The usefulness of this gene as a new target for the detection of Leishmania was demonstrated. Because of its potential, it is recommended to improve the sensitivity of the method and to evaluate it in different endemic regions.
Asunto(s)
Proteínas del Choque Térmico HSP20/genética , Leishmania/genética , Leishmania/aislamiento & purificación , Leishmania/clasificación , Reacción en Cadena de la Polimerasa , Especificidad de la EspecieRESUMEN
BACKGROUND: The Hsp20 genes are associated with stress caused by HS and other abiotic factors, but have recently been found to be associated with the response to biotic stresses. These genes represent the most abundant class among the HSPs in plants, but little is known about this gene family in soybean. Because of their apparent multifunctionality, these proteins are promising targets for developing crop varieties that are better adapted to biotic and abiotic stresses. Thus, in the present study an in silico identification of GmHsp20 gene family members was performed, and the genes were characterized and subjected to in vivo expression analysis under biotic and abiotic stresses. RESULTS: A search of the available soybean genome databases revealed 51 gene models as potential GmHsp20 candidates. The 51 GmHsp20 genes were distributed across a total of 15 subfamilies where a specific predicted secondary structure was identified. Based on in vivo analysis, only 47 soybean Hsp20 genes were responsive to heat shock stress. Among the GmHsp20 genes that were potentials HSR, five were also cold-induced, and another five, in addition to one GmAcd gene, were responsive to Meloidogyne javanica infection. Furthermore, one predicted GmHsp20 was shown to be responsive only to nematode infection; no expression change was detected under other stress conditions. Some of the biotic stress-responsive GmHsp20 genes exhibited a divergent expression pattern between resistant and susceptible soybean genotypes under M. javanica infection. The putative regulatory elements presenting some conservation level in the GmHsp20 promoters included HSE, W-box, CAAT box, and TA-rich elements. Some of these putative elements showed a unique occurrence pattern among genes responsive to nematode infection. CONCLUSIONS: The evolution of Hsp20 family in soybean genome has most likely involved a total of 23 gene duplications. The obtained expression profiles revealed that the majority of the 51 GmHsp20 candidates are induced under HT, but other members of this family could also be involved in normal cellular functions, unrelated to HT. Some of the GmHsp20 genes might be specialized to respond to nematode stress, and the predicted promoter structure of these genes seems to have a particular conserved pattern related to their biological function.
Asunto(s)
Glycine max/genética , Proteínas del Choque Térmico HSP20/genética , Respuesta al Choque Térmico/genética , Proteínas de Plantas/genética , Transcriptoma , Animales , Secuencia de Bases , Mapeo Cromosómico , Secuencia Conservada , Resistencia a la Enfermedad/genética , Duplicación de Gen , Genoma de Planta , Proteínas del Choque Térmico HSP20/metabolismo , Interacciones Huésped-Parásitos , Cadenas de Markov , Datos de Secuencia Molecular , Filogenia , Enfermedades de las Plantas/parasitología , Proteínas de Plantas/metabolismo , Regiones Promotoras Genéticas , Sitios de Carácter Cuantitativo , Análisis de Secuencia de ADN , Glycine max/parasitología , Glycine max/fisiología , Tylenchoidea/fisiologíaRESUMEN
The Leishmania genus comprises up to 35 species, of which 20 are responsible for human disease. However, the taxonomic status for many of them is under discussion. The small Heat Shock Proteins (sHSPs) are physiologically relevant, protecting cellular proteins from aggregation and maintaining cellular viability under intensive stress conditions. In Leishmania, a protein of this class was previously described, the 20-kDa heat-shock protein (HSP20), which is encoded by a single gene. In the present study, we used this target, alone or in combination with hsp70 gene, to investigate the phylogenetic relationships among Leishmania species. Using a pair of degenerate primers it was possible amplifying a 370bp fragment of the hsp20 coding region in 39 strains of very different geographic origins, representing in total 16 Leishmania species (14 if L. chagasi and L. archibaldi are considered synonymous names of L. infantum and L. donovani, respectively). Nucleotide sequences were readily obtained by direct sequencing of the amplification products. Both phylogenetic trees and networks based on either hsp20 sequences or combined datasets of hsp20 and hsp70 sequences were constructed. These phylogenic analyses supported the division of the Leishmania genus into nine species: L. (L.) donovani, L. (L.) major, L. (L.) tropica, L. (L.) aethiopica, L. (L.) mexicana, L. (V.) lainsoni, L. (V.) naiffi, L. (V.) guyanensis and L. (V.) braziliensis. Additionally, by network analysis, the subspecies L. (L.) donovani infantum and L. (V.) braziliensis peruviana were recognized within the L. (L.) donovani and L. (V.) braziliensis species, respectively. Therefore, hsp20 gene was found to be a suitable molecular marker for Leishmania typing and classification purposes. In addition, this study represents a solid contribution to the objective of establishing a more reliable taxonomy for the genus Leishmania.
Asunto(s)
Genes Protozoarios , Proteínas del Choque Térmico HSP20/genética , Leishmania/genética , Secuencia de Bases , Análisis por Conglomerados , Proteínas HSP70 de Choque Térmico/genética , Leishmania/clasificación , Datos de Secuencia Molecular , Filogenia , Alineación de SecuenciaRESUMEN
Toxoplasma gondii is an obligate intracellular parasite and the causative agent of toxoplasmosis. Protein palmitoylation is known to play roles in signal transduction and in enhancing the hydrophobicity of proteins thus contributing to their membrane association. Global inhibition of protein palmitoylation has been shown to affect T. gondii physiology and invasion of the host cell. However, the proteins affected by this modification have been understudied. This paper shows that the small heat shock protein 20 from T. gondii (TgHSP20) is synthesized as a mature protein in the cytosol and is palmitoylated in three cysteine residues. However, its localization at the inner membrane complex (IMC) is dependent only on N-terminal palmitoylation. Absence or incomplete N-terminal palmitoylation causes TgHSP20 to partially accumulate in a membranous structure. Interestingly, TgHSP20 palmitoylation is not responsible for its interaction with the daughter cells IMCs. Together, our data describe the importance of palmitoylation in protein targeting to the IMC in T. gondii.
Asunto(s)
Fibroblastos/metabolismo , Proteínas del Choque Térmico HSP20/metabolismo , Membranas Intracelulares/metabolismo , Lipoilación , Toxoplasma/metabolismo , Toxoplasmosis/parasitología , Western Blotting , Células Cultivadas , Cisteína/química , Cisteína/genética , Cisteína/metabolismo , Citosol/metabolismo , Fibroblastos/citología , Técnica del Anticuerpo Fluorescente , Proteínas del Choque Térmico HSP20/genética , Humanos , Inmunoprecipitación , Mutación/genética , Transporte de Proteínas , Transducción de Señal , Toxoplasma/crecimiento & desarrollo , Toxoplasmosis/metabolismoRESUMEN
Toxoplasma gondii Hsp20 is a pellicle-associated functional chaperone whose biological role is still unknown. Hsp20 is present in different apicomplexan parasites, showing a high degree of conservation across the phylum, with Neospora caninum Hsp20 presenting an 82% identity to that of T. gondii. Hence rabbit anti-T. gondii Hsp20 serum was able to recognize the N. caninum counterpart. Interestingly, both N. caninum and T. gondii Hsp20 localized to the inner membrane complex and to the plasma membrane. Incubation of T. gondii and N. caninum tachyzoites with an anti-TgHsp20 serum reduced parasite invasion at rates of 57.23% and 54.7%, respectively. This anti-serum also reduced T. gondii gliding 48.7%. Together, all this data support a role for Hsp20 in parasite invasion and gliding motility.
Asunto(s)
Anticuerpos Antiprotozoarios/inmunología , Proteínas del Choque Térmico HSP20/inmunología , Neospora/fisiología , Toxoplasma/fisiología , Secuencia de Aminoácidos , Animales , Anticuerpos Antiprotozoarios/sangre , Células Cultivadas , Coccidiosis/parasitología , Proteínas del Choque Térmico HSP20/química , Humanos , Datos de Secuencia Molecular , Movimiento , Neospora/clasificación , Neospora/genética , Neospora/inmunología , Conejos , Alineación de Secuencia , Toxoplasma/clasificación , Toxoplasma/genética , Toxoplasma/inmunología , Toxoplasmosis/parasitologíaRESUMEN
Recombinant Toxoplasma gondii small heat shock protein HSP20, surface antigen SAG1 and dense granule GRA7 were analyzed by IgG-ELISA with serum samples of Toxoplasma infected humans grouped as I (IgG+, IgM+), II (IgG+, IgM-) and III (IgG-, IgM-). rHSP20 reacted against 80% and 62.5% of serum samples from groups I and II, respectively. rSAG1 was recognized by 85% of the samples from group I and 70.8% from group II, whereas rGRA7 was recognized by 85% and 66.6% of the serum samples from groups I and II, respectively. When a combination of two or three recombinant antigens was used, the sensitivity values improved to 85-95% for group I and 87.5-91.7% for group II. All combinations tested produced similar reactivity profiles. None of the recombinant proteins reacted against group III serum samples. In conclusion, we demonstrated that T. gondii HSP20 elicits an important B-cell response during human infection, and could be suitable for the development of serodiagnosis tools.
Asunto(s)
Proteínas del Choque Térmico HSP20 , Inmunoglobulina G/sangre , Complicaciones Parasitarias del Embarazo/diagnóstico , Proteínas Recombinantes , Toxoplasma/inmunología , Toxoplasmosis/diagnóstico , Anticuerpos Antiprotozoarios/sangre , Antígenos de Protozoos/genética , Antígenos de Protozoos/inmunología , Linfocitos B/inmunología , Ensayo de Inmunoadsorción Enzimática/métodos , Femenino , Regulación de la Expresión Génica , Proteínas del Choque Térmico HSP20/genética , Proteínas del Choque Térmico HSP20/inmunología , Humanos , Inmunoglobulina M/sangre , Embarazo , Complicaciones Parasitarias del Embarazo/inmunología , Proteínas Protozoarias/genética , Proteínas Protozoarias/inmunología , Proteínas Recombinantes/genética , Proteínas Recombinantes/inmunología , Sensibilidad y Especificidad , Toxoplasma/química , Toxoplasmosis/inmunologíaRESUMEN
Protozoa of the genus Leishmania are causative agents of leishmaniasis, an important health problem in both human and veterinary medicine. Here, we describe a new heat shock protein (HSP) in Leishmania, belonging to the small HSP (sHSP) family in kinetoplastids. The protein is highly conserved in different Leishmania species, showing instead significant divergence with sHSP's from other organisms. The humoral response elicited against this protein during Leishmania infection has been investigated in natural infected humans and dogs, and in experimentally infected hamsters. Leishmania HSP20 is a prominent antigen for canine hosts; on the contrary, the protein seems to be a poor antigen for human immune system. Time-course analysis of appearance of anti-HSP20 antibodies in golden hamsters indicated that these antibodies are produced at late stages of the infection, when clinical symptoms of disease are patent. Finally, the protective efficacy of HSP20 was assessed in mice using a DNA vaccine approach prior to challenge with Leishmania amazonensis.
Asunto(s)
Antígenos de Protozoos/administración & dosificación , Proteínas del Choque Térmico HSP20/administración & dosificación , Proteínas del Choque Térmico HSP20/inmunología , Leishmania/inmunología , Leishmaniasis/inmunología , Leishmaniasis/prevención & control , Vacunas de ADN/administración & dosificación , Animales , Antígenos de Protozoos/inmunología , Modelos Animales de Enfermedad , Ratones , Ratones Endogámicos BALB C , Resultado del Tratamiento , Vacunas de ADN/inmunologíaRESUMEN
BACKGROUND INFORMATION: Toxoplasma gondii is among the most successful parasites, with nearly half of the human population chronically infected. T. gondii has five sHsps [small Hsps (heat-shock proteins)] located in different subcellular compartments. Among them, Hsp20 showed to be localized at the periphery of the parasite body. sHsps are widespread, constituting the most poorly conserved family of molecular chaperones. The presence of sHsps in membrane structures is unusual. RESULTS: The localization of Hsp20 was further analysed using high-resolution fluorescent light microscopy as well as electron microscopy, which revealed that Hsp20 is associated with the outer surface of the IMC (inner membrane complex), in a set of discontinuous stripes following the same spiralling trajectories as the subpellicular microtubules. The detergent extraction profile of Hsp20 was similar to that of GAP45 [45 kDa GAP (gliding-associated protein)], a glideosome protein associated with the IMC, but was different from that of IMC1 protein. Although we were unable to detect interacting protein partners of Hsp20 either in normal or stressed tachyzoites, an interaction of Hsp20 with phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate phospholipids could be observed. CONCLUSIONS: Hsp20 was shown to be associated with a specialized membranous structure of the parasite, the IMC. This discontinuous striped-arrangement is unique in T. gondii, indicating that the topology of the outer leaflet of the IMC is not homogeneous.
Asunto(s)
Estructuras Celulares/metabolismo , Proteínas del Choque Térmico HSP20/metabolismo , Membranas/metabolismo , Chaperonas Moleculares/metabolismo , Proteínas Protozoarias/metabolismo , Animales , Estructuras Celulares/química , Estructuras Celulares/inmunología , Estructuras Celulares/ultraestructura , Electroporación , Técnica del Anticuerpo Fluorescente , Proteínas del Choque Térmico HSP20/química , Proteínas del Choque Térmico HSP20/genética , Proteínas del Choque Térmico HSP20/inmunología , Proteínas del Choque Térmico HSP20/aislamiento & purificación , Proteínas del Choque Térmico HSP20/ultraestructura , Membranas/química , Membranas/inmunología , Membranas/ultraestructura , Chaperonas Moleculares/química , Chaperonas Moleculares/genética , Chaperonas Moleculares/inmunología , Chaperonas Moleculares/aislamiento & purificación , Chaperonas Moleculares/ultraestructura , Fosfolípidos/metabolismo , Proteínas Protozoarias/química , Proteínas Protozoarias/inmunología , Proteínas Protozoarias/aislamiento & purificación , Proteínas Protozoarias/ultraestructura , Toxoplasma/citología , Toxoplasma/genética , Toxoplasma/metabolismoRESUMEN
Protozoa of the genus Leishmania are causative agents of leishmaniasis, an important health problem in both human and veterinary medicine. Here, we describe a new heat shock protein (HSP) in Leishmania, belonging to the small HSP (sHSP) family in kinetoplastids. The protein is highly conserved in different Leishmania species, showing instead significant divergence with sHSP's from other organisms. The humoral response elicited against this protein during Leishmania infection has been investigated in natural infected humans and dogs, and in experimentally infected hamsters. Leishmania HSP20 is a prominent antigen for canine hosts; on the contrary, the protein seems to be a poor antigen for human immune system. Time-course analysis of appearance of anti-HSP20 antibodies in golden hamsters indicated that these antibodies are produced at late stages of the infection, when clinical symptoms of disease are patent. Finally, the protective efficacy of HSP20 was assessed in mice using a DNA vaccine approach prior to challenge with Leishmania amazonensis(AU)
Protozoos del género Leishmania son agentes causantes de la leishmaniasis, que es un importante problema de salud tanto en medicina humana y veterinaria. Aquí se describe una nueva proteína de choque térmico (HSP) de Leishmania, que pertenecen a la pequeña HSP (sHSP) en kinetoplastids familia. La proteína está altamente conservada en diferentes especies de Leishmania, mostrando importantes diferencias en lugar sHSP con la de otros organismos. La respuesta humoral contra esta proteína suscitado durante la infección por Leishmania se ha investigado en seres humanos infectados naturales y perros, y en hámsters infectados experimentalmente. Leishmania HSP20 es un antígeno para canina anfitriones, por el contrario, la proteína parece ser un pobre de antígenos para el sistema inmunitario humano. Curso de análisis de tiempo de aparición de anticuerpos anti-HSP20 en hamsters dorados indicó que estos anticuerpos se producen en etapas tardías de la infección, cuando los síntomas clínicos de la enfermedad son patentes. Por último, la eficacia protectora de HSP20 se evaluó en ratones utilizando una vacuna de ADN antes de enfoque desafío con Leishmania amazonensis
Asunto(s)
Animales , Ratones , Antígenos de Protozoos/administración & dosificación , Modelos Animales de Enfermedad , Proteínas del Choque Térmico HSP20/administración & dosificación , Leishmania/inmunología , Leishmaniasis/inmunología , Leishmaniasis/prevención & control , Modelos Moleculares , ADN Protozoario/administración & dosificaciónRESUMEN
The results of this study describe the identification and characterization of the Toxoplasma gondii alpha-crystallin/small heat shock protein (sHsp) family. By database (www.toxodb.org) search, five parasite sHsps (Hsp20, Hsp21, Hsp28, Hsp29, and the previously characterized Hsp30/Bag1) were identified. As expected, they share the homologous alpha-crystallin domain, which is the key characteristic of sHsps. However, the N-terminal segment of each protein contains unique characteristics in size and sequence. Most T. gondii sHsps are constitutively expressed in tachyzoites and fully differentiated bradyzoites, with the exception of Hsp30/Bag1. Interestingly, by subcellular localization we observed that T. gondii sHsps are located in different compartments. Hsp20 is located at the apical end of the cell, Hsp28 is located inside the mitochondrion, Hsp29 showed a membrane-associated labeling, and Hsp21 appeared throughout the cytosol of the parasites. These particular differences in the immunostaining patterns suggest that their targets and functions might be different.