Giardipain-1, a protease secreted by Giardia duodenalis trophozoites, causes junctional, barrier and apoptotic damage in epithelial cell monolayers.

The adhesion of Giardia duodenalis trophozoites to intestinal epithelial cells allows the onset and maintenance of giardiasis. During these interactions, epithelial cells can be committed to apoptosis by enzymes secreted by the parasites, including cysteine proteases that are increasingly identified as virulence factors in parasitic protozoa. In this work, a monoclonal antibody (mAb1G3) raised against G. duodenalis surface components was found to react with a 25 kDa protein expressed in the cell surface and flagella of G. duodenalis trophozoites. When trophozoites expressing this protein were cultured with IEC-6 intestinal epithelial cell monolayers, a dynamic release of this protein was observed with mAbIG3. Proteomic analysis identified the protein as a mature cathepsin B-like (gCatB) enzyme, whose proteolytic activity, detected in zymograms, was eliminated by CatB inhibitor E-64. This protein was named giardipain-1 due to its functional papain-like features and was purified by affinity chromatography using mAbIG3. Upon exposure to the purified, mature and secreted forms of giardipain-1, IEC-6 epithelial cell monolayers displayed membrane blebbing and phosphatidylserine exposure on the outer cell surface, indicating an apoptotic process. In Madin Darby Canine Kidney (MDCK) cell monolayers, giardipain-1 leads to the appearance of pore-like regions and of gaps along cell-cell junctions, to decreased transepithelial electrical resistance (TER), caspase-3 activation and poly-ADP-ribose polymerase (PARP) fragmentation. At early times during exposure, giardipain-1 co-localized at cell-cell junctions, associated with occludin and induced the delocalization and degradation of tight junction proteins occludin and claudin-1. The damage caused to epithelial monolayers by giardipain-1 was blocked by pre-incubation with the CatB B Inhibitor E-64. Furthermore, silencing the giardipain-1 gene in trophozoites lowered the proteolytic activity of giardipain-1 and reduced the damage in IEC-6 monolayers. The damage observed appears to be specific to giardipain activity since almost no damage was observed when IEC-6 monolayers were incubated with papain, a non-related cysteine protease. Hence this study suggests that giardipain-1 triggers, in epithelial cells, degradation of cell-cell junctional components and apoptotic damage, supporting the notion of giardiapain-1 as a virulence factor of Giardia.

Expression and secretion of the Giardia duodenalis variant surface protein 9B10A by transfected trophozoites causes damage to epithelial cell monolayers mediated by protease activity.

Giardia duodenalis is the protozoan parasite responsible for most cases of parasitic diarrhea worldwide. The pathogenic mechanisms of giardiasis have not yet been fully characterized. In this context parasite's excretory/secretory products have been related to the damage induced by the parasite on enterocytes. Among these is the Variable Surface Proteins (VSPs) family involved in antigenic variation and in the induction of protective response. In proteomic analyses carried out to identify the proteases with high molecular weight secreted by Giardia trophozoites during the initial phase of interaction with IEC-6 cell monolayers we identified the VSP9B10A protein. In silico bioinformatics analyses predicted a central region in residues 324-684 displaying the catalytic triad and the substrate binding pocket of cysteine proteases. The analysis of the effect of the VSP9B10A protein on epithelial cell monolayers using trophozoites that were transfected with a plasmid carrying the vsp9b10a gene sequence under the control of a constitutive promoter showed that transfected trophozoites expressing the VSP9B10A protein caused cytotoxic damages on IEC-6 and MDCK cell monolayers. This was characterized by loss of cell-cell contacts and cell detachment from the substrate while no damage was observed with trophozoites that did not express the VSP9B10A protein. The same cytotoxic effect was detected when IEC-6 cell monolayers were incubated only with supernatants from co-cultures of IEC-6 cell monolayers with VSP9B10A transfected trophozoites and this effect was not observed when transfected trophozoites were incubated with a monospecific polyclonal antibody anti-VSP9B10A previous to interaction with IEC-6 monolayers. These results demonstrate that the VSP9B10A protein secreted upon interaction with epithelial cells caused damage in these cells. Thus this protein might be considered as a conditional virulence factor candidate. To our knowledge this is the first report on the proteolytic activity from a Giardia VSP opening new research lines on these proteins.

Albendazole induces oxidative stress and DNA damage in the parasitic protozoan Giardia duodenalis.

The control of Giardia duodenalis infections is carried out mainly by drugs, among these albendazole (ABZ) is commonly used. Although the cytotoxic effect of ABZ usually involves binding to ß-tubulin, it has been suggested that oxidative stress may also play a role in its parasiticidal mechanism. In this work the effect of ABZ in Giardia clones that are susceptible or resistant to different concentrations (1.35, 8, and 250 µM) of this drug was analyzed. Reactive oxygen species (ROS) were induced by ABZ in susceptible clones and this was associated with a decrease in growth that was alleviated by cysteine supplementation. Remarkably, ABZ-resistant clones exhibited partial cross-resistance to H2O2, whereas a Giardia H2O2-resistant strain can grow in the presence of ABZ. Lipid oxidation and protein carbonylation in ABZ-treated parasites did not show significant differences as compared to untreated parasites; however, ABZ induced the formation of 8OHdG adducts and DNA degradation, indicating nucleic acid oxidative damage. This was supported by observations of histone H2AX phosphorylation in ABZ-susceptible trophozoites treated with 250 µM ABZ. Flow cytometry analysis showed that ABZ partially arrested cell cycle in drug-susceptible clones at G2/M phase at the expense of cells in G1 phase. Also, ABZ treatment resulted in phosphatidylserine exposure on the parasite surface, an event related to apoptosis. All together these data suggest that ROS induced by ABZ affect Giardia genetic material through oxidative stress mechanisms and subsequent induction of apoptotic-like events.

Giardia fatty acyl-CoA synthetases as potential drug targets.

Giardiasis caused by Giardia intestinalis (syn. G. lamblia, G. duodenalis) is one of the leading causes of diarrheal parasitic diseases worldwide. Although limited drugs to treat giardiasis are available, there are concerns regarding toxicity in some patients and the emerging drug resistance. By data-mining genome sequences, we observed that G. intestinalis is incapable of synthesizing fatty acids (FA) de novo. However, this parasite has five long-chain fatty acyl-CoA synthetases (GiACS1 to GiACS5) to activate FA scavenged from the host. ACS is an essential enzyme because FA need to be activated to form acyl-CoA thioesters before they can enter subsequent metabolism. In the present study, we performed experiments to explore whether some GiACS enzymes could serve as drug targets in Giardia. Based on the high-throughput datasets and protein modeling analyses, we initially studied the GiACS1 and GiACS2, because genes encoding these two enzymes were found to be more consistently expressed in varied parasite life cycle stages and when interacting with host cells based on previously reported transcriptome data. These two proteins were cloned and expressed as recombinant proteins. Biochemical analysis revealed that both had apparent substrate preference toward palmitic acid (C16:0) and myristic acid (C14:0), and allosteric or Michaelis-Menten kinetics on palmitic acid or ATP. The ACS inhibitor triacsin C inhibited the activity of both enzymes (IC50 = 1.56 µM, K i = 0.18 µM for GiACS1, and IC50 = 2.28 µM, K i = 0.23 µM for GiACS2, respectively) and the growth of G. intestinalis in vitro (IC50 = 0.8 µM). As expected from giardial evolutionary characteristics, both GiACSs displayed differences in overall folding structure as compared with their human counterparts. These observations support the notion that some of the GiACS enzymes may be explored as drug targets in this parasite.

An antioxidant response is involved in resistance of Giardia duodenalis to albendazole.

Albendazole (ABZ) is a therapeutic benzimidazole used to treat giardiasis that targets ß-tubulin. However, the molecular bases of ABZ resistance in Giardia duodenalis are not understood because ß-tubulin in ABZ-resistant clones lacks mutations explaining drug resistance. In previous work we compared ABZ-resistant (1.35, 8, and 250 µM) and ABZ-susceptible clones by proteomic analysis and eight proteins involved in energy metabolism, cytoskeleton dynamics, and antioxidant response were found as differentially expressed among the clones. Since ABZ is converted into sulphoxide (ABZ-SO) and sulphone (ABZ-SOO) metabolites we measured the levels of these metabolites, the antioxidant enzymes and free thiols in the susceptible and resistant clones. Production of reactive oxygen species (ROS) and levels of ABZ-SO/ABZ-SOO induced by ABZ were determined by fluorescein diacetate-based fluorescence and liquid chromatography respectively. The mRNA and protein levels of antioxidant enzymes (NADH oxidase, peroxiredoxin 1a, superoxide dismutase and flavodiiron protein) in these clones were determined by RT-PCR and proteomic analysis. The intracellular sulfhydryl (R-SH) pool was quantified using dinitrobenzoic acid. The results showed that ABZ induced ROS accumulation in the ABZ-susceptible Giardia cultures but not in the resistant ones whilst the accumulation of ABZ-SO and ABZ-SOO was lower in all ABZ-resistant cultures. Consistent with these findings, all the antioxidant enzymes detected and analyzed were upregulated in ABZ-resistant clones. Likewise the R-SH pool increased concomitantly to the degree of ABZ-resistance. These results indicate an association between accumulation of ABZ metabolites and a pro-oxidant effect of ABZ in Giardia-susceptible clones. Furthermore the antioxidant response involving ROS-metabolizing enzymes and intracellular free thiols in ABZ-resistant parasites suggest that this response may contribute to overcome the pro-oxidant cytotoxicity of ABZ.

Proteomic and transcriptional analyses of genes differentially expressed in Giardia duodenalis clones resistant to albendazole.

In this study we performed proteomic and transcriptional analyses to identify and characterize genes differentially expressed in Giardia duodenalis clones resistant to albendazole. The expression of proteins and their corresponding mRNAs was analyzed in clones resistant in vitro to different concentrations of albendazole (1.35, 8.0 and 250 µM) and these were compared with albendazole-sensitive clones using two approaches: (1) two-dimensional protein electrophoresis to analyze the proteome by the LC-MS/MS technique, and (2) semi-quantitative RT-PCR to assess the mRNA levels of proteins with the highest levels of differential expression .This strategy allowed the identification of eight proteins differentially expressed in albendazole resistant clones with roles in: (a) the cytoskeletal system (alpha 2-giardin and RanBP1), (b) the antioxidant metabolism (NADH oxidase) and (c) energy metabolism (triosephosphate isomerase, phosphoglycerate kinase and ornithine carbamoyltransferase). Gene expression analyses of these genes correlated well with the proteomics results. These observations suggest that resistance to albendazole in Giardia encompasses a complex response involving an altered expression of genes regulated at the transcriptional level that might have an important role in maintaining cell structural stability, coping with oxidative stress and adapting energy supply to a new metabolic status. These molecules are indeed promising targets for drug development.

A helminth cestode parasite express an estrogen-binding protein resembling a classic nuclear estrogen receptor.

The role of an estrogen-binding protein similar to a known mammalian estrogen receptor (ER) is described in the estradiol-dependent reproduction of the helminth parasite Taenia crassiceps. Previous results have shown that 17-ß-estradiol induces a concentration-dependent increase in bud number of in vitro cultured cysticerci. This effect is inhibited when parasites are also incubated in the presence of an ER binding-inhibitor (tamoxifen). RT-PCR assays using specific oligonucleotides of the most conserved ER sequences, showed expression by the parasite of a mRNA band of molecular weight and sequence corresponding to an ER. Western blot assays revealed reactivity with a 66 kDa protein corresponding to the parasite ER protein. Tamoxifen treatment strongly reduced the production of the T. crassiceps ER-like protein. Antibody specificity was demonstrated by immunoprecipitating the total parasite protein extract with anti-ER-antibodies. Cross-contamination by host cells was discarded by flow cytometry analysis. ER was specifically detected on cells expressing paramyosin, a specific helminth cell marker. Parasite cells expressing the ER-like protein were located by confocal microscopy in the subtegumental tissue exclusively. Analysis of the ER-like protein by bidimensional electrophoresis and immunoblot identified a specific protein of molecular weight and isoelectric point similar to a vertebrates ER. Sequencing of the spot produced a small fragment of protein similar to the mammalian nuclear ER. Together these results show that T. crassiceps expresses an ER-like protein which activates the budding of T. crassiceps cysticerci in vitro. To the best of our knowledge, this is the first report of an ER-like protein in parasites. This finding may have strong implications in the fields of host-parasite co-evolution as well as in sex-associated susceptibility to this infection, and could be an important target for the design of new drugs.

Hypochlorous acid scavenging activities of thioallyl compounds from garlic.

The hypochlorous acid (HOCl) scavenging capacities of 10 garlic compounds containing modifications in the thioallyl group (-S-CH2CH ═ CH2) were determined by a catalase protection assay, and the corresponding structure-activity relationships using molecular descriptors were calculated. This scavenging activity was enhanced by increasing the number of S atoms or by the alanyl group (-CH2CH-NH2-COOH) and decreased in the absence of the C ═ C bond or in the presence of a sulfoxide group in the thioallyl group. Interestingly, S-allylcysteine and its corresponding sulfoxide (alliin) showed the highest and lowest HOCl-scavenging capacities, respectively. Quantitative modeling by multiple regression analysis and partial least-squares projections showed that the topological descriptor polar surface area and two electronic properties, namely, highest occupied molecular orbital and total energy, contributed mainly to variations in the HOCl scavenging activity of thioallyl compounds. These observations provide new insights on the antioxidant mechanism of garlic derivatives in processes involving HOCl production.

A new MAP kinase protein involved in estradiol-stimulated reproduction of the helminth parasite Taenia crassiceps.

MAP kinases (MAPK) are involved in the regulation of cellular processes such as reproduction and growth. In parasites, the role of MAPK has been scarcely studied. Here, we describe the participation of an ERK-like protein in estrogen-dependent reproduction of the helminth parasite Taenia crassiceps. Our results show that 17beta-estradiol induces a concentration-dependent increase in the bud number of in vitro cultured cysticerci. If parasites are also incubated in presence of an ERK-inhibitor, the stimulatory effect of estrogen is blocked. The expression of ERK-like mRNA and its corresponding protein was detected in the parasite. The ERK-like protein was over-expressed by all treatments. Nevertheless, a strong induction of phosphorylation of this protein was observed only in response to 17beta-estradiol. Cross-contamination by host cells was discarded by flow cytometry analysis. Parasite cells expressing the ERK-like protein were exclusively located at the subtegument tissue by confocal microscopy. Finally, the ERK-like protein was separated by bidimensional electrophoresis and then sequenced, showing the conserved TEY activation motif, typical of all known ERK 1/2 proteins. Our results show that an ERK-like protein is involved in the molecular signalling during the interaction between the host and T. crassiceps, and may be considered as target for anti-helminth drugs design.

New tools provide further insights into Giardia and Cryptosporidium biology.

The ubiquity and importance of Giardia and Cryptosporidium as pathogens are reflected in the increasing number of publications concerning these organisms, but they are not the only reason why researchers are increasingly turning their attention to studying Giardia and Cryptosporidium. As new tools and databases become available, it is now possible to investigate fundamental issues related to their biology and relationship with their hosts. In this article, we highlight recent advances in research and outline questions arising that need to be addressed as a way of focusing the attention of the research and health communities and encouraging further dialogue and collaboration.

On the spread of the novel influenza A (H1N1) virus in Mexico.

A novel influenza A H1N1 virus of swine origin is responsible for the influenza epidemic affecting Mexico, the United States of America (USA), and 39 other countries. While the origin of this emerging pathogen remains uncertain, an increase in the reported incidence of respiratory diseases was noted during March 2009 at the town of La Gloria, in the southeastern state of Veracruz, Mexico. So far, this is the first community in which a case of novel influenza A H1N1 virus has been identified. Further cases were rapidly detected in other areas of Mexico and elsewhere. Initially, the atypical respiratory disease outbreak caused great uncertainty posing a challenge to the Mexican health system. Control measures such as social distancing, timely medical care, and personal hygiene have so far proven effective in containing the outbreak, resulting in a decline of the number of new cases. To the best of our knowledge, it appears that the virus might not be as virulent or contagious as previously thought. Here we provide a description of the influenza epidemic spread in Mexico. As the virus disseminates worldwide, there is concern about the possibility of a new reassortment resulting in a more pathogenic strain that will pose a threat for every country. The influenza epidemic provided lessons that underscore the importance of epidemiologic surveillance and preparedness. Further investigation to address questions about this new virus and conditions for its spread is warranted.

In vitro resistance to 5-nitroimidazoles and benzimidazoles in Giardia duodenalis: variability and variation in gene expression.

The susceptibility of Giardia duodenalis trophozoites exposed in vitro to sublethal concentrations of metronidazole (MTZ) and albendazole (ABZ) may exhibit inter-culture (variability) and intra-culture (variation) differences in drug susceptibility. It was previously reported that MTZ-resistant trophozoites may display changes in pyruvate:ferredoxin oxidoreductase (PFOR) expression while changes at the beta-tubulin molecule are apparently absent in ABZ-resistant cultures. To assess the levels of gene expression of these molecules, we obtained cloned cultures growing at concentrations up to 23 microM MTZ (WBRM23) and up to 8muM ABZ (WBRA8) and gene sequence and expression of pfor and beta-tubulin loci were compared with these of drug-susceptible clone WB1. Neither the pfor nor the beta-tubulin genes showed changes at sequence level but the MTZ-resistant clones WBRM21 and WBRM23 showed up-regulation of the pfor RNA using the gdh gene as reference. By using WB1 and WBRA8 clones in representational difference analyses of gene expression (RDA) an insert referred to as ARR-VSP was selected and sequenced. It showed the highest homology to one VSP molecule in the Giardia Genome Database (orf GL50803_101765). This isogene was up-regulated in five ABZ-resistant clones and the clone WBRA8 exhibited the highest RNA expression level. When successive progenies of clones WB1, WBRM23 and WBRA8 were analyzed in Northern blot assays to detect pfor and ARR-VSP RNAs respectively, the expression patterns showed variation for both genes but it was much lower in the clone WBRA8. These results suggest that G. duodenalis cultures either susceptible or resistant to MTZ and ABZ may display variability and variation at RNA expression levels albeit these were more marked in the MTZ-resistant parasites. These data might have further implications defining major mechanisms involved in drug resistance of Giardia.

Ultrastructural study of the encystation and excystation processes in Naegleria sp.

An important aspect of the biology of Naegleria sp. is the differentiation processes that occur during encystation and excystation. We studied these using both fluorescence and transmission electron microscopy techniques. In the initial stages of encystation, the cisternae of the endoplasmic reticulum became densely filled with a fibrillar material. Vesicles with a similar content that appeared to be derived from the cisternae were also observed in close contact with the plasma membrane. As encystation progressed, the fibrillar material became localized on the surface of the amoeba. An irregular compaction was observed in some areas of the cyst wall, which contained thin extensions of the cyst wall fibrillar material. Completely formed cysts had two to three ostioles, each sealed by an operculum. The operculum contained two areas in which a differential compaction of the fibrillar structure was observed. When excystation was induced, small dense granules (DGs), which were in close contact with fibrillar material were observed in the cyst cytoplasm and in the peritrophic space. During excystation, the more compact component of the operculum moves to enable the pseudopod of the emerging trophozoite to penetrate the ostiole. Vacuoles containing a fibrillar material, probably derived from the cyst wall, were observed in the cytoplasm of the pseudopodia. Our results provide a platform for further studies using biochemical markers to investigate the origin of the cyst wall as well as the role of DGs during excystation in Naegleria.

Seroepidemiology of giardiasis in Mexico.

Prevalence of antibodies against Giardia duodenalis was determined by enzyme-linked immunosorbent assay in serum samples from a national serologic survey of Mexico that included all geographic areas and socioeconomic and demographic data for each person sampled. The country was divided into four regions on the basis of development (high, medium high, medium low, and low). Of 3,461 serum samples tested, 1,914 (55.3%) were positive for IgG antibodies against Giardia duodenalis. Seropositivity was age-specific; the probability of seropositivity increased 4.9-fold (95% confidence interval [CI] = 3.16-7.64) in adolescents 10-19 years of age, 8.0-fold (95% CI = 5.19-12.53) in young adults 20-39 years of age, and 12.6-fold (95% CI = 7.93-20.28) in adults more than 40 years of age. Giardia duodenalis seropositivity was associated with male sex (odds ratio = 1.40, 95% CI = 1.22-1.61). No association was found between seropositivity and socioeconomic variables or regional development status.

Identification of nucleoli in the early branching protist Giardia duodenalis.

Giardia duodenalis has been described as 'anucleolated'. In this work we analysed the subcellular distribution of several nucleolar markers in Giardia nuclei using silver and immunostaining techniques for electron and confocal laser microscopy as well as expression of epitope-tagged proteins in transgenic trophozoites. We identified anteronuclear fibrogranular structures corresponding to nucleolar organising regions with recruited ribonucleoprotein complexes, rRNA and epitope-tagged fibrillarin and rRNA-pseudouridine synthase (CBF5). Recombinant fibrillarin and CBF5 were targeted to this subcompartment. This study demonstrates the presence of nucleoli in G. duodenalis and provides a model to analyse minimal requirements for nucleolar assembly and maintenance in eukaryotic cells.

Giardia duodenalis: adhesion-deficient clones have reduced ability to establish infection in Mongolian gerbils.

The role of Giardia duodenalis surface molecules in the attachment of trophozoites to epithelial cells has been established through the dual strategies of characterizing G. duodenalis clones with deficient adhesion and blocking experiments with surface-specific monoclonal antibodies. Also, the infectivity of the analyzed clones was tested using Mongolian gerbils as experimental model. Two adhesion-deficient G. duodenalis clones, C6 and C7, were isolated from the wild type C5 clone which in turn was obtained from the WB strain. The adhesion efficiencies of C6 and C7 clones (48.2+/-4.9 and 32.6+/-2.4, respectively) were significantly lower as compared with WB strain or C5 clone (82.8+/-6.4 and 79.9+/-7.9). Analysis of radiolabel surface proteins by 1D and 2D SDS-PAGE revealed prominently labelled 28 and 88 kDa components in C6 and C7 clones and a major 200 kDa protein in the C5 clone and the WB strain. The 88 and 200 kDa components are acidic proteins by two-dimensional electrophoretic analyses. The most striking difference between wild-type and adhesion-deficient Giardia trophozoites was the reduced expression of a 200 kDa surface protein in the latter. Significantly, a mAb (IG3) specific for the 200 kDa protein that reacted with more than 99% of WB and C5 trophozoites and less than 1% of C6 and C7 trophozoites as determined by indirect immunofluorescence inhibited the adhesion of trophozoites from WB and C5 clone to Madin Darby Canine Kidney cells by 52% and 40.9%, respectively, suggesting a participation of this antigen in adherence. Finally, the functional relevance of trophozoite adhesion to epithelial cells was indicated by the reduced capacity of the adhesion-deficient clones to establish the infection in Mongolian gerbils.

Identification, molecular characterisation and differential expression of caveolin-1 in Trichinella spiralis maturing oocytes and embryos.

Caveolins are integral membrane proteins implicated in cholesterol homeostasis and transport, endocytosis mechanisms and regulation of signal transduction in differentiated cells. In this work a caveolin-1 gene from the nematode Trichinella spiralis (Ts-cav-1) was cloned and identified as an adult-specific antigen. For this, a cDNA library of T. spiralis 3-day-old adult worms was screened using a stage-specific cDNA-labelled probe. One positive clone contained a cDNA insert of 1427-bp and a full-length open reading frame (ORF) of 687-bp, which encodes for a 229 amino acid polypeptide with a theoretical molecular weight of 26kDa. BLAST and FASTA searches revealed a 36% and 57% identity with Caenorhabditis elegans caveolin-1, respectively. Confocal laser microscopy analysis using antibodies generated against Ts-CAV-1 protein and cross-sections of adult parasites showed that Ts-CAV-1 gradually accumulates on the surface of Trichinella oocytes and embryos, reaching a maximum at 3days p.i., and decreasing during new-born larvae (NBL) development. RT-PCR assays of parasites from 1 to 4days p.i. showed a similar gene expression profile to that observed for Ts-CAV-1 which suggests a specific developmental regulation. Free cholesterol was mainly distributed in the female germ line and it displayed increasing membrane accumulation, similar to the pattern obtained for Ts-CAV-1 protein, which suggests a temporal membrane association with Ts-CAV-1 that in turn will perform the functions mentioned above. Our results strongly indicate that Ts-cav-1 from T. spiralis plays a role in oocyte maturation and embryogenesis during development, demonstrating gender-specific expression.

Protein kinase C isoforms from Giardia duodenalis: identification and functional characterization of a beta-like molecule during encystment.

Protein kinase C (PKC) is a family of serine/threonine kinases that regulate many different cellular processes such as cell growth and differentiation in eukaryotic cells. Using specific polyclonal antibodies raised against mammalian PKC isoforms, it was demonstrated here for the first time that Giardia duodenalis expresses several PKC isoforms (beta, delta, epsilon, theta and zeta). All PKC isoforms detected showed changes in their expression pattern during encystment induction. In addition, selective PKC inhibitors blocked the encystment in a dose-dependent manner, suggesting that PKC isozymes may play important roles during this differentiation process. We have characterized here the only conventional-type PKC member found so far in Giardia, which showed an increased expression and changes in its intracellular localization pattern during cyst formation. The purified protein obtained by chromatography on DEAE-cellulose followed by size-exclusion chromatography, displayed in vitro kinase activity using histone HI-IIIS as substrate, which was dependent on cofactors required by conventional PKCs, i.e., phospholipids and calcium. An open reading frame in the Giardia Genome Database that encodes a homolog of PKCbeta catalytic domain was identified and cloned. The expressed recombinant protein was also recognized by a mammalian anti-PKCbeta antibody and was referred as giardial PKCbeta on the basis of all these experimental evidence.