Raúl Argüello-García

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.

Cyst and encystment in protozoan parasites: optimal targets for new life-cycle interrupting strategies?

Certain protozoan parasites use survival strategies to reside outside the host such as the formation of cysts. This dormant and resistant stage results from the complex process of encystment that involves diverse molecular and cellular modifications. The stimuli and changes associated with cyst biogenesis are a matter of ongoing studies in human and animal protozoan parasites such as amoeba and Giardia species because blocking every step in the encystment pathway should, in theory, interrupt their life cycles. The present review thoroughly examines this essential process in those protozoan parasites and discusses the possibility of using that information to develop new kinds of anti-parasite specific and life cycle-interrupting drugs, aimed at holding back the dissemination of these infections.

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.

Relationship of spermatoscopy, prostatic acid phosphatase activity and prostate-specific antigen (p30) assays with further DNA typing in forensic samples from rape cases

In the forensic laboratory the biological analyses for rape investigation commonly include vaginal swabs as sample material combined to biochemical tests including sperm cytology (SC) and detection of acid phosphatase activity (AP) and prostate-specific antigen (PSA, p30) for the conclusive identification of semen components. Most reports comparing these tests relied on analysis of semen samples or donor swabs taken under controlled conditions; however their individual or combined efficacy under real live sampling conditions in different laboratories is largely unknown. We carried out SC, APA and PSA analyses in vaginal swabs collected from casework rapes submitted to Mexican Forensic Laboratories at Texcoco and Toluca. On the basis of positive and negative results from each assay and sample, data were classified into eight categories (I-VIII) and compared with those obtained in the two only similar studies reported in Toronto, Canada and Hong Kong, China. SC and APA assays had the higher overall positivity in Toluca and Texcoco samples respectively and otherwise PSA had a lower but very similar positivity between these two laboratories. When compared to the previous studies some similarities were found, namely similar frequencies (at a ratio of approximately 1 out of 3) of samples being positive or negative by all techniques (Categories I and VI respectively) and a comparable overall positivity of APA and SC but higher than that of PSA. Indeed the combined results of using SC, APA and PSA tests was considered as conclusive for semen detection from approximately 1 out of 3 cases (Category I) to approximately 1 out of 2 cases in a scenario where at least SC is positive, strongly presumptive in 2 out of 3 cases (with at least one test positive) and the remainder 1 out of 3 cases (Category VI) suggested absence of semen. By determining Y-STR polymorphisms (12-loci) in additional samples obtained at Toluca laboratory, complete DNA profiles were determined from all Category I samples, none marker was detected from all Category VI samples and mostly partial profiles were obtained from samples of other categories. These observations give an overview on the variability in efficacy of each test performed at different laboratories and provide a general notion about the in praxis contribution of SC, APA and PSA tests for further DNA typing in the forensic analysis of rape.

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.

Induction of protection in murine experimental models against Trichinella spiralis : an up-to-date review

The parasitic nematode Trichinella spiralis, an aetiological agent of the disease known as trichinellosis, infects wild and domestic animals through contaminated pig meat, which is the major source for Trichinella transmission. Prevention of this disease by interrupting parasite transmission includes vaccine development for livestock; however, major challenges to this strategy are the complexity of the T. spiralis life cycle, diversity of stage-specific antigens, immune-evasion strategies and the modulatory effect of host responses. Different approaches have been taken to induce protective immune responses by T. spiralis immunogens. These include the use of whole extracts or excretory-secretory antigens, as well as recombinant proteins or synthesized epitopes, using murine experimental models for trichinellosis. Here these schemes are reviewed and discussed, and new proposals envisioned to block the zoonotic transmission of this parasite.

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 β-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.

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

Albendazole (ABZ) is a therapeutic benzimidazole used to treat giardiasis that targets β-tubulin. However, the molecular bases of ABZ resistance in Giardia duodenalis are not understood because β-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.

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, Ki = 0.18 μM for GiACS1, and IC50 = 2.28 μM, Ki = 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.

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.