ß(2 → 1)-ß(2 → 6) and ß(2 → 1) fructans protect from impairment of intestinal tight junction's gene expression and attenuate human dendritic cell responses in a fructan-dependent fashion.

ß(2 â†’ 1)-ß(2 â†’ 6) branched graminan-type fructans (GTFs) and ß(2 â†’ 1) linear fructans (ITFs) possess immunomodulatory properties and protect human intestinal barrier function, however the mechanisms underlying these effects are not well studied. Herein, GTFs and ITFs effects with different degree of polymerization (DP) values on tight junctions (TJs) genes CLDN-1, -2 and -3, CDH1, OCLN and TJP1 were studied in Caco-2 gut epithelial cells, under homeostatic and inflammatory conditions. Also, cytokine production in dendritic cells (DCs) was studied. Higher DP fructans decreased the expression of the pore forming CLDN-2. Higher DP GTFs enhanced CLDN-3, OCLN, and TJP-1. Fructans prevented mRNA dysregulation of CLDN-1, -2 and -3 induced by the barrier disruptors A23187 and deoxynivalenol in a fructan-type dependent fashion. The production of pro-inflammatory cytokines MCP-1/CCL2, MIP-1α/CCL3 and TNFα by DCs was also attenuated in a fructan-type dependent manner and was strongly attenuated by DCs cultured with medium of Caco-2 cells which were pre-exposed to fructans. Our data show that specific fructans have TJs and DCs modulating effects and contribute to gut homeostasis. This might serve to design effective dietary means to prevent intestinal inflammation.

Human Triosephosphate Isomerase Is a Potential Target in Cancer Due to Commonly Occurring Post-Translational Modifications.

Cancer involves a series of diseases where cellular growth is not controlled. Cancer is a leading cause of death worldwide, and the burden of cancer incidence and mortality is rapidly growing, mainly in developing countries. Many drugs are currently used, from chemotherapeutic agents to immunotherapy, among others, along with organ transplantation. Treatments can cause severe side effects, including remission and progression of the disease with serious consequences. Increased glycolytic activity is characteristic of cancer cells. Triosephosphate isomerase is essential for net ATP production in the glycolytic pathway. Notably, some post-translational events have been described that occur in human triosephosphate isomerase in which functional and structural alterations are provoked. This is considered a window of opportunity, given the differences that may exist between cancer cells and their counterpart in normal cells concerning the glycolytic enzymes. Here, we provide elements that bring out the potential of triosephosphate isomerase, under post-translational modifications, to be considered an efficacious target for treating cancer.

Repurposing of rabeprazole as an anti-Trypanosoma cruzi drug that targets cellular triosephosphate isomerase.

Trypanosoma cruzi is the causative agent of American trypanosomiasis, which mainly affects populations in Latin America. Benznidazole is used to control the disease, with severe effects in patients receiving this chemotherapy. Previous studies have demonstrated the inhibition of triosephosphate isomerase from T. cruzi, but cellular enzyme inhibition has yet to be established. This study demonstrates that rabeprazole inhibits both cell viability and triosephosphate isomerase activity in T. cruzi epimastigotes. Our results show that rabeprazole has an IC50 of 0.4 µM, which is 14.5 times more effective than benznidazole. Additionally, we observed increased levels of methyl-glyoxal and advanced glycation end products after the inhibition of cellular triosephosphate isomerase by rabeprazole. Finally, we demonstrate that the inactivation mechanisms of rabeprazole on triosephosphate isomerase of T. cruzi can be achieved through the derivatization of three of its four cysteine residues. These results indicate that rabeprazole is a promising candidate against American trypanosomiasis.

Is Your Kid Actin Out? A Series of Six Patients With Inherited Actin-Related Protein 2/3 Complex Subunit 1B Deficiency and Review of the Literature.

BACKGROUND: Hereditary actin-related protein 2/3 complex subunit 1B deficiency is characterized clinically by ear, skin, and lung infections, bleeding, eczema, food allergy, asthma, skin vasculitis, colitis, arthritis, short stature, and lymphadenopathy. OBJECTIVE: We aimed to describe the clinical, laboratory, and genetic features of six patients from four Mexican families. METHODS: We performed exome sequencing in patients of four families with suspected actinopathy, collected their data from medical records, and reviewed the literature for reports of other patients with actin-related protein 2/3 complex subunit 1B deficiency. RESULTS: Six patients from four families were included. All had recurrent infections, mainly bacterial pneumonia, and cellulitis. A total of 67% had eczema whereas 50% had food allergies, failure to thrive, hepatomegaly, and bleeding. Eosinophilia was found in all; 84% had thrombocytopenia, 67% had abnormal-size platelets and anemia. Serum levels of IgG, IgA, and IgE were highly increased in most; IgM was normal or low. T cells were decreased in 67% of patients, whereas B and NK cells were increased in half of patients. Two of the four probands had compound heterozygous variants. One patient was successfully transplanted. We identified 28 other patients whose most prevalent features were eczema, recurrent infections, failure to thrive, bleeding, diarrhea, allergies, vasculitis, eosinophilia, platelet abnormalities, high IgE/IgA, low T cells, and high B cells. CONCLUSION: Actin-related protein 2/3 complex subunit 1B deficiency has a variable and heterogeneous clinical spectrum, expanded by these cases to include keloid scars and Epstein-Barr virus chronic hepatitis. A novel deletion in exon 8 was shared by three unrelated families and might be the result of a founder effect.

ß(2 → 1)-ß(2 → 6) branched graminan-type fructans and ß(2 → 1) linear fructans impact mucus-related and endoplasmic reticulum stress-related genes in goblet cells and attenuate inflammatory responses in a fructan dependent fashion.

Dietary fibers such as fructans have beneficial effects on intestinal health but it is unknown whether they impact goblet cells (GCs). Here we studied the effects of inulin-type fructans (ITFs) and graminan-type fructans (GTFs) with different molecular weights on mucus- and endoplasmic reticulum (ER) stress-related genes in intestinal GCs. To that end, GCs were incubated in the presence of ITFs or GTFs, or ITFs and GTFs + TNFα or the N-glycosylation inhibitor tunicamycin (Tm). IL-8 production by GCs was studied as a marker of inflammation. Effects between ITFs and GTFs were compared. We found a beneficial impact of GTFs especially on the expression of RETNLB. GTF II protects from the TNFα-induced gene expression dysregulation of MUC2, TFF3, GAL3ST2, and CHST5. Also, all the studied fructans prevented Tm-induced dysregulation of GAL3ST2. Interestingly, only the short chain fructans ITF I and GTF I have anti-inflammatory properties on GCs. All the studied fructans except ITF I decreased the expression of the ER stress-related HSPA5 and XBP1. All these benefits were fructan-structure and chain length dependent. Our study contributes to a better understanding of chemical structure-dependent beneficial effects of ITFs and GTFs on gut barrier function, which could contribute to prevention of gut inflammatory disorders.

TLR 2/1 interaction of pectin depends on its chemical structure and conformation.

Citrus pectins have demonstrated health benefits through direct interaction with Toll-like receptor 2. Methyl-ester distribution patterns over the homogalacturonan were found to contribute to such immunomodulatory activity, therefore molecular interactions with TLR2 were studied. Molecular-docking analysis was performed using four GalA-heptamers, GalA7Me0, GalA7Me1,6, GalA7Me1,7 and GalA7Me2,5. The molecular relations were measured in various possible conformations. Furthermore, commercial citrus pectins were characterized by enzymatic fingerprinting using polygalacturonase and pectin-lyase to determine their methyl-ester distribution patterns. The response of 12 structurally different pectic polymers on TLR2 binding and the molecular docking with four pectic oligomers clearly demonstrated interactions with human-TLR2 in a structure-dependent way, where blocks of (non)methyl-esterified GalA were shown to inhibit TLR2/1 dimerization. Our results may be used to understand the immunomodulatory effects of certain pectins via TLR2. Knowledge of how pectins with certain methyl-ester distribution patterns bind to TLRs may lead to tailored pectins to prevent inflammation.

Not enough by half: NFAT5 haploinsufficiency in two patients with Epstein-Barr virus susceptibility.

Introduction: The transcription factor Nuclear factor of activated T cells 5 (NFAT5), pivotal in immune regulation and function, can be induced by osmotic stress and tonicity-independent signals. Objective: We aimed to investigate and characterize two unrelated patients with Epstein-Barr virus susceptibility and no known genetic etiology. Methods: After informed consent, we reviewed the electronic charts, extracted genomic DNA, performed whole-exome sequencing, filtered, and prioritized their variants, and confirmed through Sanger sequencing, family segregation analysis, and some functional assays, including lymphoproliferation, cytotoxicity, and characterization of natural killer cells. Results: We describe two cases of pediatric Mexican patients with rare heterozygous missense variants in NFAT5 and EBV susceptibility, a school-age girl with chronic-active infection of the liver and bowel, and a teenage boy who died of hemophagocytic lymphohistiocytosis. Discussion: NFAT5 is an important regulator of the immune response. NFAT5 haploinsufficiency has been described as an immunodeficiency syndrome affecting both innate and adaptive immunity. EBV susceptibility might be another manifestation in the spectrum of this disease.

The Giardial Arginine Deiminase Participates in Giardia-Host Immunomodulation in a Structure-Dependent Fashion via Toll-like Receptors.

Beyond the problem in public health that protist-generated diseases represent, understanding the variety of mechanisms used by these parasites to interact with the human immune system is of biological and medical relevance. Giardia lamblia is an early divergent eukaryotic microorganism showing remarkable pathogenic strategies for evading the immune system of vertebrates. Among various multifunctional proteins in Giardia, arginine deiminase is considered an enzyme that plays multiple regulatory roles during the life cycle of this parasite. One of its most important roles is the crosstalk between the parasite and host. Such a molecular "chat" is mediated in human cells by membrane receptors called Toll-like receptors (TLRs). Here, we studied the importance of the 3D structure of giardial arginine deiminase (GlADI) to immunomodulate the human immune response through TLRs. We demonstrated the direct effect of GlADI on human TLR signaling. We predicted its mode of interaction with TLRs two and four by using the AlphaFold-predicted structure of GlADI and molecular docking. Furthermore, we showed that the immunomodulatory capacity of this virulent factor of Giardia depends on the maintenance of its 3D structure. Finally, we also showed the influence of this enzyme to exert specific responses on infant-like dendritic cells.

ß(2→1) chicory and ß(2→1)-ß(2→6) agave fructans protect the human intestinal barrier function in vitro in a stressor-dependent fashion.

Dietary fibers such as fructans can protect the intestinal epithelial barrier integrity, but the mechanisms underlying this protection are not completely understood. We aimed to study the protective effect of ß(2→1)-ß(2→6) branched graminan-type fructans (GTFs) on gut epithelial barrier function that was disrupted by three different agents which impact the barrier function via different cellular mechanisms. The effects of GTFs were compared with those of linear ß(2→1) inulin-type fructans (ITFs). T84 intestinal epithelial monolayers were incubated with GTFs and ITFs. Afterwards, the monolayers were challenged with the barrier disruptors calcium ionophore A23187, 12-myristate 13-acetate (PMA) and deoxynivalenol (DON). Transepithelial resistance was measured with an electric cell-substrate impedance sensing system. All fructans studied prevented the barrier disruption induced by A23187. ITF II protected from the disruptive effects of PMA. However, none of the studied fructans influenced the disruption induced by DON. As a measure of disruption-induced inflammation, interleukin-8 (IL-8) production by the intestinal epithelium was determined by ELISA. The production of IL-8 induced by A23187 was decreased by all fructans, whereas IL-8 production induced by DON decreased only upon pre-treatment with ITF II. None of the studied fructans prevented PMA induced IL-8 production. GTFs just like ITFs can influence the barrier function and inflammatory processes in gut epithelial cells in a structure-dependent fashion. These distinct protective effects are dependent on the different signaling pathways that lead to gut barrier disruption.

Naturally occurring deamidated triosephosphate isomerase is a promising target for cell-selective therapy in cancer.

Human triosephosphate isomerase (HsTIM) is a central glycolytic enzyme and is overexpressed in cancer cells with accelerated glycolysis. Triple-negative breast cancer is highly dependent on glycolysis and is typically treated with a combination of surgery, radiation therapy, and chemotherapy. Deamidated HsTIM was recently proposed as a druggable target. Although thiol-reactive drugs affect cell growth in deamidated HsTIM-complemented cells, the role of this protein as a selective target has not been demonstrated. To delve into the usefulness of deamidated HsTIM as a selective target, we assessed its natural accumulation in breast cancer cells. We found that deamidated HsTIM accumulates in breast cancer cells but not in noncancerous cells. The cancer cells are selectively programmed to undergo cell death with thiol-reactive drugs that induced the production of methylglyoxal (MGO) and advanced glycation-end products (AGEs). In vivo, a thiol-reactive drug effectively inhibits the growth of xenograft tumors with an underlying mechanism involving deamidated HsTIM. Our findings demonstrate the usefulness of deamidated HsTIM as target to develop new therapeutic strategies for the treatment of cancers and other pathologies in which this post translationally modified protein accumulates.

Probiotic improves symptomatic and viral clearance in Covid19 outpatients: a randomized, quadruple-blinded, placebo-controlled trial.

Intestinal bacteria may influence lung homeostasis via the gut-lung axis. We conducted a single-center, quadruple-blinded, randomized trial in adult symptomatic Coronavirus Disease 2019 (Covid19) outpatients. Subjects were allocated 1:1 to probiotic formula (strains Lactiplantibacillus plantarum KABP022, KABP023, and KAPB033, plus strain Pediococcus acidilactici KABP021, totaling 2 × 109 colony-forming units (CFU)) or placebo, for 30 days. Co-primary endpoints included: i) proportion of patients in complete symptomatic and viral remission; ii) proportion progressing to moderate or severe disease with hospitalization, or death; and iii) days on Intensive Care Unit (ICU). Three hundred subjects were randomized (median age 37.0 years [range 18 to 60], 161 [53.7%] women, 126 [42.0%] having known metabolic risk factors), and 293 completed the study (97.7%). Complete remission was achieved by 78 of 147 (53.1%) in probiotic group compared to 41 of 146 (28.1%) in placebo (RR: 1.89 [95 CI 1.40-2.55]; P < .001), significant after multiplicity correction. No hospitalizations or deaths occurred during the study, precluding the assessment of remaining co-primary outcomes. Probiotic supplementation was well-tolerated and reduced nasopharyngeal viral load, lung infiltrates and duration of both digestive and non-digestive symptoms, compared to placebo. No significant compositional changes were detected in fecal microbiota between probiotic and placebo, but probiotic supplementation significantly increased specific IgM and IgG against Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV2) compared to placebo. It is thus hypothesized this probiotic primarily acts by interacting with the host's immune system rather than changing colonic microbiota composition. Future studies should replicate these findings and elucidate its mechanism of action (Registration: NCT04517422).Abbreviations: AE: Adverse Event; BMI: Body Mass Index; CONSORT: CONsolidated Standards of Reporting Trials; CFU: Colony-Forming Units; eDRF: Electronic Daily Report Form; GLA: Gut-Lung Axis; GSRS: Gastrointestinal Symptoms Rating Scale; hsCRP: High-sensitivity C-Reactive Protein; HR: Hazard Ratio; ICU: Intensive Care Unit; OR: Odds Ratio; PCoA: Principal Coordinate Analysis; RR: Relative Risk; RT-qPCR: Real-Time Quantitative Polymerase Chain Reaction; SARS-CoV2: Severe acute respiratory syndrome coronavirus 2; SpO2: Peripheral Oxygen Saturation; WHO: World Health Organization.

Improved yield, stability, and cleavage reaction of a novel tobacco etch virus protease mutant.

The protease catalytic subunit of the nuclear inclusion protein A from tobacco etch virus (TEVp) is widely used to remove tags and fusion proteins from recombinant proteins. Some intrinsic drawbacks to its recombinant production have been studied for many years, such as low solubility, auto-proteolysis, and instability. Some point mutations have been incorporated in the amino acid protease sequence to improve its production. Here, a comprehensive review of each mutation reported so far has been made to incorporate them into a mutant called TEVp7M with a total of seven changes. This mutant with a His7tag at N-terminus was produced with remarkable purification yields (55 mg/L of culture) from the soluble fraction in a single step affinity purification. The stability of His7-TEVp7M was analyzed and compared with the single mutant TEVp S219V, making evident that His7-TEVp7M shows very constant thermal stability against pH variation, whereas TEVp S219V is highly sensitive to this change. The cleavage reaction was optimized by determining the amount of protease that could cleave a 100-fold excess substrate in the shortest possible time at 30 °C. Under these conditions, His7-TEVp7M was able to cleave His-tag in the buffers commonly used for affinity purification. Finally, a structural analysis of the mutations showed that four of them increased the polarity of the residues involved and, consequently, showed increased solubility of TEVp and fewer hydrophobic regions exposed to the solvent. Taken together, the seven changes studied in this work improved stability, solubility, and activity of TEVp producing enough protease to digest large amounts of tags or fusion proteins. KEY POINTS: • Production of excellent yields of a TEVp (TEVp7M) by incorporation of seven changes. • His-tag removal in an excess substrate in the common buffers used for purification. • Incorporated mutations improve polarity, stability, and activity of TEVp7M.

Multilevel Approach for the Treatment of Giardiasis by Targeting Arginine Deiminase.

Giardiasis represents a latent problem in public health due to the exceptionally pathogenic strategies of the parasite Giardia lamblia for evading the human immune system. Strains resistant to first-line drugs are also a challenge. Therefore, new antigiardial therapies are urgently needed. Here, we tested giardial arginine deiminase (GlADI) as a target against giardiasis. GlADI belongs to an essential pathway in Giardia for the synthesis of ATP, which is absent in humans. In silico docking with six thiol-reactive compounds was performed; four of which are approved drugs for humans. Recombinant GlADI was used in enzyme inhibition assays, and computational in silico predictions and spectroscopic studies were applied to follow the enzyme's structural disturbance and identify possible effective drugs. Inhibition by modification of cysteines was corroborated using Ellman's method. The efficacy of these drugs on parasite viability was assayed on Giardia trophozoites, along with the inhibition of the endogenous GlADI. The most potent drug against GlADI was assayed on Giardia encystment. The tested drugs inhibited the recombinant GlADI by modifying its cysteines and, potentially, by altering its 3D structure. Only rabeprazole and omeprazole decreased trophozoite survival by inhibiting endogenous GlADI, while rabeprazole also decreased the Giardia encystment rate. These findings demonstrate the potential of GlADI as a target against giardiasis.

Delivery mode-associated gut microbiota in the first 3 months of life in a country with high obesity rates: A descriptive study.

Delivery methods during childbirth and their related gut microbiota profiles have important impacts on health later in life, they can contribute to the development of diseases such as obesity, whose highest prevalence rate is found among the Mexican child population. Coincidentally, Mexico has one of the highest global average annual rate increase in cesarean births (C-section). Since Mexico leads the world in childhood obesity, studying the relationship between childbirth delivery methods and gut microbiota profiles in this vulnerable population may be used to identify early risk factors for obesity in other developed and developing countries. The objective of this study is to determine the association between child delivery method and gut microbiota profiles in healthy Mexican newborns.Fecal samples of 57 term infants who participated in a randomized clinical trial in 2013 to study the safety of Agave fructans in newborns, were used in this study. DNA samples were extracted and used to characterize the microbiota composition using high-throughput 16S rRNA gene sequencing. The samples were further divided based on childbirth delivery method, as well as early diet. Gut microbiota profiles were determined and analyzed using cluster analysis followed by multiple correspondence analysis.An unusual high abundance of Proteobacteria was found in the gut microbiota of all Mexican infants studied, regardless of delivery method. Feces from infants born by C-section had low levels of Bacteroidetes, high levels of Firmicutes, especially Clostridium and Enterococcus, and a strikingly high ratio of Firmicutes/Bacteroidetes (F:B). Profiles enriched in Bacteroidetes and low F:B ratios, were strongly associated with vaginal delivery.The profile of gut microbiota associated with feces from Mexican infants born by C-section, may be added to the list of boosting factors for the worrying obesity epidemic in Mexico.

Rabeprazole inhibits several functions of Entamoeba histolytica related with its virulence.

Amoebiasis is a human parasitic disease caused by Entamoeba histolytica. The parasite can invade the large intestine and other organs such as liver; resistance to the host tissue oxygen is a condition for parasite invasion and survival. Thioredoxin reductase of E. histolytica (EhTrxR) is a critical enzyme mainly involved in maintaining reduced the redox system and detoxifying the intracellular oxygen; therefore, it is necessary for E. histolytica survival under both aerobic in vitro and in vivo conditions. In the present work, it is reported that rabeprazole (Rb), a drug widely used to treat heartburn, was able to inhibit the EhTrxR recombinant enzyme. Moreover, Rb affected amoebic proliferation and several functions required for parasite virulence such as cytotoxicity, oxygen reduction to hydrogen peroxide, erythrophagocytosis, proteolysis, and oxygen and complement resistances. In addition, amoebic pre-incubation with sublethal Rb concentration (600 µM) promoted amoebic death during early liver infection in hamsters. Despite the high Rb concentration used to inhibit amoebic virulence, the wide E. histolytica pathogenic-related functions affected by Rb strongly suggest that its molecular structure can be used as scaffold to design new antiamoebic compounds with lower IC50 values.

Deamidated Human Triosephosphate Isomerase is a Promising Druggable Target.

Therapeutic strategies for the treatment of any severe disease are based on the discovery and validation of druggable targets. The human genome encodes only 600-1500 targets for small-molecule drugs, but posttranslational modifications lead to a considerably larger druggable proteome. The spontaneous conversion of asparagine (Asn) residues to aspartic acid or isoaspartic acid is a frequent modification in proteins as part of the process called deamidation. Triosephosphate isomerase (TIM) is a glycolytic enzyme whose deamidation has been thoroughly studied, but the prospects of exploiting this phenomenon for drug design remain poorly understood. The purpose of this study is to demonstrate the properties of deamidated human TIM (HsTIM) as a selective molecular target. Using in silico prediction, in vitro analyses, and a bacterial model lacking the tim gene, this study analyzed the structural and functional differences between deamidated and nondeamidated HsTIM, which account for the efficacy of this protein as a druggable target. The highly increased permeability and loss of noncovalent interactions of deamidated TIM were found to play a central role in the process of selective enzyme inactivation and methylglyoxal production. This study elucidates the properties of deamidated HsTIM regarding its selective inhibition by thiol-reactive drugs and how these drugs can contribute to the development of cell-specific therapeutic strategies for a variety of diseases, such as COVID-19 and cancer.

On the molecular and cellular effects of omeprazole to further support its effectiveness as an antigiardial drug.

Research on Giardia lamblia has accumulated large information about its molecular cell biology and infection biology. However, giardiasis is still one of the commonest parasitic diarrheal diseases affecting humans. Additionally, an alarming increase in cases refractory to conventional treatment has been reported in low prevalence settings. Consequently, efforts directed toward supporting the efficient use of alternative drugs, and the study of their molecular targets appears promising. Repurposing of proton pump inhibitors is effective in vitro against the parasite and the toxic activity is associated with the inhibition of the G. lamblia triosephosphate isomerase (GlTIM) via the formation of covalent adducts with cysteine residue at position 222. Herein, we evaluate the effectiveness of omeprazole in vitro and in situ on GlTIM mutants lacking the most superficial cysteines. We studied the influence on the glycolysis of Giardia trophozoites treated with omeprazole and characterized, for the first time, the morphological effect caused by this drug on the parasite. Our results support the effectiveness of omeprazole against GlTIM despite of the possibility to mutate the druggable amino acid targets as an adaptive response. Also, we further characterized the effect of omeprazole on trophozoites and discuss the possible mechanism involved in its antigiardial effect.

Mechanisms of natural resistance of Balb/c mice to experimental liver amoebiasis.

Entamoeba histolytica is the parasite responsible for human amoebiasis. The analysis of the natural resistance mechanisms of some rodents to amoebic liver abscess (ALA) may reveal alternative pathogenicity mechanisms to those previously discovered in the experimental model of ALA in hamsters. In this work the natural resistance of BALB/c mice to ALA was explored by performing: (i) in vivo chemotaxis analysis with a specifically designed chamber; (ii) in vitro amoebic survival in fresh and decomplemented serum; (iii) histological temporal course analysis of ALA development in mice with different treatments (hypocomplementemic, hyperimmune and treated with iNOS and NADPH oxidase inhibitors) and (iv) mouse liver amoebic infection by both in situ implantation of ALA from hamsters and inoculation of parasites into the peritoneal cavity. The results show that E. histolytica clearance from the mouse liver is related to a low chemotactic activity of complement, which results in poor inflammatory response and parasite inability to cause tissue damage. Also, the absence of amoebic tropism for the mouse liver is correlated with resistance to experimental liver amoebiasis.

Characterization of proteolytic activities of Giardia lamblia with the ability to cleave His-tagged N-terminal sequences.

Giardia lamblia is one of the most common protozoan infectious agents in the world and is responsible for diarrheal disease and chronic postinfectious illness. During the host-parasite interaction, proteases are important molecules related to virulence, invasion, and colonization, not only for Giardia but also for other parasites. We aimed to characterize the cysteine protease activity detected in trophozoite lysates. This proteolytic activity showed the ability to cleave NH-terminal sequences with either a recognition sequence for a viral protease or a recognition sequence for thrombin. This cleavage activity was detected in nonencysting trophozoites and increased with the progression of encystation. This activity was also detected in excretion/secretion products of axenic trophozoites and in trophozoites cocultured with differentiated Caco-2 cells. Based on size exclusion chromatography, we obtained a fraction enriched in low- to medium-molecular-weight proteins that was capable of exerting this cleavage activity and aggregating human platelets. Finally, our results suggest that this proteolytic activity is shared with other protozoan parasites.

Complement is a rat natural resistance factor to amoebic liver infection.

Amoebiasis is a parasitic disease caused by Entamoeba histolytica This illness is prevalent in poor countries causing 100,000 deaths worldwide. Knowledge of the natural resistance mechanisms of rats to amoebic liver abscess (ALA) development may help to discover new pathogenic factors and to design novel therapeutic strategies against amoebiasis. In this work, histologic analyses suggested that the complement system may play a central role in rat natural resistance to ALA. E. histolytica trophozoites disappeared from rat liver within 6 h post-infection with minimal or no inflammatory infiltrate. In vitro findings indicate that rat complement was lethal for the parasite. Furthermore, hamsters became resistant to ALA by intravenous administration of fresh rat serum before infection. The amoebicidal potency of rat complement was 10 times higher than hamster complement and was not related to their respective CH50 levels. The alternative pathway of complement plays a central role in its toxicity to E. histolytica since trypan blue, which is a C3b receptor inhibitor, blocks its amoebicidal activity. These results suggest that amoebic membrane affinity, high for C3b and/or low for Factor H, in comparison with the hamster ones, may result in higher deposition of membrane complex attack on parasite surface and death.