Intestinal microbiota - A modulator of the Trypanosoma cruzi-vector-host triad.

Chagas disease affects millions of people, and it is a major cause of death in Latin America. Prevention and development of an effective treatment for this infection can be favored by a more thorough understanding of T. cruzi interaction with the microbiome of vectors and hosts. Next-generation sequencing technology vastly broadened the knowledge about intestinal bacteria composition, showing that microbiota within each host (triatomines and mammals) is composed by high diversity of species, although few dominant phyla. This fact may represent an ecological balance that was acquired during the evolutionary process of the microbiome-host complex, and that serves to perpetuate this system. In this context, commensal microbiota is also essential to protect hosts, conferring them resistance to pathogens colonization. However, in some situations, the microbiota is not able to prevent infection but only modulate it. Here we will review the role of the microbiota on the parasite-vector-host triad with a focus on the kinetoplastida of medical importance Trypanosoma cruzi. Novel strategies to control Chagas disease based on intestinal microbiome will also be discussed.

High-Level Expression in Escherichia coli, Purification and Kinetic Characterization of LAPTc, a Trypanosoma cruzi M17-Aminopeptidase.

The M17 leucyl-aminopeptidase of Trypanosoma cruzi (LAPTc) is a novel drug target for Chagas disease. The objective of this work was to obtain recombinant LAPTc (rLAPTc) in Escherichia coli. A LAPTc gene was designed, optimized for its expression in E. coli, synthesized and cloned into the vector pET-19b. Production of rLAPTc in E. coli BL21(DE3)pLysS, induced for 20 h at 25 °C with 1 mM IPTG, yielded soluble rLAPTC that was catalytically active. The rLAPTc enzyme was purified in a single step by IMAC. The recombinant protein was obtained with a purity of 90% and a volumetric yield of 90 mg per liter of culture. The enzymatic activity has an optimal pH of 9.0, and preference for Leu-p-nitroanilide (appKM = 74 µM, appkcat = 4.4 s-1). The optimal temperature is 50 °C, and the cations Mg2+, Cd2+, Ba2+, Ca2+ and Zn2+ at 4 mM inhibited the activity by 60% or more, while Mn2+ inhibited by only 15% and addition of Co2+ activated by 40%. The recombinant enzyme is insensitive toward the protease inhibitors PMSF, TLCK, E-64 and pepstatin A, but is inhibited by EDTA and bestatin. Bestatin is a non-competitive inhibitor of the enzyme with a Ki value of 881 nM. The enzyme is a good target for inhibitor identification.

The fecal, oral, and skin microbiota of children with Chagas disease treated with benznidazole.

BACKGROUND: Chagas disease is still prevalent in rural areas of South America. In endemic areas of Bolivia, school children are screened for the program of Chagas disease eradication of the Ministry of Health, and positive children are treated. Here, we compared the fecal, oral and skin microbiomes of children with or without Chagas disease, and before and after benznidazol treatment of infected children. METHODS: A total of 543 Bolivian children (5-14 years old) were tested for Chagas disease, and 20 positive children were treated with Benznidazole. Fecal samples and oral and skin swabs were obtained before and after treatment, together with samples from a group of 35 uninfected controls. The 16S rRNA genes were sequenced and analyzed using QIIME to determine Alpha diversity differences and community distances, and linear discriminant analyses to determine marker taxa by infection status or treatment. RESULTS: Twenty out of 543 children screened were seropositive for Chagas disease (3.7%) and were included in the study, together with 35 control children that were seronegative for the disease. Fecal samples, oral and skin swabs were taken at the beginning of the study and after the anti-protozoa therapy with Benznidazole to the chagasic children. Infected children had higher fecal Firmicutes (Streptococcus, Roseburia, Butyrivibrio, and Blautia), and lower Bacteroides and also showed some skin -but not oral- microbiota differences. Treatment eliminated the fecal microbiota differences from control children, increasing Dialister (class Clostridia) and members of the Enterobacteriaceae, and decreasing Prevotella and Coprococcus, with minor effects on the oral and skin bacterial diversity. CONCLUSIONS: The results of this study show differences in the fecal microbiota associated with Chagas disease in children, and also evidence that treatment normalizes fecal microbiota (makes it more similar to that in controls), but is associated with oral and skin microbiota differences from control children. Since microbiota impacts in children, it is important to determine the effect of drugs on the children microbiota, since dysbiosis could lead to physiological effects which might be avoidable with microbiota restoration interventions.

IL-17RA-Signaling Modulates CD8+ T Cell Survival and Exhaustion During Trypanosoma cruzi Infection.

The IL-17 family contributes to host defense against many intracellular pathogens by mechanisms that are not fully understood. CD8+ T lymphocytes are key elements against intracellular microbes, and their survival and ability to mount cytotoxic responses are orchestrated by several cytokines. Here, we demonstrated that IL-17RA-signaling cytokines sustain pathogen-specific CD8+ T cell immunity. The absence of IL-17RA and IL-17A/F during Trypanosoma cruzi infection resulted in increased tissue parasitism and reduced frequency of parasite-specific CD8+ T cells. Impaired IL-17RA-signaling in vivo increased apoptosis of parasite-specific CD8+ T cells, while in vitro recombinant IL-17 down-regulated the pro-apoptotic protein BAD and promoted the survival of activated CD8+ T cells. Phenotypic, functional, and transcriptomic profiling showed that T. cruzi-specific CD8+ T cells derived from IL-17RA-deficient mice presented features of cell dysfunction. PD-L1 blockade partially restored the magnitude of CD8+ T cell responses and parasite control in these mice. Adoptive transfer experiments established that IL-17RA-signaling is intrinsically required for the proper maintenance of functional effector CD8+ T cells. Altogether, our results identify IL-17RA and IL-17A as critical factors for sustaining CD8+ T cell immunity to T. cruzi.

ROS and Trypanosoma cruzi: Fuel to infection, poison to the heart.

The activation of macrophage respiratory burst in response to infection with Trypanosoma cruzi inflicts oxidative damage to the host's tissues. For decades, the role of reactive oxygen species (ROS) in the elimination of T. cruzi was taken for granted, but recent evidence suggests parasite growth is stimulated in oxidative environments. It is still a matter of debate whether indeed oxidative environments provide ideal conditions (e.g., iron availability in macrophages) for T. cruzi growth and whether indeed ROS signals directly to stimulate growth. Nitric oxide (NO) and ROS combine to form peroxynitrite, participating in the killing of phagocytosed parasites by activated macrophages. In response to infection, mitochondrial ROS are produced by cardiomyocytes. They contribute to oxidative damage that persists at the chronic stage of infection and is involved in functional impairment of the heart. In this review, we discuss how oxidative stress helps parasite growth during the acute stage and how it participates in the development of cardiomyopathy at the chronic stage.

Identification of di-substituted ureas that prevent growth of trypanosomes through inhibition of translation initiation.

Some 1,3-diarylureas and 1-((1,4-trans)-4-aryloxycyclohexyl)-3-arylureas (cHAUs) activate heme-regulated kinase causing protein synthesis inhibition via phosphorylation of the eukaryotic translation initiation factor 2 (eIF2) in mammalian cancer cells. To evaluate if these agents have potential to inhibit trypanosome multiplication by also affecting the phosphorylation of eIF2 alpha subunit (eIF2α), we tested 25 analogs of 1,3-diarylureas and cHAUs against Trypanosoma cruzi, the agent of Chagas disease. One of them (I-17) presented selectivity close to 10-fold against the insect replicative forms and also inhibited the multiplication of T. cruzi inside mammalian cells with an EC50 of 1-3 µM and a selectivity of 17-fold. I-17 also prevented replication of African trypanosomes (Trypanosoma brucei bloodstream and procyclic forms) at similar doses. It caused changes in the T. cruzi morphology, arrested parasite cell cycle in G1 phase, and promoted phosphorylation of eIF2α with a robust decrease in ribosome association with mRNA. The activity against T. brucei also implicates eIF2α phosphorylation, as replacement of WT-eIF2α with a non-phosphorylatable eIF2α, or knocking down eIF2 protein kinase-3 by RNAi increased resistance to I-17. Therefore, we demonstrate that eIF2α phosphorylation can be engaged to develop trypanosome-static agents in general, and particularly by interfering with activity of eIF2 kinases.

Engineering Oral and Parenteral Amorphous Amphotericin B Formulations against Experimental Trypanosoma cruzi Infections.

Chagas disease (CD) is a parasitic zoonosis endemic in most mainland countries of Central and South America affecting nearly 10 million people, with 100 million people at high risk of contracting the disease. Treatment is only effective if received at the early stages of the disease. Only two drugs (benznidazole and nifurtimox) have so far been marketed, and both share various limitations such as variable efficacy, many side effects, and long duration of treatment, thus reducing compliance. The in vitro and in vivo efficacy of poly-aggregated amphotericin B (AmB), encapsulated poly-aggregated AmB in albumin microspheres (AmB-AME), and dimeric AmB-sodium deoxycholate micelles (AmB-NaDC) was evaluated. Dimeric AmB-NaDC exhibited a promising selectivity index (SI = 3164) against amastigotes, which was much higher than those obtained for licensed drugs (benznidazole and nifurtimox). AmB-AME, but not AmB-NaDC, significantly reduced the parasitemia levels (3.6-fold) in comparison to the control group after parenteral administration at day 7 postinfection. However, the oral administration of AmB-NaDC (10-15 mg/kg/day for 10 days) resulted in a 75% reduction of parasitemia levels and prolonged the survival rate in 100% of the tested animals. Thus, the results presented here illustrate for the first time the oral efficacy of AmB in the treatment of trypanosomiasis. AmB-NaDC is an easily scalable, affordable formulation prepared from GRAS excipients, enabling treatment access worldwide, and therefore it can be regarded as a promising therapy for trypanosomiasis.

Antibody profiles induced by Trypanosoma cruzi in chagasic patients with previous or current exposure to mycobacteria.

Since the immune response mounted by the host to a particular microorganism might be influenced by the acquired immunological experience due to previous contact with other microorganisms, we performed a cross-sectional study to explore the pattern of Trypanosoma cruzi infection-related antibodies in T. cruzi-seropositive individuals presenting concomitant tuberculosis, or the antecedent of BCG vaccination. Sampled individuals were grouped as follows: patients with Chagas disease, not vaccinated with BCG, who further developed pulmonary tuberculosis; individuals with Chagas disease, BCG-vaccinated; and subjects with Chagas disease, presenting neither BCG scar nor tuberculosis disease. Non-vaccinated individuals or without tuberculosis, presented the highest values of anti-PH (P < 0.001), anti-FRA (P < 0.001), anti-p2ß (P = 0.0023) and anti-B13 (P < 0.001) antibodies. The present findings constitute the first demonstration of the potential influence of concomitant tuberculosis on Chagas disease.

Hydroxamic acid derivatives: a promising scaffold for rational compound optimization in Chagas disease.

This work describes the antitrypanocidal activity of two hydroxamic acid derivatives containing o-ethoxy (HAD1) and p-ethoxy (HAD2) as substituent in the aromatic ring linked to the isoxazoline ring. HAD1 and HAD2 induced a significant reduction in the number of intracellular parasites and consequently showed activity on the multiplication of the parasite. Treatment of cardiomyocytes and macrophages with the compounds revealed no significant loss in cell viability. Ultrastructural alterations after treatment of cardiomyocytes or macrophages infected by Trypanosoma cruzi with the IC50 value of HAD1 revealed alterations to amastigotes, showing initial damage seen as swelling of the kinetoplast. This gave a good indication of the ability of the drug to permeate through the host cell membrane as well as its selectivity to the parasite target. Both compounds HAD1 and 2 were able to reduce the cysteine peptidases and decrease the activity of metallopeptidases.

Avaliação da expressão de syndecan-4 e de seu papel como biomarcados na cardiomiopatia chagásica crônica / Evaluation of syndecan-4 expression and its role as biomarkers in chronic chagasic cardiomyopathy

INTRODUÇÃO: A cardiomiopatia chagásica crônica (CCC), doença de elevada morbimortalidade, associada à grave disfunção ventricular e a arritmias cardíacas, caracteriza-se histologicamente por intensa reação inflamatória multifocal, com pronunciada fibrose miocárdica. Diante da ausência de uma terapia eficaz para os pacientes com as formas mais graves da doença, torna-se crucial a descoberta de biomarcadores que possam identificar pacientes em estágios mais precoces, sob risco mais elevado para a progressão da doença. Neste contexto, surge a syndecan-4, uma glicoproteína transmembrana associada à inflamação e fibrose, cujos níveis estão aumentados em indivíduos com insuficiência cardíaca. OBJETIVO: Neste trabalho, avaliamos o padrão de expressão da syndecan-4 no tecido cardíaco de camundongos e de indivíduos com cardiomiopatia chagásica e a possível correlação entre a concentração sérica de syndecan-4 com grau de fibrose miocárdica e com fração de ejeção do ventrículo esquerdo em indivíduos com doença de Chagas...

INTRODUCTION: The hallmark of chronic Chagas cardiomyopathy (CCC) is the presence of a multifocal inflammatory reaction, which leads to myocardial fibrosis, often followed by ventricular dysfunction and arrhythmias. Syndecan-4 is a transmembrane glycoprotein associated with inflammation and fibrosis. Syndecan-4 levels are increased in subjects with heart failure and it has been proposed as a biomarker to predict cardiovascular events. The expression of syndecan-4 is increased in the hearts of mice chronically infected with Trypanosoma cruzi, suggesting a role of this protein in the pathogenesis of CCC. OBJETIVE: Here we aimed to evaluate the pattern of expression of syndecan-4 in heart tissue of mice and subjects with Chagas cardiomyopathy, and to correlate with the degree of inflammation and fibrosis, as well as to determine the correlation of syndecan-4 serum concentration with the degree of myocardial fibrosis and with left ventricular ejection fraction in subjects with Chagas disease...

Histological and endoscopic features of the stomachs of patients with Chagas disease in the era of Helicobacter pylori

Introduction Most studies that have evaluated the stomachs of patients with Chagas disease were performed before the discovery of Helicobacter pylori and used no control groups. This study compared the gastric features of chagasic and non-chagasic patients and assessed whether gastritis could be associated with Chagas disease. Methods Gastric biopsy samples were taken from patients who underwent endoscopy for histological analysis according to the Updated Sydney System. H. pylori infection was assessed by histology, 16S ribosomal ribonucleic acid (rRNA) polymerase chain reaction (PCR), serology and the 13C-urea breath test. Patients were considered H. pylori-negative when all of these diagnostic tests were negative. Clinical and socio-demographic data were obtained by reviewing medical records and using a questionnaire. Results The prevalence of H. pylori infection (70.3% versus 71.7%) and chronic gastritis (92.2% versus 85%) was similar in the chagasic and non-chagasic groups, respectively; such as peptic ulcer, atrophy and intestinal metaplasia. Gastritis was associated with H. pylori infection independent of Chagas disease in a log-binomial regression model. However, the chagasic H. pylori-negative patients showed a significantly higher grade of mononuclear (in the corpus) and polymorphonuclear ...

Histological and endoscopic features of the stomachs of patients with Chagas disease in the era of Helicobacter pylori.

INTRODUCTION: Most studies that have evaluated the stomachs of patients with Chagas disease were performed before the discovery of Helicobacter pylori and used no control groups. This study compared the gastric features of chagasic and non-chagasic patients and assessed whether gastritis could be associated with Chagas disease. METHODS: Gastric biopsy samples were taken from patients who underwent endoscopy for histological analysis according to the Updated Sydney System. H. pylori infection was assessed by histology, 16S ribosomal ribonucleic acid (rRNA) polymerase chain reaction (PCR), serology and the 13C-urea breath test. Patients were considered H. pylori-negative when all of these diagnostic tests were negative. Clinical and socio-demographic data were obtained by reviewing medical records and using a questionnaire. RESULTS: The prevalence of H. pylori infection (70.3% versus 71.7%) and chronic gastritis (92.2% versus 85%) was similar in the chagasic and non-chagasic groups, respectively; such as peptic ulcer, atrophy and intestinal metaplasia. Gastritis was associated with H. pylori infection independent of Chagas disease in a log-binomial regression model. However, the chagasic H. pylori-negative patients showed a significantly higher grade of mononuclear (in the corpus) and polymorphonuclear (PMN) (in the antrum) cell infiltration. Additionally, the patients with the digestive form of Chagas disease showed a significantly lower prevalence of corpus atrophy than those with other clinical forms. CONCLUSIONS: The prevalence of H. pylori infection and of gastric histological and endoscopic features was similar among the chagasic and non-chagasic patients. Additionally, this is the first controlled study to demonstrate that H. pylori is the major cause of gastritis in patients with Chagas disease.

Purinergic system ecto-enzymes participate in the thromboregulation of patients with indeterminate form of Chagas disease.

Chagas disease (CD) is a chronic and endemic illness caused by the parasite Trypanosoma cruzi. Microvascular disturbances play an important role in the progress of the disease. The purinergic signaling system participates in regulatory functions, such as immunomodulation, neuroprotection, and thromboregulation. This study aimed to investigate the activities of purinergic system ecto-enzymes present on the platelet surface and the platelet aggregation profile from patients with indeterminate form of Chagas disease (IFCD). Thirty patients diagnosed with IFCD and 30 healthy subjects were selected. Ecto-nucleoside triphosphate diphosphohydrolase (E-NTPDase), ecto-nucleotide pyrophosphatase/phosphodiesterase (E-NPP), ecto-5'-nucleotidase (E-5'-NT) and ecto-adenosine deaminase (E-ADA) activities were measured in platelets isolated from these individuals as well as the platelet aggregation. Results demonstrated an increase of 21 % in the E-NPP activity and 30 % in the E-5'-NT activity in IFCD group (P < 0.05); however, a decrease of 34 % in the E-ADA activity was determined in the same group (P < 0.001). A significant decrease of 12.7 % and 12.8 % in the platelet aggregation of IFCD group in two different concentrations of ADP (5 and 10 µM) was observed, respectively (P < 0.05). Increased E-NPP and E-5-NT activities as well as decreased E-ADA activity in platelets of patients with IFCD contributed to decrease platelet aggregation, suggesting that the purinergic system is involved in the thromboregulation process in these patients, since adenosine (the final product of ATP hydrolysis) has cardioprotective and vasodilator effects that prevent the clinical progress of the disease.

Liver transplantation using Chagas-infected donors in uninfected recipients: a single-center experience without prophylactic therapy.

Organ shortage is the first cause of death on liver transplant waiting lists. As a consequence, we recently decided to expand liver acceptance to those organs that could potentially transmit infectious diseases to their recipients. On January 2010, we initiated a prospective protocol using livers from Chagas-infected donors for transplanting uninfected recipients without using prophylactic therapy. During a 13-month period, 9 of 37 (24%) liver transplants were performed within this protocol. After transplant, each recipient was sequentially and strictly monitored for infection transmission using the Strout method and promptly treated with benznidazole if this occurs. During follow-up, two patients died without Chagas infection and only two (donor-derived T. cruzi transmission rate: 2/9; 22%) patients developed donor-derived Chagas transmission without clinical symptoms. The median follow-up time of the seven live patients was 15 months (range: 13-20). At present, all are symptoms-free with excellent allograft function and without evidence of Chagas disease. In conclusion, we consider that Chagas-infected donors are a promising source of liver grafts that could reduce the growing mortality on liver waiting lists in America. Relevant data from larger prospective studies are required to confirm these preliminary excellent results.

Trypanosoma cruzi immune response modulation decreases microbiota in Rhodnius prolixus gut and is crucial for parasite survival and development.

Trypanosoma cruzi in order to complete its development in the digestive tract of Rhodnius prolixus needs to overcome the immune reactions and microbiota trypanolytic activity of the gut. We demonstrate that in R. prolixus following infection with epimastigotes of Trypanosoma cruzi clone Dm28c and, in comparison with uninfected control insects, the midgut contained (i) fewer bacteria, (ii) higher parasite numbers, and (iii) reduced nitrite and nitrate production and increased phenoloxidase and antibacterial activities. In addition, in insects pre-treated with antibiotic and then infected with Dm28c, there were also reduced bacteria numbers and a higher parasite load compared with insects solely infected with parasites. Furthermore, and in contrast to insects infected with Dm28c, infection with T. cruzi Y strain resulted in a slight decreased numbers of gut bacteria but not sufficient to mediate a successful parasite infection. We conclude that infection of R. prolixus with the T. cruzi Dm28c clone modifies the host gut immune responses to decrease the microbiota population and these changes are crucial for the parasite development in the insect gut.

A new endemic focus of Chagas disease in the northern region of Veraguas Province, Western Half Panama, Central America.

BACKGROUND: Chagas disease was originally reported in Panama in 1931. Currently, the best knowledge of this zoonosis is restricted to studies done in historically endemic regions. However, little is known about the distribution and epidemiology of Chagas disease in other rural areas of the country. METHODS AND FINDINGS: A cross-sectional descriptive study was carried out between May 2005 - July 2008 in four rural communities of the Santa Fe District, Veraguas Province. The study included an entomologic search to collect triatomines, bloodmeal type identification and infection rate with trypanosomes in collected vectors using a dot- blot and PCR analysis, genotyping of circulating Trypanosoma cruzi (mini-exon gene PCR analysis) and the detection of chagasic antibodies among inhabitants. The vector Rhodnius pallescens was more frequently found in La Culaca and El Pantano communities (788 specimens), where it was a sporadic household visitor. These triatomines presented darker coloration and larger sizescompared with typical specimens collected in Central Panama. Triatoma dimidiata was more common in Sabaneta de El Macho (162 specimens). In one small sub-region (El Macho), 60% of the houses were colonized by this vector. Of the examined R. pallescens, 54.7.0% (88/161) had fed on Didelphis marsupialis, and 24.6% (34/138) of T. dimidiata specimens collected inside houses were positive for human blood. R. pallescens presented an infection index with T. cruzi of 17.7% (24/136), with T. rangeli of 12.5% (17/136) and 50.7% (69/136) were mixed infections. In 117 T. dimidiata domestic specimens the infection index with T. cruzi was 21.4%. Lineage I of T. cruzi was confirmed circulating in these vectors. A T. cruzi infection seroprevalence of 2.3% (24/1,056) was found in this population. CONCLUSIONS: This is the first report of Chagas disease endemicity in Santa Fe District, and it should be considered a neglected public health problem in this area of Panama.

Cytotoxic, trypanocidal, and antifungal activities of Eugenia jambolana L.

Chagas' disease, caused by Trypanosoma cruzi, is considered a public health problem. Nowadays, chemotherapy is the only available treatment for this disease, and the drugs currently used, nifurtimox and benzonidazole, present high toxicity levels. Alternatives for replacing these drugs are natural extracts from Eugenia jambolana, a plant used in traditional medicine because of its antimicrobial and biological activities. An ethanol extract from E. jambolana was prepared. To research in vitro anti-epimastigote activity, T. cruzi CL-B5 clone was used. Epimastigotes were inoculated at a concentration of 1×10(5)/mL in 200 µL of tryptose-liver infusion. For the cytotoxicity assay J774 macrophages were used. To examine antifungal activity, Candida albicans, Candida tropicalis, and Candida krusei were used. This is the first record of trypanocide activity for E. jambolana. The effective concentration capable of killing 50% of the parasites was 56.42 µg/mL. The minimum inhibitory concentration was ≤1,024 µg/mL. Metronidazole showed a potentiation of its antifungal effect when combined with the ethanol extract of E. jambolana. Thus our results indicate that E. jambolana could be a source of plant-derived natural products with anti-epimastigote and antifungal modifying activity with moderate toxicity.

Mexican Trypanosoma cruzi T. cruzi I strains with different degrees of virulence induce diverse humoral and cellular immune responses in a murine experimental infection model.

It is has been shown that the majority of T. cruzi strains isolated from Mexico belong to the T. cruzi I (TCI). The immune response produced in response to Mexican T. cruzi I strains has not been well characterized. In this study, two Mexican T. cruzi I strains were used to infect Balb/c mice. The Queretaro (TBAR/MX/0000/Queretaro)(Qro) strain resulted in 100% mortality. In contrast, no mortality was observed in mice infected with the Ninoa (MHOM/MX/1994/Ninoa) strain. Both strains produced extended lymphocyte infiltrates in cardiac tissue. Ninoa infection induced a diverse humoral response with a higher variety of immunoglobulin isotypes than were found in Qro-infected mice. Also, a stronger inflammatory TH1 response, represented by IL-12p40, IFNgamma, RANTES, MIG, MIP-1beta, and MCP-1 production was observed in Qro-infected mice when compared with Ninoa-infected mice. We propose that an exacerbated TH1 immune response is a likely cause of pathological damage observed in cardiac tissue and the primary cause of death in Qro-infected mice.