Involvement of TLR6 in the induction of COX-2, PGE2 and IL-10 in macrophages by lipids from virulent S2P and attenuated R1A Babesia bovis strains.

Toll like receptors (TLRs) are involved in the modulation of diverse host genes expression through a complex network of signalling events that allow for an appropriate response to a microbial pathogen. In the present work we used TLR6KO mice in order to study the role of TLR6 in the immune discrimination of lipids from two Babesia bovis strains, attenuated R1A (LA) and virulent S2P (LV), and the consequent macrophage activation. We demonstrated that TLR6 is required for lipid body induction in murine peritoneal macrophages by both LA and LV. Interestingly, as regards IL-10 and COX-2/PGE2 pathway induction by LA and LV, we observed differences in the biological effects produced by these lipid extracts. Our results indicate a role of TLR6 in the down-modulation of these immunoregulators only in the case of LA, whereas this receptor was not implicated in pro-inflammatory TNFα, IL-6 and KC release induced by LA. Remarkably, LV did not exert the down-modulatory effect observed for LA, supporting the notion that LA and LV possess different lipid composition that could correlate with the polar pathogenic effect of both B. bovis strains.

Babesia bovis: lipids from virulent S2P and attenuated R1A strains trigger differential signalling and inflammatory responses in bovine macrophages.

The intra-erythrocytic protozoan Babesia bovis is an economically important pathogen that causes an acute and often fatal infection in adult cattle. Babesiosis limitation depends on the early activation of macrophages, essential cells of the host innate immunity, which can generate an inflammatory response mediated by cytokines and nitric oxide (NO). Herein, we demonstrate in bovine macrophages that lipids from B. bovis attenuated R1A strain (LA) produced a stronger NO release, an early TNFα mRNA induction and 2-fold higher IL-12p35 mRNA levels compared to the lipids of virulent S2P strain (LV). Neither LA nor LV induced anti-inflammatory IL-10. Regarding signalling pathways, we here report that LA induced a significant phosphorylation of p38 and extracellular signal-regulated kinases 1 and 2 (ERK1/2) whereas LV only induced a reduced activation of ERK1/2. Besides, NF-κB was activated by LA and LV, but LA produced an early degradation of the inhibitor IκB. Interestingly, LV and the majority of its lipid fractions, exerted a significant inhibition of concanavalin A-induced peripheral blood mononuclear cell proliferation with respect to LA and its corresponding lipid fractions. In addition, we determined that animals infected with R1A developed a higher increase in IgM anti-phosphatidylcholine than those inoculated with S2P. Collectively, S2P lipids generated a decreased inflammatory response contributing to the evasion of innate immunity. Moreover, since R1A lipids induced a pro-inflammatory profile, we propose these molecules as good candidates for immunoprophylactic strategies against babesiosis.

Lipids from attenuated and virulent Babesia bovis strains induce differential TLR2-mediated macrophage activation.

Babesia bovis is an intraerythrocytic apicomplexan protozoa of cattle that causes an acute infection with parasite persistence. Babesiosis limitation depends on macrophages, essential effector cells of the host innate defense, which generate inflammatory cytokines and nitric oxide. Herein, we report quantitative differences in the lipid composition of merozoites from two B. bovis strains with polar behaviour: attenuated R1A and virulent S2P. Accordingly, we observed a distinct inflammatory response induced by the total lipids of R1A (L(A)) and S2P (L(V)) in murine peritoneal macrophages. L(A) and particularly its fractions phosphatidic acid and phosphatidylserine+phosphatidylinositol (PS+PI), produced a strong activation of these cells with lipid body formation, cyclooxygenase-2 expression and pro-inflammatory TNFalpha, IL-6 and KC secretion. Although L(V) did not activate these cells, the corresponding PS+PI fraction induced TNFalpha, IL-6 and KC release. Therefore, these facts might be suggesting the presence of an inhibitor in L(V). Furthermore, the employment of wild type and toll like receptor 2 knockout (TLR2KO) mice allowed us to demonstrate that macrophage activation by the stimulating lipid fractions was mediated through TLR2. Interestingly, only L(A) activated the extracellular signal-regulated kinases 1 and 2 (ERK1/2). Inhibitory studies employing UO126, indicated that the ERK pathway was required for TNFalpha, IL-6 and KC release. In conclusion, the absence of inflammatory response observed with the lipids of S2P virulent strain could constitute an evasion mechanism of the innate immune response enabling parasite establishment in the host.

Phospholipase A1 from Trypanosoma cruzi infective stages generates lipid messengers that activate host cell protein kinase c.

Here we have studied phospholipase A1 (Plase A1) from Trypanosoma cruzi infective stages and it's possible role regarding the interaction with mammalian host cells. Plase A1 was mainly detected as a membrane-bound activity in the infective amastigote and trypomastigote stages, being remarkably higher with respect to the non-infective epimastigotes. It is noteworthy that only the infective stages secreted Plase A1. Moreover, along the differentiation process from epimastigotes into metacyclic trypomastigotes, the secreted enzyme activity increased simultaneously with the appearance of metacyclic forms, as expected. Since this enzyme is predominantly membrane-associated and secreted by the infective stages, Vero cell lipid profile modifications were analysed after interaction with either intact infective parasites or purified T. cruzi Plase A1. Significant changes in Vero cell lipid composition were observed, with the appearance of free fatty acids, diacylglycerol and lysophosphatidylcholine. Concomitantly with the generation of second lipid messengers, host cell protein kinase C activation was demonstrated. These results indicate that T. cruzi Plase A1 could play a critical role in the early events of parasite-host cell interaction that precede invasion.

Novel cysteine proteinase in Trypanosoma cruzi metacyclogenesis.

With the aim to study proteinases released to the culture medium during Trypanosoma cruzi metacyclogenesis, the presence of cysteine proteinases (CPs) was analysed in culture supernatants obtained throughout the differentiation induced by stimulation of epimastigotes with Triatoma infestans hindgut homogenate. In SDS-gelatin containing gels, an important endopeptidase activity with apparent molecular weight range between 97 and 116 kDa was encountered at pH 6, which was abolished by the specific cysteine proteinase inhibitor E-64 and TLCK, but not by pepstatin, 1,10 phenantroline or PMSF. This novel CP, named TcCPmet, showed affinity to cystatin-Sepharose, denoting its thiol-proteinase character as well as to ConA-Sepharose, indicating it contains N-linked oligosaccharides. However, it presented a different elution pattern on ConA-Sepharose than cruzipain and, in addition, it was not recognized by anti-cruzipain serum, facts that strongly suggest the different nature of both CPs. Moroever, evidence is presented indicating that TcCPmet was able to hydrolyse the same chromogenic peptides as cruzipain at optimal alkaline pH values, although with a different order of effectiveness. Our results indicate the presence of a novel CP secreted by metacyclic trypomastigotes and reinforces the important role of these enzymes in metacyclogenesis.

Regulation of Trypanosoma cruzi invasion of nonphagocytic cells by the endocytically active GTPases dynamin, Rab5, and Rab7.

During invasion of nonphagocytic cells by Trypanosoma cruzi (T. cruzi), host cell lysosomes are recruited to the plasma membrane attachment site followed by lysosomal enzyme secretion. The membrane trafficking events involved in invasion have not been delineated. We demonstrate here that T. cruzi invasion of nonphagocytic cells was completely abolished by overexpression of a dominant negative mutant of dynamin. Likewise, overexpression of a dominant negative mutant of Rab5, the rate-limiting GTPase for endocytosis, resulted in reduced infection rates compared with cells expressing Rab5 wild-type. Moreover, cells expressing the activated mutant of Rab5 experienced higher infection rates. A similar pattern was also observed when Rab7-transfected cells were examined. Confocal microscopy experiments showed that parasites colocalized with green fluorescent protein-Rab5-positive early endosomes after 5 min of invasion. These data clearly indicate that newly forming T. cruzi phagosomes first interact with an early endosomal compartment and subsequently with other late component markers before lysosomal interaction occurs.

Trypanosoma cruzi: conditions required to improve metacyclic differentiation in axenic culture

En el presente trabajo se estudió el comportamieto de varias cepas y clones de Epi de T. cruzi en medios estimulantes de la diferenciación: M 16, TAUP, TAUS, LIT-hemina y G-IH. Los resultados mostraron que en nuestras condiciones experimentales el medio G-IH fue el único adecuado para producir morfogénesis Epi-Mtc. La temperatura óptima del proceso tanto de multiplicación como de diferenciación en ese medio fue 28-C. el empleo G-IH empobrecido en nutrientes por dilución no indujo diferenciación. Los parásitos que se obtuvieron de medio bifásico entre 24 a 48 h de cultivados fueron los óptimos para obtener morfogénesis. El bloqueo de la oferta de Ca++ externo afectó la capacidad de diferenciarse de las cepas Tul y RA en grado variable

Citocromos en diferentes estadíos, cepas y poblaciones de Trypanosoma cruzi / Cytochromes in different stages, strains and populations of Trypanosoma cruzi

Los espectros diferenciales de muestras "reducida menos oxidada" (Red-Ox) y "reducida. CO menos reducida" (Re. CO-Red) de tripomastigotes metacíclicos del Trypanosoma cruzi (cepa Tulahuen) revelaron la presencia de los citocrómos aa3, b, c555 y o. Espectros concordantes se obtuvieron con la fracción mitocondrial de la forma epimastigote de la misma cepa, después de filtrar las membranas por Sephadex G-50 para excluir hemoproteínas espúreas provenientes del medio de cultivo o hemoproteínas solubles (hemoglobina, peroxidasa, catalasa, etc.), o citocrómos P-420 y P-450. El tratamiento de la fracción mitocondrial cruda con guanidina y colato de Na y la formación de complejos con cianuro, revelaron que el citocrómo o es un constituyente integral de esas membranas. El análisis de los piridinahemocrómos provenientes de los citocrómos mitocondriales demostró la presencia de los hemos A, B y C. Una banda espectral a 625 nm en los espectros de los tripomastigotes, en las membranas mitocondriales, y en los piridina-hemocrómos indicó la presencia de un citocrómo d. Un examen comparativo del contenido en citocrómos o y b de una serie de poblaciones de T. cruzi, provenientes originariamente en su mayoría de pacientes con formas agudas o crónicas de la enfermedad de Chagas, demostró variabilidad en el contenido en citocrómos y falta de correlación de este parámetro con las propiedades biológicas del parásito, en particular con la letalidad para el ratón