The parasite Trypanosoma brucei possesses a large family of transmembrane receptor–like adenylate cyclases. Activation of these enzymes requires the dimerization of the catalytic domain and typically occurs under stress. Using a dominant-negative strategy, we found that reducing adenylate cyclase activity by about 50% allowed trypanosome growth but reduced the parasite’s ability to control the early innate immune defense of the host. Specifically, activation of trypanosome adenylate cyclase resulting from parasite phagocytosis by liver myeloid cells inhibited the synthesis of the trypanosome-controlling cytokine tumor necrosis factor–α through activation of protein kinase A in these cells. Thus, adenylate cyclase activity of lyzed trypanosomes favors early host colonization by live parasites. The role of adenylate cyclases at the host-parasite interface could explain the expansion and polymorphism of this gene family.

Here we report the monitoring of the digestive tract colonization of Rhodnius prolixus by Trypanosoma cruzi using an accurate determination of the parasite load by qPCR coupled with fluorescence and bioluminescence imaging (BLI). These complementary methods revealed critical steps necessary for the parasite population to colonize the insect gut and establish vector infection. qPCR analysis of the parasite load in the insect gut showed several limitations due mainly to the presence of digestive-derived products that are thought to degrade DNA and inhibit further the PCR reaction. We developed a real-time PCR strategy targeting the T. cruzi repetitive satellite DNA sequence using as internal standard for normalization, an exogenous heterologous DNA spiked into insect samples extract, to precisely quantify the parasite load in each segment of the insect gut (anterior midgut, AM, posterior midgut, PM, and hindgut, H). Using combined fluorescence microscopy and BLI imaging as well as qPCR analysis, we showed that during their journey through the insect digestive tract, most of the parasites are lysed in the AM during the first 24 hours independently of the gut microbiota. During this short period, live parasites move through the PM to establish the onset of infection. At days 3–4 post-infection (p.i.), the parasite population begins to colonize the H to reach a climax at day 7 p.i., which is maintained during the next two weeks. Remarkably, the fluctuation of the parasite number in H remains relatively stable over the two weeks after refeeding, while the populations residing in the AM and PM increases slightly and probably constitutes the reservoirs of dividing epimastigotes. These data show that a tuned dynamic control of the population operates in the insect gut to maintain an equilibrium between non-dividing infective trypomastigote forms and dividing epimastigote forms of the parasite, which is crucial for vector competence. Although the key aspects of the T. cruzi life cycle were described more than one century ago, the development and interactions of T. cruzi with its vector are poorly characterized. By dissection of different compartments of the triatomine gut (prototype Rhodnius prolixus) (i.e., AM, PM and H) at regular time intervals, we evaluated trypanosome development within the insect using an accurate qPCR assay. qPCR analysis of trypanosomal colonization and clearance dynamics in real-time were confirmed in vivo using both fluorescence and bioluminescence imaging, which revealed massive parasite lysis during the first 24 hours post-feeding (p.f.). After one week, the parasite succeeded in establishing a resident population in each compartment of the gut, albeit at varying levels. From one week after the onset of infection in the AM and PM, some resident forms agglomerated into rosettes, clustering in close association with the vector tissue and constituting potential parasite reservoirs of the bug. For the first time, we have described a methodology to accurately quantify parasites in the insect gut that would be a useful tool for evaluating the impact of RNAi silencing of insect genes during the course of infection by T. cruzi.

Trypanosoma brucei, etiological agent of Sleeping Sickness in Africa, is the prototype of African trypanosomes, protozoan extracellular flagellate parasites transmitted by saliva (Salivaria). In these parasites the molecular controls of the cell cycle and environmental sensing are elaborate and concentrated at the flagellum. Genomic analyses suggest that these parasites appear to differ considerably from the host in signaling mechanisms, with the exception of receptor-type adenylate cyclases (AC) that are topologically similar to receptor-type guanylate cyclase (GC) of higher eukaryotes but control a new class of cAMP targets of unknown function, the cAMP response proteins (CARPs), rather than the classical protein kinase A cAMP effector (PKA). T. brucei possesses a large polymorphic family of ACs, mainly associated with the flagellar membrane, and these are involved in inhibition of the innate immune response of the host prior to the massive release of immunomodulatory factors at the first peak of parasitemia. Recent evidence suggests that in T. brucei several insect-specific AC isoforms are involved in social motility, whereas only a few AC isoforms are involved in cytokinesis control of bloodstream forms, attesting that a complex signaling pathway is required for environmental sensing. In this review, after a general update on cAMP signaling pathway and the multiple roles of cAMP, I summarize the existing knowledge of the mechanisms by which pathogenic microorganisms modulate cAMP levels to escape immune defense.

Chagas disease is a vector-borne parasitic disease caused by the flagellated protozoan Trypanosoma cruzi and transmitted to humans by a large group of bloodsucking triatomine bugs. Triatomine insects, such as Rhodnius prolixus, ingest a huge amount of blood in a single meal. Their midgut represents an important interface for triatomine–trypanosome interactions. Furthermore, the development of parasites and their vectorial transmission are closely linked to the blood feeding and digestion; thus, an understanding of their physiology is essential for the development of new strategies to control triatomines. In this study, we used label-free quantitative proteomics to identify and analyze the early effect of blood feeding on protein expression in the midgut of Rhodnius prolixus. We both identified and quantified 124 proteins in the anterior midgut (AM) and 40 in the posterior midgut (PM), which vary significantly 6 h after feeding. The detailed analysis of these proteins revealed their predominant involvement in the primary function of hematophagy, including proteases, proteases inhibitors, amino acids metabolism, primary metabolites processing, and protein folding. Interestingly, our proteomics data show a potential role of the AM in protein digestion. Moreover, proteins related to detoxification processes and innate immunity, which are largely accepted to be triggered by blood ingestion, were mildly modulated. Surprisingly, one third of blood-regulated proteins in the AM have unknown function. This work contributes to the improvement of knowledge on the digestive physiology of triatomines in the early hours post-feeding. It provides key information for selecting new putative targets for the development of triatomine control tools and their potential role in the vector competence, which could be applied to other vector species.

Chagas disease is one of the most common parasitic infections in Latin America, which is transmitted by hematophagous triatomine bugs, of which Rhodnius prolixus is the vector prototype for the study of this disease. The protozoan parasite Trypanosoma cruzi, the etiologic agent of this disease, is transmitted by the vector to humans through the bite wound or mucosa. The passage of the parasite through the digestive tract of its vector constitutes a key step in its developmental cycle. Herewith, by a using high-throughput proteomic tool in order to characterize the midgut proteome of R. prolixus, we describe a set of functional groups of proteins, as well as the biological processes in which they are involved. This is the first proteomic analysis showing an elaborated hematophagy machinery involved in the digestion of blood, among which, several families of proteases have been characterized. The evaluation of the activity of cathepsin D proteases in the anterior part of the digestive tract of the insect suggested the existence of a proteolytic activity within this compartment, suggesting that digestion occurs early in this compartment. Moreover, several heat shock proteins, blood clotting inhibitors, and a powerful antioxidant enzyme machinery against reactive oxygen species (ROS) and cell detoxification have been identified. Highlighting the complexity and importance of the digestive physiology of insects could be a starting point for the selection of new targets for innovative control strategies of Chagas disease.