Trypanosoma cruzi is the agent of Chagas disease, transmitted by hematophagous triatomine vectors. Establishing transmission cycles is key to understand the epidemiology of the disease, but integrative assessments of ecological interactions shaping parasite transmission are still limited. Current approaches also lack sensitivity to assess the full extent of this ecological diversity. Here we developed a metabarcoding approach based on next-generation sequencing to identify triatomine gut microbiome, vertebrate feeding hosts, and parasite diversity and their potential interactions. We detected a dynamic microbiome in Triatoma dimidiata, including 23 bacterial orders, which differed according to blood sources. Fourteen vertebrate species served as blood sources, corresponding to domestic, synantropic and sylvatic species, although four (human, dog, cow and mice) accounted for over 50% of blood sources. Importantly, bugs fed on multiple hosts, with up to 11 hosts identified per bug, indicating very frequent host-switching. A high clonal diversity of T. cruzi was detected, with up to 20 haplotypes per bug. This analysis provided much greater sensitivity to detect multiple blood meals and multiclonal infections with T. cruzi, which should be taken into account to develop transmission networks, and characterize the risk for human infection, eventually leading to a better control of disease transmission.

The diversity of Trypanosoma cruzi parasites infecting humans is still poorly understood. We used deep sequencing to analyze this diversity in chagasic patients from Mexico. Such information is crucial to understand transmission cycles and to identify determinants of epidemiological and clinical characteristics of the infection. We analyzed parasite mini-exon spliced-leader sequences following amplification of blood DNA by polymerase chain reaction and deep sequencing. Chagasic patients presented a diverse assemblage of parasite haplotypes covering TcI, TcII, TcV, and TcVI discrete typing units, with a mean (±SEM) of 3.9 ± 0.7 haplotypes/patient, and 47% harbored infections with multiple discrete typing units. Most parasite haplotypes from patients were identical or similar to those for Triatoma dimidiata from the same region, confirming their local circulation. Infection with multiple T. cruzi strains may influence serological diagnostic test results and disease progression in patients and should be taken into account to evaluate associations between parasite diversity and clinical aspects of T. cruzi infections.

Introduction: Chagas disease, caused by the protozoan parasite Trypanosoma cruzi, is a major public health problem in the Americas, and existing drugs have severe limitations. In this context, a vaccine would be an attractive alternative for disease control. One of the difficulties in developing an effective vaccine lies in the high genetic diversity of T. cruzi. In this study, we evaluated the level of sequence diversity of the leading vaccine candidate Tc24 in multiple parasite strains.Methods and Results: We quantified its level of polymorphism within and between T. cruzi discrete typing units (DTUs) and how this potential polymorphism is structured by different selective pressures. We observed a low level of polymorphism of Tc24 protein, weakly associated with parasite DTUs, but not with the geographic origin of the strains. In particular, Tc24 was under strong purifying selection pressure and predicted CD8⁺ T‐cell epitopes were mostly conserved. Tc24 strong conservation may be associated with structural/functional constrains to preserve EF hand domains and their calcium‐binding loops, and Tc24 is likely important for the parasite fitness.Discussion: Together, these results show that a vaccine based on Tc24 is likely to be effective against a wide diversity of parasite strains across the American continent, and further development of this vaccine candidate should be a high priority.

Chagas disease is caused by the protozoan parasite Trypanosoma cruzi, and activation of CD8⁺ T cells is crucial for a protective immune response. Therefore, the identification of antigens with major histocompatibility complex class I epitopes is a crucial step for vaccine development against T. cruzi. Our aim was to identify novel antigens and epitopes by immunoinformatics analysis of the parasite proteome (12 969 proteins) and to validate their immunotherapeutic potential in infected mice. We identified 172 predicted epitopes, using NetMHC and RANKPEP. The corresponding protein sequences were reanalyzed to generate a consensus prediction, and 26 epitopes were selected for in vivo validation. The interferon γ (IFN-γ) recall response of splenocytes from T. cruzi–infected mice confirmed that 10 of 26 epitopes (38%) induced IFN-γ production. The immunotherapeutic potential of a mixture of all 10 peptides was evaluated in infected mice. The therapeutic vaccine was able to control T. cruzi infection, as evidenced by reduced parasitemia, cardiac tissue inflammation, and parasite burden and increased survival. These findings illustrate the benefits of this approach for the rapid development of a vaccine against pathogens with large genomes. The identified peptides and the proteins from which they are derived are excellent candidates for the development of a vaccine against T. cruzi.

Parasitic diseases cause ∼ 500,000 deaths annually and remain a major challenge for therapeutic development. Using a rational design based approach, we developed peptide inhibitors with anti-parasitic activity that were derived from the sequences of parasite scaffold proteins LACK (Leishmania's receptor for activated C-kinase) and TRACK (Trypanosoma receptor for activated C-kinase). We hypothesized that sequences in LACK and TRACK that are conserved in the parasites, but not in the mammalian ortholog, RACK (Receptor for activated C-kinase), may be interaction sites for signaling proteins that are critical for the parasites' viability. One of these peptides exhibited leishmanicidal and trypanocidal activity in culture. Moreover, in infected mice, this peptide was also effective in reducing parasitemia and increasing survival without toxic effects. The identified peptide is a promising new anti-parasitic drug lead, as its unique features may limit toxicity and drug-resistance, thus overcoming central limitations of most anti-parasitic drugs.