João Luís Reis-Cunha

Evasion of the Immune Response by Trypanosoma cruzi during Acute Infection

Trypanosoma cruzi is the etiologic agent of Chagas disease, a neglected tropical disease that affects millions of people mainly in Latin America. To establish a life-long infection, T. cruzi must subvert the vertebrate host’s immune system, using strategies that can be traced to the parasite’s life cycle. Once inside the vertebrate host, metacyclic trypomastigotes rapidly invade a wide variety of nucleated host cells in a membrane-bound compartment known as the parasitophorous vacuole, which fuses to lysosomes, originating the phagolysosome. In this compartment, the parasite relies on a complex network of antioxidant enzymes to shield itself from lysosomal oxygen and nitrogen reactive species. Lysosomal acidification of the parasitophorous vacuole is an important factor that allows trypomastigote escape from the extremely oxidative environment of the phagolysosome to the cytoplasm, where it differentiates into amastigote forms. In the cytosol of infected macrophages, oxidative stress instead of being detrimental to the parasite, favors amastigote burden, which then differentiates into bloodstream trypomastigotes. Trypomastigotes released in the bloodstream upon the rupture of the host cell membrane express surface molecules, such as calreticulin and GP160 proteins, which disrupt initial and key components of the complement pathway, while others such as glycosylphosphatidylinositol-mucins stimulate immunoregulatory receptors, delaying the progression of a protective immune response. After an immunologically silent entry at the early phase of infection, T. cruzi elicits polyclonal B cell activation, hypergammaglobulinemia, and unspecific anti-T. cruzi antibodies, which are inefficient in controlling the infection. Additionally, the coexpression of several related, but not identical, epitopes derived from trypomastigote surface proteins delays the generation of T. cruzi-specific neutralizing antibodies. Later in the infection, the establishment of an anti-T. cruzi CD8+ immune response focused on the parasite’s immunodominant epitopes controls parasitemia and tissue infection, but fails to completely eliminate the parasite. This outcome is not detrimental to the parasite, as it reduces host mortality and maintains the parasite infectivity toward the insect vectors.

Chromosomal copy number variation reveals differential levels of genomic plasticity in distinct Trypanosoma cruzi strains

BackgroundTrypanosoma cruzi, the etiologic agent of Chagas disease, is currently divided into six discrete typing units (DTUs), named TcI–TcVI. CL Brener, the reference strain of the T. cruzi genome project, is a hybrid with a genome assembled into 41 putative chromosomes. Gene copy number variation (CNV) is well documented as an important mechanism to enhance gene expression and variability in T. cruzi. Chromosomal CNV (CCNV) is another level of gene CNV in which whole blocks of genes are expanded simultaneously. Although the T. cruzi karyotype is not well defined, several studies have demonstrated a significant variation in the size and content of chromosomes between different T. cruzi strains. Despite these studies, the extent of diversity in CCNV among T. cruzi strains based on a read depth coverage analysis has not been determined.ResultsWe identify the CCNV in T. cruzi strains from the TcI, TcII and TcIII DTUs, by analyzing the depth coverage of short reads from these strains using the 41 CL Brener chromosomes as reference. This study led to the identification of a broader extent of CCNV in T. cruzi than was previously speculated. The TcI DTU strains have very few aneuploidies, while the strains from TcII and TcIII DTUs present a high degree of chromosomal expansions. Chromosome 31, which is the only chromosome that is supernumerary in all six T. cruzi samples evaluated in this study, is enriched with genes related to glycosylation pathways, highlighting the importance of glycosylation to parasite survival.ConclusionsIncreased gene copy number due to chromosome amplification may contribute to alterations in gene expression, which represents a strategy that may be crucial for parasites that mainly depend on post-transcriptional mechanisms to control gene expression.

Comparative genomic analysis of Leishmania (Viannia) peruviana and Leishmania (Viannia) braziliensis.

BackgroundThe Leishmania (Viannia) braziliensis complex is responsible for most cases of New World tegumentary leishmaniasis. This complex includes two closely related species but with different geographic distribution and disease phenotypes, L. (V.) peruviana and L. (V.) braziliensis. However, the genetic basis of these differences is not well understood and the status of L. (V.) peruviana as distinct species has been questioned by some.Here we sequenced the genomes of two L. (V.) peruviana isolates (LEM1537 and PAB-4377) using Illumina high throughput sequencing and performed comparative analyses against the L. (V.) braziliensis M2904 reference genome. Comparisons were focused on the detection of Single Nucleotide Polymorphisms (SNPs), insertions and deletions (INDELs), aneuploidy and gene copy number variations.ResultsWe found 94,070 variants shared by both L. (V.) peruviana isolates (144,079 in PAB-4377 and 136,946 in LEM1537) against the L. (V.) braziliensis M2904 reference genome while only 26,853 variants separated both L. (V.) peruviana genomes.Analysis in coding sequences detected 26,750 SNPs and 1,513 indels shared by both L. (V.) peruviana isolates against L. (V.) braziliensis M2904 and revealed two L. (V.) braziliensis pseudogenes that are likely to have coding potential in L. (V.) peruviana. Chromosomal read density and allele frequency profiling showed a heterogeneous pattern of aneuploidy with an overall disomic tendency in both L. (V.) peruviana isolates, in contrast with a trisomic pattern in the L. (V.) braziliensis M2904 reference.Read depth analysis allowed us to detect more than 368 gene expansions and 14 expanded gene arrays in L. (V.) peruviana, and the likely absence of expanded amastin gene arrays.ConclusionsThe greater numbers of interspecific SNP/indel differences between L. (V.) peruviana and L. (V.) braziliensis and the presence of different gene and chromosome copy number variations support the classification of both organisms as closely related but distinct species.The extensive nucleotide polymorphisms and differences in gene and chromosome copy numbers in L. (V.) peruviana suggests the possibility that these may contribute to some of the unique features of its biology, including a lower pathology and lack of mucosal development.

Genome-Wide Screening and Identification of New Trypanosoma cruzi Antigens with Potential Application for Chronic Chagas Disease Diagnosis

The protozoan Trypanosoma cruzi is the etiologic agent of Chagas disease, an infection that afflicts approximately 8 million people in Latin America. Diagnosis of chronic Chagas disease is currently based on serological tests because this condition is usually characterized by high anti-T. cruzi IgG titers and low parasitemia. The antigens used in these assays may have low specificity due to cross reactivity with antigens from related parasite infections, such as leishmaniasis, and low sensitivity caused by the high polymorphism among T. cruzi strains. Therefore, the identification of new T. cruzi-specific antigens that are conserved among the various parasite discrete typing units (DTUs) is still required. In the present study, we have explored the hybrid nature of the T. cruzi CL Brener strain using a broad genome screening approach to select new T. cruzi antigens that are conserved among the different parasite DTUs and that are absent in other trypanosomatid species. Peptide arrays containing the conserved antigens with the highest epitope prediction scores were synthesized, and the reactivity of the peptides were tested by immunoblot using sera from C57BL/6 mice chronically infected with T. cruzi strains from the TcI, TcII or TcVI DTU. The two T. cruzi proteins that contained the most promising peptides were expressed as recombinant proteins and tested in ELISA experiments with sera from chagasic patients with distinct clinical manifestations: those infected with T. cruzi from different DTUs and those with cutaneous or visceral leishmaniasis. These proteins, named rTc_11623.20 and rTc_N_10421.310, exhibited 94.83 and 89.66% sensitivity, 98.2 and 94.6% specificity, respectively, and a pool of these 2 proteins exhibited 96.55% sensitivity and 98.18% specificity. This work led to the identification of two new antigens with great potential application in the diagnosis of chronic Chagas disease.

Epitope Mapping of the HSP83.1 Protein of Leishmania braziliensis Discloses Novel Targets for Immunodiagnosis of Tegumentary and Visceral Clinical Forms of Leishmaniasis

ABSTRACT Gold standard serological diagnostic methods focus on antigens that elicit a strong humoral immune response that is specific to a certain pathogen. In this study, we used bioinformatics approaches to identify linear B-cell epitopes that are conserved among Leishmania species but are divergent from the host species Homo sapiens and Canis familiaris and from Trypanosoma cruzi, the parasite that causes Chagas disease, to select potential targets for the immunodiagnosis of leishmaniasis. Using these criteria, we selected heat shock protein 83.1 of Leishmania braziliensis for this study. We predicted three linear B-cell epitopes in its sequence. These peptides and the recombinant heat shock protein 83.1 (rHSP83.1) were tested in enzyme-linked immunosorbent assays (ELISAs) against serum samples from patients with tegumentary leishmaniasis (TL) and visceral leishmaniasis (VL) and from dogs infected with Leishmania infantum (canine VL [CVL]). Our data show that rHSP83.1 is a promising target in the diagnosis of TL. We also identified specific epitopes derived from HSP83.1 that can be used in the diagnosis of human TL (peptide 3), both human and canine VL (peptides 1 and 3), and all TL, VL, and CVL clinical manifestations (peptide 3). Receiver operating characteristic (ROC) curves confirmed the superior performance of rHSP83.1 and peptides 1 and 3 compared to that of the soluble L. braziliensis antigen and the reference test kit for the diagnosis of CVL in Brazil (EIE-LVC kit; Bio-Manguinhos, Fiocruz). Our study thus provides proof-of-principle evidence of the feasibility of using bioinformatics to identify novel targets for the immunodiagnosis of parasitic diseases using proteins that are highly conserved throughout evolution.

Comparative genomics of canine-isolated Leishmania (Leishmania) amazonensis from an endemic focus of visceral leishmaniasis in Governador Valadares, southeastern Brazil.

Leishmaniasis is a highly diverse group of diseases caused by kinetoplastid of the genus Leishmania. These parasites are taxonomically diverse, with human pathogenic species separated into two subgenera according to their development site inside the alimentary tract of the sand fly insect vector. The disease encompasses a variable spectrum of clinical manifestations with tegumentary or visceral symptoms. Among the causative species in Brazil, Leishmania (Leishmania) amazonensis is an important etiological agent of human cutaneous leishmaniasis that accounts for more than 8% of all cases in endemic regions. L. (L.) amazonensis is generally found in the north and northeast regions of Brazil. Here, we report the first isolation of L. (L.) amazonensis from dogs with clinical manifestations of visceral leishmaniasis in Governador Valadares, an endemic focus in the southeastern Brazilian State of Minas Gerais where L. (L.) infantum is also endemic. These isolates were characterized in terms of SNPs, chromosome and gene copy number variations, confirming that they are closely related to a previously sequenced isolate obtained in 1973 from the typical Northern range of this species. The results presented in this article will increase our knowledge of L. (L.) amazonensis-specific adaptations to infection, parasite survival and the transmission of this Amazonian species in a new endemic area of Brazil.

Gene and Chromosomal Copy Number Variations as an Adaptive Mechanism Towards a Parasitic Lifestyle in Trypanosomatids

Trypanosomatids are a group of kinetoplastid parasites including some of great public health importance, causing debilitating and life-long lasting diseases that affect more than 24 million people worldwide. Among the trypanosomatids, Trypanosoma cruzi, Trypanosoma brucei and species from the Leishmania genus are the most well studied parasites, due to their high prevalence in human infections. These parasites have an extreme genomic and phenotypic variability, with a massive expansion in the copy number of species-specific multigene families enrolled in host-parasite interactions that mediate cellular invasion and immune evasion processes. As most trypanosomatids are heteroxenous, and therefore their lifecycles involve the transition between different hosts, these parasites have developed several strategies to ensure a rapid adaptation to changing environments. Among these strategies, a rapid shift in the repertoire of expressed genes, genetic variability and genome plasticity are key mechanisms. Trypanosomatid genomes are organized into large directional gene clusters that are transcribed polycistronically, where genes derived from the same polycistron may have very distinct mRNA levels. This particular mode of transcription implies that the control of gene expression operates mainly at post-transcriptional level. In this sense, gene duplications/losses were already associated with changes in mRNA levels in these parasites. Gene duplications also allow the generation of sequence variability, as the newly formed copy can diverge without loss of function of the original copy. Recently, aneuploidies have been shown to occur in several Leishmania species and T. cruzi strains. Although aneuploidies are usually associated with debilitating phenotypes in superior eukaryotes, recent data shows that it could also provide increased fitness in stress conditions and generate drug resistance in unicellular eukaryotes. In this review, we will focus on gene and chromosomal copy number variations and their relevance to the evolution of trypanosomatid parasites.

Genetic Polymorphisms and Phenotypic Profiles of Sulfadiazine-Resistant and Sensitive Toxoplasma gondii Isolates Obtained from Newborns with Congenital Toxoplasmosis in Minas Gerais, Brazil

Background Previous Toxoplasma gondii studies revealed that mutations in the dhps (dihydropteroate synthase) gene are associated with resistance to sulfonamides. Although Brazilian strains are genotypically different, very limited data are available regarding the susceptibility of strains obtained from human to sulfonamides. The aim of this study was to evaluate the efficacy of sulfadiazine (SDZ) against Brazilian isolates of T. gondii and verify whether isolates present polymorphisms in the dhps gene. We also investigated whether the virulence-phenotype and/or genotype were associated with the profile of susceptibility to SDZ. Methods Five T. gondii isolates obtained from newborns with congenital toxoplasmosis were used to verify susceptibility. Mice were infected with 104 tachyzoites and orally treated with different doses of SDZ. The mortality curve was evaluated by the Log-rank test. The presence of polymorphisms in the dhps gene was verified using sequencing. A descriptive analysis for 11 Brazilian isolates was used to assess the association between susceptibility, genotype, and virulence-phenotype. Results Statistical analysis showed that TgCTBr03, 07, 08, and 16 isolates were susceptible to SDZ, whereas TgCTBr11 isolate presented a profile of resistance to SDZ. Nineteen polymorphisms were identified in dhps exons. Seven polymorphisms corresponded to non-synonymous mutations, with four being new mutations, described for the first time in this study. No association was found between the profile of susceptibility and the virulence-phenotype or genotype of the parasite. Conclusions There is a high variability in the susceptibilities of Brazilian T. gondii strains to SDZ, with evidence of drug resistance. Despite the large number of polymorphisms identified, the profile of susceptibility to SDZ was not associated with any of the dhps variants identified in this study. Other genetic factors, not yet determined, may be associated with the resistance to SDZ; thus, further studies are needed as a basis for a more adequate toxoplasmosis treatment.

Repeat-driven generation of antigenic diversity in a major human pathogen, Trypanosoma cruzi

Trypanosoma cruzi, a zoonotic kinetoplastid protozoan with a complex genome, is the causative agent of American trypanosomiasis (Chagas disease). The parasite uses a highly diverse repertoire of surface molecules, with roles in cell invasion, immune evasion and pathogenesis. Thus far, the genomic regions containing these genes have been impossible to resolve and it has been impossible to study the structure and function of the several thousand repetitive genes encoding the surface molecules of the parasite. We here present an improved genome assembly of a T. cruzi clade I (TcI) strain using high coverage PacBio single molecule sequencing, together with Illumina sequencing of 34 T. cruzi TcI isolates and clones from different geographic locations, sample sources and clinical outcomes. Resolution of the surface molecule gene structure reveals an unusual duality in the organisation of the parasite genome, a core genomic region syntenous with related protozoa flanked by unique and highly plastic subtelomeric regions encoding surface antigens. The presence of abundant interspersed retrotransposons in the subtelomeres suggests that these elements are involved in a recombination mechanism for the generation of antigenic variation and evasion of the host immune response. The comparative genomic analysis of the cohort of TcI strains revealed multiple cases of such recombination events involving surface molecule genes and has provided new insights into T. cruzi population structure.

Detection of multiple circulating Leishmania species in Lutzomyia longipalpis in the city of Governador Valadares, southeastern Brazil

Background Leishmaniasis encompasses a group of diverse clinical diseases caused by protozoan parasites of the Leishmania genus. This disease is a major public health problem in the New World affecting people exposed in endemic regions. The city of Governador Valadares (Minas Gerais/Brazil) is a re-emerging area for visceral leishmaniasis, with 191 human cases reported from 2008 to 2017 and a lethality rate of 14.7%. The transmission of the parasite occurs intensely in this region with up to 22% of domestic dogs with positive serology for the visceral form. Lu. longipalpis is one of the most abundant sand fly species in this area. Despite this scenario, so far there is no information regarding the circulating Leishmania species in the insect vector Lutzomyia longipalpis in this focus. Methodology/Principal Findings We collected 616 female Lutzomyia longipalpis sand flies between January and September 2015 in the Vila Parque Ibituruna neighborhood (Governador Valadares/MG), which is located on a transitional area between the sylvatic and urban environments with residences built near a preserved area. After DNA extraction of individual sand flies, the natural Leishmania infections in Lu. longipalpis were detected by end-point PCR, using primers derived from kDNA sequences, specific for L. (Leishmania) or L. (Viannia) subgenus. The sensitivity of these PCR reactions was 0.1 pg of DNA for each Leishmania subgenus and the total infection rate of 16.2% (100 positive specimens). Species-specific PCR detected the presence of multiple Leishmania species in infected Lu. longipalpis specimens in Governador Valadares, including L. amazonensis (n=3), L. infantum (n=28), L. (Viannia) spp. (n=20), coinfections with L. infantum and L. (Viannia) spp. (n=5), and L. (Leishmania) spp (n=44). Conclusions Our results demonstrate that multiple Leishmania species circulate in Lu. longipalpis in Governador Valadares and reveal a potential increasing risk of transmission of the different circulating parasite species. This information is a key factor for planning surveillance and effective control strategies against leishmaniasis in this endemic focus. Author summary Leishmaniasis is a neglected tropical disease transmitted to mammals by the bite of sand flies infected with parasites of the Leishmania genus. This disease affects millions of people in various regions of the world, including Brazil. The municipality of Governador Valadares (Minas Gerais/Brazil) is a re-emergent focus of intense transmission of leishmaniasis, with a high number of human cases and a high prevalence of infected domestic dogs. To develop better leishmaniasis control strategies for the region, we performed a surveillance study of Lu. longipalpis, the main vector of visceral leishmaniasis in Brazil, and identified circulating species of Leishmania in this insect vector. We estimate that the natural infection rate of Lu. longipalpis for these parasites was of 16.2% in the study area. We also detected the presence of multiple circulating Leishmania species (L. amazonensis, L. infantum and Viannia subgenus) in Lu. longipalpis in Governador Valadares city, including 5 sand flies coinfected with L. infantum and L. (Viannia). Thus, our results reinforce the need for a rigid and systematic control of the sand flies monitoring in this area, due to the potential risk of transmission of different species of the Leishmania parasites.