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The Trypanosoma cruzi adenylyl cyclase (AC) multigene family encodes different isoforms (around 15) sharing a variable large N-terminal domain, which is extracellular and receptor-like, followed by a transmembrane helix and a conserved C-terminal catalytic domain. It was proposed that these key enzymes in the cAMP signalling pathway allow the parasite to sense its changing extracellular milieu in order to rapidly adapt to its new environment, which is generally achieved through a differentiation process. One of the critical differentiation events the parasitic protozoan T. cruzi undergoes during its life cycle, known as metacyclogenesis, occurs in the digestive tract of the insect and corresponds to the differentiation from noninfective epimastigotes to infective metacyclic trypomastigote forms. By in vitro monitoring the activity of AC during metacyclogenesis, we showed that both the activity of AC and the intracellular cAMP content follow a similar pattern of transient stimulation in a two-step process, with a first activation peak occurring during the first hours of nutritional stress and a second peak between 6 and 48 h, corresponding to the cellular adhesion. During this differentiation process, a general mechanism of upregulation of AC expression of both mRNA and protein is triggered and in particular for a major subclass of these enzymes that are present in various gene copies commonly associated to the THT gene clusters. Although the scattered genome distribution of these gene copies is rather unusual in trypanosomatids and seems to be a recent acquisition in the evolution of the T. cruzi clade, their encoded product redistributed on the flagellum of the parasite upon differentiation could be important to sense the extracellular milieu.

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DNA topoisomerases are enzymes that modulate DNA topology. Among them, topoisomerase 3α is engaged in genomic maintenance acting in DNA replication termination, sister chromatid separation, and dissolution of recombination intermediates. To evaluate the role of this enzyme in Trypanosoma cruzi, the etiologic agent of Chagas disease, a topoisomerase 3α knockout parasite (TcTopo3α KO) was generated, and the parasite growth, as well as its response to several DNA damage agents, were evaluated. There was no growth alteration caused by the TcTopo3α knockout in epimastigote forms, but a higher dormancy rate was observed. TcTopo3α KO trypomastigote forms displayed reduced invasion rates in LLC-MK2 cells when compared with the wild-type lineage. Amastigote proliferation was also compromised in the TcTopo3α KO, and a higher number of dormant cells was observed. Additionally, TcTopo3α KO epimastigotes were not able to recover cell growth after gamma radiation exposure, suggesting the involvement of topoisomerase 3α in homologous recombination. These parasites were also sensitive to drugs that generate replication stress, such as cisplatin (Cis), hydroxyurea (HU), and methyl methanesulfonate (MMS). In response to HU and Cis treatments, TcTopo3α KO parasites showed a slower cell growth and was not able to efficiently repair the DNA damage induced by these genotoxic agents. The cell growth phenotype observed after MMS treatment was similar to that observed after gamma radiation, although there were fewer dormant cells after MMS exposure. TcTopo3α KO parasites showed a population with sub-G1 DNA content and strong γH2A signal 48 h after MMS treatment. So, it is possible that DNA-damaged cell proliferation due to the absence of TcTopo3α leads to cell death. Whole genome sequencing of MMS-treated parasites showed a significant reduction in the content of the multigene families DFG-1 and RHS, and also a possible erosion of the sub-telomeric region from chromosome 22, relative to non-treated knockout parasites. Southern blot experiments suggest telomere shortening, which could indicate genomic instability in TcTopo3α KO cells owing to MMS treatment. Thus, topoisomerase 3α is important for homologous recombination repair and replication stress in T. cruzi, even though all the pathways in which this enzyme participates during the replication stress response remains elusive.

The widespread occurrence of anthelmintic-resistant gastrointestinal nematodes (GINs), particularly Haemonchus contortus, in sheep production systems has magnified the need to identify and develop alternative control strategies. Strategies include the selection of genetically GIN-resistant sheep and the implementation of biological parasite control to reduce dependence on anthelmintic drugs. In this study, we aimed to establish the molecular identity of bacterial communities present in the abomasum of sheep classified as resistant or susceptible to H. contortus. Thirty-eight sheep were experimentally infected with L3 Haemonchus contortus and analyzed for fecal egg count (FEC), and hematocrit (Ht) to establish haemonchosis resistance or susceptibility. Four resistant sheep (RS) and four susceptible sheep (SS) were selected for microbial sampling and subsequent phylogenetic analysis. Molecular identification of the bacteria was based on amplification of the bacterial 16S rRNA gene, construction of a 16S rDNA clone library, and subsequent gene sequencing. Significant differences (p = 0.05) were observed in the occurrence of different phyla identified in RS and SS libraries: Firmicutes (61.4% and 37.2%, respectively), Proteobacteria (10.2% and 37.2%, respectively), Bacteroidetes (12.8% and 5.8%, respectively), and unclassified bacteria (12.8% and 17%, respectively). Differences between the proportions of bacterial communities present in the RS and SS pool samples were observed, contributing as a first step toward the assessment of the association between the gastrointestinal tract microbiota and nematode resistance in sheep. ResumoNa produção de ovinos, a disseminação de nematódeos gastrintestinais (NGI) resistentes aos antihelmínticos, em especial Haemonchus contortus, tem levado à busca de estratégias de controle alternativo, como a seleção de animais geneticamente resistentes aos NGI e o controle biológico. Neste trabalho, buscou-se identificar molecularmente as comunidades bacterianas presentes no abomaso de animais classificados como resistentes ou susceptíveis ao H. contortus. Foram utilizados 38 ovinos para classificação em resistentes ou susceptíveis à hemoncose, por meio de infecções experimentais com L3 de H. contortus e posterior análise de variações na contagem de ovos por grama de fezes (∆OPG) e hematócrito (∆Ht). Destes, foram selecionados para colheita de material e posterior análise filogenética, quatro ovinos resistentes (OR) e quatro susceptíveis (OS). A identificação molecular de bactérias foi realizada por técnicas moleculares a partir da amplificação do gene RNAr 16S bacteriano, construção de bibliotecas de clones de RNAr 16S e posterior sequenciamento gênico. O trabalho mostrou diferença significativa (p=0,05) na porcentagem dos filos predominantes para as bibliotecas OR e OS, respectivamente: Firmicutes (61,4% e 37,2%), Proteobacterias (10,2% e 37,2%), Bacteroidetes (12,8% e 5,8%) e Bactérias não classificadas (12,8% e 17%). Diferenças entre os pools OR e OS com relação à proporção de...