The heterologous expression of Escherichia coli MutT enzyme is involved in the protection against oxidative stress in Leishmania braziliensis

BACKGROUND Oxidative stress is responsible for generating DNA lesions and the 8-oxoguanine (8-oxoG) is the most commonly lesion found in DNA damage. When this base is incorporated during DNA replication, it could generate double-strand DNA breaks and cellular death. MutT enzyme hydrolyzes the 8-oxoG from the nucleotide pool, preventing its incorporation during DNA replication. OBJECTIVES To investigate the importance of 8-oxoG in Leishmania infantum and L. braziliensis, in this study we analysed the impact of heterologous expression of Escherichia coli MutT (EcMutT) enzyme in drug-resistance phenotype and defense against oxidative stress. METHODS Comparative analysis of L. braziliensis and L. infantum H2O2 tolerance and cell cycle profile were performed. Lines of L. braziliensis and L. infantum expressing EcMutT were generated and evaluated using susceptibility tests to H2O2 and SbIII, cell cycle analysis, γH2A western blotting, and BrdU native detection assay. FINDINGS Comparative analysis of tolerance to oxidative stress generated by H2O2 showed that L. infantum is more tolerant to exogenous H2O2 than L. braziliensis. In addition, cell cycle analysis showed that L. infantum, after treatment with H2O2, remains in G1 phase, returning to its normal growth rate after 72 h. In contrast, after treatment with H2O2, L. braziliensis parasites continue to move to the next stages of the cell cycle. Expression of the E. coli MutT gene in L. braziliensis and L. infantum does not interfere in parasite growth or in susceptibility to SbIII. Interestingly, we observed that L. braziliensis EcMutT-expressing clones were more tolerant to H2O2 treatment, presented lower activation of γH2A, a biomarker of genotoxic stress, and lower replication stress than its parental non-transfected parasites. In contrast, the EcMutT is not involved in protection against oxidative stress generated by H2O2 in L. infantum. MAIN CONCLUSIONS Our results showed that 8-oxoG clearance in L. braziliensis is important to avoid misincorporation during DNA replication after oxidative stress generated by H2O2.

Avaliação de IFN-γ e IL-10 em cães naturalmente infectados com Leishmania (Leishmania) chagasi com e sem manifestações clínicas / Evaluation of INF-γ and IL-10 in dogs naturally infected by Leishmania (Leishmania) chagasi with and without clinic events

As leishmanioses têm como agentes etiológicos parasitas intracelulares obrigatórios pertencentes ao gênero Leishmania capazes de infectar diferentes espécies de mamíferos e nestes se reproduzirem dentro do sistema fagocítico mononuclear. Os cães domésticos são os principais responsáveis pela manutenção da cadeia epidemiológica da doença, podendo apresentar uma grande variedade de perfis clínicos, desde aparentemente sadios a severamente acometidos. Avaliou-se a expressão das citocinas de cães naturalmente infectados com Leishmania (Leishmania) chagasi. Foram coletadas 50 amostras, sendo 20 de animais positivos e sintomáticos para Leishmaniose Visceral Canina (LVC), 20 de animais positivos e assintomáticos e 10 de animais sabidamente negativos para a LVC. As amostras foram analisadas pelo teste imunocromatográfico rápido Dual Path Platform (DPP/Biomanguinhos®) e pelo ELISA (EIE/Biomanguinhos®) indireto para detecção de anticorpos anti-Leishmania. Após as confirmações dos testes, foi realizado o ELISA de captura (R & D Systems) para quantificação das citocinas IL-10 e IFN-γ. Houve diferença estatística entre os grupos observando um aumento nos níveis de IFN-γ nos animais assintomáticos e um aumento de IL-10 nos sintomáticos.(AU)

Leishmaniasis has as obligatory intracellular parasitic etiological agents belonging to the genus Leishmania capable of infecting different species of mammals and reproducing them within the mononuclear phagocytic system. Domestic dogs are the main responsible for maintaining the epidemiological chain of the disease, presenting a wide variety of clinical profiles, from apparently healthy to severely affected. The expression of the cytokines from dogs naturally infected with Leishmania (Leishmania) chagasi was evaluated. Blood samples were collected from 50 animals, 20 from positive and symptomatic dogs for Leishmaniasis Canine (CVL), 20 from positive asymptomatic animals and 10 negative. Samples were analyzed by immunochromatographic test Dual Path Platform (DPP/Biomanguinhos®) and by indirect ELISA (EIE/Biomanguinhos®) for detection of anti-Leishmania antibodies. There was statistical difference between the groups observing an increase in IFN-γ levels in asymptomatic animals and an IL-10 increase in symptomatic.(AU)

Isolation of Leishmania infantum, zymodeme MON-1 from canine and human visceral leishmaniasis on Margarita Island, Venezuela

An increase in the incidence of human visceral leishmaniasis (HVL) has been detected in recent years on Margarita Island, located off the NE coast of Venezuela. Recent studies have revealed reactivity to rK39 antigen (Leishmania chagasi) in 20 percent of 541 sera from domestic dogs in endemic communities; PCR reactions were positive using primers for the L. donovani complex. Here we report that isolates from human and canine infection, identified by isoenzyme analysis, correspond to L. infantum, zymodeme MON-1. This appears to be the first isolation and identification of an isolate from HVL on Margarita Island and demonstrates the presence of this zymodeme in the canine population