Expression and cellular localisation of Trypanosoma cruzi calpains.

BACKGROUND: Calpains are present in almost all organisms and comprise a family of calcium-dependent cysteine peptidases implicated in crucial cellular functions. Trypanosoma cruzi, the causative agent of Chagas disease, presents an expansion on this gene family with unexplored biological properties. OBJECTIVES: Here, we searched for calpains in the T. cruzi genome, evaluated the mRNA levels, calpain activity and the protein expression and determined the cellular localisation in all three parasite life cycle forms. METHODS/FINDINGS: Sixty-three calpain sequences were identified in T. cruzi CL Brener genome, with fourteen domain arrangements. The comparison of calpain mRNA abundance by quantitative polymerase chain reaction (qPCR) revealed seven up-regulated sequences in amastigotes and/or bloodstream trypomastigotes and five in epimastigotes. Western Blotting analysis revealed seven different molecules in the three parasite forms, and one amastigote-specific, while no proteolytic activity could be detected. Flow cytometry assays revealed a higher amount of intracellular calpains in amastigotes and/or trypomastigotes in comparison to epimastigotes. Finally, ultrastructural analysis revealed the presence of calpains in the cytoplasm, vesicular and plasma membranes of the three parasite forms, and in the paraflagellar rod in trypomastigotes. CONCLUSION: Calpains are differentially expressed and localised in the T. cruzi life cycle forms. This study adds data on the calpain occurrence and expression pattern in T. cruzi.

Novel molecular evidence of population structure in Anopheles (Kerteszia) bellator from Brazilian Atlantic Forest.

Anopheles bellator is a primary malaria vector in the Atlantic Forest. Partial sequences of timeless and Clock genes were used to assess the genetic differentiation of five Brazilian populations, which showed strong population structure (e.g. high F ST values and fixed differences) in all pairwise comparisons between Bahia sample and the others from Paraná, São Paulo and Rio de Janeiro states. Also, the resulting phylogenetic trees clearly grouped the sequences from Bahia in a different cluster with high bootstrap values. Among southern and southeastern populations low levels of genetic differentiation were found suggesting a general stability of the genetic structure.

Calpains of Leishmania braziliensis: genome analysis, differential expression, and functional analysis.

BACKGROUND: Calpains are proteins belonging to the multi-gene family of calcium-dependent cysteine peptidases that undergo tight on/off regulation, and uncontrolled proteolysis of calpains is associated with severe human pathologies. Calpain orthologues are expanded and diversified in the trypanosomatids genome. OBJECTIVES: Here, we characterised calpains in Leishmania braziliensis, the main causative agent of cutaneous leishmaniasis in Brazil. METHODS/FINDINGS: In total, 34 predicted calpain-like genes were identified. After domain structure evaluation, reverse transcription-quantitative polymerase chain reaction (RT-qPCR) during in vitro metacyclogenesis revealed (i) five genes with enhanced expression in the procyclic stage, (ii) one augmented gene in the metacyclic stage, and (iii) one procyclic-exclusive transcript. Western blot analysis revealed that an antibody against a consensus-conserved peptide reacted with multiple calpain-like proteins, which is consistent with the multi-gene family characteristic. Flow cytometry and immunocytochemistry analyses revealed the presence of calpain-like molecules mainly in the cytoplasm, to a lesser extent in the plasma membrane, and negligible levels in the nucleus, which are all consistent with calpain localisation. Eventually, the calpain inhibitor MDL28170 was used for functional studies revealing (i) a leishmaniostatic effect, (ii) a reduction in the association index in mouse macrophages, (iii) ultra-structural alterations conceivable with autophagy, and (iv) an enhanced expression of the virulence factor GP63. CONCLUSION: This report adds novel insights into the domain structure, expression, and localisation of L. braziliensis calpain-like molecules.

Influence of the fish oil in the gut microbiota of wistar rats (Rattus norvegicus) treated with dexamethasone and amoxicillin.

The widespread use of antibiotics and anti-inflammatory has been more and more prominent. In spite of the proven pharmacological potential, its collateral effects are still being described. The fish oil is made of acids fatty polyunsaturated, as the omega 3. The aim of this paper is to check if there would be interference of this fish oil in the gut microbiota of rats when treated with dexamethasone and amoxicillin, joining with the parameter lipids and glycemic. This study was done with 42 Wistar rats, divided into 6 groups with 7 animals each: naive (CTL), amoxicillin (AMOX), dexamethasone (DEX), fish oil (OLP), associated amoxicillin and fish oil (AMOX + OLP) association dexamethasone and fish oil (DEX + OLP). The results show that the fish oil influenced in the concentration of blood glucose in the animals, keeping stable levels even after a pool of glucose. Differently, the fish oil increased the levels of LDL in the animals. The amoxicillin changed the mass of liver and spleen, changed the levels of triglyceride and changed the gut microbiota. The dexamethasone influenced the lipids parameters and mass of the spleen as well as it slightly increased the amount of cholesterol LDL. It is possible to conclude that fish oil increases the levels of LDL in the tested model and the dose tested, but is able to maintain glucose levels even after a pool of the same, and can be a preventive model with hyperglycemia.

Leishmania infantum detection in Nyssomyia neivai and dogs in Southern Brazil.

BACKGROUND: The sand fly Nyssomyia neivai is one of the most abundant species in Southern Brazil. It is frequently found in areas that are foci of visceral leishmaniasis in the state of Santa Catarina, caused by Leishmania infantum. In this region, the main vector of L. infantum, Lutzomyia longipalpis, has not been detected. In the absence of L. longipalpis, this study aimed to identify the sand fly fauna and diagnose any potential Leishmania spp. infection in sand flies and in dogs in a region of Southern Brazil that experienced a recent canine visceral leishmaniasis outbreak. METHODS: This report includes a survey of the sand fly fauna at the Zoonosis Control Center of the Municipality of Tubarão (Santa Catarina, Brazil). Molecular tests were conducted to investigate Leishmania spp. natural infection in sand flies using polymerase chain reaction (PCR). In positive females, in addition to morphological identification, molecular analysis through DNA barcoding was performed to determine the sand fly species. Additionally, the dogs were tested for the presence of Leishmania spp. using a non-invasive technique for the collection of biological material, to be assessed by PCR. RESULTS: A total of 3419 sand flies, belonging to five genera, were collected. Nyssomyia neivai was the most abundant species (85.8%), followed by Migonemyia migonei (13.3%), Pintomyia fischeri (0.8%), Evandromyia edwardsi (< 0.1%), and species of the genus Brumptomyia. (0.1%). Out of the 509 non-engorged females analyzed by PCR, two (0.4%) carried L. infantum DNA. The naturally infected females were identified as Ny. neivai, in both morphological and molecular analysis. In addition, two out of 47 conjunctival swabs from dogs tested positive for L. infantum, yielding an infection rate of 4.2%. CONCLUSIONS: These results confirm the presence of Ny. neivai naturally infected with L. infantum in an area where dogs were also infected by the parasite, suggesting its potential role as a vector in Southern Brazil.

Evidence of a conserved mammalian immunosuppression mechanism in Lutzomyia longipalpis upon infection with Leishmania.

Introduction: Sand flies (Diptera: Phlebotominae) belonging to the Lutzomyia genus transmit Leishmania infantum parasites. To understand the complex interaction between the vector and the parasite, we have been investigating the sand fly immune responses during the Leishmania infection. Our previous studies showed that genes involved in the IMD, Toll, and Jak-STAT immunity pathways are regulated upon Leishmania and bacterial challenges. Nevertheless, the parasite can thrive in the vectors' gut, indicating the existence of mechanisms capable of modulating the vector defenses, as was already seen in mammalian Leishmania infections. Methods results and discussion: In this study, we investigated the expression of Lutzomyia longipalpis genes involved in regulating the Toll pathway under parasitic infection. Leishmania infantum infection upregulated the expression of two L. longipalpis genes coding for the putative repressors cactus and protein tyrosine phosphatase SHP. These findings suggest that the parasite can modulate the vectors' immune response. In mammalian infections, the Leishmania surface glycoprotein GP63 is one of the inducers of host immune depression, and one of the known effectors is SHP. In L. longipalpis we found a similar effect: a genetically modified strain of Leishmania amazonensis over-expressing the metalloprotease GP63 induced a higher expression of the sand fly SHP indicating that the L. longipalpis SHP and parasite GP63 increased expressions are connected. Immuno-stained microscopy of L. longipalpis LL5 embryonic cells cultured with Leishmania strains or parasite conditioned medium showed cells internalization of parasite GP63. A similar internalization of GP63 was observed in the sand fly gut tissue after feeding on parasites, parasite exosomes, or parasite conditioned medium, indicating that GP63 can travel through cells in vitro or in vivo. When the sand fly SHP gene was silenced by RNAi and females infected by L. infantum, parasite loads decreased in the early phase of infection as expected, although no significant differences were seen in late infections of the stomodeal valve. Conclusions: Our findings show the possible role of a pathway repressor involved in regulating the L. longipalpis immune response during Leishmania infections inside the insect. In addition, they point out a conserved immunosuppressive effect of GP63 between mammals and sand flies in the early stage of parasite infection.

Lutzomyia longipalpis Antimicrobial Peptides: Differential Expression during Development and Potential Involvement in Vector Interaction with Microbiota and Leishmania.

Antimicrobial peptides (AMPs) are produced to control bacteria, fungi, protozoa, and other infectious agents. Sand fly larvae develop and feed on a microbe-rich substrate, and the hematophagous females are exposed to additional pathogens. We focused on understanding the role of the AMPs attacin (Att), cecropin (Cec), and four defensins (Def1, Def2, Def3, and Def4) in Lutzomyia longipalpis, the main vector of visceral leishmaniasis in the Americas. Larvae and adults were collected under different feeding regimens, in addition to females artificially infected by Leishmania infantum. AMPs' gene expression was assessed by qPCR, and gene function of Att and Def2 was investigated by gene silencing. The gene knockdown effect on bacteria and parasite abundance was evaluated by qPCR, and parasite development was verified by light microscopy. We demonstrate that L. longipalpis larvae and adults trigger AMPs expression during feeding, which corresponds to an abundant presence of bacteria. Att and Def2 expression were significantly increased in Leishmania-infected females, while Att suppression favored bacteria growth. In conclusion, L. longipalpis AMPs' expression is tuned in response to bacteria and parasites but does not seem to interfere with the Leishmania cycle.

Cryptic diversity in an Atlantic Forest malaria vector from the mountains of South-East Brazil.

BACKGROUND: Anopheles (Kerteszia) cruzii is the primary vector of human and simian malarias in Brazilian regions covered by the Atlantic Rainforest. Previous studies found that An. cruzii presents high levels of behavioural, chromosomal and molecular polymorphisms, which led to the hypothesis that it may be a complex of cryptic species. Here, An. cruzii specimens were collected in five sites in South-East Brazil located at different altitudes on the inner and coastal slopes of two mountain ranges covered by Atlantic Rainforest, known as Serra do Mar and Serra da Mantiqueria. Partial sequences for two genes (Clock and cpr) were generated and compared with previously published sequences from Florianópolis (southern Brazil). Genetic diversity was analysed with estimates of population structure (F ST ) and haplotype phylogenetic trees in order to understand how many species of the complex may occur in this biome and how populations across the species distribution are related. RESULTS: The sequences from specimens collected at sites located on the lower coastal slopes of Serra do Mar (Guapimirim, Tinguá and Sana) clustered together in the phylogenetic analysis, while the major haplotypes from sites located on higher altitude and at the continental side of the same mountains (Bocaina) clustered with those from Serra da Mantiqueira (Itatiaia), an inner mountain range. These two An. cruzii lineages showed statistically significant genetic differentiation and fixed characters, and have high F ST values typical of between species comparisons. Finally, in Bocaina, where the two lineages occur in sympatry, we found deviations from Hardy-Weinberg equilibrium due to a deficit of heterozygotes, indicating partial reproductive isolation. These results strongly suggest that at least two distinct lineages of An. cruzii (provisorily named "Group 1" and "Group 2") occur in the mountains of South-East Brazil. CONCLUSIONS: At least two genetically distinct An. cruzii lineages occur in the Atlantic Forest covered mountains of South-East Brazil. The co-occurrence of distinct lineages of An. cruzii (possibly incipient species) in those mountains is an interesting biological phenomenon and may have important implications for malaria prevalence, Plasmodium transmission dynamics and control.

The sandfly Lutzomyia longipalpis LL5 embryonic cell line has active Toll and Imd pathways and shows immune responses to bacteria, yeast and Leishmania.

BACKGROUND: Lutzomyia longipalpis is the main vector of visceral leishmaniasis in Latin America. Sandfly immune responses are poorly understood. In previous work we showed that these vector insects respond to bacterial infections by modulating a defensin gene expression and activate the Imd pathway in response to Leishmania infection. Aspects of innate immune pathways in insects (including mosquito vectors of human diseases) have been revealed by studying insect cell lines, and we have previously demonstrated antiviral responses in the L. longipalpis embryonic cell line LL5. METHODS: The expression patterns of antimicrobial peptides (AMPs) and transcription factors were evaluated after silencing the repressors of the Toll pathway (cactus) and Imd pathway (caspar). AMPs and transcription factor expression patterns were also evaluated after challenge with heat-killed bacteria, heat-killed yeast, or live Leishmania. RESULTS: These studies showed that LL5 cells have active Toll and Imd pathways, since they displayed an increased expression of AMP genes following silencing of the repressors cactus and caspar, respectively. These pathways were also activated by challenges with bacteria, yeast and Leishmania infantum chagasi. CONCLUSIONS: We demonstrated that L. longipalpis LL5 embryonic cells respond to immune stimuli and are therefore a good model to study the immunological pathways of this important vector of leishmaniasis.

Constitutive and blood meal-induced trypsin genes in Lutzomyia longipalpis.

Trypsins constitute some of the most abundant midgut digestive proteases expressed by hematophagous insects upon blood feeding. In addition to their role in the digestion of the blood meal, these proteases also have been implicated in the ability of certain pathogens to infect their natural vector. In sand flies, digestive proteases including trypsins were associated with early killing of Leishmania and are believed to play a role in the species-specificity dictating sand fly vectorial capacity. Our group is involved in studies of midgut digestive proteases in the sand fly Lutzomyia longipalpis, the principal vector of visceral leishmaniasis in Brazil. Here we report on the identification of two cDNAs, Lltryp1 and Lltryp2, which code for putative midgut trypsins in L. longipalpis. Analyses of RNA abundance using semi-quantitative RT-PCR show a different pattern of expression between the two genes. Lltryp1 expression remains undetected until blood feeding and reaches a peak at 12 h post-blood meal (PBM), returning to pre-blood meal levels at 72 h PBM. Additionally, Lltryp1 expression is undetected during larval development. Lltryp2, on the other hand, is constitutively expressed as high levels in the non-blood fed female, but is reduced upon blood feeding. At the end of the digestive cycle, Lltryp2 regains its pre-blood meal levels. This cDNA also is present in all developmental stages and in adult males. This pattern of expression is reminiscent of what is seen in mosquitoes and Old World sand flies, but has characteristics that are unique to L. longipalpis.

Aspectos moleculares da imunidade de Lutzomyia longipalpis (Díptera: Psychodidae) / Molecular aspects of immunity of Lutzomyia longipalpis (Díptera: Psychodidae)

Os flebotomíneos são conhecidos pela transmissão da leishmaniose, porém transmitem também doenças ligadas a bactérias e vírus. Além disso, estes insetos entram em contato com diversos patógenos como fungos e helmintos. As defesas contra patógenos em flebotomíneos são quase totalmente desconhecidas. A principal via de infecção se dá pela alimentação sangüínea, sendo o intestino o primeiro órgão da resposta imune. Baseado nisso, o transcriptoma deste tecido foi analisado. Foram seqüenciados ESTs de 3 bibliotecas de cDNA, construídas a partir de tubos digestivos de fêmeas dissecadas 6 (6h) ou 72 horas (72h) após alimentação sangüínea e após 72 horas de infecção com Leishmania chagasi (Inf). Dentre os genes encontrados vários são ligados ao processo de digestão e de defesa. A comparação entre as bibliotecas mostra aumento na quantidade de transcritos ligados à defesa em Inf (ex. defensina, serpina, PGRP) e a digestão em 6h (ex. tripsinas, V-ATPases e Kazal). Foram identificadas anteriormente duas formas de tripsina: Lltryp1, expressa entre 2 e 48 horas após a alimentação sangüínea e Lltryp2, constitutiva, porém regulada negativamente durante a digestão. Lltryp1 é ausente no desenvolvimento e Lltryp2 é expressa principalmente a partir do estágio larval L3. V-ATPase também é modulada pela alimentação sangüínea. Após alimentação sua expressão aumenta até 48 horas decaindo após 72 horas e nas fases do desenvolvimento é crescente atingindo o máximo em pupa e machos. Dentre os transcritos envolvidos em defesa, um gene de defensina foi caracterizado. Aparentemente a defensina é modulada pela digestão sangüínea, com expressão aumentada nas primeiras horas e decaindo após 72 horas, não sendo detectada nas fases larvais iniciais. Na tentativa de silenciar genes relacionados à imunidade em células de L. longipalpis (LL5) utilizando o mecanismo de RNAi, foi identificado uma resposta antiviral inespecífica. A replicação de partículas virais do vírus do oeste do...

Aspectos moleculares da imunidade de Lutzomyia longipalpis (Díptera: Psychodidae) / Molecular aspects of immunity of Lutzomyia longipalpis (Díptera: Psychodidae)

Os flebotomíneos são conhecidos pela transmissão da leishmaniose, porém transmitem também doenças ligadas a bactérias e vírus. Além disso, estes insetos entram em contato com diversos patógenos como fungos e helmintos. As defesas contra patógenos em flebotomíneos são quase totalmente desconhecidas. A principal via de infecção se dá pela alimentação sangüínea, sendo o intestino o primeiro órgão da resposta imune. Baseado nisso, o transcriptoma deste tecido foi analisado. Foram seqüenciados ESTs de 3 bibliotecas de cDNA, construídas a partir de tubos digestivos de fêmeas dissecadas 6 (6h) ou 72 horas (72h) após alimentação sangüínea e após 72 horas de infecção com Leishmania chagasi (Inf). Dentre os genes encontrados vários são ligados ao processo de digestão e de defesa. A comparação entre as bibliotecas mostra aumento na quantidade de transcritos ligados à defesa em Inf (ex. defensina, serpina, PGRP) e a digestão em 6h (ex. tripsinas, V-ATPases e Kazal). Foram identificadas anteriormente duas formas de tripsina: Lltryp1, expressa entre 2 e 48 horas após a alimentação sangüínea e Lltryp2, constitutiva, porém regulada negativamente durante a digestão. Lltryp1 é ausente no desenvolvimento e Lltryp2 é expressa principalmente a partir do estágio larval L3. V-ATPase também é modulada pela alimentação sangüínea. Após alimentação sua expressão aumenta até 48 horas decaindo após 72 horas e nas fases do desenvolvimento é crescente atingindo o máximo em pupa e machos. Dentre os transcritos envolvidos em defesa, um gene de defensina foi caracterizado. Aparentemente a defensina é modulada pela digestão sangüínea, com expressão aumentada nas primeiras horas e decaindo após 72 horas, não sendo detectada nas fases larvais iniciais. Na tentativa de silenciar genes relacionados à imunidade em células de L. longipalpis (LL5) utilizando o mecanismo de RNAi, foi identificado uma resposta antiviral inespecífica. A replicação de partículas virais do vírus do oeste do...imunes.(AU)