Alternative splicing originates different domain structure organization of Lutzomyia longipalpis chitinases.

BACKGROUND The insect chitinase gene family is composed by more than 10 paralogs, which can codify proteins with different domain structures. In Lutzomyia longipalpis, the main vector of visceral leishmaniasis in Brazil, a chitinase cDNA from adult female insects was previously characterized. The predicted protein contains one catalytic domain and one chitin-binding domain (CBD). The expression of this gene coincided with the end of blood digestion indicating a putative role in peritrophic matrix degradation. OBJECTIVES To determine the occurrence of alternative splicing in chitinases of L. longipalpis. METHODS We sequenced the LlChit1 gene from a genomic clone and the three spliced forms obtained by reverse transcription polymerase chain reaction (RT-PCR) using larvae cDNA. FINDINGS We showed that LlChit1 from L. longipalpis immature forms undergoes alternative splicing. The spliced form corresponding to the adult cDNA was named LlChit1A and the two larvae specific transcripts were named LlChit1B and LlChit1C. The B and C forms possess stop codons interrupting the translation of the CBD. The A form is present in adult females post blood meal, L4 larvae and pre-pupae, while the other two forms are present only in L4 larvae and disappear just before pupation. Two bands of the expected size were identified by Western blot only in L4 larvae. MAIN CONCLUSIONS We show for the first time alternative splicing generating chitinases with different domain structures increasing our understanding on the finely regulated digestion physiology and shedding light on a potential target for controlling L. longipalpis larval development.

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 TGF-ß Has a Role in Leishmania infantum chagasi Survival in the Vector.

Despite the increasing number of studies concerning insect immunity, Lutzomyia longipalpis immune responses in the presence of Leishmania infantum chagasi infection has not been widely investigated. The few available studies analyzed the role of the Toll and IMD pathways involved in response against Leishmania and microbial infections. Nevertheless, effector molecules responsible for controlling sand fly infections have not been identified. In the present study we investigated the role a signal transduction pathway, the Transforming Growth Factor-beta (TGF-ß) pathway, on the interrelation between L. longipalpis and L. i. chagasi. We identified an L. longipalpis homolog belonging to the multifunctional cytokine TGF-ß gene family (LlTGF-ß), which is closely related to the activin/inhibin subfamily and potentially involved in responses to infections. We investigated this gene expression through the insect development and in adult flies infected with L. i. chagasi. Our results showed that LlTGF-ß was expressed in all L. longipalpis developmental stages and was upregulated at the third day post L. i. chagasi infection, when protein levels were also higher as compared to uninfected insects. At this point blood digestion is finished and parasites are in close contact with the insect gut. In addition, we investigated the role of LlTGF-ß on L. longipalpis infection by L. i. chagasi using either gene silencing by RNAi or pathway inactivation by addition of the TGF-ß receptor inhibitor SB431542. The blockage of the LlTGF-ß pathway increased significantly antimicrobial peptides expression and nitric oxide levels in the insect gut, as expected. Both methods led to a decreased L. i. chagasi infection. Our results show that inactivation of the L. longipalpis TGF-ß signal transduction pathway reduce L. i. chagasi survival, therefore suggesting that under natural conditions the parasite benefits from the insect LlTGF-ß pathway, as already seen in Plamodium infection of mosquitoes.

The flagellar protein FLAG1/SMP1 is a candidate for Leishmania-sand fly interaction.

Leishmaniasis is a serious problem that affects mostly poor countries. Various species of Leishmania are the agents of the disease, which take different clinical manifestations. The parasite is transmitted by sandflies, predominantly from the Phlebotomus genus in the Old World and Lutzomyia in the New World. During development in the gut, Leishmania must survive various challenges, which include avoiding being expelled with blood remnants after digestion. It is believed that attachment to the gut epithelium is a necessary step for vector infection, and molecules from parasites and sand flies have been implicated in this attachment. In previous work, monoclonal antibodies were produced against Leishmania. Among these an antibody was obtained against Leishmania braziliensis flagella, which blocked the attachment of Leishmania panamensis flagella to Phlebotomus papatasi guts. The protein recognized by this antibody was identified and named FLAG1, and the complete FLAG1 gene sequence was obtained. This protein was later independently identified as a small, myristoylated protein and called SMP1, so from now on it will be denominated FLAG1/SMP1. The FLAG1/SMP1 gene is expressed in all developmental stages of the parasite, but has higher expression in promastigotes. The anti-FLAG1/SMP1 antibody recognized the flagellum of all Leishmania species tested and generated the expected band by western blots. This antibody was used in attachment and infection blocking experiments. Using the New World vector Lutzomyia longipalpis and Leishmania infantum chagasi, no inhibition of attachment ex vivo or infection in vivo was seen. On the other hand, when the Old World vectors P. papatasi and Leishmania major were used, a significant decrease of both attachment and infection were seen in the presence of the antibody. We propose that FLAG1/SMP1 is involved in the attachment/infection of Leishmania in the strict vector P. papatasi and not the permissive vector L. longipalpis.

Alternative splicing originates different domain structure organization of Lutzomyia longipalpis chitinases

BACKGROUND The insect chitinase gene family is composed by more than 10 paralogs, which can codify proteins with different domain structures. In Lutzomyia longipalpis, the main vector of visceral leishmaniasis in Brazil, a chitinase cDNA from adult female insects was previously characterized. The predicted protein contains one catalytic domain and one chitin-binding domain (CBD). The expression of this gene coincided with the end of blood digestion indicating a putative role in peritrophic matrix degradation. OBJECTIVES To determine the occurrence of alternative splicing in chitinases of L. longipalpis. METHODS We sequenced the LlChit1 gene from a genomic clone and the three spliced forms obtained by reverse transcription polymerase chain reaction (RT-PCR) using larvae cDNA. FINDINGS We showed that LlChit1 from L. longipalpis immature forms undergoes alternative splicing. The spliced form corresponding to the adult cDNA was named LlChit1A and the two larvae specific transcripts were named LlChit1B and LlChit1C. The B and C forms possess stop codons interrupting the translation of the CBD. The A form is present in adult females post blood meal, L4 larvae and pre-pupae, while the other two forms are present only in L4 larvae and disappear just before pupation. Two bands of the expected size were identified by Western blot only in L4 larvae. MAIN CONCLUSIONS We show for the first time alternative splicing generating chitinases with different domain structures increasing our understanding on the finely regulated digestion physiology and shedding light on a potential target for controlling L. longipalpis larval development.

Papel das moléculas FLAG de leishmania e TGF-beta de lutzomyia longipalpis na interação parasito - vetor / Role of leishmania FLAG molecules and TGF-beta of Lutzomyia longipalpis in parasite interaction - vector

As leishmanioses constituem um grave problema de saúde. São causadas por protozoários do gênero Leishmania e sua transmissão ao hospedeiro vertebrado se dá pela picada de fêmeas infectadas do gênero Lutzomyia (Novo Mundo) e Phlebotominae (Velho Mundo). Na alimentação, as leishmanias são ingeridas pelo flebotomíneo junto com sangue e uma etapa fundamental para a manutenção da infecção no vetor refere-se à adesão dos parasitos ao epitélio intestinal com o término da digestão. Para isso, o flagelo das formas promastigotas desempenha papel essencial no processo de fixação dos parasitos às células intestinais. Estudos prévios do nosso grupo identificaram um gene codificante para uma proteína flagelar denominada FLAG que pode estar diretamente envolvida na adesão do parasito ao tubo digestivo do flebotomíneo. Visando a melhor caracterização do gene, realizou-se no presente trabalho a análise da transcrição do gene FLAG nos diferentes estágios de desenvolvimento de Leishmania infantum chagasi e Leishmania pifanoi por PCR em tempo real. Tanto L. i. chagasi quanto L. pifanoi não apresentaram diferença de expressão entre promastigota de fase exponencial de crescimento (procíclica) e de fase estacionária (metacíclica). Entretanto, em L.pifanoi, as promastigotas apresentaram uma maior expressão de FLAG do que as amastigotas que têm um flagelo interno curto. Com o intuito de verificarmos o papel de FLAG na adesão ao tubo digestivo do vetor, realizamos ensaios de inibição de adesão ex vivo e in vivo utilizando anticorpo monoclonal anti-FLAG. Os ensaios foram feitos com os pares L. i. chagasi X Lutzomyia longipalpis, Leishmania amazonensis X L. longipalpis e Leishmania major X Phlebotomus papatasi, incubando previamente os parasitos com o anticorpo anti-FLAG. Os resultados não mostraram diferenças significativas no número de parasitos aderidos entre os grupos teste e controle, nos experimentos com espécies do Novo Mundo. Entretanto, com o par natural L. major e P. papatasi, foi possível observar uma redução significativa de parasitos presentes no tubo digestivo quando os mesmos foram pré-incubados com o anticorpo. Logo, em espécies do Novo Mundo, a proteína FLAG não parece estar relacionada com a adesão de Leishmania ao tubo digestivo do flebotomíneo enquanto que no par do Velho Mundo L. major e P. papatasi, ela parece ter um papel na interação. Em paralelo foram feitos experimentos de “pull down” para a identificação de moléculas potencialmente capazes de interagir com FLAG no intestino de L. longipalpis. Para tal, foram usados extratos protéicos de células embrionárias LL5 de L. longipalpis. Observamos um grande número de ligações inespecíficas à resina ou à proteína de fusão, mas nenhuma banda pôde ser detectada ligando-se especificamente a FLAG. Isto está de acordo com os resulltados de inibição de interação ex vivo e in vivo. Os insetos são organismos que conseguem se adaptar a diversas condições ambientais. Essa capacidade de sobrevivência está apoiada em um sistema de resposta imune inata bem desenvolvido. Em nosso laboratório, uma sequência de cDNA similar a TGF-beta (Transforming Growth Factor – beta) foi identificada em fêmeas de L. longipalpis infectadas com L. i. chagasi através de experimentos de Differential Display RT-PCR (DDRT-PCR). A molécula foi seqüenciada e classificada como pertencente à família das ativinas. As citocinas da super-familia das TGF-beta são responsáveis pela sinalização e regulação de uma série de processos biológicos em diversos organismos. Essas proteínas são agrupadas em diferentes sub-familias, dentre elas a da ativina/inibina está relacionada a processo de desenvolvimento neuronal e processos de diferenciação. A análise da expressão do RNA de TGF-beta revelou um aumento significativo 72 horas após a infecção com L. i. chagasi quando comparado com fêmeas alimentadas apenas com sangue. Para avaliar o possível papel de TGF-beta na infecção por Leishmania, realizamos experimentos de infecção de L. longipalpis com L. i. infantum contendo ou não anticorpo anti-TGF-beta. Observamos um aumento de parasitos no grupo alimentado com anticorpo, indicando um possível papel de TGF-beta na ativação da resposta imune do vetor contra a Leishmania. A interação entre parasitos e seus vetores é uma etapa fundamental do ciclo infectivo. Por isso, o estudo de moléculas – chave para o desenvolvimento do ciclo de transmissão torna-se cada vez mais importante.