Lutzomyia longipalpis TGF-β 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-β) pathway, on the interrelation between L. longipalpis and L. i. chagasi. We identified an L. longipalpis homolog belonging to the multifunctional cytokine TGF-β gene family (LlTGF-β), 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-β 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-β on L. longipalpis infection by L. i. chagasi using either gene silencing by RNAi or pathway inactivation by addition of the TGF-β receptor inhibitor SB431542. The blockage of the LlTGF-β 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-β signal transduction pathway reduce L. i. chagasi survival, therefore suggesting that under natural conditions the parasite benefits from the insect LlTGF-β pathway, as already seen in Plamodium infection of mosquitoes

A complexidade da interação Leishmania-flebotomíneo: Do estudo de moléculas envolvidas na adesão do parasita ao tubo digestivo à análise do papel de citocinas-like na modulação da resposta imune do vetor

Submitted by Gilvan Almeida ([email protected]) on 2017-01-02T12:22:52Z No. of bitstreams: 1 license.txt: 1748 bytes, checksum: 8a4605be74aa9ea9d79846c1fba20a33 (MD5)Approved for entry into archive by Anderson Silva ([email protected]) on 2017-06-05T19:08:03Z (GMT) No. of bitstreams: 2 Tatiana Di Blasi.docx: 14191 bytes, checksum: f7b9b8c09b76446c622b6b818ebe7500 (MD5) license.txt: 1748 bytes, checksum: 8a4605be74aa9ea9d79846c1fba20a33 (MD5)Made available in DSpace on 2017-06-05T19:08:03Z (GMT). No. of bitstreams: 2 Tatiana Di Blasi.docx: 14191 bytes, checksum: f7b9b8c09b76446c622b6b818ebe7500 (MD5) license.txt: 1748 bytes, checksum: 8a4605be74aa9ea9d79846c1fba20a33 (MD5) Previous issue date: 2016Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Rio de Janeiro, RJ, BrasilAs leishmanioses são causadas pelo parasita Leishmania e transmitidas ao vertebrado pela picada do flebotomíneo. Durante o ciclo no vetor, uma etapa crucial ocorre ao fim da digestão com a adesão dos parasitas ao epitélio intestinal do inseto. Nosso grupo anteriormente determinou o papel da proteína flagelar FLAG1/SMP1 de L. major na adesão ao tubo digestivo de P. papatasi. Neste trabalho foram feitos ensaios de interação entre L. major e L. i. chagasi X células embrionárias LL5 e PP9, e também experimentos de inibição da interação entre células PP9 e L. major com adição de proteína recombinante FLAG/MBP. Os resultados mostraram que apesar de haver adesão, a proteína FLAG1/SMP1 parece não ter um envolvimento nesse processo de interação com células embrionárias. Em paralelo, fizemos uma busca por possíveis moléculas ligantes de FLAG1/SMP1, através de experimentos de pull down tanto com lisados de células PP9 quanto de intestinos dissecados de P. papatasi. Nos ensaios com células PP9 identificamos 6 genes de interesse codificantes para proteínas associadas à membrana. Todos esses genes são expressos tanto em intestino quanto em carcaça de P. papatasi. Nos ensaios de pull down com intestinos, identificamos um gene que codifica para uma proteína ligante de ácido hialurônico (AH). Esse gene tem uma tendência a aumento da expressão em intestinos em relação à carcaça de fêmeas não alimentadas. Realizamos também ensaios de microscopia confocal em tubos digestivos pré-incubados com a proteína recombinante FLAG/MBP. Confirmamos que FLAG1/SMP1 interage com o epitélio intestinal de P. papatasi Outra etapa importante da interação parasita-vetor é a resposta imune ativada pela Leishmania dentro do inseto. Nosso grupo detectou previamente a superexpressão de uma TGF-\03B2 de L. longipalpis (LlTGF-\03B2) 72h após infecção por L. i. chagasi. Esse gene foi silenciado com o objetivo de testar o efeito da depleção de LlTGF-\03B2 sobre a resposta imune do inseto. Observamos que a redução de LlTGF-\03B2 gerou um aumento do peptídeo antimicrobiano (PAM) cecropina. Investigamos o efeito do silenciamento em fêmeas infectadas com L. i. chagasi. Com a redução de LlTGF-\03B2, há uma redução drástica de infecção a partir de 48h, além de um aumento da expressão dos PAMs cecropina e defensina 2, além de iNOS indicando que LlTGF-\03B2 pode regular negativamente a expressão de moléculas efetoras da imunidade. Para confirmar os dados obtidos no silenciamento, alimentamos L. longipalpis com L. i. chagasi e anticorpo anti-TGF-\03B2. Observamos uma maior infecção no grupo alimentado com anticorpo após 96h. Posteriormente, verificamos por western blot que o anticorpo utilizado reconhece TGF-\03B2 presente no sangue usado nas alimentações e, possivelmente, o bloqueio dessas moléculas causou o aumento do número de parasitas. Com isso, concluímos que a TGF-\03B2 do sangue tem um efeito inicial de ativação da resposta imune com produção de moléculas efetoras. E que, posteriormente, o contato da Leishmania com o intestino pode estar causando uma ativação do gene LlTGF-\03B2 e uma regulação negativa desses efetores imunesLeishmaniasis are caused by the protozoan Leishmania and transmitted to the vertebrate host through the bite of sandflies. Inside the vector a crucial step occurs at the end of digestion, with the adhesion of the parasite\2019s flagellum to the midgut epithelium. Previously, our group observed that a L. major flagellar protein named FLAG1/SMP1 has a role in parasite adhesion to the P. papatasi midgut. In the present work, we performed interaction assays between Leishmania and embryonic LL5 and PP9 cells and also experiments of adhesion inhibition between PP9 cells and L. major by adding different concentrations of FLAG/MBP recombinant protein. The results showed that, although we observed adhesion, the protein FLAG1/SMP1 is not involved in this interaction process. In parallel, we performed pull down experiments in order to search for FLAG1/SMP1 ligands in PP9 cells and dissected guts of P. papatasi lysates. In the assays with PP9 cells we identified 6 putative candidate genes coding for membrane proteins. All of the selected genes are expressed in both gut and carcass. From the pull down assays with dissected guts, we identified a gene coding for a hyaluronan-binding protein. This gene has an increased expression in guts of nonfed females comparing to carcass. We also carried out confocal microscopy experiments using midguts pre-incubated with the recombinant protein FLAG/MBP, and confirmed that FLAG1/SMP1 directly interacts with the epithelium of P. papatasi Another important step of the parasite-vector interaction is the immune response that is activated by Leishmania infection. Our group previously detected the overexpression of a TGF-\03B2 from L. longipalpis (LlTGF-\03B2) at 72h after infection with L. i. chagasi. Aiming to test the effect of LlTGF-\03B2 depletion we performed a gene silencing experiment, and observed that LlTGF-\03B2 reduction causes an increase of the antimicrobial peptide (AMP) cecropin and also iNOS, indicating that LlTGF-\03B2 is probably regulating negatively the expression of immunity effector genes. LlTGF-\03B2 silencing in infected flies caused a strong reduction in the L. i. chagasi infection after 48h and also an increased expression of the AMPs cecropin and defensin 2, and also iNOS. To confirm the data obtained with the silencing, we fed L. longipalpis with L. i. chagasi plus antibody anti- LlTGF-\03B2, and observed that the antibody fed group had an increased infection load at 96h. We then performed western blot assays and determined that the antibody used in the previous experiments recognized the mammalian TGF-\03B2 present in the blood used during the artificial feedings. Consequently, the blocking of this mammalian molecule was probably responsible for the increase in the number of parasites. Thereby we can assume that in L. longipalpis the mammalian TGF-\03B2 caused an initial activation of the immune response and that, at a later time point, the Leishmania contact with the gut or the increase of NOS might be causing an activation of LLTGF-\03B2 and a negative regulation of immune effector

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

Alternative splicing originates different domain structure organization of Lutzomyia longipalpis chitinases

<div><p> 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.</p></div