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

Trypsin-Like Serine Proteases in Lutzomyia longipalpis – Expression, Activity and Possible Modulation by Leishmania infantum chagasi

Background: Midgut enzymatic activity is one of the obstacles that Leishmania must surpass to succeed in establishing infection. Trypsins are abundant digestive enzymes in most insects. We have previously described two trypsin cDNAs of L. longipalpis: one (Lltryp1) with a bloodmeal induced transcription pattern, the other (Lltryp2) with a constitutive transcription pattern. We have now characterized the expression and activity of trypsin-like proteases of Lutzomyia longipalpis, the main vector of visceral leishmaniasis in Brazil. Methodology and Principal Findings: In order to study trypsin expression profiles we produced antibodies against peptides specific for Lltryp1 and Lltryp2. The anti-Lltryp1-peptide antibody revealed a band of 28 kDa between 6 and 48 hours. The anti-Lltryp2 peptide antibody did not evidence any band. When proteinaceous substrates (gelatin, hemoglobin, casein or albumin) were co-polymerized in polyacrylamide gels, insect midguts obtained at 12 hours after feeding showed a unique proteolytic pattern for each substrate. All activity bands were strongly inhibited by TLCK, benzamidine and 4-amino-benzamidine, indicating that they are trypsin-like proteases. The trypsin-like activity was also measured in vitro at different time points after ingestion of blood or blood containing Leishmania infantum chagasi, using the chromogenic substrate BArNA. L. longipalpis females fed on blood infected with L. i. chagasi had lower levels of trypsin activity after 12 and 48 hours than non-infected insects, suggesting that the parasite may have a role in this modulation. Conclusions and Significance: Trypsins are important and abundant digestive enzymes in L. longipalpis. Protein production and enzymatic activity followed previously identified gene expression of a blood modulated trypsin gene. A decrease of enzymatic activity upon the parasite infection, previously detected mostly in Old World vectors, was detected for the first time in the natural vector-parasite pair L. longipalpis-L. i. chagasi

Identification of Secreted Proteins Involved in Nonspecific dsRNA-Mediated Lutzomyia longipalpis LL5 Cell Antiviral Response

Submitted by Sandra Infurna ([email protected]) on 2019-02-14T13:43:03Z No. of bitstreams: 1 yaramariat_cseko_etal_IOC_2018.pdf: 2048797 bytes, checksum: b315ee0381efe6114c33e4d8e230ef8b (MD5)Approved for entry into archive by Sandra Infurna ([email protected]) on 2019-02-14T13:52:25Z (GMT) No. of bitstreams: 1 yaramariat_cseko_etal_IOC_2018.pdf: 2048797 bytes, checksum: b315ee0381efe6114c33e4d8e230ef8b (MD5)Made available in DSpace on 2019-02-14T13:52:25Z (GMT). No. of bitstreams: 1 yaramariat_cseko_etal_IOC_2018.pdf: 2048797 bytes, checksum: b315ee0381efe6114c33e4d8e230ef8b (MD5) Previous issue date: 2018Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Biologia Molecular de Parasitas e Vetores. Rio de Janeiro, RJ. Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Biologia Molecular de Parasitas e Vetores. Rio de Janeiro, RJ. Brasil.Fundação Oswaldo Cruz. Instituto Carlos Chagas. Laboratório de Genômica Funcional. Curitiba, PR, Brasil / Fundação Oswaldo Cruz. Instituto Carlos Chagas. Plataforma Espectrometria de Massas. Curitiba, PR, Brasil.Fundação Oswaldo Cruz. Instituto Carlos Chagas. Laboratório de Genômica Funcional. Curitiba, PR, Brasil / Fundação Oswaldo Cruz. Instituto Carlos Chagas. Plataforma Espectrometria de Massas. Curitiba, PR, Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Biologia Molecular de Parasitas e Vetores. Rio de Janeiro, RJ. Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Biologia Molecular de Parasitas e Vetores. Rio de Janeiro, RJ. Brasil.Hematophagous insects transmit infectious diseases. Sand flies are vectors of leishmaniasis, but can also transmit viruses. We have been studying immune responses of Lutzomyia longipalpis, the main vector of visceral leishmaniasis in the Americas. We identified a non-specific antiviral response in L. longipalpis LL5 embryonic cells when treated with non-specific double-stranded RNAs (dsRNAs). This response is reminiscent of interferon response in mammals. We are investigating putative effectors for this antiviral response. Secreted molecules have been implicated in immune responses, including interferon-related responses. We conducted a mass spectrometry analysis of conditioned medium from LL5 cells 24 and 48 h after dsRNA or mock treatment. We identified 304 proteins. At 24 h, 19 proteins had an abundance equal or greater than 2-fold change, while the levels of 17 proteins were reduced when compared to control cells. At the 48 h time point, these numbers were 33 and 71, respectively. The two most abundant secreted peptides at 24 h in the dsRNA-transfected group were phospholipid scramblase, an interferon-inducible protein that mediates antiviral activity, and forskolin-binding protein (FKBP), a member of the immunophilin family, which mediates the effect of immunosuppressive drugs. The transcription profile of most candidates did not follow the pattern of secreted protein abundance

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

Submitted by Sandra Infurna ([email protected]) on 2016-12-29T16:22:07Z No. of bitstreams: 1 bruno_nunes_etal_IOC_2016.pdf: 1229712 bytes, checksum: 11bb095573e0fd7b51b849adad88342a (MD5)Approved for entry into archive by Sandra Infurna ([email protected]) on 2016-12-29T16:33:06Z (GMT) No. of bitstreams: 1 bruno_nunes_etal_IOC_2016.pdf: 1229712 bytes, checksum: 11bb095573e0fd7b51b849adad88342a (MD5)Made available in DSpace on 2016-12-29T16:33:06Z (GMT). No. of bitstreams: 1 bruno_nunes_etal_IOC_2016.pdf: 1229712 bytes, checksum: 11bb095573e0fd7b51b849adad88342a (MD5) Previous issue date: 2016Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Biologia Molecular de Parasitas e Vetores. Rio de Janeiro, RJ, Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Biologia Molecular de Parasitas e Vetores. Rio de Janeiro, RJ, Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Biologia Molecular de Parasitas e Vetores. Rio de Janeiro, RJ, Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Biologia Molecular de Parasitas e Vetores. Rio de Janeiro, RJ, Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Biologia Molecular de Parasitas e Vetores. Rio de Janeiro, RJ, Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Biologia Molecular de Parasitas e Vetores. Rio de Janeiro, RJ, Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Biologia Molecular de Parasitas e Vetores. Rio de Janeiro, RJ, Brasil.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

Enzymatic assay performed in solution with trypsin synthetic substrate BAρNA.

<p>Horizontal axis indicates insect midgut samples obtained at different hours ABF. Vertical axis indicate enzyme units detected per midgut. <b>0</b>: sugar-fed females; <b>6</b> to <b>72</b>: females obtained at 6, 12, 24, 48 and 72 hours ABF. White bars indicate samples obtained from non infected insect midguts. Gray bars indicate samples obtained from infected insect midguts. Samples corresponding to half of one midgut were used in each assay. Statistically significant difference (<i>P</i><0.05) is marked with an asterisk.</p