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

The Sand Fly Salivary Protein Lufaxin Inhibits the Early Steps of the Alternative Pathway of Complement by Direct Binding to the Proconvertase C3b-B

Saliva of the blood feeding sand fly Lutzomyia longipalpis was previously shown to inhibit the alternative pathway (AP) of the complement system. Here, we have identified Lufaxin, a protein component in saliva, as the inhibitor of the AP. Lufaxin inhibited the deposition of C3b, Bb, Properdin, C5b, and C9b on agarose-coated plates in a dose-dependent manner. It also inhibited the activation of factor B in normal serum, but had no effect on the components of the membrane attack complex. Surface plasmon resonance (SPR) experiments demonstrated that Lufaxin stabilizes the C3b-B proconvertase complex when passed over a C3b surface in combination with factor B. Lufaxin was also shown to inhibit the activation of factor B by factor D in a reconstituted C3b-B, but did not inhibit the activation of C3 by reconstituted C3b-Bb. Proconvertase stabilization does not require the presence of divalent cations, but addition of Ni2+ increases the stability of complexes formed on SPR surfaces. Stabilization of the C3b-B complex to prevent C3 convertase formation (C3b-Bb formation) is a novel mechanism that differs from previously described strategies used by other organisms to inhibit the AP of the host complement system

The Sand Fly Salivary Protein Lufaxin Inhibits the Early Steps of the Alternative Pathway of Complement by Direct Binding to the Proconvertase C3b-B

Submitted by Sandra Infurna ([email protected]) on 2017-11-14T12:07:24Z No. of bitstreams: 1 vladimir_vale_ertal_IOC_2017.pdf: 2840461 bytes, checksum: 613bd3eac5d01125f45aebbca1826ed9 (MD5)Approved for entry into archive by Sandra Infurna ([email protected]) on 2017-11-14T12:22:52Z (GMT) No. of bitstreams: 1 vladimir_vale_ertal_IOC_2017.pdf: 2840461 bytes, checksum: 613bd3eac5d01125f45aebbca1826ed9 (MD5)Made available in DSpace on 2017-11-14T12:22:52Z (GMT). No. of bitstreams: 1 vladimir_vale_ertal_IOC_2017.pdf: 2840461 bytes, checksum: 613bd3eac5d01125f45aebbca1826ed9 (MD5) Previous issue date: 2017Universidade Federal de Minas Gerais. Instituto de Ciências Biológicas. Departamento de Parasitologia. Laboratório de Fisiologia de Insetos Hematófagos. Belo Horizonte, MG, Brasil / Universidade Federal do Piauí. Campus Senador Helvídio Nunes de Barros. Picos, PI, BrasilUniversidade Federal de Minas Gerais. Instituto de Ciências Biológicas. Departamento de Parasitologia. Laboratório de Fisiologia de Insetos Hematófagos. Belo Horizonte, MG, Brasil / Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Simulídeos, Oncocercose e Infecções Simpátricas: Mansonelose e Malária. Rio de Janeiro, RJ, Brasil.Universidade Federal de Minas Gerais. Instituto de Ciências Biológicas. Departamento de Parasitologia. Laboratório de Fisiologia de Insetos Hematófagos. Belo Horizonte, MG, Brasil.National Institutes of Health. National Institute of Allergy and Infectious Diseases. Laboratory of Malaria and Vector Research. Vector Molecular Biology Section. Rockville, MD, USA.Universidade Federal de Minas Gerais. Instituto de Ciências Biológicas. Departamento de Parasitologia. Laboratório de Fisiologia de Insetos Hematófagos. Belo Horizonte, MG, Brasil / Instituto Nacional de Ciência e Tecnologia em Entomologia Molecular. Rio de Janeiro,RJ, Brasil.Universidade Federal de Minas Gerais. Instituto de Ciências Biológicas. Departamento de Parasitologia. Laboratório de Fisiologia de Insetos Hematófagos. Belo Horizonte, MG, Brasil / Instituto Nacional de Ciência e Tecnologia em Entomologia Molecular. Rio de Janeiro,RJ, Brasil.National Institutes of Health. National Institute of Allergy and Infectious Diseases. Laboratory of Malaria and Vector Research. Vector Molecular Biology Section. Rockville, MD, USA.National Institutes of Health. National Institute of Allergy and Infectious Diseases. Laboratory of Malaria and Vector Research. Vector Molecular Biology Section. Rockville, MD, USA.National Institutes of Health. National Institute of Allergy and Infectious Diseases. Laboratory of Malaria and Vector Research. Vector Molecular Biology Section. Rockville, MD, USA.National Institutes of Health. National Institute of Allergy and Infectious Diseases. Laboratory of Malaria and Vector Research. Vector Molecular Biology Section. Rockville, MD, USA.National Institutes of Health. National Institute of Allergy and Infectious Diseases. Laboratory of Malaria and Vector Research. Vector Molecular Biology Section. Rockville, MD, USA.Universidade Federal de Minas Gerais. Instituto de Ciências Biológicas. Departamento de Parasitologia. Laboratório de Fisiologia de Insetos Hematófagos. Belo Horizonte, MG, Brasil / Instituto Nacional de Ciência e Tecnologia em Entomologia Molecular. Rio de Janeiro,RJ, Brasil.Saliva of the blood feeding sand fly Lutzomyia longipalpis was previously shown to inhibit the alternative pathway (AP) of the complement system. Here, we have identified Lufaxin, a protein component in saliva, as the inhibitor of the AP. Lufaxin inhibited the deposition of C3b, Bb, Properdin, C5b, and C9b on agarose-coated plates in a dose-dependent manner. It also inhibited the activation of factor B in normal serum, but had no effect on the components of the membrane attack complex. Surface plasmon resonance (SPR) experiments demonstrated that Lufaxin stabilizes the C3b-B proconvertase complex when passed over a C3b surface in combination with factor B. Lufaxin was also shown to inhibit the activation of factor B by factor D in a reconstituted C3b-B, but did not inhibit the activation of C3 by reconstituted C3b-Bb. Proconvertase stabilization does not require the presence of divalent cations, but addition of Ni(2+) increases the stability of complexes formed on SPR surfaces. Stabilization of the C3b-B complex to prevent C3 convertase formation (C3b-Bb formation) is a novel mechanism that differs from previously described strategies used by other organisms to inhibit the AP of the host complement system

Serine protease activities in Oxysarcodexia thornax (Walker) (Diptera: Sarcophagidae) first instar larva

We report for the first time the expression of multiple protease activities in the first instar larva (L1) of the flesh fly Oxysarcodexia thornax (Walker). Zymographic analysis of homogenates from freshly obtained L1 revealed a complex proteolytic profile ranging from 21.5 to 136 kDa. Although some activities were detected at pH 3.5 and 5.5, the optimum pH for most of the proteolytic activities was between pH 7.5 and 9.5. Seven of 10 proteases were completely inactivated by phenyl-methyl sulfonyl-fluoride, suggesting that main proteases expressed by L1 belong to serine proteases class. Complete inactivation of all enzymatic activities was obtained using N-p-Tosyl-L-phenylalanine chloromethyl ketone (100 µM), a specific inhibitor of chymotrypsin-like serine proteases