Lutzomyia longipalpis Saliva Induces Heme Oxygenase-1 Expression at Bite Sites

Sand flies bite mammalian hosts to obtain a blood meal, driving changes in the host inflammatory response that support the establishment of Leishmania infection. This effect is partially attributed to components of sand fly saliva, which are able to recruit and activate leukocytes. Our group has shown that heme oxygenase-1 (HO-1) favors Leishmania survival in infected cells by reducing inflammatory responses. Here, we show that exposure to sand fly bites is associated with induction of HO-1 in vivo. Histopathological analyses of skin specimens from human volunteers experimentally exposed to sand fly bites revealed that HO-1 and Nrf2 are produced at bite sites in the skin. These results were recapitulated in mice ears injected with a salivary gland sonicate (SGS) or exposed to sand fly bites, indicating that vector saliva may be a key factor in triggering HO-1 expression. Resident skin macrophages were the main source HO-1 at 24–48 h after bites. Additionally, assays in vivo after bites and in vitro after stimulation with saliva both demonstrated that HO-1 production by macrophages was Nrf2-dependent. Collectively, our data demonstrates that vector saliva induces early HO-1 production at the bite sites, representing a major event associated with establishment of naturally-transmitted Leishmania infections

Lutzomyia longipalpis Saliva Induces Heme Oxygenase-1 Expression at Bite Sites

Submitted by Ana Maria Fiscina Sampaio ([email protected]) on 2018-12-19T16:23:43Z No. of bitstreams: 1 Luz NF. Lutzomyia longipalpis saliva...2018.pdf: 1955626 bytes, checksum: f84660a8c004e77cec0bb334c37bb074 (MD5)Approved for entry into archive by Ana Maria Fiscina Sampaio ([email protected]) on 2018-12-19T16:46:42Z (GMT) No. of bitstreams: 1 Luz NF. Lutzomyia longipalpis saliva...2018.pdf: 1955626 bytes, checksum: f84660a8c004e77cec0bb334c37bb074 (MD5)Made available in DSpace on 2018-12-19T16:46:42Z (GMT). No. of bitstreams: 1 Luz NF. Lutzomyia longipalpis saliva...2018.pdf: 1955626 bytes, checksum: f84660a8c004e77cec0bb334c37bb074 (MD5) Previous issue date: 2018Fundação de Amparo a Pesquisa do Estado da Bahia (FAPESB) e Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), and by the Intramural Research Program of the NIH, National Institute of Allergy and Infectious Diseases. This study was financed in part by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - Brasil (CAPES) - Finance Code 001. NL is a fellowship recipient from CAPES Brazil; PM is a fellowship recipient from FAPESB; AV is a fellowship recipient from Programa Nacional de Pós-Doutorado/CAPES; CdO, UL, CB and VMB are senior investigators of CNPq.Fundação Oswaldo Cruz. Instituto Gonçalo Moniz. Salvador, BA, Brasil.National Institutes of Health. National Institute of Allergy and Infectious Diseases. Laboratory of Malaria and Vector Research. Vector Molecular Biology Section. Rockville, MD, United States.National Institutes of Health. National Institute of Allergy and Infectious Diseases. Laboratory of Malaria and Vector Research. Vector Molecular Biology Section. Rockville, MD, USA.Federal University of Rio de Janeiro. Carlos Chagas Filho Biophysics Institute. Laboratory of Molecular Parasitology, Center of Health Science. Rio de Janeiro, RJ, Brazil.National Institutes of Health. National Institute of Allergy and Infectious Diseases. Laboratory of Malaria and Vector Research. Vector Molecular Biology Section. Rockville, MD, USA.Fundação Oswaldo Cruz. Instituto Gonçalo Moniz. Salvador, BA, Brasil / Universidade Federal da Bahia. Faculdade de Medicina. Salvador, BA, Brasil.Fundação Oswaldo Cruz. Instituto Gonçalo Moniz. Salvador, BA, Brasil / Universidade Federal da Bahia. Faculdade de Medicina. Salvador, BA, Brasil.National Institutes of Health. National Institute of Allergy and Infectious Diseases. Laboratory of Parasitic Diseases, Immunobiology Section. Bethesda, MD, USA.Fundação Oswaldo Cruz. Teresina, PI, 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.Fundação Oswaldo Cruz. Instituto Gonçalo Moniz. Salvador, BA, Brasil / Universidade Federal da Bahia. Faculdade de Medicina. Salvador, BA, Brasil.Fundação Oswaldo Cruz. Instituto Gonçalo Moniz. Salvador, BA, Brasil / Universidade Federal da Bahia. Faculdade de Medicina. Salvador, BA, Brasil.Federal University of Rio de Janeiro. Carlos Chagas Filho Biophysics Institute. Laboratory of Molecular Parasitology, Center of Health Science. Rio de Janeiro, RJ, Brazil.Uniformed Services University of the Health Sciences. Infectious Diseases Division. Bethesda, MD, USA.Fundação Oswaldo Cruz. Instituto Gonçalo Moniz. Salvador, BA, Brasil / Universidade Federal da Bahia. Faculdade de Medicina. Salvador, BA, Brasil / Fundação José Silveira, Bahia. Multinational Organization Network Sponsoring Translational and Epidemiological Research. Salvador, BA, Brasil.Fundação Oswaldo Cruz. Instituto Gonçalo Moniz. Salvador, BA, Brasil / Universidade Federal da Bahia. Faculdade de Medicina. Salvador, BA, 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, United States.Fundação Oswaldo Cruz. Instituto Gonçalo Moniz. Salvador, BA, Brasil / Universidade Federal da Bahia. Faculdade de Medicina. Salvador, BA, Brasil.Sand flies bite mammalian hosts to obtain a blood meal, driving changes in the host inflammatory response that support the establishment of Leishmania infection. This effect is partially attributed to components of sand fly saliva, which are able to recruit and activate leukocytes. Our group has shown that heme oxygenase-1 (HO-1) favors Leishmania survival in infected cells by reducing inflammatory responses. Here, we show that exposure to sand fly bites is associated with induction of HO-1 in vivo. Histopathological analyses of skin specimens from human volunteers experimentally exposed to sand fly bites revealed that HO-1 and Nrf2 are produced at bite sites in the skin. These results were recapitulated in mice ears injected with a salivary gland sonicate (SGS) or exposed to sand fly bites, indicating that vector saliva may be a key factor in triggering HO-1 expression. Resident skin macrophages were the main source HO-1 at 24-48 h after bites. Additionally, assays in vivo after bites and in vitro after stimulation with saliva both demonstrated that HO-1 production by macrophages was Nrf2-dependent. Collectively, our data demonstrates that vector saliva induces early HO-1 production at the bite sites, representing a major event associated with establishment of naturally-transmitted Leishmania infections

Structure of SALO, a leishmaniasis vaccine candidate from the sand fly <i>Lutzomyia longipalpis</i>

<div><p>Background</p><p>Immunity to the sand fly salivary protein SALO (<b>S</b>alivary <b>A</b>nticomplement of <i>Lutzomyia</i> <b><i>lo</i></b><i>ngipalpis</i>) protected hamsters against <i>Leishmania infantum</i> and <i>L</i>. <i>braziliensis</i> infection and, more recently, a vaccine combination of a genetically modified <i>Leishmania</i> with SALO conferred strong protection against <i>L</i>. <i>donovani</i> infection. Because of the importance of SALO as a potential component of a leishmaniasis vaccine, a plan to produce this recombinant protein for future scale manufacturing as well as knowledge of its structural characteristics are needed to move SALO forward for the clinical path.</p><p>Methodology/Principal findings</p><p>Recombinant SALO was expressed as a soluble secreted protein using <i>Pichia pastoris</i>, rSALO(P), with yields of 1g/L and >99% purity as assessed by SEC-MALS and SDS-PAGE. Unlike its native counterpart, rSALO(P) does not inhibit the classical pathway of complement; however, antibodies to rSALO(P) inhibit the anti-complement activity of sand fly salivary gland homogenate. Immunization with rSALO(P) produces a delayed type hypersensitivity response in C57BL/6 mice, suggesting rSALO(P) lacked anti-complement activity but retained its immunogenicity. The structure of rSALO(P) was solved by S-SAD at Cu-K_alpha to 1.94 Å and refined to <i>R</i>_{<i>factor</i>} 17%. SALO is ~80% helical, has no appreciable structural similarities to any human protein, and has limited structural similarity in the C-terminus to members of insect odorant binding proteins. SALO has three predicted human CD4⁺ T cell epitopes on surface exposed helices.</p><p>Conclusions/Significance</p><p>The results indicate that SALO as expressed and purified from <i>P</i>. <i>pastoris</i> is suitable for further scale-up, manufacturing, and testing. SALO has a novel structure, is not similar to any human proteins, is immunogenic in rodents, and does not have the anti-complement activity observed in the native salivary protein which are all important attributes to move this vaccine candidate forward to the clinical path.</p></div

Predicted MHC class II T cell epitopes for the SALO protein in humans.

<p>Predicted MHC class II T cell epitopes for the SALO protein in humans.</p

Antibodies against rSALO(P) block anti-complement activity present in the salivary glands of the sand fly <i>Lutzomyia longipalpis</i> (SGH).

<p>(A) Hemolytic assays using SGH (0.5 salivary gland pairs) in the presence of different dilutions of anti-rSALO(P) antibodies (1:10; 1:100; 1:1000 or 1:10000, in PBS). The data represents the mean ± standard deviation of three independent repetitions (ANOVA and Tukey test). Hemolysis was measured at 414nm. (B) Western blot showing rSALO(P) antibodies recognizing rSALO(P) (rSALO Pichia), native SALO from the salivary gland homogenate of <i>Lutzomyia longipalpis</i> (SGH), and rSALO(H) (rSALO HEK). SDS-PAGE was run under reducing conditions. Pre-immune sera was used as a control.</p

Statistics for data collection and model refinement.

<p>Statistics for data collection and model refinement.</p

Comparison of SALO to PdSP15.

<p>(A) Ribbon diagram of a SALO monomer. (B) Ribbon diagram of pdsp15 (C) C-terminus of SALO (grey) superposes well with PdSP15 (aqua marine). (D) The amino acid sequence alignment comparing SALO to PdSP15 generated with <i>ESPript</i>3.0 [<a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0005374#pntd.0005374.ref036" target="_blank">36</a>] reveals a conserved C-terminal odorant-binding domain. The location of the three predicted T-cell epitopes are shown as black lines. Secondary-structure elements are as follows: α-helices (α), 3₁₀-helices (η), β-strands (β) and β-turns (TT). Identical residues are shown on a red background; conserved residues are shown in red; and conserved regions are shown in blue boxes.</p

The three predicted T cell epitopes on SALO are located on exposed helices.

<p>The position of peptide sequences given at the bottom is shown in red on the SALO structural surface (gray).</p