Vladimir Fazito do Vale

Universidade Federal de Minas Gerais

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Nelder F. Gontijo

Universidade Federal de Minas Gerais

Marcos H. Pereira

Universidade Federal de Minas Gerais

Ricardo N. Araújo

Universidade Federal de Minas Gerais

Jesús G. Valenzuela

Vector Oncology (United States)

Antonio Ferreira Mendes‐Sousa

Universidade Federal do Piauí

José M. C. Ribeiro

Vector Oncology (United States)

Maurício Roberto Viana Sant’Anna

Universidade Federal de Minas Gerais

Daniel Costa Queiroz

Universidade Federal de Minas Gerais

John F. Andersen

National Institute of Allergy and Infectious Diseases

Naylene C. S. Silva

Universidade Federal de Minas Gerais

Cooperative Institutions

Universidade Federal de Minas Gerais

Fundação Oswaldo Cruz

National Institute of Allergy and Infectious Diseases

National Institutes of Health

Universidade Federal do Rio de Janeiro

Vector (United States)

Instituto Nacional de Ciência e Tecnologia em Entomologia Molecular

Universidade de São Paulo

Vector Oncology (United States)

Government of the United States of America

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Estudo da atividade inibidora do sistema do complemento humano presente na saliva e conteúdo intestinal de Lutzomyia longipalpis (Diptera: Psychodidae)

Vladimir Fazito do Vale

O sistema do complemento desempenha um importante papel na imunidade inata e adaptativa, principalmente atraves da opsonizacao de organismos invasores, producao de anafilotoxinas e formacao de poros na superficie de patogenos. A saliva de Lutzomyia longipalpis, o principal vetor de Leishmania infantum nas Americas, e capaz de inibir tanto a via classica como a via alternativa do sistema do complemento humano. Este estudo teve o objetivo de ampliar o conhecimento sobre a inibicao do complemento por L. longipalpis. Em relacao a via classica, a saliva de L. longipalpis nao inibiu a deposicao do primeiro componente do complemento, o C1q. Porem, uma expressiva inibicao dos componentes seguintes C4b, C3b, C5b e C9 ocorreu quando a saliva estava presente. A formacao do complexo de ataque a membrana nao foi influenciada pela presenca da saliva, indicando um ponto de inibicao nas etapas iniciais da cascata. O extrato de glândula salivar foi capaz de inibir a clivagem do C4, mas nao atuou inibindo a atividade enzimatica do C1s. O modo de atuacao da saliva de L. longipalpis parece estar relacionado com o fato do extrato de glândula salivar ser capaz de se ligar ao C1q humano. A proteina salivar responsavel pela inibicao da via classica foi identificada como sendo a LJM19, uma proteina recombinante de aproximadamente 11 kDa que e encontrada na saliva como um dimero de 22,3 kDa. A inibicao da via alternativa tambem foi relacionada as etapas iniciais da cascata do complemento. O extrato de glândula salivar foi capaz de inibir a deposicao de C3b, fator Bb, C5b e C9. A inibicao da deposicao do fator Bb, e provavelmente de todos os outros componentes da via alternativa, foi relacionada a capacidade da saliva em impedir a clivagem do fator B. Porem, a saliva de L. longipalpis nao foi capaz de inibir diretamente a atividade enzimatica do fator D. Alem da inibicao do complemento pela saliva de L. longipalpis, o conteudo intestinal tambem foi testado. Nesse caso, os ensaios de deposicao nao constataram inibicao do C5b para a via alternativa. Para a via classica, o conteudo intestinal de L. longipalpis inibiu a deposicao de C3b e C5b, sem inibir a deposicao de C4b. A presenca de inibidores do complemento na saliva e intestino de flebotomineos deve estar relacionada com a protecao do epitelio intestinal contra os efeitos liticos presentes no sangue ingerido. Alem disso, considerando que a opsonizacao de moleculas (mesmo moleculas soluveis como as proteinas salivares) por C3b aumenta consideravelmente sua imunogenicidade, a inibicao do complemento acabaria por proteger essas moleculas salivares de uma resposta imunologica dirigida contra elas

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Estudo sobre a anatomia, pH e digestão de proteínas no intestino de larvas de Lutzomyia longipalpis (Diptera: Psychodidae).

Vladimir Fazito do Vale

O flebotomineo Lutzomyia longipalpis e o principal vetor de Leishmania infantum, o agente etiologico da Leishmaniose Visceral Americana. Apesar da importância desta especie em saude publica, nao existem na literatura descricoes sobre a anatomia interna do tubo digestivo e pouco se sabe sobre os processos digestivos em larvas de flebotomineos. Sendo assim, este trabalho teve o objetivo de descrever a anatomia do tubo digestivo, determinar o pH na luz intestinal e ainda investigar a digestao de proteinas em larvas de Lutzomyia longipalpis. O intestino medio constitui a maior parte do tubo digestivo e os cecos gastricos estao ausentes. Com o uso de corantes vitais indicadores de pH, foi observado um gradiente de pH, variando de > 9 no interior do intestino medio anterior a 6,5-7 no interior do intestino medio posterior. As endoproteases sao secretadas no intestino medio anterior e sao capazes de digerir azocaseina em diversos valores de pH, especialmente no pH 11. O uso de varios inibidores mostrou que as endoproteases digestivas sao enzimas semelhantes a tripsina e a quimotripsina. Esse resultado foi confirmado com o uso dos substratos L-BApNA e N-CBZ-L-PpNA, especificos para tripsinas e quimotripsinas, respectivamente. Foram observadas 11 bandas (102 a 33 kDa) de atividade de endoproteases no gel de poliacrilamida contendo gelatina como substrato. As tripsinas encontradas no trato digestivo de larvas de L. longipalpis estao soluveis na luz intestinal. Aminopeptidases tambem foram estudadas com o uso de substratos sinteticos. Essas enzimas estao localizadas na porcao posterior do intestino medio, ligadas ao epitelio e com pH otimo entre 6,5 e 8. Estes resultados estao de acordo com a proposta de que as proteinas sao digeridas ate pequenos peptideos dentro do espaco endoperitrofico no intestino medio anterior e estes peptideos sao digeridos em aminoacidos no espaco ectoperitrofico no intestino medio posterior.

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

Frontiers in Immunology (2017)

Antonio Ferreira Mendes‐Sousa Vladimir Fazito do Vale Naylene C. S. Silva Anderson B. Guimarães-Costa Marcos H. Pereira

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.

Complement

Salivary Proteins

10.3389/fimmu.2017.01065

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Citations (23)

Midgut pH profile and protein digestion in the larvae of Lutzomyia longipalpis (Diptera: Psychodidae)

Journal of Insect Physiology (2007)

Vladimir Fazito do Vale Marcos H. Pereira Nelder F. Gontijo

Digestion

10.1016/j.jinsphys.2007.06.005

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Citations (38)

Inhibition of the classical pathway of the complement system by saliva of Amblyomma cajennense (Acari: Ixodidae)

Experimental Parasitology (2016)

Paula Ferreira Franco Naylene C. S. Silva Vladimir Fazito do Vale Jéssica F. Abreu Vânia Cristina dos Santos

Amblyomma americanum

Lectin pathway

10.1016/j.exppara.2016.03.002

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An Inhibitor of the Alternative Pathway of Complement in Saliva of New World Anopheline Mosquitoes

The Journal of Immunology (2016)

Antonio Ferreira Mendes‐Sousa Daniel Costa Queiroz Vladimir Fazito do Vale José M. C. Ribeiro Jesús G. Valenzuela

Abstract The complement system present in circulating blood is an effective mechanism of host defense, responsible for the killing of pathogens and the production of potent anaphylatoxins. Inhibitors of the complement system have been described in the saliva of hematophagous arthropods that are involved in the protection of digestive tissues against complement system–mediated damage. In this study, we describe albicin, a novel inhibitor of the alternative pathway of complement from the salivary glands of the malaria vector, Anopheles albimanus. The inhibitor was purified from salivary gland homogenates by reverse-phase HPLC and identified by mass spectrometry as a small (13.4-kDa) protein related to the gSG7 protein of Anopheles gambiae and Anopheles stephensi. Recombinant albicin was produced in Escherichia coli and found to potently inhibit lysis of rabbit erythrocytes in assays of the alternative pathway while having no inhibitory effect on the classical or lectin pathways. Albicin also inhibited the deposition of complement components on agarose-coated plates, although it could not remove previously bound components. Antisera produced against recombinant albicin recognized both the native and recombinant inhibitors and also blocked their activities in in vitro assays. Using surface plasmon resonance and enzymatic assays, we found that albicin binds and stabilizes the C3-convertase complex (C3bBb) formed on a properdin surface and inhibits the convertase activity of a reconstituted C3bBb complex in solution. The data indicate that albicin specifically recognizes the activated form of the complex, allowing more efficient inhibition by an inhibitor whose quantity is limited.

C3-convertase

Anopheles stephensi

Properdin

Lectin pathway

10.4049/jimmunol.1600020

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Citations (19)

LJM19, a salivary protein from the sand fly vector that transmits leishmaniasis, is a novel inhibitor of the classical pathway of complement

Immunobiology (2012)

Viviana P. Ferreira Gurpanna Saggu Marcos H. Pereira Ricardo N. Araújo Vladimir Fazito do Vale

Complement

10.1016/j.imbio.2012.08.153

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SALO, a novel classical pathway complement inhibitor from saliva of the sand fly Lutzomyia longipalpis

Scientific Reports (2016)

Viviana P. Ferreira Vladimir Fazito do Vale Michael K. Pangburn Maha Abdeladhim Antonio Ferreira Mendes‐Sousa

Blood-feeding insects inject potent salivary components including complement inhibitors into their host's skin to acquire a blood meal. Sand fly saliva was shown to inhibit the classical pathway of complement; however, the molecular identity of the inhibitor remains unknown. Here, we identified SALO as the classical pathway complement inhibitor. SALO, an 11 kDa protein, has no homology to proteins of any other organism apart from New World sand flies. rSALO anti-complement activity has the same chromatographic properties as the Lu. longipalpis salivary gland homogenate (SGH)counterparts and anti-rSALO antibodies blocked the classical pathway complement activity of rSALO and SGH. Both rSALO and SGH inhibited C4b deposition and cleavage of C4. rSALO, however, did not inhibit the protease activity of C1s nor the enzymatic activity of factor Xa, uPA, thrombin, kallikrein, trypsin and plasmin. Importantly, rSALO did not inhibit the alternative or the lectin pathway of complement. In conclusion our data shows that SALO is a specific classical pathway complement inhibitor present in the saliva of Lu. longipalpis. Importantly, due to its small size and specificity, SALO may offer a therapeutic alternative for complement classical pathway-mediated pathogenic effects in human diseases.

Lectin pathway

Complement component 2

10.1038/srep19300

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Citations (47)

Host Modulation by a Parasite: How Leishmania infantum Modifies the Intestinal Environment of Lutzomyia longipalpis to Favor Its Development

PLoS ONE (2014)

Vânia Cristina dos Santos Vladimir Fazito do Vale Sydnei Magno Silva Alexandre Alves Sousa Nascimento Natália Alvim Araújo Saab

Some reports have described the interference of Leishmania on sand flies physiology, and such behavior most likely evolved to favor the development and transmission of the parasite. Most of these studies showed that Leishmania could modulate the level of proteases in the midgut after an infective blood meal, and decreased proteolytic activity is indeed beneficial for the development of promastigotes in the gut of sand flies. In the present study, we performed a detailed investigation of the intestinal pH in Lutzomyia longipalpis females naturally infected with Leishmania infantum and investigated the production of trypsin by these insects using different approaches. Our results allowed us to propose a mechanism by which these parasites interfere with the physiology of L. longipalpis to decrease the production of proteolytic enzymes. According to our hypothesis L. infantum promastigotes indirectly interfere with the production of trypsin by modulating the mechanism that controls the intestinal pH via the action of a yet non-identified substance released by promastigote forms inside the midgut. This substance is not an acid, whose action would be restrict on to release H+ to the medium, but is a substance that is able to interfere with midgut physiology through a mechanism involving pH control. According to our hypothesis, as the pH decreases, the proteolytic enzymes efficiency is also reduced, leading to a decline in the supply of amino acids to the enterocytes: this decline reduces the stimulus for protease production because it is regulated by the supply of amino acids, thus leading to a delay in digestion.

Blood meal

Proteolytic enzymes

Digestion

10.1371/journal.pone.0111241

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Citations (17)

Carbohydrate digestion in Lutzomyia longipalpis’ larvae (Diptera – Psychodidae)

Journal of Insect Physiology (2012)

Vladimir Fazito do Vale Bruno H. Moreira Caroline da Silva Moraes Marcos H. Pereira Fernando Ariel Genta

Lutzomyia longipalpis is the principal species of phlebotomine incriminated as vector of Leishmania infantum, the etiological agent of visceral leishmaniasis in the Americas. Despite its importance as vector, almost nothing related to the larval biology, especially about its digestive system has been published. The objective of the present study was to obtain an overview of carbohydrate digestion by the larvae. Taking in account that phlebotomine larvae live in the soil rich in decaying materials and microorganisms we searched principally for enzymes capable to hydrolyze carbohydrates present in this kind of substrate. The principal carbohydrases encountered in the midgut were partially characterized. One of them is a α-amylase present in the anterior midgut. It is probably involved with the digestion of glycogen, the reserve carbohydrate of fungi. Two other especially active enzymes were present in the posterior midgut, a membrane bound α-glucosidase and a membrane bound trehalase. The first, complete the digestion of glycogen and the other probably acts in the digestion of trehalose, a carbohydrate usually encountered in microorganisms undergoing hydric stress. In a screening done with the use of p-nitrophenyl-derived substrates other less active enzymes were also observed in the midgut. A general view of carbohydrate digestion in L. longipalpis was presented. Our results indicate that soil microorganisms appear to be the main source of nutrients for the larvae.

Digestion

Trehalase

10.1016/j.jinsphys.2012.07.005

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Citations (22)