Heterologous expression of the chitinase enzyme of Leishmania (L.) infantum chagasi, Leishmania (V.) braziliensis and Leishmania (V.) amazonensis: serological diagnosis and comparative molecular study using expression systems of insects cell and bacteria

• Grant number: 13/26096-4
• Support Opportunities: Scholarships in Brazil - Post-Doctoral
• Start date: May 01, 2014
• End date: August 31, 2017
• Field of knowledge: Health Sciences - Medicine - Medical Clinics
• Agreement: Coordination of Improvement of Higher Education Personnel (CAPES)
• Principal Investigator: Marcia Aparecida Speranca
• Grantee: Aline Diniz Cabral
• Host Institution: Centro de Ciências Naturais e Humanas (CCNH). Universidade Federal do ABC (UFABC). Ministério da Educação (Brasil). Santo André , SP, Brazil

Abstract

Leishmaniasis, a neglected infectious disease expanding in Brazil, caused by Leishmania species, Class Kinetoplasta and Family Trypanosomatidae, is transmitted by insects (sandfly - Lutzomyia) and affects humans, domestic dogs and wild animals. The clinical presentation of this disease depends on the species of the parasite and the host immune response, ranging from asymptomatic, cutaneous and mucocutaneous lesions (MCL) with disfiguring and disabling sequelae, and visceral leishmaniasis (VL) that is the most serious clinical manifestation since if not properly treated, can lead to death. The main endemic Leishmania species in Brazil involved in MCL is L. (Viannia) braziliensis and L. (V.) amazonensis; and in VL is Leishmania (L.) infantum chagasi . The ineffectiveness of current methods of treatment and control of leishmaniasis is related to the difficulty in diagnosing and identifying Leishmania species in circulation, which is essential for the correct treatment and also to the management of animal reservoirs, particularly in endemic areas for several species of the parasite, and for other trypanosomes, which results in cross-reactions in diagnostic tests. Thus, this research project has as its main objective the evaluation of Leishmania chitinase produced in heterologous protein expression systems, as antigens to detect specific antibodies to L. i . chagasi in serum samples from humans and dogs infected by this parasite species. The specificity of nucleotide and amino acid sequence of chitinase in different species of Leishmania, the chitinase expression in both vertebrate and invertebrate stages of parasite development, and its exportation to the extracellular environment, support the hypothesis that Leishmania chitinase constitutes on excellent tool for specific antibodies detection produced for each parasite species. Initially, this specificity will be investigated by serological tests on samples of individuals and dogs infected with L. i . chagasi with recombinant derivatives of chitinase from L. i. chagasi, L. braziliensis and L. amazonensis produced in heterologous prokaryotic and eukaryotic expression systems. The expression of this enzyme in eukaryotic system is unique and will be held due to the biochemical properties based on Leishmania chitinase primary amino acid sequence, which suggests that this protein is post-translationaly modified specifically by eukaryotic cells. Therefore, this study also will provide a comparative analysis of the biochemical and molecular properties of the recombinant chitinases produced in both heterologous systems, in order to understand its role in the hosts-parasite biological interactions. (AU)

Scientific publications

(8)

(References retrieved automatically from Web of Science and SciELO through information on FAPESP grants and their corresponding numbers as mentioned in the publications by the authors)

VERDE OLIVEIRA ROCHA, ANA VITORIA; SODRE MORENO, BRENDA FERNANDA; CABRAL, ALINE DINIZ; LOUZEIRO, NAYARA MENDES; MIRANDA, LEANDRO MACEDO; BRANDAO DOS SANTOS, VIVIAN MAGALHAES; COSTA, FRANCISCO BORGES; SEABRA NOGUEIRA, RITA DE MARIA; MARCILI, ARLEI; SPERANCA, MARCIA APARECIDA; et al. Diagnosis and epidemiology of Leishmania infantum in domestic cats in an endemic area of the Amazon region, Brazil. Veterinary Parasitology, v. 273, p. 80-85, SEP 2019. (13/26096-4, 11/00147-6, 16/14514-4, 10/50886-7)

SUZUKI, RODRIGO BUZINARO; CABRAL, ALINE DINIZ; ALVES MARTINS, LUCIAMARE PERINETTI; SPERANCA, MARCIA APARECIDA. A highly sensitive Leishmania infantum chagasi isolation method from bone marrow and peripheral blood of adults and children. JOURNAL OF INFECTION IN DEVELOPING COUNTRIES, v. 10, n. 11, p. 1275-1277, NOV 2016. (13/26096-4, 12/20221-9)

DA SILVA, VIVIAM M.; CABRAL, ALINE D.; SPERANCA, MARCIA A.; SQUINA, FABIO M.; MUNIZ, JOAO RENATO C.; MARTIN, LYDIE; NICOLET, YVAIN; GARCIA, WANIUS. High-resolution structure of a modular hyperthermostable endo-beta-1,4-mannanase from Thermotoga petrophila: The ancillary immunoglobulin-like module is a thermostabilizing domain. BIOCHIMICA ET BIOPHYSICA ACTA-PROTEINS AND PROTEOMICS, v. 1868, n. 8, AUG 2020. (13/26096-4, 16/15836-5, 17/17275-3, 17/16291-5, 14/02065-5, 16/14514-4, 14/50897-0, 15/50590-4)

GARCIA, FELIPE BAENA; CABRAL, ALINE DINIZ; FUHLENDORF, MAX MARIO; DA CRUZ, GEOMAR FEITOSA; DOS SANTOS, JULIETE VITORINO; FERREIRA, GRAZIELE CRISTINA; CARNEIRO DE REZENDE, BERNARD ROBIN; SANTANA, CARLA MOREIRA; PUZER, LUCIANO; SASAKI, SERGIO DAISHI; et al. Functional and structural characterization of an ecotin-like serine protease inhibitor from Trypanosoma cruzi. International Journal of Biological Macromolecules, v. 151, p. 459-466, MAY 15 2020. (13/26096-4, 17/17275-3, 16/14514-4)

CABRAL, ALINE DINIZ; GARCIA, FELIPE BAENA; SUZUKI, RODRIGO BUZINARO; GOIS FILHO, TANIL LACERDA; DA COSTA, RENATA TORRES; PEREIRA VASCONCELOS, LIGIA MARINHO; SANTOS, EDMAR SILVA; SPERANCA, MARCIA APARECIDA. Dataset on recombinant expression of an ancient chitinase gene from different species of Leishmania parasites in bacteria and in Spodoptera frugiperda cells using baculovirus. DATA IN BRIEF, v. 32, OCT 2020. (13/26096-4, 16/14514-4, 12/20221-9)

SANTOS, DAUBIAN; RIBEIRO, GUILHERME CUNHA; CABRAL, ALINE DINIZ; SPERANCA, MARCIA APARECIDA. A non-destructive enzymatic method to extract DNA from arthropod specimens: Implications for morphological and molecular studies. PLoS One, v. 13, n. 2, FEB 1 2018. (16/14514-4, 13/26096-4)

MURILLO, JULIANA LONDONO; CABRAL, ALINE DINIZ; UEHARA, MABEL; DA SILVA, VIVIAM MOURA; DOS SANTOS, JULIETE VITORINO; CARVALHO MUNIZ, JOAO RENATO; ESTROZI, LEANDRO FARIAS; FENEL, DAPHNA; GARCIA, WANIUS; SPERANCA, MARCIA APARECIDA. Nucleoprotein from the unique human infecting Orthobunyavirus of Simbu serogroup (Oropouche virus) forms higher order oligomers in complex with nucleic acids in vitro. Amino Acids, v. 50, n. 6, p. 711-721, JUN 2018. (13/26096-4, 09/11347-6, 12/03503-0, 15/02897-3)

CABRAL, ALINE DINIZ; GARCIA, FELIPE BAENA; DA COSTA, RENATA TORRES; PEREIRA VASCONCELOS, LIGIA MARINHO; UEHARA, MABEL; SANTOS, EDMAR SILVA; SPERANCA, MARCIA APARECIDA. A scalable suspension insect cell transfection method for production of baculoviruses with low amplification passages. METHODSX, v. 7, 2020. (09/11347-6, 13/26096-4, 16/14514-4, 12/20221-9)