Insights into the tracking of the cysteine proteinase B COOH-terminal polypeptide of Leishmania (Leishmania) amazonensis by surface plasmon resonance
Leishmania (Leishmania) amazonensis has adaptive mechanisms to the host environment that are guided by its proteinases, including cysteine proteinase B (CPB), and primarily its COOH-terminal region (Cyspep). This work aimed to track the fate of Cyspep by surface plasmon resonance (SPR) of promastigotes and amastigotes to gain a greater understanding of the adaptation of this parasite in both hosts. This strategy consisted of antibody immobilization on a COOH1 surface, followed by interaction with parasite proteins and epoxysuccinyl-L-leucylamido(4-guanidino)butane (E-64). Pro-CPB and Cyspep were detected using specific polyclonal antibodies against a recombinant Cyspep in both parasite forms. The parasitic supernatants from amastigotes and promastigotes exhibited higher anti-Cyspep recognition compared with that in the subcellular fractions. As the supernatant of the promastigote cultures exhibited resonance unit values indicative of an effective with to E-64, this result was assumed to be Pro-CPB detection. Finally, after using three sequential SPR assay steps, we propose that amastigotes and promastigotes release Cyspep into the extracellular environment, but only promastigotes release this polypeptide as Pro-CPB.
KeywordsLeishmania (L.) amazonensis Cysteine proteinase Trans-epoxysuccinyl-L-leucylamido(4-guanidino)butane Surface plasmon resonance
We are grateful to the facilities and technical support of the Fundação Oswaldo Cruz Technological Platforms, the Surface Resonance Plasmonic Platform (RPT03E), Real-Time PCR-RJ (RPT09A), Multiuser Research Facility of Flow Cytometry-Parametric Analysis (IOC), and Culture Medium Platform (IOC).
This study was financed in part by the Conselho Nacional de Desenvolvimento Científico e Tecnológico—Brasil (CNPq) (509737/2010-2; 401356/2014-0), the Fundação de Amparo à Pesquisa do Estado do Rio de Janeiro—Brasil (FAPERJ) (E-26/102.413/2010; E-26/111.954/2011; E-26/110.592/2012), and the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior—Brasil (CAPES)—Finance Code 001. Bernardo A. S. Pereira and Francisco O.R. Oliveira Junior are research fellows of Fiocruz. Carlos R. Alves and Mirian C.S. Pereira are research fellows of the CNPq institution.
Compliance with ethical standards
Conflict of interest
The authors declare that they have no competing interests.
- Alves CR, Pontes de Carvalho LC, Souza AL, De Simone SG (2001a) A strategy for the identification of T-cell epitopes on Leishmania cysteine proteinases. Cytobios 104:33–41Google Scholar
- Da Silveira-Júnior LS, Souza-Silva F, Pereira BA, Cysne-Finkelstein L, Cavalcanti Júnior GB, Alves CR (2017) Exploring the association of surface plasmon resonance with recombinant MHC:Ig hybrid protein as a tool for detecting T lymphocytes in mice infected with Leishmania (Leishmania) amazonensis. Biomed Res Int 2017:9089748CrossRefGoogle Scholar
- De Côrtes LMC, De Souza Pereira MC, De OFOJ, Corte-Real S, Da Silva FS, Pereira BA, De Fátima Madeira M, De Moraes MT, Brazil RP, Alves CR (2012) Leishmania (Viannia) braziliensis: insights on subcellular distribution and biochemical properties of heparin-binding proteins. Parasitology 139:200–207CrossRefGoogle Scholar
- Gomes CB, Silva FS, Charret KD, Pereira BA, Finkelstein LC, Santos-de-Souza R, de Castro Côrtes LM, Pereira MC, Rodrigues de Oliveira FO Jr, Alves CR (2017) Increasing in cysteine proteinase B expression and enzymatic activity during in vitro differentiation of Leishmania (Viannia) braziliensis: first evidence of modulation during morphological transition. Biochimie 133:28–36CrossRefGoogle Scholar
- Nakhaee A, Taheri T, Taghikhani M, Mohebali M, Salmanian AH, Fasel N, Rafati S (2004) Humoral and cellular immune responses against type I cysteine proteinase of Leishmania infantum are higher in asymptomatic than symptomatic dogs selected from a naturally infected population. Vet Parasitol 119:107–123CrossRefGoogle Scholar
- Pereira BA, Silva FS, Rebello KM, Marín-Villa M, Traub-Cseko YM, Andrade TC, Bertho AL, Caffarena ER, Alves CR (2011) In silico predicted epitopes from the COOH-terminal extension of cysteine proteinase B inducing distinct immune responses during Leishmania (Leishmania) amazonensis experimental murine infection. BMC Immunol 8(12):44CrossRefGoogle Scholar
- Pral EM, da Moitinho ML, Balanco JM, Teixeira VR, Milder RV, Alfieri SC (2003) Growth phase and medium ph modulate the expression of proteinase activities and the development of megasomes in axenically cultivated Leishmania (Leishmania) amazonensis amastigote-like organisms. J Parasitol 89:35–43CrossRefGoogle Scholar