title: Susceptibility of Phytomonas serpens to calpain inhibitors in vitro: interference on the proliferation, ultrastructure, cysteine peptidase expression and interaction with the invertebrate host.
abstract: A pleiotropic response to the calpain inhibitor MDL28170 was detected in the tomato parasite Phytomonas serpens. Ultrastructural studies revealed that MDL28170 caused mitochondrial swelling, shortening of flagellum and disruption of trans Golgi network. This effect was correlated to the inhibition in processing of cruzipain-like molecules, which presented an increase in expression paralleled by decreased proteolytic activity. Concomitantly, a calcium-dependent cysteine peptidase was detected in the parasite extract, the activity of which was repressed by pre-incubation of parasites with MDL28170. Flow cytometry and Western blotting analyses revealed the differential expression of calpain-like proteins (CALPs) in response to the pre-incubation of parasites with the MDL28170, and confocal fluorescence microscopy confirmed their surface location. The interaction of promastigotes with explanted salivary glands of the insect Oncopeltus fasciatus was reduced when parasites were pre-treated with MDL28170, which was correlated to reduced levels of surface cruzipain-like and gp63-like molecules. Treatment of parasites with anti-Drosophila melanogaster (Dm) calpain antibody also decreased the adhesion process. Additionally, parasites recovered from the interaction process presented higher levels of surface cruzipain-like and gp63-like molecules, with similar levels of CALPs cross-reactive to anti-Dm-calpain antibody. The results confirm the importance of exploring the use of calpain inhibitors in studying parasites' physiology.
authors.affiliation: Universidade Federal do Rio de Janeiro, Instituto de Microbiologia Paulo de Góes, Departamento de Microbiologia Geral, Laboratório de Investigação de Peptidases, Rio de Janeiro, RJ, Brasil.
authors.forename: Simone Santiago Carvalho de
authors.initials: SS
authors.lastname: Oliveira
authors.affiliation: Universidade Federal do Rio de Janeiro, Instituto de Química, Programa de Pós-Graduação em Bioquímica, Rio de Janeiro, RJ, Brasil.
authors.forename: Diego de Souza
authors.initials: DS
authors.lastname: Gonçalves
authors.affiliation: Fundação Oswaldo Cruz, Instituto Oswaldo Cruz, Laboratório de Estudos Integrados em Protozoologia, Coleção de Protozoários, Rio de Janeiro, RJ, Brasil.|Instituto Federal de Educação, Ciência e Tecnologia, Laboratório de Microbiologia, Rio de Janeiro, RJ, Brasil.
authors.forename: Aline Dos Santos
authors.initials: AD
authors.lastname: Garcia-Gomes
authors.affiliation: Universidade Federal do Rio de Janeiro, Instituto de Microbiologia Paulo de Góes, Departamento de Microbiologia Geral, Laboratório de Bioquímica de Microrganismos, Rio de Janeiro, RJ, Brasil.
authors.forename: Inês Correa
authors.initials: IC
authors.lastname: Gonçalves
authors.affiliation: Centro Universitário Estadual da Zona Oeste, Laboratório de Tecnologia em Cultura de Células, Rio de Janeiro, RJ, Brasil.
authors.forename: Sergio Henrique
authors.initials: SH
authors.lastname: Seabra
authors.affiliation: Fundação Oswaldo Cruz, Instituto Oswaldo Cruz, Laboratório de Biologia Celular, Rio de Janeiro, RJ, Brasil.
authors.forename: Rubem Figueiredo
authors.initials: RF
authors.lastname: Menna-Barreto
authors.affiliation: Universidade Federal do Rio de Janeiro, Instituto de Microbiologia Paulo de Góes, Departamento de Microbiologia Geral, Laboratório de Bioquímica de Microrganismos, Rio de Janeiro, RJ, Brasil.
authors.forename: Angela Hampshire de Carvalho Santos
authors.initials: AH
authors.lastname: Lopes
authors.affiliation: Fundação Oswaldo Cruz, Instituto Oswaldo Cruz, Laboratório de Estudos Integrados em Protozoologia, Coleção de Protozoários, Rio de Janeiro, RJ, Brasil.
authors.forename: Claudia Masini
authors.initials: CM
authors.lastname: D'Avila-Levy
authors.affiliation: Universidade Federal do Rio de Janeiro, Instituto de Microbiologia Paulo de Góes, Departamento de Microbiologia Geral, Laboratório de Investigação de Peptidases, Rio de Janeiro, RJ, Brasil.|Universidade Federal do Rio de Janeiro, Instituto de Química, Programa de Pós-Graduação em Bioquímica, Rio de Janeiro, RJ, Brasil.
authors.forename: André Luis Souza Dos
authors.initials: AL
authors.lastname: Santos
authors.affiliation: Universidade Federal do Rio de Janeiro, Instituto de Microbiologia Paulo de Góes, Departamento de Microbiologia Geral, Laboratório de Investigação de Peptidases, Rio de Janeiro, RJ, Brasil.
authors.forename: Marta Helena
authors.initials: MH
authors.lastname: Branquinha
mesh_terms: D000818:Animals; D015153:Blotting, Western; D003545:Cysteine / Q000187:drug effects* / Q000378:metabolism; D004151:Dipeptides; D056898:Euglenozoa / Q000187:drug effects* / Q000201:enzymology / Q000648:ultrastructure; D005434:Flow Cytometry; D020062:Heteroptera / Q000469:parasitology*; D006790:Host-Parasite Interactions / Q000502:physiology*; D007928:Lethal Dose 50; D008854:Microscopy, Electron; D012469:Salivary Glands / Q000469:parasitology
chemical_list: D004151:Dipeptides; D003545:Cysteine; C058076:calpain inhibitor III