Bodo sp., a Free-Living Flagellate, Expresses Divergent Proteolytic Activities from the Closely Related Parasitic Trypanosomatids CLAUDIA M. D’AVILA-LEVY, a ALINE C. C. VOLOTA ˜ O, b FERNANDA M. ARAU ´ JO, c JOSE ´ B. DE JESUS, a,d MARIA CRISTINA M. MOTTA, e ALANE B. VERMELHO, c ANDRE ´ L. S. SANTOS c and MARTA H. BRANQUINHA c a Laborato´rio de Biologia Molecular e Doenc ¸as Endeˆmicas, Instituto Oswaldo Cruz (IOC), Fiocruz, Rio de Janeiro, RJ 21045-900, Brazil, and b Laborato´rio de Epidemiologia Molecular de Doenc ¸as Infecciosas, Instituto Oswaldo Cruz (IOC), Fiocruz, Rio de Janeiro, RJ 21045-900, Brazil, and c Departamento de MicrobiologiaGeral, Instituto de Microbiologia Prof. Paulo de Go´es, Centro de Cieˆncias da Sau´de, Bloco I, Universidade Federal do Rio de Janeiro, Cidade Universita´ria, Rio de Janeiro, RJ 21941-902, Brazil, and d Departamento de Cieˆncias Naturais, Universidade Federal de Sa˜o Joa˜o Del Rei, Sa˜o Joa˜o Del Rei, Minas Gerais, MG 36300-000, Brazil, and e Instituto de Biofı´sica Carlos Chagas Filho, Centro de Cieˆncias da Sau´de, UniversidadeFederal do Rio de Janeiro, Cidade Universita´ria, Rio de Janeiro, RJ 21941-590, Brazil ABSTRACT. We report the characterization of cell-associated and extracellular peptidases of Bodo sp., a free-living flagellate of the Bodonidae family, order Kinetoplastida, which is considered ancestral to the trypanosomatids. This bodonid isolate is phylogenetically related to Bodo caudatus and Bodo curvifilus. The proteolytic activity profiles of Bodo sp. were determined by sodium dodecyl sulfate- polyacrylamide gel electrophoresis containing co-polymerized gelatin, casein, hemoglobin, or bovine serum albumin as substrates. The enzymatic complex degraded gelatin better in acidic pH, and under these conditions four proteolytic bands (120, 100, 90, and 75 kDa) were detected in the cellular or extracellular extracts. Two peptidases (250 and 200 kDa) were exclusively detected with the substrate casein. All these enzymes belong to the serine peptidase class, based on inhibition by aprotinin and phenylmethylsulfonyl fluoride. This is the first biochemical characterization of peptidases in a free-living Bodo sp., potentially providing insight into the physiology of these protozoa and the evolutionary importance of peptidases to the order Kinetoplastida as some of these enzymes are important virulence factors in patho- genic trypanosomatids. Key Words. Bodonidae, evolution, Kinetoplastida, peptidase, Trypanosomatidae. K INETOPLASTIDA, including the families Trypanosomat- idae and Bodonidae, is one of the most interesting groups of eukaryotic microorganisms. The family Trypanosomatidae in- cludes parasites responsible for major human diseases, such as sleeping sickness (Trypanosoma brucei), Chagas’ disease (Try- panosoma cruzi), and leishmaniasis (Leishmania spp.). Some try- panosomatids are also parasites of a wide variety of vertebrates, invertebrates, ciliates, and plants (McGhee and Cosgrove 1980; Vickerman 1994). The family Bodonidae includes free-living spe- cies that are common in aquatic environments, playing major roles in microbial food webs by consuming bacteria and other small eukaryotes (Arndt et al. 2000). Peptidases are ubiquitous enzymes that hydrolyze peptide bonds in proteins, being classified as endo- or exopeptidases. Endopeptidases are subdivided into five classes according to the catalytic mechanism (Barrett, Rawlings, and O’Brien 2001). Only threonine endopeptidase has not been described yet in the try- panosomatids (Branquinha et al. 1996; Santos et al. 2005; Silva- Lopez et al. 2004; Valdivieso, Dagger, and Rasco ´n 2007; Vermelho et al. 2007), while the most prominent proteolytic ac- tivities displayed by these flagellates are cysteine and metallo- endopeptidases (Branquinha et al. 1996; d’Avila-Levy et al. 2001; Santos et al. 2005). These enzymes have been implicated in a number of aspects of host–trypanosomatid interactions, including tissue and cell invasion, parasite differentiation, inactivation of deleterious host proteins, and catabolism of exogenous proteins for cell nutrition (McKerrow et al. 1993; Vermelho et al. 2007). It is important to increase our knowledge of peptidase distri- bution in the family Trypanosomatidae because these enzymes could constitute chemotherapeutic, vaccine, and diagnostic tar- gets. In the family Cryptobiidae (suborder Bodonina), the eco- nomically important parasite of fishes, Cryptobia salmositica, which causes cryptobiosis, characterized by anemia in salmonids, expresses cysteine peptidases (49, 60, 66, and 97 kDa) and a 200-kDa metallopeptidase (Zuo and Woo 1998). However, to our knowledge, the peptidases of the family Bodonidae (suborder Bodonina) have never been characterized. Comparison of pepti- dase profile between free-living and classically pathogenic proto- zoa might provide some clues on evolution and function of peptidases in general. Therefore we used zymographic analysis to investigate the cellular and secreted peptidases of a Bodo sp. These enzymes were characterized in relation to pH and temper- ature dependence, sensitivity to specific inhibitors, and their abil- ity to hydrolyze distinct proteinaceous substrates. MATERIALS AND METHODS Microorganisms and cultivation of cells. Bodonid flagellates were isolated from the sap of Cocos nucifera (Tavares da Silva et al. 1988) and were maintained in a monoxenic culture of bacteria for 7 d at 28 1C in brain–heart infusion (BHI) medium (3% [w/v] BHI and 0.002% hemin [w/v]) supplemented with 2% (v/v) fetal calf serum. The monoxenic culture was prepared by treatment of the original Bodo sp. isolate with 50 mg/ml gentamicin for 3 d, followed by centrifugation at 1,500 g, extensive washing with BHI, and supplementation with monoxenic feeder bacteria. The feeder bacteria were isolated from the original Bodo sp. culture by selecting a single colony grown on BHI-agar plates at 28 1C. Thus, while the bacterial culture is monoxenic the bacterial feeder is undefined. Polymerase chain reaction (PCR) amplification and se- quencing of kinetoplastid 18S ribosomal RNA (rRNA) genes. The bodonid DNA was extracted using the standard phenol– chloroform method (Sambrook, Fritsch, and Maniatis 1982). The 18S rRNA gene from culture sample DNA was PCR- amplified using kinetoplastid-specific primers: kineto14F (5 0 -CTGCCAGTAGTCATATGCTTGTTTCAAGGA-3 0 and kin- eto2026R (5 0 -GATCCTTCTGCAGGTTCACCTACAGCT-3 0 ), and the PCR conditions described previously (von der Heyden and Corresponding Author: Claudia M. D’Avila-Levy, Laborato ´rio de Biologia Molecular e Doenc ¸as Ende ˆmicas, Instituto Oswaldo Cruz, Fundac ¸a ˜o Oswaldo Cruz, Av. Brazil, 4365 - Pavilha ˜o Leo ˆnidas Deane, Manguinhos - Rio de Janeiro – RJ, CEP 21040-900, Brazil—Telephone number: 3865 8232; FAX number: 2290 0479; e-mail: davila.levy@ioc.fiocruz.br 454 J. Eukaryot. Microbiol., 56(5), 2009 pp. 454–458 r 2009 The Author(s) Journal compilation r 2009 by the International Society of Protistologists DOI: 10.1111/j.1550-7408.2009.00424.x