Parasitol Res (2003) 89: 320–322 DOI 10.1007/s00436-002-0792-3
SH O RT CO MM U N IC A T IO N
Fla´via V.S. Almeida Æ Marta H. Branquinha Salvatore Giovanni-De-Simone Æ Alane B. Vermelho
Extracellular metalloproteinase activity in Phytomonas franc¸ai
Received: 7 October 2002 / Accepted: 23 October 2002 / Published online: 17 December 2002 Springer-Verlag 2002
Abstract Extracellular proteolytic activities were detected in Phytomonas franc¸ai culture supernatant. A 67-kDa enzyme was purified by ammonium sulfate precipitation and gel filtration in a HPLC system. This proteinase was optimally active at 28 C and pH 5.0; and the use of proteolytic inhibitors indicated that it belongs to the metalloproteinase class. This is the first report on the purification of an extracellular metalloproteinase from a Phytomonas species.
Roitman 1986). However, further studies on transmission are required to demonstrate conclusively the organism’s pathogenicity. Studies carried out in recent years analyzed the extracellular proteolytic activities in trypanosomatids, since the role of proteases in the interaction with the host occurs at a variety of levels, from the acquisition of nutrients to the establishment of the infection (McKerrow et al. 1993). In order to assess the occurrence of extracellular proteinases in phytomonads, the present study focuses on the proteolytic profile of P. franc¸ai. We also present the purification of an extracellular metalloproteinase and biochemically characterize this enzyme.
Introduction Phytomonas spp comprise heteroxenous trypanosomatids that are supposedly transmitted between plant hosts by phytophagous insects and have been isolated from the latex of latificerous plants, the phloem of trees, mature fruits and the seeds of many plant families (Dollet 1984; Camargo 1999). P. franc¸ai is found associated with a disease known as ‘‘chochamento das raı´ zes’’, which means ‘‘empty roots’’, in the latex of cassava (Manihot esculenta Crantz); and the disease is characterized by poor root system development and general chlorosis of the aerial part of the plant (Vainstein and F.V.S. Almeida Æ M.H. Branquinha Æ A.B. Vermelho (&) Departamento de Microbiologia Geral, Instituto de Microbiologia Prof. Paulo de Go´es, Universidade Federal do Rio de Janeiro, CCS Bloco I, 21941-590, Rio de Janeiro, Brazil E-mail:
[email protected] Fax: +55-21-25608344 S. Giovanni-De-Simone Laborato´rio de Bioquı´ mica de Proteı´ nas e Peptı´ deos, Departamento de Bioquı´ mica e Biologia Molecular, Fundac¸a˜o Oswaldo Cruz, Rio de Janeiro, Brazil S. Giovanni-De-Simone Departamento de Biologia Celular e Molecular, Universidade Federal Fluminense, Nitero´i, Brazil
Materials and methods The trypanosomatid, Phytomonas franc¸ai, provided by Dr. Maria Auxiliadora de Souza, Fundac¸a˜o Oswaldo Cruz, Rio de Janeiro, was cultivated in 3% brain/heart infusion medium (BHI) supplemented with 4 mg% hemin, 2% fetal bovine serum and 2 mg% folic acid. Cell viability was assessed by trypan blue cell dye exclusion (Barankiewicz et al. 1988). For proteinase purification, 4·105 cells/ml were inoculated into 1 l of medium and maintained at 28 C for 96 h. The cell-free supernatant was precipitated with (NH4)2SO4 and subjected to gel filtration, using a Shinpack Diol-150 column as previously described by our group (Melo et al. 2001). SDS-PAGE was performed using 10% polyacrylamide gels under reduction conditions (Laemmli 1970); and protein bands were identified by silver staining (Melo et al. 2001). Gelatinolytic activity was assayed using 10% SDS-PAGE with 0.1% gelatin incorporated into the gel (Heussen and Dowdle 1980). Proteinase activity was measured spectrophotometrically, using the substrate gelatin as described by Jones et al. (1998; Table 1). The enzyme unit was defined as described by Melo et al. (2001). The effect of pH on the proteolytic activity was determined with the following buffer systems: 0.1 M sodium citrate (pH 4.0–5.0) and 0.1 M sodium phosphate (pH 6.0–8.0). Proteolytic activity was also analyzed at different temperatures (25, 28, 37, 50 C). The proteinase inhibitors used are listed in Table 2. In order to verify the effect of metal ions on the purified proteinase, an apoenzyme was prepared by dialysis in the presence of 250 lM 1,10-phenanthroline, followed by the addition of 100 lM ZnSO4 or CaCl2 to ascertain the recovery of the proteolytic activity. The purified enzyme was incubated for 1 h at room temperature with
321 Table 1 Purification of an extracellular metalloproteinase from Phytomonas franc¸ai Purification step
Total protein (mg)
Total activity (units)
Specific activity (units/mg)
Purification factor (x-fold)
Activityyield (%)
Culture supernatant Ammoniumsulfate precipitation Shinpack Diol
5,200 440 16
2,3512.0 2,892.0 869.9
4.5 6.6 54.4
1.0 1.5 12.0
100.0 12.3 3.7
the inhibitors and metal ions. Following incubation, the substrate gelatin was added and the remaining activity was measured under standard assay conditions (Jones et al. 1998). Results are expressed as the relative percentage of activity with inhibitors or metal ions, subtracted from activity without inhibitors or metal ions.
Results and discussion Previous results from our group showed that maximal liberation of proteinases into the culture medium was detected on day 4 of culture, as Phytomonas franc¸ai reached the late-log and stationary phases. On day 4 of growth, the extracellular proteinase profile showed bands migrating in the 90–94 kDa range and at 70 kDa (Fig. 1B). A combination of ammonium sulfate precipitation and gel filtration chromatography was carried out with the aim of purifying the lower-molecular-mass extracellular proteinase from P. franc¸ai. A protein band with a molecular mass of 67 kDa (Fig. 1 A) in SDS-PAGE and one band of gelatinolytic activity (Fig. 1B) was identified after the purification. It is interesting to note that similar enzymes migrating at 60–90 kDa in SDSPAGE/gelatin were identified by our group, such as in Crithidia guilhermei (Melo et al. 2001), C. fasciculata and
C. oncopelti (d’Avila-Levy et al. 2001) and are also reported for Blastocrithidia culicis (Santos et al. 2001). The yield of the purified proteolytic activity was low, being approximately 3.7% with 12-fold purification (Table 1). The optimum pH for the purified proteinase was 5.0 and the enzyme activity decreased markedly at pH values above 6.0. The optimum temperature for the proteolytic activity was 28 C, retaining approximately 70% of its maximum activity at 37 C (data not shown). The extracellular proteinase purified from the culture supernatant of P. franc¸ai was completely blocked by the metalloproteinase inhibitor, 1,10-phenanthroline, and strongly inhibited by EDTA. Other inihibitors weakly inhibited the enzyme. The proteolytic activity was partially inhibited by the divalent ion, Ca2+, while Zn2+ stimulated the enzyme. This inhibition profile suggested that the extracellular proteolytic activity purified in this study corresponds to a metalloproteinase, probably Zn2+-dependent (Table 2). In trypanosomatids, acidic metalloproteinases were previously detected in Leishmania mexicana amastigote lysosomes, which are antigenically related to gp63 (McKerrow et al. 1993), in the culture supernatant of C. guilhermei (Melo et al. 2001) and in the endosymbiont-harboring Crithidia species (d’Avila-Levy et al. 2001), while cell-associated metalloproteinases in many trypanosomatid species are usually active across a broad pH range (Clayton et al. 1995; Branquinha et al. 1996). Latex is a metabolically and developmentally heterogeneous fluid. In cassava plants, it is composed of very high concentrations of sugars, particularly sucrose, amino acids, lipids and hydrolases (Lynn and ClevetteRadford 1987). Interestingly, many plants differ widely in the protein content of their latex, with pHs between 4.5 and 5.5 (Lynn and Clevette-Radford 1987). The extracellular metalloproteinase from P. franc¸ai was Table 2 Effect of proteinase inhibitors on purified metalloproteinase. See materials and methods for details of inhibitors
Fig. 1A, B Analysis of the purification steps. Silver-stained SDSPAGE (A) and SDS-PAGE/gelatin (B) were used to analyze the purification steps of Phytomonas franc¸ai extracellular proteinase activity. The ammonium sulfate precipitation fraction (lane 1) and gel-filtration fraction (lane 2) are shown. About 100 lg of protein was analyzed in lane 1 and 50 lg of protein in lane 2. The gel strip on the left indicates molecular mass markers (in kiloDaltons)
Inhibitor/metal ions
Effective concentration
Residual activity (%)
Control PMSF STI E-64 1,10-Phenanthroline EDTA ZnSO4 CaCl2
–
100.0 78.3 52.8 55.0 0.0 8.6 109.7 51.3
1 100 20 250 5 100 100
mM lg/ml lM lM mM lM lM
322
mostly active at pH 5.0, indicating that latex is a favorable environment for its activity. So, proteins and amino acids present in latex and in the insect could probably be used for nutritional purposes by the trypanosomatid. This is the first report on the detection of extracellular proteinases in Phytomonas spp, but further work is necessary to gain a better understanding of the proteinase functions in these trypanosomatids. The characterization of similar extracellular proteinases in other phytomonads may determine the importance of these enzymes in the parasite interaction with their insect and plant hosts. Acknowledgements We thank Ieˆda Coleto Miguel de Castro e Silva for technical assistance. This study was supported by the Conselho Nacional de Desenvolvimento Cientı´ fico e Tecnolo´gico (MCT/ CNPq), Conselho de Ensino para Graduados e Pesquisas (CEPG/ UFRJ), Fundac¸a˜o Oswaldo Cruz (FIOCRUZ) and Fundac¸a˜o Carlos Chagas Filho de Amparo a` Pesquisa do Estado do Rio de Janeiro (FAPERJ). The experiments comply with the current laws of Brazil.
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