Journal of
INVERTEBRATE PATHOLOGY Journal of Invertebrate Pathology 85 (2004) 9–17 www.elsevier.com/locate/yjipa
Protease and phospholipase inhibition protect Veneza zonata (Hemiptera Coreidae) against septicemia caused by parasite trypanosomatid 563DT Daniele de Oliveira,a Tatiana de Arruda Campos Brasil de Souza,a Letıcia Sayuri Murate,a Jose Vitor Jankevicius,b Luiz Carlos Jabur Gaziri,c and Shiduca Itow Jankeviciusa,* a
Departamento de Microbiologia, Centro de Ci^ encias Biol ogicas, Universidade Estadual de Londrina, CEP 86051-970, Londrina, Paran a, Brazil b Universidade do Norte do Paran a-UNOPAR-Av.Paris, 675, CEP 86041-140, Londrina, Paran a, Brazil. c Departamento de Fisiologia, Centro de Ci^ encias Biol ogicas, Universidade Estadual de Londrina, CEP 86051-970, Londrina, Paran a, Brazil Received 14 April 2003; accepted 1 December 2003
Abstract Veneza zonata (Hemiptera Coreidae) is an insect which causes losses in several crops, and it is also an important vector of lower trypanosomatids. V. zonata specimens were collected on rural properties in Londrina, state of Paran a, Southern Brazil. Inoculation of Leptomonas 563DT into V. zonata hemocoel caused insect death within approximately 24 h, with large bacterial proliferation into their hemocoels. Some bacteria which were found in the digestive tract of those insects, such as Escherichia coli, Providencia rettgeri, and Kluyveria ascorbata, were also found in their hemolymph, which suggests that trypanosomatid crossing into hemocoel caused mechanical lesions in the digestive tract that allowed intestinal bacteria to infect the hemolymph, thereby leading to lethal septicemia. In this study we analysed proteolytic activities from the 563DT Leptomonas strain, which is pathogenic for V. zonata, aiming at evaluating the potential use of this Leptomonas strain for the biocontrol of the insect. The proteolytic action was evaluated on cells and on culture supernatants of trypanosomatids. We also evaluated the gelatinolytic activities, the action over natural and synthetic substrates for aminopeptidases, and the action of protease inhibitors during all trypanosomatid growth stages. A significant reduction in the number of insect deaths was observed when Leptomonas 563DT were incubated with inhibitors of proteases and phospholipases before being inoculated into the insects, which suggests that those enzymes are involved in the pathogenic mechanism. Ó 2003 Elsevier Inc. All rights reserved. Keywords: Trypanosomatids; Proteases and phospholipases; Veneza zonata; Bacteriological pathogenesis; Biological control
1. Introduction Veneza zonata is a hemipteran insect of the Coreidae family, and it is an important vector of lower trypanosomatids. Its geographic distribution is wide, ranging from the USA to South America (King and Saunders, 1984; Morril, 1913). These insects feed on corn, sorghum, bean, tomato, soy, pigeon pea, and various legumes and fruits (King and Saunders, 1984). In Londrina region (Southern Brazil), the presence of this insect in cornfields is predominant, and it is considered * Corresponding author. Fax: +11-55-43-332-72979. E-mail address:
[email protected] (S. I. Jankevicius).
0022-2011/$ - see front matter Ó 2003 Elsevier Inc. All rights reserved. doi:10.1016/j.jip.2003.12.002
an important plague in agriculture. Adults and nymphs feed on developing seeds and fruits causing colour loss, rot and fruit falls. Orange crops are intensely affected, with feeding lesions causing spots on fruits that lower their commercial value (Kubo and Batista, 1992). Besides its agricultural importance, V. zonata is frequently infected with trypanosomatids of the Phytomonas, Leptomonas, Herpetomonas, and Crithidia genera (Batistoti et al., 2001). Although numerous species of protozoa are pathogenic for insects (Henry, 1981), only a few have been used as insect control agents. Nosema locustae is the single commercially registered protozoa, and it has been used for many years in the United States of America, Canada, and Africa in herbage grasshoppers
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control (Mason and Erlandson, 1994) and currently considered a type of fungus. Some trypanosomatids can produce alterations in insect behaviour, decreasing their adaptability, changing their alimentary habits, and altering their motor control. This was reported in the systems Rhodnius prolixus/Trypanosoma rangeli (Grewal, 1957), Pyrrhocoris apterus/Leptomonas pyrrhocoris (Lipa, 1963), Hippelates pusio/Herpetomonas muscarum (Bailey and Brooks, 1972), and Triatoma infestans/ Blastocrithidia triatomae (Schaub, 1992). According to Dias et al. (1994), there are only a few studies about use of protozoa in microbial control of insects, which concerned mainly phytophagous hemipterans. Baccan et al. (2001) observed that, among 60 different isolates of trypanosomatids, only 563 DT strain (Leptomonas), which was isolated from Euchistus heros, was lethal to V. zonata. These flagellates colonize the digestive tract, cross the intestinal barriers into the hemocoel, and allow invasion of the hemocoel by intestinal bacteria, which probably causes the insect death. The simple hemocoel invasion by trypanosomatids apparently is not the cause of death, since insect death is reduced and septicemia do not occur in the migration to hemocoel of V. zonata of another Leptomonas, strain 714DT, isolated from the digestive tract of the V. zonata itself. The mechanism of V. zonata hemocoel invasion seems to be different in the strains 714DT and 563DT of Leptomonas (Baccan et al., 2001). This study aimed at elucidating the pathogenic mechanism of 563DT trypanosomatid strain to V. zonata, through identification of bacteria present in V. zonata digestive tract and hemocoel, and through the characterization of protozoan proteases and phospholipases in the search of differences between the Leptomonas strains 563DT and 714DT that could explain their behaviour in the V. zonata hemocoel infection.
2. Materials and methods 2.1. Collection of insects Veneza zonata were collected in rural properties in Londrina, state of Paran a, Southern Brazil, and immediately transported in cages to the laboratory. After oviposition, the eggs were separated and the nymphs fed on laboratory produced cherry tomatoes and corn, which were not infected with trypanosomatids. The insects were reared and maintained at 28 °C in an incubator with a 12-h light/12-h dark schedule. 2.2. Trypanosomatids Two strains of trypanosomatids isolated in our laboratory, were analysed: 714DT, a Leptomonas isolated
from the digestive tract of V. zonata, which establishes infection in the salivary glands of this insect and in plants (Cavazzana, 1999); and 563DT, a Leptomonas isolated from the digestive tract of E. heros (Hemiptera Pentatomidae) (Batistoti et al., 2001). The 714DT and 563DT strains of Leptomonas were grown in GYPMI medium (Itow Jankevicius et al., 1993) at 28 °C. 2.3. Bacteriological identification of the digestive tract and hemocoel microbiota Veneza zonata experimentally infected with trypanosomatids and non-infected insects were dissected on a sterile environment, for hemocoel and digestive tract bacterial isolation. The insect were killed by chloroform, externally disinfected with ethanol (70%) and in biological safety cabinets, the hemolymph aliquots were collected by leg amputation, and inoculated in petri plates containing blood agar, nutrient agar or MacConkey agar medium. The digestive tract were aseptically dissected and macerated in sterile petri plates and inoculated in other plates of blood agar, nutrient agar or MacConkey agar medium. After colony growth at 37 °C, samples of the colonies were transferred to GYPMI medium. As control of possible contamination during the surgical procedures, samples from the same hemocoel and digestive tract preparations were immediately inoculated into GYPMI liquid medium. Standard biochemical tests according to BergeyÕs Manual of Systematic Bacteriology (1992) were employed for bacterial identification. 2.4. Preparations employed for protease assays Trypanosomatid cells (2 109 ) were centrifuged at 3.86 103 g for 10 min at 4 °C and resuspended in 1 ml of cold 0.15 M NaCl. This cell suspension and the culture medium supernatant were used in the assays of protease activity. The experiments were accomplished in duplicate, repeated thrice, and as control, blank tubes without cell suspension or supernatant were used. For the determination of optimum pH of each enzymatic assay, the buffers sodium citrate (pH 4.0 and 5.0), Tris–maleate (pH 6.0–7.0), and Tris–HCl (pH 7.0–14.0) were used. After determination of the optimum pH, it was determined the optimum temperature, from 20 to 60 °C, tested in the optimum pH of each assay. 2.5. Enzymatic assays on cell suspensions 2.5.1. Azocasein and azoalbumine as substrates The method used was based on the description of Charney and Tomarelli (1947). Azocasein and azoalbumin are non-specific substrates for a broad range of proteases, which upon hydrolysis liberate small peptides not precipitable by strong acids, as an orange compound
D. de Oliveira et al. / Journal of Invertebrate Pathology 85 (2004) 9–17
that can be quantified on alkaline pH. The incubation mixture tested contained 0.5 M of different buffers: sodium citrate (pH 4.0 and 5.0), or Tris–maleate (pH 6.0–7.0), or Tris–HCl (pH 7.0–14.0). One hundred microliters of 1% (wt/vol.) azocasein aqueous solution were added to 200 ll of cellular suspension, in a total volume of 400 ll. After 30 min of incubation at 37 °C, the reaction was stopped by addition of 0.8 ml of 5% (vol./ vol.) perchloric acid (PCA) and the tubes kept on ice for 30 min. After centrifuging at 1.39 103 g for 5 min, the supernatant fluid was neutralized with 0.8 ml of NaOH 1 N. The absorbance of the orange compound obtained was measured in spectrophotometer at 440 nm. 2.6. b-Naphthylamide as substrate The method used was essentially as described by Goldbarg et al. (1959). b-Naphthylamide is a substrate for aminopeptidases. The enzymatic activity is determined by measurement of the b-naphthylamine liberated from b-naphthylamide. After enzymatic hydrolysis, the liberated naphthylamine is diazoted and converted to an azodye by a modification of the Bratton–Marshall reaction. The incubation mixture consisted of the same buffers described above; with the addition of 1 mM leucine-, lysine-, serine-, and n-a-benzoyl-D L -arginine-b naphthylamide (Leu-NA, Lys-NA, Ser-NA, and BANA, respectively); plus 50 ll of living cell suspension of either strains. After 30 min at 37 °C, the reaction was stopped by the addition of 0.5 ml of 40% (wt/vol.) trichloroacetic acid (TCA). After centrifugation at 1.39 103 g for 5 min, to 0.5 ml of the supernatants were added successively, at 2 min intervals, 0.5 ml of a 1 mg/ml sodium nitrite solution (NaNO2 ), 0.5 ml of a 5 mg/ml ammonium sulfamate solution, and 1.0 ml of 0.5 mg/ml ethanolic solution of N-1-naphthylethylenediamine-di HCl. After 30 min, the absorbance of the azodye formed was measured in a spectrophotometer, at 550 nm. 2.7. Esterasic activity Esterasic activity was assayed with carbobenzoxy-L tyrosine-p-nitrophenylester (CTN) as substrate, by quantification of the p-nitrophenol liberated from CTN by protozoan esterases. The incubation mixture consisted of 2 ml buffer, 0.4 ml of 0.1 mM CTN, and 0.6 ml of cell suspension, as described by Walsh and Wilcox (1970).
11
jars at 28 °C, until colony formation. TCA 20% (wt/vol.) was then added, and the cleared zones around the colonies indicated that the protein was degraded by trypanosomatid proteases. 2.9. Enzymatic assays with supernatants The determination of enzymatic activities of culture medium supernatants on synthetic substrates used the same method described by Goldbarg et al. (1959), and over azocasein and azoalbumine, also the method of Charney and Tomarelli (1947). The incubation mixture consisted of the same buffers described above; approximately 2 ml of supernatant were obtained from the GYPMI culture medium of 563DT or 714DT strains at exponential growth and used instead of cell suspension as above. 2.10. Action of protease and phospholipase inhibitors on enzymatic activities The inhibitors utilized in this assay were N-tosyl-L phenylalanine chloromethyl ketone (TPCK, 100 lg/ml), ethylenediamine tetraacetic acid (EDTA, 10 mM), pepstatin (1.0 lg/ml), aprotinin (0.09 lg/ml), or 2 mM of either lead acetate, Fe2 SO4 , CuCl2 , KCl, MnSO4 , ZnSO4 , CoSO4 , CaSO4 , HgCl2 , Al(OH)2 , N-a-p-tosyl-L lysine chloromethyl ketone (TLCK), iodoacetamide, leupeptin, ethyleneglycol-bis-(b-aminoethyl)-N,N,N0 ,N0 tetraacetic acid (EGTA), phenylmethylsulfonyl fluoride (PMSF), ortho-phenanthroline (O-Phe), and palmitoyl D L -carnitine. After incubating the cell suspensions with enzyme inhibitors for 10 min, at the previously determined optimum temperature and pH, the assays were performed as described above, carried out in duplicate, with three repetitions. The enzymatic activity assayed in presence of inhibitors was expressed as percent of the values obtained from the same cell preparation and in absence of added inhibitor, which absorbance was taken as 100% of activity. Trypanosomatids at different growth stages were also utilized on these experiments. The direct action of protease and phospholipase inhibitors were determined in the same way except for the absence of addition of trypanosomatids or culture medium supernatant. 2.11. Inoculation of trypanosomatids treated with inhibitors of proteases and phospholipases into insects
2.8. Gelatinolytic activities The gelatinolytic activities of both strains were assayed in petri plates containing 20 ml of GYPMI medium (Itow Jankevicius et al., 1993) supplemented with 2% of each agar and gelatine. After spreading the trypanosomatids, the plates were maintained in anaerobe
For the experimental infection with trypanosomatids treated with protease inhibitors, 10 insects were used in each test, with 10 replications. The experimental system, developed by Baccan et al. (2001) consist of the inoculation of culture trypanosomatid forms into the hemocoel of V. zonata, were the protozoans rapidly multiply,
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invade the gut wall, opening a way to bacterial invasion of hemocoel in 24 h, instead of 12–14 days in the natural oral infection. The abdominal wall of the insects was externally cleaned with ethanol (70%), and 10 lL of an otherwise sterile trypanosomatid suspension (104 cells) treated with protease and/or phospholipase inhibitors were inoculated by means of an insulin syringe. The needle puncture was closed with sterile glycerine. This procedure was carried out under strict aseptic conditions, into a laminar flow biological safety cabinet and insects which received only sterile 0.15 M NaCl or each enzyme inhibitor separately were used as controls. The insect survival rates were then daily observed.
3. Results The bacteria identified in the digestive tract and hemocoel of insects experimentally infected with the strain 563DT are listed in Table 1. No bacteria were detected in the hemocoel of insects that were not experimentally infected with the 563DT strain, nor in those infected with strain 714DT. The bacteria identified in the digestive tract of infected and not infected insects were mostly the same. The supernatants from GYPMI culture media presented no enzymatic activities on either the synthetic or natural substrates employed.
Table 1 Bacteriological identification of digestive tract and hemocoel microbiota of Veneza zonata infected with 563DT strain trypanosomatid Bacteria identified in insects experimentally infected with strain 563DT Digestive tract
Hemocoel
Citrobacter amalonaticus Edwardsiella ictaluri Enterobacter aerogenes Erwinia ananas Erwinia chrysanthemi Erwinia nigrifluens Erwinia uredovona Escherichia blattae Escherichia coli Klebsiella oxytoca Kluyvera ascorbata Proteus myxofasciens Proteus penneri Providencia rettgeri Providencia rustigiane Salmonella bongori Xenorhabdus nematophilus
Citrobacter amalonaticus Edwardsiella ictaluri Enterobacter aerogenes Erwinia ananas Erwinia chrysanthemi Erwinia uredovona Escherichia blattae Escherichia coli Kluyvera ascorbata Proteus penneri Providencia rettgeri Providencia rustigiane
The results were from a representative experiment, repeated many times with other inoculated V. zonata specimens, with almost similar results. Insects not infected or infected with strain 714DT shows the same bacteria in digestive tract, but none in hemocoel. The identification of bacteria was based on morphological and biochemical data described in BergeyÕs Systematic Bacteriology (1992).
All substrates and inhibitors as the conditions (pH and temperature) in the enzymatic determinations and insect inoculations did not affect the viability of flagellates, as demonstrated by motility of trypanosomes in contrast microscopy. The enzymatic assay with 563DT and 714DT trypanosomatid living cells on natural substrate azocasein presented an optimum pH about 13–14, and on azoalbumine at pH 12, at 37 °C, for both strains. The enzymatic activity on Leu-NA presented an optimum at pH 6.75 for the 563DT strain and 8.0 for the 714DT strain. On Lys-NA, pH 10 was optimum for both isolates, and for BANA, the optimum pH was 10.0 for 563DT and 11.0 for 714DT. For Ser-NA, pH 6.7 was the optimum for both strains. The enzymatic activity at lower pHs was minimal, and no enzymatic activities were detected in citrate buffer at pH 4.0–5.0 in both strains. The optimum temperature for Leu-NA was 35 °C for both strains. On Lys-NA, 35 °C was found for the 563DT strain, and 25 °C for the 714DT. In BANA, the temperature value was 45 °C for both strains and in Ser-NA, 45 °C was found also for both strains. Enzymatic activity on CTN demonstrated optimum pH 7.0 and optimum temperature of 25 °C for both strains. Gelatinolytic activities were detected for both tested strains, although they were higher in 563DT. The use of protease inhibitors allow classification of the enzymatic activities into four types: cysteine-, metallo-, serine-, and aspartic-proteases. The characterization of proteases by means of inhibitors in all growth phases of trypanosomatids is shown in Tables 2 and 3. The action of protease and phospholipase inhibitors on the survival rate of V. zonata experimentally infected with treated trypanosomatids is shown in Table 4. The direct action of protease and phospholipase inhibitors on V. zonata is shown in Table 5.
4. Discussion Naturally occurring entomopathogens are important regulatory factors of insect populations. Many species are employed as biological control agents of insect pests in row and glasshouse crops, orchards, ornamental plants, range turf and lawn, store products, and forestry and for abatement of pest and vector insects of veterinary and medical importance (Burges, 1981; Lacey and Kaya, 2000; Tanaka and Kaya, 1993). Protozoan diseases of insects are ubiquitous and have an important regulatory role in the growth of insect populations (Brooks, 1974; Maddox, 1987; Schaub, 1994). Ingested entomopathogens commonly invade the bodies of insects by penetrating the midgut and entering the hemocoel. These include useful microbial agents for
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Table 2 Characterization of proteolytic enzymes in 563DT trypanosomatid growth phases by utilization of proteases inhibitors Substrate
Proteolytic enzyme
% Enzymatic activity in presence of specific inhibitor Growth phase 563DT strain Lag phase
Log phase
Stationary phase
Lys-NA, pH 8.0, h ¼ 35 °C
Cysteine-protease Serine-protease Metallo-protease Aspartic-protease
26.2 34.0 84.6 79.4
18.6 0.2 0.4 11.3
39.5 17.0 33.7 0
Leu-NA, pH 6.75, h ¼ 35 °C
Cysteine-protease Serine-protease Metallo-protease Aspartic-protease
73.5 44.6 47.2 61.5
51.3 52.5 67.8 83.1
39.4 48.4 87.8 80.0
Ser-NA, pH 6.75, h ¼ 45 °C
Cysteine-protease Serine-protease Metallo-protease Aspartic-protease
67.1 45.3 70.4 42.7
66.4 43.4 63.9 41.5
62.5 38.6 56.1 7.3
BANA, pH 10.0, h ¼ 45 °C
Cysteine-protease Serine-protease Metallo-protease Aspartic-protease
54.4 40.3 83.4 61.2
40.3 36.9 65.2 70.7
69.5 39.8 89.2 62.7
The temperature and pH utilized was the optimum for each substrate and it was previously determined. Growth phase: corresponding growth phase of 563DT strain in GYPMI medium, at 28 °C, without agitation. The values are an average of assays in duplicate, repeated thrice. Tested substrates: Lys-NA, Leu-NA, Ser-NA, and BANA. Proteolytic enzyme: enzyme characterized by action of protease inhibitors*. % Enzymatic activities in presence inhibitors: inhibition value calculated according the same reaction in absence of inhibitor (100%). *Tested inhibitors: TPCK, TLCK, leupeptin, aprotinin, PMSF, EGTA, EDTA, O-Phe, pepstatin, and iodoacetamide.
Table 3 Characterization of proteolytic enzymes in 714DT trypanosomatid growth phases by utilization of proteases inhibitors Substrate
Proteolytic enzyme
% Enzymatic activity in presence of specific inhibitor Growth phase 563DT strain Lag phase
Log phase
Stationary phase
Lys-NA, pH 10.0, h ¼ 25 °C
Cysteine-protease Serine-protease Metallo-protease Aspartic-protease
29.7 44.8 53.4 99
36.5 15.6 16 16.7
16.8 32.6 33.6 30.2
Leu-NA, pH 11.0, h ¼ 35 °C
Cysteine-protease Serine-protease Metallo-protease Aspartic-protease
36.1 34.6 77.8 81.5
53.6 52.4 53.6 79.3
16.4 43.5 63.5 65.1
Ser-NA, pH 6.7, h ¼ 45 °C
Cysteine-protease Serine-protease Metallo-protease Aspartic-protease
61.8 37.1 39.2 24.4
57.6 52.7 43.4 58.4
42.5 60.8 45.3 65.2
BANA, pH 11.0, h ¼ 45 °C
Cysteine-protease Serine-protease Metallo-protease Aspartic-protease
53.4 72.4 74.2 97.8
53.1 39.7 74.4 55.1
27.6 18.3 97.5 57.9
The temperature and pH used was the optimum for each substrate and it was previously determined. Growth phase: corresponding growth phase of 714DT strain in GYPMI medium, at 28 °C, without agitation. The values are an average of assays in duplicate, repeated thrice. Tested substrates: Lys-NA, Leu-NA, Ser-NA, and BANA. Proteolytic enzyme: enzyme characterized by action of protease inhibitors*. % Enzymatic activities in presence inhibitors: inhibition value calculated according the same reaction in absence of inhibitor (100%). *Tested inhibitors: TPCK, TLCK, leupeptin, aprotinin, PMSF, EGTA, EDTA, O-Phe, pepstatin, and iodoacetamide.
biological control of pests, as well as agents of harmful human diseases carried by insect vectors. The peritrophic membrane is a critical barrier to the invasion
and may effectively exclude certain microbes; its pore sizes are usually smaller than bacteria, yet some bacteria, viruses, trypanosomes, and filarial worms are able to
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Table 4 Percentual survival rate of Veneza zonata inoculated with trypanosomatids treated and not treated with protease and phospholipase inhibitors Trypanosomatid
563
563
563
563
563
563
563
563
563
563
563
563
563
563 C1
714 C2
None C3
Inhibitor
A
B
C
D
E
F
G
H
I
J
K
L
M
None
None
None
80 70 60 50 50 40 40 30 30 30 30 20 20 20 20 10 10 10 10 10 10 10 10 10 10 10 10 10 0
70 70 60 60 50 40 30 20 20 20 20 20 20 10 10 0
70 70 60 60 60 60 50 40 40 40 40 40 40 40 40 40 30 20 20 10 10 10 10 10 10 10 10 10 0
80 40 30 30 20 0
80 60 40 40 40 40 40 40 40 40 40 20 20 20 20 20 20 10 10 10 10 10 10 10 10 10 10 10 10 10 0
80 40 40 40 40 40 30 30 30 30 30 10 10 10 10 10 10 10 10 10 0
10 0
90 80 60 60 50 50 50 50 40 40 40 30 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 10 10 10 10 10 0
50 20 20 20 20 20 20 20 20 20 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 0
80 50 50 50 50 50 50 50 50 50 50 40 40 40 30 30 20 10 10 10 10 10 10 10 10 10 10 10 10 0
40 40 40 20 20 20 20 10 10 10 10 10 0
80 30 30 20 20 20 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 0
40 0
10 0
80 80 80 70 70 60 50 50 40 40 30 20 20 20 20 20 20 20 20 10 10 10 0
100 100 100 100 100 100 100 100 90 90 90 90 80 70 70 60 60 60 60 60 50 50 40 40 40 40 40 40 40 40 30 20 10 0
% Survival 1 (24 h) 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34
Inhibitors: A—EDTA, O-Phe, EGTA; B—PMSF, TLCK.; C—pepstatin; D—aprotinin; E—EDTA, O-Phe, EGTA, PMSF, TPCK, TLCK, pepstatin, aprotinin; F—palmitoyl-D L -carnitine; G—palmitoyl-D L -carnitine, TLCK, leupeptin; H—TPCK, TLCK, leupeptin; I—aprotinin, palmitoylD L -carnitine, leupeptin, TLCK; J—pepstatin, leupeptin, iodoacetamide, palmitoyl-D L -carnitine; K—iodoacetamide, leupeptin, pepstatin, TPCK; L—palmitoyl-D L -carnitine, EDTA, O-Phe, PMSF; M—palmitoyl-D L -carnitine, iodoacetamide, PMSF, leupeptin; C1—563DT/inhibitor absence; C2-714DT/inhibitor absence; C3—0.15 M of NaCl. Values 1–34: days of insect survival; % Survival: percentual rate of live insects.
penetrate the membrane either mechanically or by action of their enzymes (Daly et al., 1998). Inoculation of 563DT (Leptomonas) trypanosomatid into hemocoel of V. zonata causes insect death by septicemia within approximately 24 h, probably because trypanosomatid crossing the intestinal barrier causes a perforation which allows bacteria from the digestive tract to reach the hemocoel (Baccan et al., 2001). Insects fed on tomatoes inoculated with 563DT exhibited multiplying forms in the digestive tract (6 days after) and hemocoel (two or three additional days) and they died 12–14 days after exposure by septicemia. When the bugs fed a pool of antibiotics (penicillin, nalidixic acid, tetracyclines, sulphonamides, and erythromycin, 100 mg/ml each) and then strain 563DT, the protozoan infected the hemocoel but no bacterial sep-
ticemia ocurred and the bugs survived, indicating that the probable mechanism is bacterial septicemia (Maddox et al., 1981) and not trypanosomatid direct pathogenic action. We observed an increase in the survival of V. zonata which received an inoculum of 563DT trypanosomatids pre-treated with protease inhibitors, evidencing that proteases and phospholipases participate in the pathogenicity (McKerrow et al., 1993) of this trypanosomatid. The action of protease inhibitors over 714DT and 563DT strains presented differences during protozoan growth stages and when tested with different synthetic substrates, indicating that there are differentiated cellular and secreted enzymatic activities, as there are also differences in protease activity in all growth stages of these trypanosomatids.
Table 5 Action of protease and phospholipase inhibitors inoculated in Veneza zonata None EDTA
EGTA
O-FE
PMSF
TLCK
TPCK
Pepstatin
Aprotinin
Palmitoyl Carnitine
Iodoacetamide
Leupeptin
1 (24 h) 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34
90 90 80 70 70 70 70 70 70 60 60 60 60 40 40 40 40 40 40 40 40 40 30 30 20 10 10 10 0
100 100 90 90 80 80 80 70 70 50 50 50 40 40 40 30 30 20 20 20 10 10 0
90 90 80 80 70 70 60 50 50 40 20 20 20 10 10 10 10 10 10 10 10 10 10 10 10 10 0
90 90 90 80 80 80 80 80 70 70 70 60 50 50 50 40 30 20 10 10 10 0
90 90 80 80 70 60 60 50 50 40 40 30 20 10 0
100 100 90 90 90 90 90 90 90 80 80 70 70 70 60 60 40 40 40 20 20 10 0
100 100 100 90 90 90 50 50 50 50 50 50 50 50 50 40 40 40 40 40 30 30 30 30 20 20 20 10 10 10 0
80 80 80 70 70 70 70 70 70 70 70 60 60 60 60 60 50 50 50 40 40 30 30 20 20 10 0
100 100 100 100 100 80 80 80 80 70 70 70 50 50 50 50 50 50 50 50 50 50 40 40 40 40 40 40 30 30 20 0
80 70 60 50 50 50 50 50 50 50 50 40 40 40 30 30 30 30 20 20 10 10 10 0
90 90 80 80 80 80 80 60 60 60 60 50 50 50 50 50 50 50 50 50 50 50 40 50 50 40 40 40 30 30 20 20 0
563DT None
714DT None
NaCl, 0.15 M None
10 0
80 80 80 70 70 60 50 50 40 40 30 20 20 20 20 20 20 20 20 10 10 10 0
100 100 100 100 100 100 100 100 90 90 90 90 80 70 70 60 60 60 60 60 50 50 40 40 40 40 40 40 40 40 30 20 10 0
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Trypanosomatid Inhibitor
Percentual rate survival of V. zonata treated with only protease and phospholipase inhibitors (without strain 563DT Leptomonas). 1–34: days of insect survival. Values in columns: percentage of survival of insects in each day after inoculation.
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Trypanosomatid living cells showed enzymatic activity on azocasein, azoalbumine, and gelatine. This kind of protease activity presented by the 563DT strain seems to be bound to the cell surface. The 563DT gelatinolytic activity was higher than that of the 714DT strain. Itow Jankevicius and Camargo (1977) described activity over azocasein and also over gelatine in Trypanosoma cruzi cells homogenate, but the same was not observed in other lower trypanosomatids. Few studies have been done to identify extracellular proteinases in trypanosomatids. It has been shown that T. cruzi and Trypanosoma brucei release cysteine-proteases (Okenu et al., 1999). Extracellular metallo-proteases have been detected in Crithidia guillermei (De Mello et al., 2001) and in the genus Herpetomonas (Santos et al., 1999). Neither of the strains tested in this study produced detectable extracellular proteinases in the GYPMI culture medium supernatant. It was observed for the Lys-NA substrate that the enzymatic activity predominating in the lag phase of 563DT was cysteine-protease, due to its inhibition by chelant agents and iodoacetamide. Serine-proteases were observed in higher quantities in the lag phase, showing in presence of TPCK, PMSF, leupeptin and aprotinin, 34.0% of original enzymatic activity (without inhibitors). In the log phase, an increase in the percentage rate of cysteine-, metallo-, serine-, and asparticproteases production was observed, demonstrating the presence of a ‘‘pool’’ of protease production by 563DT trypanosomatids. The highest enzymatic activity inhibition was of serine-proteases, with 0.2% of the original enzymatic activity when in the presence of TPCK, PMSF, leupeptin, and aprotinin. The main proteolytic enzyme product in stationary phase was aspartic-protease, that showed 100% inhibition in the presence of pepstatin. Serine-proteases were detected in larger quantities in this phase. The action of protease inhibitors on Leu-NA substrate presented some differences in the same strain, maybe because of enzyme specificity for substrate. Serine- and metallo-proteases were present in larger quantities in the lag phase, and the same ‘‘pool’’ of proteases can be observed during log phase. Aspartic-proteases can be observed in lower proportion during all the growth phases. In the stationary phase the main protease produced was a cysteine-protease that revealed only 39.4% of enzymatic activity when in the presence of chelant agents and iodoacetamide. The characterization of proteolytic enzymes on the BANA substrate showed the presence of cysteine-, serine-, and aspartic-proteases in major proportion on the lag phase, and a ‘‘pool’’ of proteases in the log phase. Serine-proteases were present in major proportion in the stationary phase. The bond of the enzyme on BANA substrate is very specific, which might explain why the aspartic-inhibitors were not so effective as on other substrates.
Comparison of the enzymatic activity on Lys-NA between 714DT and 563DT strains shows that in the log phase the cysteine-protease presented higher activity in the 714DT strain and lower in the 563DT strain. The main protease characterized in the lag phase on Lys-NA and BANA was cysteine-proteases, on Leu-NA, serineprotease and on Ser-NA, aspartic-protease. In the log phase it was serine- and metallo-proteases for Lys-NA, similar values for all enzymes in Leu-NA and Ser-NA, and serine-proteases on BANA. Cysteine-protease was present in major proportion on the stationary phase when tested with Lys-NA, Leu-NA, and Ser-NA substrates, and in BANA serine-proteases could also be detected in high proportion. The increase in V. zonata survival rate was higher when 563 DT trypanosomatids were pre-treated with a ‘‘cocktail’’ of inhibitors containing EDTA, EGTA, PMSF, TPCK, TLCK, O-Phe, pepsin, and aprotinin, which is a confirmation of the participation of a ‘‘pool’’ of proteases in the pathogenesis mechanism. The insects treated with a ‘‘cocktail’’ of proteolytic inhibitors presented a survival rate similar to that of control insects inoculated only with sterile 0.15 M NaCl (Table 5). The results revealed the presence of serine-proteases in all growth phases of the 563DT strain and in all tested substrates. Their involvement in the pathogenicity mechanism to V. zonata is evidenced by the observation that insects which received an inoculum of trypanosomatids pre-treated with serine-protease inhibitors (mainly TLCK) showed higher survival rate than those treated with other proteases inhibitors, except on G and M combinations (Table 4), where increase in survival rate was not observed, which can be attributed to the absence of aspartic-protease inhibitors and low stability of these serine-protease inhibitors, suggesting that an aspartic-protease was the main enzyme involved in the mechanism of 563DT strain to the insect pathogenicity. Survival rate of insects inoculated with trypanosomatids treated only with pepstatin confirmed this supposition. The treatment of trypanosomatids with a mixture of aspartic-, serine-, cysteine-, and metallo-protease inhibitors plus phospholipase inhibitors was the most efficient in increasing insect survival rate. The presence of bacteria in the hemocoel of insects infected with strain 563DT, and their absence on insects infected with 714DT, in agreement with the differences of enzymatic activities presented by those strains, allows us to suggest that the cause of crossing of bacteria from the digestive tract to the hemocoel is the digestive tract perforation by aspartic-proteases, serine-proteases (and others in lower proportion), and phospholipases present on the surface of 563DT trypanosomatid living cells. The presence of the same bacteria in the digestive tract of infected and non-infected insects shows that hemocoel invasion is accomplished by self-microbiota of digestive tract. The bacteria found in the digestive tract
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and in the hemocoel of infected insects were not always the same, since the bacteria were not cloned at the first phase of isolation. Until the present moment, out of the four proteinase types (cysteine-, metallo-, serine-, and aspartic-proteinases), only aspartic-proteinases had not been detected yet in trypanosomatids (DÕAvila-Levi et al., 2001). The present study is the first to describe an asparticprotease in trypanosomatids. Furthermore, we conclude that this protease, in concert with serine-proteases and phospholipases present on surface of 563DT (Leptomonas), are important factors in the pathogenicity mechanism of this strain to V. zonata. This study indicates also the possibility of using a new microbial agent for biological control of insects of economical importance.
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