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Differential expression of cruzipain- and gp63-like molecules in thephytoflagellate trypanosomatid Phytomonas serpens induced by exogenous proteins

Camila G.R. Elias a,1, Michel G. Chagas b,1, Ana Luiza Souza-Gonçalves a, Bernardo M.O. Pascarelli c,Claudia M. d’Avila-Levy d, Marta H. Branquinha b, André L.S. Santos a,⇑a Laboratório de Estudos Integrados em Bioquímica Microbiana, Departamento de Microbiologia Geral, Instituto de Microbiologia Paulo de Góes (IMPPG), Bloco E-subsolo,Centro de Ciências da Saúde (CCS), Universidade Federal do Rio de Janeiro (UFRJ), Av. Carlos Chagas Filho, 373, Cidade Universitária, Rio de Janeiro, RJ 21941-902, Brazilb Laboratório de Bioquímica de Proteases, Departamento de Microbiologia Geral, IMPPG, CCS, UFRJ, Rio de Janeiro, RJ, Brazilc Laboratório de Patologia, Instituto Oswaldo Cruz (IOC), Fundação Oswaldo Cruz (FIOCRUZ), Rio de Janeiro, RJ, Brazild Laboratório de Biologia Molecular e Doenças Endêmicas, IOC, FIOCRUZ, Rio de Janeiro, RJ, Brazil

a r t i c l e i n f o

Article history:Received 8 April 2011Received in revised form 7 October 2011Accepted 7 October 2011Available online 19 October 2011

Keywords:Phytomonas serpensProteasesCruzipainGp63Exogenous proteinsHemoglobin

a b s t r a c t

Phytomonas serpens synthesizes metallo- and cysteine-proteases that are related to gp63 and cruzipain,respectively, two virulence factors produced by pathogenic trypanosomatids. Here, we described the cel-lular distribution of gp63- and cruzipain-like molecules in P. serpens through immunocytochemistry andconfocal fluorescence microscopy. Both proteases were detected in distinct cellular compartments, pre-senting co-localization in membrane domains and intracellular regions. Subsequently, we showed thatexogenous proteins modulated the production of both protease classes, but in different ways. Regardingthe metalloprotease, only fetal bovine serum (FBS) influenced the gp63 expression, reducing its surfaceexposition (�30%). Conversely, the cruzipain-like molecule was differentially modulated according tothe proteins: human and bovine albumins reduced its expression around 50% and 35%, respectively;mucin and FBS did not alter its production, while IgG and hemoglobin drastically enhanced its surfaceexposition around 7- and 11-fold, respectively. Additionally, hemoglobin induced an augmentation inthe cell-associated cruzipain-like activity in a dose-dependent manner. A twofold increase of the secretedcruzipain-like protein was detected after parasite incubation with 1% hemoglobin compared to the par-asites incubated in PBS-glucose. The results showed the ability of P. serpens in modulating the expressionand the activity of proteolytic enzymes after exposition to exogenous proteins, with emphasis in its cru-zipain-like molecules.

� 2011 Elsevier Inc. All rights reserved.

1. Introduction

Phytomonas serpens is a trypanosomatid isolated for the firsttime from the sap of tomatoes (Gibbs, 1957; Jankevicius et al.,1988). There is no precise information available about the pathoge-nicity of trypanosomatids in fruits. Usually these phytoflagellatesremain compressed around the point of inoculation; however, aloss in nutritional quality and, above all, a loss in economic valueadded to the product are undoubtedly documented (Camargo,1999). With another focus, P. serpens has been used as a modelfor biochemical and genetic studies on the Trypanosomatidae fam-ily, helping to unravel several basic mechanisms of this uniquegroup of eukaryotic microorganisms (Sá-Carvalho and Traub-Cseko, 1995; Fernandez-Becerra et al., 1997; Maslov et al., 1998;Nawathean and Maslov, 2000; González-Halphen and Maslov,

2004; Lukes et al., 2006). In this context, some peculiarities of Phy-tomonas metabolism have been studied in detail. Most remarkablewas the observation that several genes of the respiratory complexwere absent in this kinetoplastid genome. Linked to the absence ofcytochromes and lack of functional Krebs cycle, evidence that ATPproduction is largely driven by glycolysis was provided (Sánchez-Moreno et al., 1992; Nawathean and Maslov, 2000; González-Hal-phen and Maslov, 2004). Furthermore, P. serpens cells presenthumoral and cellular cross-immunity against Trypanosoma cruziand Leishmania spp., which suggests similarities between theirstructural components (Breganó et al., 2003; Pinge-Filho et al.,2005; Elias et al., 2006, 2008, 2009; Santos et al., 2006a,b, 2007;Graça-de Souza et al., 2010).

Accumulated experimental evidences have demonstrated thatat least one class of bioactive molecules produced by P. serpens isresponsible for the antigenic cross-reactivity against human path-ogenic trypanosomatids: the proteolytic enzymes (Elias et al.,2006, 2008; Santos et al., 2006a,b, 2007). In this sense, two distinctclasses of proteases were described in P. serpens promastigotes: a

0014-4894/$ - see front matter � 2011 Elsevier Inc. All rights reserved.doi:10.1016/j.exppara.2011.10.005

⇑ Corresponding author. Fax: +55 21 2560 8344.E-mail addresses: andre@micro.ufrj.br, alsouzasantos@gmail.com (A.L.S. Santos).

1 These authors contributed equally to this work.

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63 kDa metalloprotease that had similarity with gp63 (synony-mous leishmanolysin) of Leishmania spp., and two cysteine prote-ases of 38 and 40 kDa that presented similar antigenic propertieswith the major cysteine protease (named cruzipain) produced byT. cruzi. Biochemical analyses have shown that the gp63-likemolecule of P. serpens was preferentially located on the parasitesurface anchored to the membrane domains via a glycosylphos-phatidylinositol (GPI) anchor (d’Avila-Levy et al., 2006). Interest-ingly, antibody against the Leishmania amazonensis gp63 partiallyinhibited the adherence of P. serpens cells to the salivary glandsexplanted from Oncopeltus fasciatus, a phytophagous insect(d’Avila-Levy et al., 2006). Possible biological functions were alsoproposed for the cysteine proteases produced by P. serpens: usingcysteine protease inhibitors and anti-cruzipain antibody, wepreviously reported that parasite proliferation, morphology andadhesion to the salivary gland of O. fasciatus were altered whenthe parasite cysteine proteases were blocked (Santos et al.,2006b, 2007; Elias et al., 2008). Furthermore, P. serpens cysteineproteases had their activities quantitatively modulated when theparasites were cultivated in distinct growth conditions: culturemedia rich in complex protein sources, like yeast extract, peptone,tryptone and liver infusion, promoted an augmentation in the pro-tease production concomitantly with increasing parasite growthrate, suggesting a possible protease modulation according to thepresence of organic nitrogenous compounds (Elias et al., 2008).

In the present paper, we have described the influence of exoge-nous proteinaceous compounds on the expression of both cruzi-pain- and gp63-like molecules produced by promastigote cells ofP. serpens when incubated in starving conditions. In addition, theco-localization of both metallo- and cysteine proteases were evi-denced in this trypanosomatid by means of immunomicroscopicalanalyses.

2. Material and methods

2.1. Parasites and cultivation

P. serpens (isolate 9T; CT–IOC–189), isolated from tomato(Lycopersicon esculentum), was provided by Coleção de Tripanosso-matídeos, Instituto Oswaldo Cruz – Fundação Oswaldo Cruz, Rio deJaneiro, Brazil. The plant flagellate (promastigote forms) wasgrown and maintained by weekly transfers in 50 ml Erlenmeyerflasks containing 20 ml of brain heart infusion (BHI) medium.The parasites were grown at 26 �C for 48 h (exponential growthphase) (Elias et al., 2008). The cells were harvested by centrifuga-tion at 500g, for 5 min at 4 �C, washed three times with cold iso-tonic phosphate-buffered saline (PBS; 150 mM NaCl; 20 mMphosphate buffer, pH 7.2), and intact and viable cells were usedalong all the experiment procedures. Cellular growth was esti-mated by counting the parasites in a Neubauer chamber.

2.2. Immunocytochemistry analyses

The parasites were collected by centrifugation, washed twice inPBS and fixed in a solution containing 0.1% glutaraldehyde, 2%formaldehyde in 0.1% cacodylate buffer, pH 7.2. After 1 h fixation,cells were washed in PBS, dehydrated in methanol and embeddedin Lowicryl K4 M resin at –20 �C. Ultrathin sections were collectedin nickel grids and incubated in 0.1 M Tris-buffered saline (TBS;150 mM NaCl, 10 mM Tris, pH 7.4) followed by a 15 min incubationin 50 mM ammonium chloride. After three washings in TBS, thegrids were incubated separately with the rabbit anti-cruzipainpolyclonal antibody raised against the native cruzipain obtainedfrom the Tul 2 strain of T. cruzi (kindly provided by Dr. Juan-JoseCazzulo, Instituto de Investigaciones Biotecnologicas, Universidad

Nacional de General San Martin, Buenos Aires, Argentina), the rab-bit anti-gp63 polyclonal antibody (H50) against the recombinantgp63 molecule from Leishmania mexicana (kindly provided by Dr.Peter Overath, Max-Planck-Institut für Biologie, AbteilungMembranbiochemie, Germany) and the respective rabbit pre-seradiluted at 1:50 in TBS supplemented with 1% bovine serum albu-min (BSA) and 1% Tween 20 (TBSBT). Grids were washed threetimes in TBSBT and subsequently incubated with gold-labeled goatanti-rabbit Immunoglobulin G (IgG) (15 nm, dilution 1:10) for 1 h(Pereira et al., 2010). Ultrathin sections were stained with uranylacetate and lead citrate and observed in a Zeiss ETM 10 C transmis-sion electron microscope.

2.3. Confocal fluorescence microscopy

The parasites (1 � 107 cells) were harvested by centrifugation(500g/4 �C/5 min), washed with PBS and fixed with 4% paraformal-dehyde in PBS (pH 7.2) for 30 min, and allowed to settle on glasscoverslips treated with 0.01% poly-L-lysine for 30 min. Subse-quently, the cells were permeabilized by 0.5% Triton X-100 inPBS for 10 min. Then, they were incubated for 1 h with a blockingsolution (2.5% BSA, 10% fetal calf serum and 1% skimmed milk di-luted in PBS) to inhibit unspecific labeling. The cells were furtherincubated overnight with the rabbit anti-cruzipain antibody di-luted 1:100 in PBS supplemented with 1% BSA, washed with PBSfor three times for 5 min each, and incubated for an hour with goatanti-rabbit IgG-labeled with Alexa 546 (Invitrogen, Oregon, USA) at1:750 dilution in PBS. These cells were then incubated overnightwith the sheep anti-gp63 polyclonal antibody (0180) against thepurified gp63 molecule from Leishmania chagasi (kindly providedby Dr. Mary Wilson, Department of Internal Medicine, Universityof Iowa, USA) diluted at 1:100 in PBS supplemented with 1% BSA,washed with PBS for three times for 5 min each, and incubatedfor an hour with donkey anti-sheep IgG-labeled with fluoresceinisothiocyanate (FITC) (Sigma, St. Louis, USA) at 1:20 dilution inPBS. They were washed three times with PBS, and the coverslipswere mounted in slides with p-phenylenediamine in buffered glyc-erin. A negative control was prepared by substituting the incuba-tion step with primary antibodies for incubation with PBS. Thematerial was analyzed and the images captured in a Carl ZeissLSM 510 META confocal laser scanning microscope (Carl Zeiss,Germany).

2.4. Viability tests

The survivability and the morphology of P. serpens cells alongthe incubation period in PBS-glucose supplemented or not withdifferent exogenous proteins was assessed by (i) direct observationof the parasite mobility through optical microscopic analyses, (ii)resazurin assay, a redox potential indicator, (iii) propidium iodidestaining, in order to measure the level of the uptake of this com-pound by damaged parasite cells due to a loss in cell membraneintegrity, (iv) measurement of lactate dehydrogenase activity, acytoplasmic enzyme, in the cell-free secretion supernatant fluids,and (v) flow cytometry analyses, in order to measure two morpho-logical parameters, cell size and granularity. In some of theseexperiments, parasites were treated with 0.4% paraformaldehydeor sodium azide (0.95 g/l) for 30 min in order to obtain non-viablecells to use as a negative control in the viability tests.

2.5. Effect of exogenous proteins on surface cruzipain- and gp63-likemolecules

First of all, P. serpens cells grown in BHI medium for 48 h wereharvested by centrifugation at 500g, for 5 min at 4 �C, and washedthree times with cold PBS. The intact cells (1 � 107) were

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resuspended in 1 ml of sterile isotonic PBS supplemented with 0.1%glucose (PBS-glucose) and then incubated for 1, 2, 3 and 4 h at26 �C. After that, the cells were removed by centrifugation (500g/5 min/4 �C) and the supernatants were passed over a 0.22-lmmembrane (Millipore, São Paulo, Brazil), to obtain the PBS-glucoseconditioned supernatants (Elias et al., 2008). Second, the parasites(1 � 107) were incubated for 3 h in 1 ml PBS-glucose supplementedor not with different proteins (at final concentration of 1%): BSA,human serum albumin (HSA), hemoglobin, mucin, IgG and fetal bo-vine serum (FBS). All the proteins were analytical grade (Sigma).After that, the mixture systems were centrifuged and cells werewashed in PBS and processed for flow cytometry analysis. Briefly,the cells were processed as described in the confocal fluorescenceassay, except for the absence of the parasite permeabilization withTriton X-100. The parasite-associated fluorescence was quantifiedon a fluorescence-activated cell sorter (FACS) (FACSCalibur, BD Bio-science, USA) equipped with a 15-mW argon laser emitting at488 nm. Non-treated cells and those treated with the secondaryantibody alone were run in parallel as controls. Each experimentalpopulation was then mapped by using at two-parameter histogramof forward-angle light scatter versus log fluorescence after reading10,000 cells. The results were expressed as percentage of fluores-cent cells that was calculated by the difference between the fluo-rescence detected in parasites treated with cruzipain antibody(either the cells pre-incubated or not with the different proteins)and the fluorescence of parasites treated only with secondary anti-body (in this case, the secondary antibody did not bind to parasitesurface generating similar results as compared to autofluores-cence). Alternatively, cells were also analyzed by confocalmicroscopy.

2.6. Proteolytic activity assay

After incubation of P. serpens cells in 1 ml PBS-glucose for 3 hwith different concentration of hemoglobin (0%, 0.1%, 1.0% and10%), the mixtures were centrifuged and cells were washed threetimes in PBS and then lysed by the addition of 0.1% sodium dodecylsulfate (SDS). The cells were broken in a vortex by alternating 1-min shaking and 2-min cooling intervals, followed by centrifuga-tion at 10,000g for 30 min at 4 �C, in order to obtain the wholeparasite cellular extracts (Santos et al., 2005). Protein concentra-tion was determined by the method described by Lowry et al.(1951), using BSA as standard. Samples containing 50 lg of proteinof each system were resuspended in SDS–polyacrylamide gel elec-trophoresis (PAGE) sample buffer (125 mM Tris, pH 6.8, 4% SDS,20% glycerol and 0.002% bromophenol blue). Proteases were as-sayed and characterized by electrophoresis on 10% SDS–PAGE with0.1% co-polymerized hemoglobin as substrate. After electrophore-sis, at a constant voltage of 120 V at 4 �C, SDS was removed byincubation with 10 volumes of 2.5% Triton X-100 for 1 h at roomtemperature under constant agitation. Then, the gels were incu-bated at 37 �C, or alternatively at 26 �C, in 50 mM sodium phos-phate buffer supplemented with 2 mM dithiothreitol (DTT), pH5.0, for 48 h, to promote the proteolysis (Elias et al., 2008), in theabsence or in the presence of 1 lM trans-epoxysuccinyl L-leucy-lamido-(4-guanidino) butane (E-64), a selective cysteine proteaseinhibitor. The gels were stained for 2 h with 0.2% Coomassie bril-liant blue R-250 in methanol–acetic acid–water (50:10:40) and de-stained overnight in a solution containing methanol–acetic acid–water (5:10:85), to intensify the digestion halos (Santos et al.,2005). The molecular masses of the proteases were estimated bycomparison with the mobility of low molecular mass standards.The gels were dried, scanned and the density profiles digitally pro-cessed. Further, the densitometric scanning analysis was per-formed with the use of the Kodak Digital Science EDAS 120software. In these analyses, bands in each gel were manually se-

lected using the free selection tool provided by the software. Bandareas were then determined by repeating this process three times,to diminish the probability of errors in these estimations. Values ofband area were further integrated with means of gray level in se-lected bands, generating densitometric values that were used inthe comparison between corresponding bands from the differentgels. For proteolytic bands analyses, first the images were invertedusing the tool provided by the software and then the measure-ments were performed (Elias et al., 2006).

2.7. Enzyme-linked immunosorbent assay (ELISA) and Westernblotting for detection of secreted cruzipain-like molecules

The supernatants obtained after the incubation of parasiteswith PBS-glucose supplemented or not with different hemoglobinconcentrations were centrifuged and then filtered in a 0.22-lmmembrane, generating the cell-free PBS-glucose supernatants.The 96-well plates were coated with 5 lg/ml of each supernatantfluid for 1 h at 37 �C. Then, the plates were blocked with 5% low-fat dried milk in 0.1 M TBS (pH 7.4) containing 0.5% Tween 20(TBS/Tween) for 1 h at room temperature. Then, plates werewashed three times (10 min each) with the blocking solution andincubated separately with the anti-cruzipain antibody and pre-serum at 1:400 dilution. Following incubation for 1 h, the plateswere washed five times with TBS/Tween and then again incubatedwith peroxidase-labeled anti-rabbit IgG (Sigma) at a 1:2500 dilu-tion. The reaction was developed with o-phenylenediamine dihy-drochloride (Sigma) and stopped with sulfuric acid. Sampleswere assessed in triplicate and plates were read at 490 nm withan ELISA reader. The final graphics were expressed as results ofthe absorbance obtained with the immunized serum subtractedfrom the corresponding non-immunized serum (Elias et al.,2009). Alternatively, the supernatants, equivalent to 20 lg of pro-teins, were separated in 12% SDS–PAGE and electrophoreticallytransferred to a nitrocellulose membrane, which was blocked andwashed as described above. Then, membranes were incubatedwith the anti-cruzipain antibody at 1:250 dilutions for 2 h. The sec-ondary antibody used was peroxidase-conjugated goat anti-rabbit IgG at 1:2500. Immunoblots were exposed to X-ray filmafter reaction with ECL reagents for chemiluminescence (Santoset al., 2006b).

2.8. Hemoglobin-binding experiment

Initially, hemoglobin (2 mg) was biotinylated using EZ-Link�

Sulfo-NHS-LC-Biotinylation kit (Pierce), according to the manufac-turer’s protocol. Non-biotinylated and biotinylated hemoglobinwere dot-blotted in order to check the efficiency of this method.The dot blot was incubated with avidin-peroxidase at 1:10,000(Sigma) and then revealed by the addition of 0.5 mg/ml diam-inobenzidine (DAB) in 1.5 M Tris–HCl buffer (pH 7.4) supple-mented with 0.01% H2O2. The color development was stopped byimmersing the membrane sheets in distilled water. Subsequently,living parasite cells were incubated with different concentrationsof biotinylated hemoglobin up to 3 h. Following this step, the cellswere washed five times in PBS, incubated with avidin-FITC andthen analyzed by flow cytometry in order to detect the percentageof hemoglobin binding to the parasite cell surface.

2.9. Statistical analysis

The experiments were performed in triplicate, in three indepen-dent experimental sets. The data were analyzed statistically bymeans of Student’s t-test using EPI-INFO 6.04 (Database and Statis-tics Program for Public Health) computer software. P values of 0.05or less were considered statistically significant.

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3. Results

Initially, we analyzed the cellular distribution of gp63- andcruzipain-like proteins in P. serpens cells by means of immunocito-chemistry analyses (Fig. 1). The results showed that both proteaseswere equally distributed in different cellular compartments,including intracellular location, membrane lining all the cell bodyand flagellum, and inside the flagellar pocket area (Fig. 1). Confocalimmunofluorescence images corroborated the wide distributionand the co-localization of both gp63- and cruzipain-like moleculesaround the cellular body and flagellar regions (Fig. 2).

As it is well-known, exogenous proteins can modulate the syn-thesis of several bioactive molecules, especially proteases, innumerous microorganisms (Banerjee et al., 1991; Monod et al.,1991; Jarai and Buxton, 1994; Kaufman et al., 2005; Leng et al.,2009). We also tested this hypothesis using P. serpens cells as a try-panosomatid model. Initially, we verified the survivability of this

phytoflagellate when incubated up to 4 h in isotonic phosphate-buffered saline supplemented with 0.1% glucose (PBS-glucose).Our results showed that the parasites began to die in a lower pro-portion (6%) after 4 h of incubation in PBS-glucose, considering theparasite lysate obtained with Triton X-100 as 100% of lysis (Fig. 3).For the subsequent tests, P. serpens cells were incubated in PBS-glucose supplemented with different proteins for 3 h. None of theproteinaceous macromolecules induced cell lysis in P. serpens(Fig. 3) or loss of viability, as assessed by different methodologiesincluding propidium iodide exclusion assay, resazurin test, parasitemobility, as well as the absence of morphological alterations byboth direct observation in optical microscopic and after measure-ment of cell size and granularity by flow cytometry (Table 1). Withrespect to gp63-like molecule, only FBS was able to significantlymodulate its expression, reducing the surface exposition byapproximately 30% (Fig. 4). Interestingly, the cruzipain-like mole-cule was differentially influenced according to each exogenousprotein as evaluated by flow cytometry analyses: BSA and HSA re-duced its expression in about 50% and 35%, respectively; mucinand FBS did not alter its production, while IgG and hemoglobindrastically enhanced its surface exposition about 7- and 11-foldwhen compared with parasites incubated only with PBS-glucose(Fig. 4).

Hemoglobin was selected to test its dose response on the cruzi-pain-related activities (which correspond to the 38 and 40 kDa cys-teine protease activities) produced by P. serpens. In this sense,viable parasites were incubated for 3 h in PBS-glucose in the pres-ence of different hemoglobin concentrations (0%, 0.1%, 1% and 10%)and the results were evidenced after SDS–PAGE containing hemo-globin as substrate (Fig. 5). A clear augmentation on the productionof both cellular cysteine proteases were observed, suggesting a di-rect influence of hemoglobin concentration, as judged by densito-metrical analyses of the E-64-sensitive proteolytic halos (Fig. 5).Interestingly, these hemoglobinase activities were better detectedat 37 �C (mammalian temperature) than 26 �C (insect vector tem-perature) (Fig. 5). Fluorescence microscopy images corroboratedthe surface detection of cruzipain-like molecules after incubationof P. serpens cells with different hemoglobin concentrations(Fig. 6A). Furthermore, we incubated the parasites with differentconcentrations of biotinylated hemoglobin (Fig. 6B, inset) up to3 h in order to demonstrate that hemoglobin does not perturbthe binding of anti-cruzipain antibody to parasite cells. The resultsshowed that only a minor fraction of parasite cells were able tobind to hemoglobin after 3 h of incubation (Fig. 6B). Taken to-gether, the low binding of hemoglobin to parasite cells and the ele-vated recognition of surface cruzipain-like molecules by anti-cruzipain antibody strongly suggests that exogenous hemoglobindid not alter the antibody binding property.

As previously reported by our group (Elias et al., 2009) andherein corroborated (Fig. 7), P. serpens cells were able to secretecruzipain-related protein to the extracellular environment. As ob-served by ELISA and Western blotting assays, the secretion of cru-zipain-like molecules was significantly increased when parasitecells were incubated with 1% hemoglobin in comparison to para-sites incubated without or with hemoglobin at both 0.1% and10% (Fig. 7).

4. Discussion

The adaptation of phytoflagellate trypanosomatids to specificconditions in their plant or invertebrate hosts is accompanied bydrastic changes in energy metabolism (Cazzulo, 1999). As a com-mon feature, trypanosomatids are capable of using carbohydratesand amino acids as carbon sources depending on their availabilityin extracellular media (Cazzulo, 1994), mainly by transport

Fig. 1. Ultrastructural immunocytochemistry analysis of Phytomonas serpens incu-bated in the presence of rabbit pre-serum (control), anti-gp63 or anti-cruzipainantibodies at 1:50 dilution, and subsequently incubated with gold-labeled second-ary antibody. Note in control system the absence of gold particles and in parasitesections treated with both primary antibodies the labeling of the membraneenclosing the cell body (black arrowheads) as well as throughout the intracellularregion (white arrowheads). Gold particles were also detected in the flagellum (f;white arrows) and in the flagellar pocket region (fp; black arrows).

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processes (Urbina, 1994). Pappas et al. (2005) reported a largenumber of expressed sequence tags corresponding to a glucosetransporter family in P. serpens. This is in agreement with the richcarbohydrate environment provided by plant hosts. However, ami-no acids and proteins are also available in plants. Furthermore, thepropagation of the parasites in the insect hosts would require theability to utilize proteins as a source of energy.

Rather than synthesize amino acids, parasitic protozoa rely ontransport systems for the acquisition of these macromolecules (Sil-ber et al., 2005). The first work on metabolite transport in phyto-monads was published by Canepa et al. (2007) using thePhytomonas strain Jma isolated from the latex of Jatropha macran-tha. Phytomonas cells showed high transport rates for glucose andfructose and lower, but significant rates, for proline, arginine, cys-teine and glutamate. Minor transport activities were also observedfor serine, glycine and aspartate. In addition, amino acid transportprocesses do not seem to be regulated by parasite subjected to car-bon starvation or during the distinct (logarithmic or stationary)growth phases (Canepa et al., 2007). On the contrary, the carbonstarvation modulated the amino acid transport in other trypanoso-matids such as T. cruzi (Canepa et al., 2004, 2005). Absolutely noth-ing is known about the pathways of amino acid uptake in P. serpensand very little is known about the degradation of macromolecularproteins and/or peptides in order to generate free amino acids forits nutrition and development.

Several trypanosomatids synthesize surface and/or secretedproteolytic enzymes that are able to degrade several proteinaceoussubstrates (Branquinha et al., 1996; Santos et al., 2005, 2008).These trypanosomatids’ proteases possess different capabilities indegrading proteins, generating small peptides that can be used asa substrate for additional cleavages in order to generate free aminoacids. This hypothesis can be corroborated by the production of dif-ferent classes of proteases by trypanosomatid cells, each one withdistinct specificities for cleaving peptide bonds and/or proteases

Fig. 2. Confocal scanning images showing the double labeling of Phytomonas serpens cells for gp63 (green labeling) and cruzipain antibody (red labeling). The labelingsuggests co-localization of both proteins in the flagellar pocket (F, white arrows) and in the membrane (F, white arrowheads). Note: (A) anti-gp63 antibody labeling, (B) anti-cruzipain labeling, (C) differential interference contrast microscopy, (D) overlay of A and B images, (E) overlay of A, B and C images, (F) zoom of the selected region from Eimage. The control was negative for both antibodies (data not shown). Scale bar indicates 20 lm. (For interpretation of the references to colour in this figure legend, the readeris referred to the web version of this article.)

Fig. 3. Viability of Phytomonas serpens cells when incubated in starving conditions(PBS supplemented with 0.1% glucose). LDH activity was measured by the oxidationof NADH and consequent decrease in the absorbance at 340 nm as a function oftime (D340nm/min) in the culture supernatants of P. serpens cells kept only in PBS-glucose for 3 and 4 h (controls) or in PBS-glucose supplemented with differentproteinaceous compounds at 1% for 3 h. The positive control of enzymatic activityconsists of a parasite lysate obtained by extraction with Triton X-100. The opticalmicroscopy images represent the Giemsa-stained intact parasites after incubationfor 3 h in PBS-glucose or in PBS-glucose added of hemoglobin and, conversely, thelysed parasites after Triton X-100 extraction. Scale bars indicate 10 lm.

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with wide substrate utilization and promiscuous sub-site specific-ities. In this sense, P. serpens cells are capable in producing two dis-tinct classes of proteases belonging to the metallo (gp63-likeproteins) and cysteine (cruzipain-like molecules) protease classes.Curiously, no metalloprotease activity was measured in P. serpenscells; however, the anti-gp63 antibody reacted with proteins of63 and 52 kDa in parasite cellular extract and with a secreted pro-tein of 60 kDa. The analysis of the partition of these reactive pro-teins after Triton X-114 extraction revealed that they werepreferentially membrane-associated, although a considerableamount was also located in cytoplasmic compartments (d’Avila-Levy et al., 2006). The gp63-like protein was linked to the P. serpensadhesion to the salivary gland of O. fasciatus, in a non-proteolytic

manner (d’Avila-Levy et al., 2006). The active proteolytic machin-ery of P. serpens was associated to the hydrolytic activities of twocysteine proteases. In fact, biochemical data had reported thatthe cruzipain-like protein of 40 kDa was located at the P. serpenssurface, attached to membrane domains via a GPI anchor, and inthe cell-free culture supernatant, in an active form, which suggestssecretion of this cysteine protease to the extracellular medium

Table 1Estimation of the parasite viability by different methods.

Parasite treatment Side scatter (SSC)a Forward scatter (FSC)b % Propidium iodide-labeled parasites Mobility Resazurinc

None 161.1 ± 7.6 509.1 ± 2.7 5.2 ± 0.3 + 7400 ± 39.0Hemoglobin 0.1% 152.1 ± 4.6 507.3 ± 4.4 5.5 ± 0.1 + 7391 ± 12.0Hemoglobin 1% 152.4 ± 2.7 508.4 ± 3.8 4.5 ± 0.2 + 7355 ± 12.7Hemoglobin 10% 162.1 ± 9.7 516.3 ± 2.1 5.1 ± 0.4 + 7322 ± 19.5Paraformaldeide 0.4% NDd ND 87.9 ± 0.6 � 220 ± 5.5Sodium azide (0.95 g/l) ND ND 9.5 ± 0.3 � 3892 ± 87.8

a SSC measurement is related to the internal granularity and/or complexity of a cell.b FSC measurement is related to cell size.c The values represent the level of resazurin reduction expressed as fluorescence intensity.d ND, non-determined.

Fig. 4. Flow cytometry showing the percentage of fluorescent cells expressingsurface cruzipain-like or gp63-like molecules in Phytomonas serpens. Parasites wereincubated for 3 h in PBS-glucose in the absence (none, control system) or in thepresence of different proteinaceous compounds (BSA, bovine serum albumin; HSA,human serum albumin; Hgb, hemoglobin; IgG, immunoglobulin G; mucin; FBS,fetal bovine serum) at 1%. After that, cells were washed, incubated with anti-cruzipain or anti-gp63 and subsequently with FITC-secondary antibody andanalyzed by flow cytometry. Symbols (�, P < 0.05 and ⁄, P < 0.001) denote valuesstatistically different in relation to the control system.

Fig. 5. Hemoglobin–SDS–PAGE showing the proteolytic activity profiles afterincubation of Phytomonas serpens cells in PBS-glucose supplemented with differenthemoglobin concentrations (0%, 0.1%, 1% and 10%). The gel was incubated in 50 mMsodium phosphate buffer supplemented with 2 mM DTT, pH 5.0, for 48 h at 37 �C.Alternatively, cellular extract (containing 50 lg proteins) obtained from cellsincubated only in PBS-glucose was electrophorezed and then incubated in the samebuffer in the presence of E-64, a selective cysteine protease inhibitor, or at 26 �C for48 h. After that, the gels were stained with Coomassie blue. The numbers on the leftindicate the apparent molecular masses of the proteases, expressed in kilodalton.The graphics show the densitometric analyses of both 38 and 40 kDa proteolytichalos, expressed in arbitrary units.

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(Santos et al., 2006b; Elias et al., 2008). In contrast, the 38 kDa cys-teine protease was exclusively partitioned in the hydrophilic frac-tion of P. serpens cells, justifying its detection in intracellularcompartments (Santos et al. 2006b; Elias et al., 2008). The immu-nocytochemistry analyses associated to the confocal images con-firmed that both cruzipain- and gp63-like molecules presentedwide cellular distribution in P. serpens, corroborating the previousbiochemical studies. Moreover, some gold particles could be visu-alized free in the flagellar pocket region, suggesting the release ofboth gp63- and cruzipain-related molecules to the extracellularenvironment. Interestingly, both cysteine protease activities wereable to degrade different co-polymerized and/or soluble proteinsubstrates, including hemoglobin, casein, IgG, mucin, albumin, gel-atin and salivary gland proteins from the phytophagous insect O.fasciatus, specially a 115 kDa surface receptor (Elias et al., 2008,2009).

Cysteine proteases are relevant to several aspects of the trypano-somatid life cycle and of parasite-host relationships (Mottram et al.,1998; Atkinson et al., 2009). The expression of surface cruzipain-likemolecules was drastically modulated when P. serpens cells wereincubated in starving conditions with exogenous proteins. Never-theless, the expression of gp63-like protein was not significantly af-fected by these exogenous macromolecules, except for FBS. Theseresults can be connected with the prior explanation, in which onlycysteine proteases were actively employed by P. serpens cells to de-grade distinct proteins. Surprisingly, the incubation of live parasiteswith hemoglobin promoted the most vigorous expression of surfacecruzipain-like proteins when compared to the other tested proteins.

Substantiating this data, the incubation of parasites with increasingconcentrations of soluble hemoglobin promoted a robust and pro-portional augmentation on both 38 and 40 kDa cell-associated cys-teine protease activities, as visualized by hemoglobin-SDS–PAGE,as well as a proportional increase in the exposition of surface cruzi-pain-like molecules. Moreover, hemoglobin at a concentration of 1%appeared to be the best inducer of the secreted cruzipain-like mole-cules. Collectively, these results demonstrated that soluble hemo-globin is able to induce the up-regulation of surface, intracellularand secreted cysteine proteases in P. serpens. A similar phenomenonwas early reported by other microorganisms, in special fungi such asAspergillus spp. (Jarai and Buxton, 1994; Monod et al., 1991), Candidaalbicans (Banerjee et al., 1991) and dermatophytes (Kaufman et al.,2005; Leng et al., 2009).

Hemoglobin is used as a major nutrient by blood parasites suchas Schistosoma mansoni, Plasmodium falciparum, Leishmania spp.and Trypanosoma spp., and is degraded by their proteases, oftenvia multi-enzyme cooperative cascades, into heme and globin.The latter is subsequently degraded into free amino acids as asource of protein biosynthesis in parasites (Chang and Chang,1985; Chappell and Dresden, 1986; Salas et al., 1995; Lara et al.,2007). Iron is also an essential nutrient to these parasites andpotentially could be also acquired from hemoglobin followinghemolysis. Moreover, contrary to the majority of eukaryotic organ-isms, trypanosomatids are unable to synthesize heme through thede novo pathway (Chang and Chang, 1985) and must acquire itfrom the surrounding environment.

A question arises from our results: how and where the parasiteswould obtain heme for its growth while inside its host plant, thetomato. Plants, like humans, contain hemoglobin. Three distinct

A

B

Fig. 6. (A) Confocal images showing the location of cruzipain-like molecules inPhytomonas serpens after incubation for 3 h in the absence (a) or in the presence of0.1% (b) and 1% (c) of hemoglobin. (B) FACS showing the binding of biotinylatedhemoglobin at the parasite cell surface. Parasite cells were incubated with differentbiotinylated hemoglobin concentrations up to 3 h and then incubated with avidin-FITC to be analyzed by flow cytometry. A representative image of the dot blot assayis shown in inset of figure B: (a) non-biotinylated and (b) biotinylated hemoglobinwere spotted at the nitrocellulose membrane and then incubated with avidin-peroxidase and revealed by addition of DAB and H2O2.

A

B

Fig. 7. ELISA showing the reactivity pattern of extracellular proteins derived fromPhytomonas serpens cells after incubation for 3 h in PBS-glucose supplemented withdifferent hemoglobin concentrations (0%, 0.1%, 1% and 10%) against the polyclonalanti-cruzipain antibody. The graphic shows the results of the absorbance obtainedwith the immunized serum subtracted from the corresponding non-immunizedserum. Symbol (�, P<0.05) denotes value statistically different in relation to thecontrol system (A). Western blotting showing the cruzipain-like molecule detectedin the released proteins from P. serpens cells. The number on the left indicates theapparent molecular mass of the reactive protein, expressed in kilodalton (B).

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types of hemoglobin exist in plants: symbiotic (leghemoglobins),non-symbiotic (nsHb) and truncated hemoglobins (Garrocho-Villegas et al., 2007; Hoy and Hargrove, 2008). Research has impli-cated these plant hemoglobins in a number of possible functionsdiffering among hemoglobin types, and possibly between plantspecies. For instance, Ioanitescu et al. (2005) characterized a class1 nsHb from tomato, as being a homodimer with high oxygen affin-ity, low oxygen dissociation rate, hexacoordination in the ferricform, and a mixture of pentacoordination and hexacoordinationin the ferrous state. Wang et al. (2003) reported the expressionof two tomato nsHb genes, SOLly GLB1 and SOLly GLB2, in differentplant organs including roots, stems and fruits. These findings led usto speculate that, once inside its host plant, P. serpens could haveincreased the expression of cruzipain-like proteases, when in con-tact with the homologous tomato hemoglobin proteins, thusinducing an increase in its proteolytic activity which could pro-mote and enhance the cleavage of proteins present in the fruit,thus increasing their nutritional intake. Our group is conductingexperiments in this field.

This is the first study conducted in trypanosomatids in order todemonstrate the effect of exogenous protein sources on proteaseproduction. Cysteine proteases produced by P. serpens possess sim-ilar biochemical and immunological properties to cruzipain mole-cules synthesized by T. cruzi, including optimum acidic pH,optimum temperature around of the human body, ability to de-grade several protein substrates for both nutritional and develop-ment purposes, broad distribution over the parasite distinctcompartments including cell surface, and adhesive capability thathelps the parasite to interact with host structures (Cazzulo et al.,1997; Santos et al., 2006b, 2007; Elias et al., 2008, 2009). The bio-chemical and/or immunological similarities between P. serpens andimportant human pathogens (Hollar and Maslov, 1997) are possi-bly reflected in the similarity of some aspects of the basic cellularmachinery (Breganó et al., 2003; Santos et al., 2007). These facts to-gether reinforce that P. serpens can be used as a trypanosomatidmodel to study basic metabolic processes in the Trypanosomatidaefamily.

Acknowledgments

We thank Dr. Juan-Jose Cazzulo (Instituto de InvestigacionesBiotecnologicas, Universidad Nacional de General San Martin, Bue-nos Aires, Argentina) for kindly supplying the anti-cruzipain anti-body Dr. Mary Wilson (Department of Internal Medicine,University of Iowa, USA) and Dr. Peter Overath (Max-Planck-Institut für Biologie, Abteilung Membranbiochemie, Germany) forkindly donating the anti-gp63 antibody and the rabbit pre-immuneantiserum. This work was supported by Grants from Fundação Car-los Chagas Filho de Amparo à Pesquisa (FAPERJ), Conselho Nacionalde Desenvolvimento Científico e Tecnológico (MCT/CNPq), Coorde-nação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES),and Fundação Oswaldo Cruz (FIOCRUZ).

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