**3. Practical examples of peptidases screening through SDS-PAGE-substrate**

#### **3.1 A first glance on** *Bodo* **sp. peptidases**

*Bodo* sp. is a free-living flagellate that belongs to the family Bodonidae, order Kinetoplastida. This bodonid isolate still has its taxonomic position unsolved, but it is phylogenetically related to *Bodo caudatus* and *Bodo curvifilus*, which are considered ancestral to the trypanosomatids. The Trypanosomatidae family comprises parasites that are of particular interest due to their medical importance, such as the etiologic agent of Chagas' disease (*Trypanosoma cruzi*), African trypanosomiasis (*Trypanosoma brucei* complex) and the various forms of leishmaniasis caused by *Leishmania* spp.. Due to their medical relevance, this family has been the focus of extensive research (Wallace, 1966; Vickerman, 1994). Peptidase characterization in *Bodo* sp. and comparison to peptidases from closely related pathogenic protozoa may help to understand peptidase function and evolution in general. The gold standard approach for such comparison would be a bioinformatic analysis of the *Bodo*

Applications of Zymography (Substrate-SDS-PAGE)

et al. unpublished data.

for Peptidase Screening in a Post-Genomic Era 271

does not tend to migrate out of the resolving gel and is inexpensive, then with an arbitrary pH and temperature (usually neutral pH at 37oC), the incubation time is varied from minutes to even 72 h, depending on the sample. After selecting the incubation time that allows the detection of the higher number of enzymes without band overlapping, variations on the pH allows the determination of this biochemical characteristic of each band. After this assay, the peptidase(s) of interest can be tested over a range of temperatures, ions, reducing agents or proteolytic inhibitors. Finally, distinct proteinaceous substrates can be co-polymerized to the gels, revealing either the ability of the detected peptidases to degrade other substrates, which ultimately gives a glance on peptidase function, or even revealing enzymes not capable of degrading gelatin (see figure 2). The proper combination of these parameters may reveal interesting enzymes, such as peptidases strictly dependable on metal ions, stimulated by reducing agents, active only at acidic or alkaline conditions and so on.

Fig. 4. Gelatin-SDS-PAGE screening of peptidases in homogenates from *Aedes albopictus* pupa. The following parameters were assessed: incubation time, pH and temperature. Peptidase activities were detected after incubation of the gels for 30, 60 or 120 min at 37oC in 100 mM Tris-HCl buffer pH 7.5. The numbers on the left indicate apparent molecular masses of the active bands expressed on kiloDaltons (kDa). Afterwards, 60 min incubation was selected and the gels were incubated in reaction buffer containing 100 mM sodium acetate at pH 3.5 or 5.5 or 100 mM Tris-HCl at pH 7.5 or 10.0. Finally, the effect of

temperature on the proteolytic activities was assayed by incubating of the gels for 60 min at 4, 10, 37, 50 or 60oC in reaction buffer containing 100 mM Tris-HCl at pH 7.5. Saboia-Vahia

genome coupled to more defined biochemical characterization of individual peptidases. However, in the absence of a *Bodo* genome, we have employed substrate-SDS-PAGE to assess peptidases in this bodonid, which presents serine peptidases ranging from 250 to 75 kDa, with a slight preference for acidic pH. This finding is dissimilar to what has been described in related pathogenic protozoa (Figure 5) (d'Avila-Levy et al., 2009). Curiously, all the analyzed closely related parasitic trypanosomatids, as well, as *Cryptobia salmonistica*, reveal through gelatin-SDS-PAGE only metallo- and cysteine peptidases, which are prototypal peptidases and virulence factors. In trypanosomatids, for instance, serine peptidases can only be detected by in-solution assays or after enrichment processes (Grellier et al. 2001). It is somewhat intriguing that cysteine and metallopeptidases are either not resistant to the denaturation/refold process and/or are not abundantly expressed by this

Fig. 3. Flowchart for peptidase screening. Several parameters must be assessed to resolve the proteolytic profile in an unknown biological sample. This scheme represents a suggestion of a step-by-step analysis of these parameters, which are: amount of sample, incubation time, pH, temperature, effect of ions, effect of reducing agents, effect of peptidase inhibitors, and ability to degrade distinct proteinaceous substrates. Usually, gelatin is the proteinaceous substrate of choice for initial screening because it is easily hydrolyzed by several peptidases,

Fig. 3. Flowchart for peptidase screening. Several parameters must be assessed to resolve the proteolytic profile in an unknown biological sample. This scheme represents a suggestion of a step-by-step analysis of these parameters, which are: amount of sample, incubation time, pH, temperature, effect of ions, effect of reducing agents, effect of peptidase inhibitors, and ability to degrade distinct proteinaceous substrates. Usually, gelatin is the proteinaceous substrate of choice for initial screening because it is easily hydrolyzed by several peptidases,

does not tend to migrate out of the resolving gel and is inexpensive, then with an arbitrary pH and temperature (usually neutral pH at 37oC), the incubation time is varied from minutes to even 72 h, depending on the sample. After selecting the incubation time that allows the detection of the higher number of enzymes without band overlapping, variations on the pH allows the determination of this biochemical characteristic of each band. After this assay, the peptidase(s) of interest can be tested over a range of temperatures, ions, reducing agents or proteolytic inhibitors. Finally, distinct proteinaceous substrates can be co-polymerized to the gels, revealing either the ability of the detected peptidases to degrade other substrates, which ultimately gives a glance on peptidase function, or even revealing enzymes not capable of degrading gelatin (see figure 2). The proper combination of these parameters may reveal interesting enzymes, such as peptidases strictly dependable on metal ions, stimulated by reducing agents, active only at acidic or alkaline conditions and so on.

Fig. 4. Gelatin-SDS-PAGE screening of peptidases in homogenates from *Aedes albopictus* pupa. The following parameters were assessed: incubation time, pH and temperature. Peptidase activities were detected after incubation of the gels for 30, 60 or 120 min at 37oC in 100 mM Tris-HCl buffer pH 7.5. The numbers on the left indicate apparent molecular masses of the active bands expressed on kiloDaltons (kDa). Afterwards, 60 min incubation was selected and the gels were incubated in reaction buffer containing 100 mM sodium acetate at pH 3.5 or 5.5 or 100 mM Tris-HCl at pH 7.5 or 10.0. Finally, the effect of temperature on the proteolytic activities was assayed by incubating of the gels for 60 min at 4, 10, 37, 50 or 60oC in reaction buffer containing 100 mM Tris-HCl at pH 7.5. Saboia-Vahia et al. unpublished data.

genome coupled to more defined biochemical characterization of individual peptidases. However, in the absence of a *Bodo* genome, we have employed substrate-SDS-PAGE to assess peptidases in this bodonid, which presents serine peptidases ranging from 250 to 75 kDa, with a slight preference for acidic pH. This finding is dissimilar to what has been described in related pathogenic protozoa (Figure 5) (d'Avila-Levy et al., 2009). Curiously, all the analyzed closely related parasitic trypanosomatids, as well, as *Cryptobia salmonistica*, reveal through gelatin-SDS-PAGE only metallo- and cysteine peptidases, which are prototypal peptidases and virulence factors. In trypanosomatids, for instance, serine peptidases can only be detected by in-solution assays or after enrichment processes (Grellier et al. 2001). It is somewhat intriguing that cysteine and metallopeptidases are either not resistant to the denaturation/refold process and/or are not abundantly expressed by this

Applications of Zymography (Substrate-SDS-PAGE)

and leupeptin (Santos et al. 2003).

biochemical machinery of these intriguing insect trypanosomatids.

with ancestral functions during the insect colonization.

for Peptidase Screening in a Post-Genomic Era 273

opisthomastigote developmental stages during its life cycle (McGhee and Cosgrove, 1980), being used as a model to study the complex events of cell differentiation process. Also, these traditionally ''non-mammalian and non-pathogenic'' microorganisms have been used as experimental models of the Trypanosomatidae family for exploring their basic mechanisms at the genetic, physiological, ultrastructural and biochemical levels. In the same way, several research groups have described common structures/molecules produced by monoxenous and heteroxenous parasites belonging to the Trypanosomatidae family (Lopes et al. 1981; Breganó et al. 2003; Santos et al. 2006, 2007; Elias et al. 2008). Interestingly, *Herpetomonas* species have been detected not only in insects, but repeatedly in plants and mammals, including immunosuppressed humans, mainly in HIV-infected individuals, in whom the parasites caused either visceral or cutaneous lesions (reviewed by Chicharro and Alvar, 2003; Morio et al. 2008), showing the ability to develop digenetic life style under certain conditions. Collectively, these studies emphasize the need for further investigation in the

Whole cellular extracts of *H. samuelpessoai* promastigotes when analyzed by gelatin-SDS-PAGE revealed two major peptidase classes: a prominent metallopeptidase of 66 kDa (actually a broad hydrolytic activity ranging from 60 to 80 kDa), inhibited by 10 mM 1,10 phenanthroline, and a minor cysteine peptidase activity of 45 kDa, restrained by 1 µM E-64

The 66 kDa metallopeptidase activity was detected in the parasite membrane fraction after Triton X-114 partition (Etges, 1992; Schneider and Glaser, 1993; Santos et al. 2003) or after treatment of living cells with phospholipase C (Santos et al. 2002; Santos et al. 2006) as well as in the extracellular environment as the major secreted peptidase component (Santos et al. 2001, 2003, 2006; Elias et al. 2006). This metallopeptidase produced by *H. samuelpessoai* cells shares common biochemical and immunological properties (Elias et al. 2006; Santos et al. 2006) with the major metallopeptidase expressed by *Leishmania* species, called leishmanolysin or gp63, a virulence factor that participates in different stages of the parasite life cycle such as adhesion and escape from host immune response (Yao, 2010). The incorporation of different proteinaceous substrates into SDS-PAGE demonstrated that leishmanolysin-like molecule from *H. samuelpessoai* was able to degrade hemoglobin, casein, immunoglobulin G, mucin, human and bovine albumins as well as the gut protein extract from *Aedes aegypti* (Figure 2) (Pereira et al. 2010a), an experimental model to study the trypanosomatids-insect interplay (reviewed by Santos et al. 2006), culminating in the generation of peptides and amino acids required for parasite growth and development, as well as it might cleave structural barriers in order to improve its dissemination. Also, the pH dependence of the 66 kDa metallopeptidase of *H. samuelpessoai* was also determined by overlay gels, presenting a broad spectrum of pH (ranging from 5 to 10) and temperature (26 to 50oC), showing maximum hydrolytic activity at pH 6.0 at 37oC (Pereira et al. 2010a). These large spectra of pH and temperature retain maximum flexibility for the trypanosomatid to survive under different environmental conditions. In this sense, the surface leishmanolysinlike molecules of *H. samuelpessoai* cells participate in adhesive properties during the interaction with invertebrate gut (Pereira et al. 2010a) and mammalian macrophages (Pereira et al. 2010b). Other *Herpetomonas* species, including *H. megaseliae* and *H. anglusteri*, produce at least one metallopeptidase similar to the leishmanolysin, which is a conserved molecule

free living bodonid, because they were not detected by zymography. This may reflect substantial differences among the peptidases from these organisms. The raw data revealed by susbtrate-zymography provided the first observation on possible differences in peptidase profile among the families Bodonidae, Trypanosomatidae, and Cryptobiidae, forming the basis for future research.

Fig. 5. Inhibition profile of cellular peptidases of *Bodo* sp. in gelatin–SDS-PAGE. In order to determine the enzymatic class, after electrophoresis, the gels were incubated for 48 h at 28oC in 50mM phosphate buffer pH 5.5 in the absence (control) or in the presence of the following proteolytic inhibitors: 1mM phenylmethylsulfonyl fluoride (PMSF), 1 mg/ml aprotinin, 10mM 1,10-phenanthroline (Phen), 1 mM pepstatin A, or 10 µM *trans*-epoxysuccinyl L-leucylamido-(4-guanidino) butane (E-64). Numbers on the left indicate relative molecular mass of the peptidases. For experimental details see d'Avila-Levy et al. 2009. Reprinted with permission of *The Journal of Eukaryotic Microbiology*.

#### **3.2 Identification of peptidases in** *Herpetomonas* **spp. and possible biological functions proposed by overlay gel approaches**

In addition to the heteroxenic parasites that are of particular interest in the Trypanosomatidae family due to their medical importance, several genera are composed of monoxenic parasites of the gut of a wide range of insects. The *Herpetomonas* genus is composed of insect trypanosomatids that display promastigote, paramastigote and

free living bodonid, because they were not detected by zymography. This may reflect substantial differences among the peptidases from these organisms. The raw data revealed by susbtrate-zymography provided the first observation on possible differences in peptidase profile among the families Bodonidae, Trypanosomatidae, and Cryptobiidae, forming the

Fig. 5. Inhibition profile of cellular peptidases of *Bodo* sp. in gelatin–SDS-PAGE. In order to determine the enzymatic class, after electrophoresis, the gels were incubated for 48 h at 28oC in 50mM phosphate buffer pH 5.5 in the absence (control) or in the presence of the following proteolytic inhibitors: 1mM phenylmethylsulfonyl fluoride (PMSF), 1 mg/ml aprotinin, 10mM 1,10-phenanthroline (Phen), 1 mM pepstatin A, or 10 µM *trans*-epoxysuccinyl L-leucylamido-(4-guanidino) butane (E-64). Numbers on the left indicate relative molecular mass of the peptidases. For experimental details see d'Avila-Levy et al. 2009. Reprinted with

**3.2 Identification of peptidases in** *Herpetomonas* **spp. and possible biological** 

In addition to the heteroxenic parasites that are of particular interest in the Trypanosomatidae family due to their medical importance, several genera are composed of monoxenic parasites of the gut of a wide range of insects. The *Herpetomonas* genus is composed of insect trypanosomatids that display promastigote, paramastigote and

permission of *The Journal of Eukaryotic Microbiology*.

**functions proposed by overlay gel approaches** 

basis for future research.

opisthomastigote developmental stages during its life cycle (McGhee and Cosgrove, 1980), being used as a model to study the complex events of cell differentiation process. Also, these traditionally ''non-mammalian and non-pathogenic'' microorganisms have been used as experimental models of the Trypanosomatidae family for exploring their basic mechanisms at the genetic, physiological, ultrastructural and biochemical levels. In the same way, several research groups have described common structures/molecules produced by monoxenous and heteroxenous parasites belonging to the Trypanosomatidae family (Lopes et al. 1981; Breganó et al. 2003; Santos et al. 2006, 2007; Elias et al. 2008). Interestingly, *Herpetomonas* species have been detected not only in insects, but repeatedly in plants and mammals, including immunosuppressed humans, mainly in HIV-infected individuals, in whom the parasites caused either visceral or cutaneous lesions (reviewed by Chicharro and Alvar, 2003; Morio et al. 2008), showing the ability to develop digenetic life style under certain conditions. Collectively, these studies emphasize the need for further investigation in the biochemical machinery of these intriguing insect trypanosomatids.

Whole cellular extracts of *H. samuelpessoai* promastigotes when analyzed by gelatin-SDS-PAGE revealed two major peptidase classes: a prominent metallopeptidase of 66 kDa (actually a broad hydrolytic activity ranging from 60 to 80 kDa), inhibited by 10 mM 1,10 phenanthroline, and a minor cysteine peptidase activity of 45 kDa, restrained by 1 µM E-64 and leupeptin (Santos et al. 2003).

The 66 kDa metallopeptidase activity was detected in the parasite membrane fraction after Triton X-114 partition (Etges, 1992; Schneider and Glaser, 1993; Santos et al. 2003) or after treatment of living cells with phospholipase C (Santos et al. 2002; Santos et al. 2006) as well as in the extracellular environment as the major secreted peptidase component (Santos et al. 2001, 2003, 2006; Elias et al. 2006). This metallopeptidase produced by *H. samuelpessoai* cells shares common biochemical and immunological properties (Elias et al. 2006; Santos et al. 2006) with the major metallopeptidase expressed by *Leishmania* species, called leishmanolysin or gp63, a virulence factor that participates in different stages of the parasite life cycle such as adhesion and escape from host immune response (Yao, 2010). The incorporation of different proteinaceous substrates into SDS-PAGE demonstrated that leishmanolysin-like molecule from *H. samuelpessoai* was able to degrade hemoglobin, casein, immunoglobulin G, mucin, human and bovine albumins as well as the gut protein extract from *Aedes aegypti* (Figure 2) (Pereira et al. 2010a), an experimental model to study the trypanosomatids-insect interplay (reviewed by Santos et al. 2006), culminating in the generation of peptides and amino acids required for parasite growth and development, as well as it might cleave structural barriers in order to improve its dissemination. Also, the pH dependence of the 66 kDa metallopeptidase of *H. samuelpessoai* was also determined by overlay gels, presenting a broad spectrum of pH (ranging from 5 to 10) and temperature (26 to 50oC), showing maximum hydrolytic activity at pH 6.0 at 37oC (Pereira et al. 2010a). These large spectra of pH and temperature retain maximum flexibility for the trypanosomatid to survive under different environmental conditions. In this sense, the surface leishmanolysinlike molecules of *H. samuelpessoai* cells participate in adhesive properties during the interaction with invertebrate gut (Pereira et al. 2010a) and mammalian macrophages (Pereira et al. 2010b). Other *Herpetomonas* species, including *H. megaseliae* and *H. anglusteri*, produce at least one metallopeptidase similar to the leishmanolysin, which is a conserved molecule with ancestral functions during the insect colonization.

Applications of Zymography (Substrate-SDS-PAGE)

trypanosomatids (Santos et al. 2005).

Hemiptera:

Sarcophagidae

Culicidae

Muscidae

Phoridae

Diptera: Calliphoridae

> Diptera: Muscidae

Hemiptera: Reduviidae

**3.4 Peptidase screening in** *Crithidia* 

b The cysteine peptidases were inhibited by 10 M E-64. c Non detected (nd).

*Liopygia* 

*Haemagogus* 

*Muscina* 

*Megaselia* 

*Phormia* 

*Musca* 

*Zelus* 

a The metallopeptidase activities were completely blocked by 10 mM 1,10-phenanthroline.

Table 1. Peptidase profiles in different *Herpetomonas* species detected in gelatin-SDS-PAGE.

Among the insect trypanosomatids, the genus *Crithidia* comprises monoxenic trypanosomatids of insects that were originally characterized by the presence of choanomastigote forms in their life cycles (Hoare and Wallace, 1966). The first studies employing zymograms in order to detect proteolytic activity in *Crithidia* spp. were performed by Frank and Ashall in 1990. In the two studies published in that year, the activity in *Crithidia fasciculata* extracts was compared to *T. cruzi*. In this sense, it is worth mentioning that *C. fasciculata*, among all non-pathogenic trypanosomatid species, has been considered an excellent model organism for many studies concerning trypanosomatids, because it can be cultivated in high yields and do not require specific bio-safety precautions

In a first approach (Ashall, 1990), parasite extracts were made by the use of 0.5% Nonidet P-40 and mixed with SDS-PAGE sample buffer in non-denaturing conditions. After

species Host

*H. anglusteri* Diptera:

*H. dendoderi* Diptera:

*H. mariadeanei* Diptera:

*H. megaseliae* Diptera:

*H. roitmani* Diptera:

*Herpetomonas*

*Herpetomonas* 

*H. muscarum ingenoplastis* 

*H. muscarum muscarum* 

*samuelpessoai* 

(Vickerman, 1994).

*H.* 

sp.

for Peptidase Screening in a Post-Genomic Era 275

be a reflection of changes in the nutritional requirements during the life-cycle of the flagellates. Therefore, the authors infer that profiles of both cellular and extracellular peptidases represent an additional criterion to be used in the identification of

> Predominant evolutive stage in culture

Family Species Metallo-

Coreidae *Phthia picta* Promastigote 4 72, 60 45, 40

Syrphidae *Ornidia obesa* Opisthomastigote 1 50 nd

*ruficornis* Promastigote 2 60 45

*janthinomys* Promastigote 5 130, 110, 95 60, 45

*stabulans* Promastigote 2 ndc 42, 38

*regina* Promastigote 2 80, 67 nd

*leucogrammus* Promastigote 2 60 45

*domestica* Promastigote 6 100, 80 95, 50, 45, 40

*scalaris* Promastigote 8 100, 80, 67, 60 95, 45, 40, 35

Number of cellassociated peptidases

Molecular masses of peptidases in kDa

> Cysteine peptidasesb

peptidasesa

The 45 kDa cysteine peptidase synthesized by *H. samuelpessoai* cells had its activity reduced during the parasite growth at 37oC in comparison to 26oC, and when cultured up to 72 h in the presence of the differentiation-eliciting agent, dimethylsulfoxide. The modulation in the 45 kDa cysteine peptidase expression is connected to the differentiation process, since both temperature and dimethylsulfoxide are able to trigger the promastigote into paramastigote transformation in *H. samuelpessoai* (Santos et al. 2003; Pereira et al. 2009). In contrast, the expression of leishmanolysin-like molecules was not modulated during the differentiation in *H. samuelpessoai* (Pereira et al. 2009, 2010b).

The cultivation of *H. megaseliae* and *H. samuelpessoai* in different growth media induced the production of distinct profiles of both cellular and extracellular peptidases as revealed by a simple inspection using substrate-SDS-PAGE (Branquinha et al. 1996; Santos et al. 2002, 2003; Nogueira de Melo et al. 2006). In addition, the incorporation of different proteinaceous substrates into SDS-PAGE allowed the identification of substrate specific proteolytic activity in a complex cellular extract. For example, cellular cysteine peptidase (115–100, 40 and 35 kDa) and metallopeptidase (70 and 60 kDa) activities of *H. megaseliae* were detected in both casein and gelatin zymograms (Nogueira de Melo et al. 2006). Additionally, the use of casein in the gel revealed a distinct acidic metallopeptidase of 50 kDa when the parasite was cultured in the modified Roitman's complex medium. However, no proteolytic activity was detected when hemoglobin was used as co-polymerized substrate (Nogueira de Melo et al. 2006).

#### **3.3 Proteases produced by** *Herpetomonas* **species: Taxonomic marker**

Insect trypanosomatids have been traditionally allocated to a number of genera that were described based on morphological features, host and geographical origin (Wallace et al. 1983; Momen, 2001). However, for identification purposes, these criteria proved to be impractical and insufficient, because the same trypanosomatid species may be recovered from diverse species of insects and the same insect species may harbor various species of trypanosomatids. In addition, the morphology of trypanosomatid cells can be modified by environmental factors (Podlipaev, 2001; Momen, 2001, 2002). Therefore, there is a need to develop more effective means of trypanosomatid identification. With this task in mind, the expression of proteolytic activities in the Trypanosomatidae family was explored as a potential marker to discriminate between the morphologically indistinguishable flagellates isolated from insects and plants (Branquinha et al. 1996; Santos et al. 1999, 2005, 2008). For instance, many trypanosomatids have been erroneously placed in the genus *Herpetomonas* or, conversely, many *Herpetomonas* spp. may remain hidden in other genera. Santos and coworkers (2005) proposed an additional tool for trypanosomatid identification, including species belonging to the *Herpetomonas* genus by using *in situ* detection of proteolytic activities on gelatin-SDS-PAGE, in association with specific peptidase inhibitors. The results showed that nine distinct *Herpetomonas* species (*H. anglusteri*, *H. samuelpessoai*, *H. mariadeanei*, *H. roitmani*, *H. muscarum ingenoplastis*, *H. muscarum muscarum*, *H. megaseliae*, *H. dendoderi* and *Herpetomonas* sp. isolated from the salivary gland of a phytophagous insect) produced species specific cellular peptidase profiles (Table 1), which can be useful in the correct identification of these parasites. The exception for this observation was seen in *H. samuelpessoai* and *H. anglusteri*, which presented a similar cell-associated proteolytic pattern. However, these two *Herpetomonas* species excreted distinct proteolytic activities, which may

The 45 kDa cysteine peptidase synthesized by *H. samuelpessoai* cells had its activity reduced during the parasite growth at 37oC in comparison to 26oC, and when cultured up to 72 h in the presence of the differentiation-eliciting agent, dimethylsulfoxide. The modulation in the 45 kDa cysteine peptidase expression is connected to the differentiation process, since both temperature and dimethylsulfoxide are able to trigger the promastigote into paramastigote transformation in *H. samuelpessoai* (Santos et al. 2003; Pereira et al. 2009). In contrast, the expression of leishmanolysin-like molecules was not modulated during the differentiation

The cultivation of *H. megaseliae* and *H. samuelpessoai* in different growth media induced the production of distinct profiles of both cellular and extracellular peptidases as revealed by a simple inspection using substrate-SDS-PAGE (Branquinha et al. 1996; Santos et al. 2002, 2003; Nogueira de Melo et al. 2006). In addition, the incorporation of different proteinaceous substrates into SDS-PAGE allowed the identification of substrate specific proteolytic activity in a complex cellular extract. For example, cellular cysteine peptidase (115–100, 40 and 35 kDa) and metallopeptidase (70 and 60 kDa) activities of *H. megaseliae* were detected in both casein and gelatin zymograms (Nogueira de Melo et al. 2006). Additionally, the use of casein in the gel revealed a distinct acidic metallopeptidase of 50 kDa when the parasite was cultured in the modified Roitman's complex medium. However, no proteolytic activity was detected when hemoglobin was used as co-polymerized substrate (Nogueira de Melo

Insect trypanosomatids have been traditionally allocated to a number of genera that were described based on morphological features, host and geographical origin (Wallace et al. 1983; Momen, 2001). However, for identification purposes, these criteria proved to be impractical and insufficient, because the same trypanosomatid species may be recovered from diverse species of insects and the same insect species may harbor various species of trypanosomatids. In addition, the morphology of trypanosomatid cells can be modified by environmental factors (Podlipaev, 2001; Momen, 2001, 2002). Therefore, there is a need to develop more effective means of trypanosomatid identification. With this task in mind, the expression of proteolytic activities in the Trypanosomatidae family was explored as a potential marker to discriminate between the morphologically indistinguishable flagellates isolated from insects and plants (Branquinha et al. 1996; Santos et al. 1999, 2005, 2008). For instance, many trypanosomatids have been erroneously placed in the genus *Herpetomonas* or, conversely, many *Herpetomonas* spp. may remain hidden in other genera. Santos and coworkers (2005) proposed an additional tool for trypanosomatid identification, including species belonging to the *Herpetomonas* genus by using *in situ* detection of proteolytic activities on gelatin-SDS-PAGE, in association with specific peptidase inhibitors. The results showed that nine distinct *Herpetomonas* species (*H. anglusteri*, *H. samuelpessoai*, *H. mariadeanei*, *H. roitmani*, *H. muscarum ingenoplastis*, *H. muscarum muscarum*, *H. megaseliae*, *H. dendoderi* and *Herpetomonas* sp. isolated from the salivary gland of a phytophagous insect) produced species specific cellular peptidase profiles (Table 1), which can be useful in the correct identification of these parasites. The exception for this observation was seen in *H. samuelpessoai* and *H. anglusteri*, which presented a similar cell-associated proteolytic pattern. However, these two *Herpetomonas* species excreted distinct proteolytic activities, which may

**3.3 Proteases produced by** *Herpetomonas* **species: Taxonomic marker** 

in *H. samuelpessoai* (Pereira et al. 2009, 2010b).

et al. 2006).

be a reflection of changes in the nutritional requirements during the life-cycle of the flagellates. Therefore, the authors infer that profiles of both cellular and extracellular peptidases represent an additional criterion to be used in the identification of trypanosomatids (Santos et al. 2005).


a The metallopeptidase activities were completely blocked by 10 mM 1,10-phenanthroline.

b The cysteine peptidases were inhibited by 10 M E-64. c Non detected (nd).

Table 1. Peptidase profiles in different *Herpetomonas* species detected in gelatin-SDS-PAGE.
