Wide Proteolytic Activity Survey Reinforces Heterogeneity Among Trypanosoma cruzi TCI and TCII Wild Populations

Abstract

Chagas disease, caused by the flagellate protozoan Trypanosoma cruzi, is characterized by considerable variation in both incidence and infection severity. This variation has been attributed to a set of complex features including the host genetic background, environmental and social factors, and the genetic heterogeneity of parasite populations. Using biochemical and molecular markers these populations can be divided into two major groups (TCI and TCII). In a previous work, our group identified cysteine and metalloprotease activities as good markers for differentiating TCI from TCII wild isolates, with a higher level of heterogeneity observed among TCII isolates. In this investigation, we applied the protease activity assay to a sample of 49 sylvatic T. cruzi isolates that had been previously assessed in terms of their Swiss mice infection patterns. Protease activity profiles were determined at pH 5.5 and 10.0 and was compared with the original host species, phylogenetic lineage, and mice infection characteristics. Substantial variability, with molecular weights ranging from 35 to 220 kDa for active proteases at pH 5.5, and of 30 to 90 kDa for active proteases at pH 10.0, was observed in gelatin substrate gels, with no phenetic separation between TCI and TCII groups or original hosts. The combinatorial expression of proteases recorded among individual isolates may account for the diverse behavior observed for parasite populations in nature.

Introduction

T he sylvatic transmission cycle of Trypanosoma cruzi, the causative agent of Chagas disease, involves invertebrate triatomine hosts and wild mammals. Social and ecologic factors bring humans into contact with T. cruzi in a domestic cycle of transmission. The parasite presents major developmental stages: epimastigotes and infective metacyclic trypomastigotes in blood-sucking vectors and intracellular amastigotes and bloodstream trypomastigotes in mammalian hosts. This parasite differentiation is linked to complex changes in gene expression (Krieger et al. 1999, Paba et al. 2004, Cestari et al, 2008). In addition to the intrinsic alterations among developmental forms, there are significant biological and epidemiological variations among parasite populations (Momen 1999, Buscaglia and Di Noia 2003), which are supported by T. cruzi clonal population structure (Tibayrenc et al. 1986, 1990).

T. cruzi has been divided into two major, well-defined phylogenetic lineages, TCI and TCII, based on markers derived from 24α rRNA and miniexon constitutive genes (Souto et al. 1996, Zingales et al. 1998). Our group is interested in dissecting additional differences between TCI and TCII groups at the molecular level. Previously, we indicated cysteine and metalloproteases as good markers for separation of TCI from TCII wild isolates, as shown by phenetic analysis (Fampa et al. 2008). Proteases represent good potential markers, based on the fact that these enzymes are proven parasite virulence factors (Sajid and McKerrow 2002). The T. cruzi cysteine protease cruzipain (Cazzulo et al. 1990) plays important roles in host cell invasion by the parasite (Aparicio et al. 2004), and treatment with cruzipain inhibitors is able to kill the parasite and cure infected mice (Meirelles et al. 1992). Metalloproteases constitute another class of protease, with the most important members in T. cruzi belonging to the gp63 family, which are involved in host cell infection (Cuevas et al. 2003, Kulkarni et al. 2009).

In this work, we analyzed 49 (24 TCI and 25 TCII) low-passage cultured field isolates that had been previously analyzed for Swiss mice infection patterns (Lisboa et al. 2007). The aim of this study was to determine the electrophoretic profile of active proteases in selected isolates and to compare those profiles with the original host species, phylogenetic lineage, and mice infection characteristics.

Materials and Methods

T. cruzi strains, hosts, and geographic location

The 49 T. cruzi field-isolated strains differed on the basis of phylogenetic grouping (TCI or TCII), hosts, and origin, as summarized in Tables 1 and 2.

Table 1.

Trypanosoma cruzi I and II Field Isolates: T. cruzi Isolates in Sylvatic Animals from Different Brazilian Biomes and Their Lineages

Isolate	Lineage	Host	Biome	Isolate	Lineage	Host	Biome
Barbeiro Elias	I	Triatoma tibimaculata	Atlantic Rainforest	C-4	II	Philander frenatus	Atlantic Rainforest
BF5	I	Rhodnius prolixus	Atlantic Rainforest	C-22	II	P. frenatus	Atlantic Rainforest
BPT-4	I	R. prolixus	Atlantic Rainforest	CPRJ 150	II	Leontopithecus chrysopygus	Atlantic Rainforest
C-45	I	P. frenatus	Atlantic Rainforest	GLT 291	II	Leontopithecus rosalia	Atlantic Rainforest
C-48	I	P. frenatus	Atlantic Rainforest	GLT 481c	II	L. rosalia	Atlantic Rainforest
CIGS-18	I	Saguinus bicolor bicolor	Amazon Forest	GLT 490	II	L. rosalia	Atlantic Rainforest
C-60	I	P. frenatus	Atlantic Rainforest	GLT 493	II	L. rosalia	Atlantic Rainforest
D7	I	Didelphis aurita	Atlantic Rainforest	GLT 543	II	L. rosalia	Atlantic Rainforest
D8	I	D. aurita	Atlantic Rainforest	GLT 564	II	L. rosalia	Atlantic Rainforest
NNB8	I	Nasua nasua	Pantanal	GLT 567	II	L. rosalia	Atlantic Rainforest
NN49	I	N. nasua	Pantanal	GLT 600	II	L. rosalia	Atlantic Rainforest
NN99	I	N. nasua	Pantanal	GLT 633	II	L. rosalia	Atlantic Rainforest
R4	I	Rattus rattus	Atlantic Rainforest	GLT 659	II	L. rosalia	Atlantic Rainforest
Thy	I	Thylamis	Pantanal	GLT 672	II	L. rosalia	Atlantic Rainforest
139	I	N. nasua	Pantanal	GLT 684	II	L. rosalia	Atlantic Rainforest
645	I	D. aurita	Atlantic Rainforest	GLT 689	II	L. rosalia	Atlantic Rainforest
6817	I	R. rattus	Caatinga	GLT 689 B	II	L. rosalia	Atlantic Rainforest
7592	I	Oecomy	Pantanal	GLT 714b	II	L. rosalia	Atlantic Rainforest
8623	I	Didelphis albiventris	Caatinga	GLT 725	II	L. rosalia	Atlantic Rainforest
8635	I	D. albiventris	Caatinga	GLT 745c	II	L. rosalia	Atlantic Rainforest
9667	I	Monodelphis sp.	Caatinga	GLT 750	II	L. rosalia	Atlantic Rainforest
10262	I	Didelphis marsupialis	Amazon Forest	GLT 778	II	L. rosalia	Atlantic Rainforest
10263	I	D. marsupialis	Amazon Forest	GLT 783	II	L. rosalia	Atlantic Rainforest
10268	I	Proechimys sp.	Amazon Forest	R4P	II	Thrichomys laurentius	Caatinga
				W20	II	L. rosalia	Atlantic Rainforest

Table 2.

Trypanosoma cruzi I and II Field Isolates: T. cruzi in Sylvatic Animals from Brazilian Biomes (Amazon Forest, Atlantic Rainforest, Pantanal, and Caatinga)

Species	Order	Biome	Genotype	Number of isolates
D. marsupialis	Marsupialia	Amazon Forest	TCI	2
Proechimys sp.	Rodentia	Amazon Forest	TCI	1
S. bicolor bicolor	Primate	Amazon Forest	TCI	1
D. aurita	Marsupialia	Atlantic Rainforest	TCI	3
L. chrysopygus	Primate	Atlantic Rainforest	TCII	1
L. rosalia	Primate	Atlantic Rainforest	TCII	21
P. frenatus	Marsupialia	Atlantic Rainforest	TCI	3
P. frenatus	Marsupialia	Atlantic Rainforest	TCII	2
R. rattus	Rodentia	Atlantic Rainforest	TCI	1
R. prolixus	Triatominae	Atlantic Rainforest	TCI	2
T. tibimaculata	Triatominae	Atlantic Rainforest	TCI	1
Thrichomys laurentius	Rodentia	Caatinga	TCII	1
Oecomys sp.	Rodentia	Pantanal	TCI	1
N. nasua	Carnivora	Pantanal	TCI	4
Thylamys	Marsupialia	Pantanal	TCI	1
D. albiventris	Marsupialia	Caatinga	TCI	2
Monodelphis sp.	Marsupialia	Caatinga	TCI	1
R. rattus	Rodentia	Caatinga	TCI	1
Total				49

Parasites' growth condition

T. cruzi epimastigotes were cultured in liver infusion tryptose (LIT) culture medium (bovine liver infusion 5 g/L, tryptose 5 g/L, NaCl 4 g/L, KCl 0.4 g/L, Na₂PO₄ 8 g/L, glucose 2 g/L; pH 7.2) supplemented with hemine at 10 mg/L and bovine fetal serum 10% with incubation at 27°C.

Parasite extracts

Five days old parasite cultures were harvested by centrifugation at 4000 g for 5 min and washed three times with cold phosphate-buffered saline (150 mM NaCl, 20 mM sodium phosphate buffer; pH 7.2). Cells were resuspended in 200 μL of distilled water, homogenized, and transferred to ice. Twenty microliters of sodium dodecyl sulfate (SDS) 10% were added, and cells submitted to three cycles of alternating between vortexing for 30 s and incubation on ice for 1 min. Extracts were then centrifuged at 15,300 g for 10 min. Supernatants were transferred to clean microcentrifuge tubes with storage at −20°C.

Protease activity assay

Proteases were assayed and characterized by electrophoresis on 10% SDS-polyacrylamide gel electrophoresis with 0.1% copolymerized gelatin as substrate (Heussen and Dowdle 1980). The gels were loaded with parasite extract derived from 5 × 10⁶ or 2 × 10⁷ per well, for pH 5.5 and 10.0, respectively. Electrophoresis was performed at a constant current of 120 V at 4°C and gels were then incubated at room temperature for 1 h in 10 volumes of 2.5% Triton X-100. The gels were then incubated for 24 h at 37°C in 50 mM sodium phosphate buffer, pH 5.5, supplemented with 2 mM DTT, or for 48 h in 50 mM glycine-NaOH buffer, pH 10.0. The gels were stained for 2 h in 0.2% Comassie Brilliant Blue R-250 in methanol–acetic acid–water (50:10:40) and destained in the same solvent. Molecular masses were calculated from mobility relative to molecular weight standards (Hanover, MD).

Phenetic analyses

Protease activity band profiles were converted into a matrix, indicating the presence or absence of specific polypeptide bands (scored as 1 or 0, respectively). Simple matrices were obtained using similarity coefficient (simple matching and Jaccard), and TCI versus TCII profile dendrograms were constructed using unweighted pair group method analysis. For these analyses, the NTSYS software package (Version 2.02; Exeter Software, Setauket, NY) was used. Only dendrograms that exhibited cophenetic correlation coefficient of >0.8 were considered.

Results

Protease activity profile in T. cruzi field-isolated strains

T. cruzi cysteine proteases, including cruzipain, are active at pH 5.5, whereas the metalloproteases are active at pH 10.0, as was recently corroborated (Fampa et al. 2008). Therefore, those pH values were employed in this study.

Proteases, independent of their pH of activity (5.0 or pH 10.0) and of group, host, and biome of origin, showed highly variable gelatinolytic profiles (Fig. 1). However, in contrast to a previous work (Lisboa et al. 2007), there was no correlation with mice infection patterns. The patterns shown in Figure 1 (a, c, e: TCI; b, d, f: TCII isolates) are representative of the observed diversity and highlights distinct TCI and TCII isolates (according to figure legends and Table 1) with variable band/activity units (Fig. 1A: a, b, 2 activities; c, d, 3 activities; and e, f, 5 activities; Fig. 1B: a, b, 1 activity; c, d, 2 activities; e, 3 activities; and f, 4 activities). It was observed that although some isolates presented the same number of active protease activities, their molecular masses were variable (Fig. 1A: c–d and e–f pairs; Fig. 1B: c–d pair).

FIG. 1.

Zymogram of Trypanosoma cruzi isolates. T. cruzi field-isolated parasites' proteolytic activities analyzed on gelatin–sodium dodecyl sulfate–polyacrylamide gel electrophoresis. Gels were incubated for 24 h at 37°C in 50 mM sodium phosphate (pH 5.5) supplemented with 2 mM DTT (A) or for 48 h in glycine-NaOH (50 mM, pH 10.0) (B). Activities from different TCI and TCII isolates are represented (A: a, TCI D8; b, TCII 672; c, TCI D7; d, TCII 564; e, TCI BPT-4; f, TCII 684; and B: a, TCI C-60; b, TCII 672; c, TCI CIGS-18; d, TCII 600; e, TCI 9967; f, TCII 567).

Zymograph divergence peculiar to pH activity values and phylogenetic lineages was observed. The number of protease activities detected by gelatinolytic analysis per isolate ranged from 0 to 5 at pH 5.5 for TCI group, 0 to 4 bands at pH 10.0 for TCI, 1 to 5 bands at pH 5.5 for TCII group, and 1 to 3 bands at pH 10 for TCII group (Table 3). Taken together, protease activity at pH 5.5 was more heterogeneous than at pH 10.0.

Table 3.

Trypanosoma cruzi I and II Field Isolates: Percentage of T. cruzi Wild Isolates (Out of 24 TCI and 25 TCII) Expressing Different Number of Protease Activities/Bands at Each pH Value

Number of bands/protease activities	% at pH 5.5 (TCI)	% at pH 10.0 (TCI)	% at pH 5.5 (TCII)	% at pH 10.0 (TCII)
5	8.3	—	12	—
4	8.3	12.5	16	—
3	16.6	16.6	4	32
2	37.5	29	52	52
1	16.6	29	16	16
0	12.5	12.5	—	—
No. of isolates	24	24	25	25

Most TCI field-isolated samples exhibited two bands at pH 5.5, and one or two bands at pH 10.0, but the majority of TCII isolates exhibited two bands at both pH 5.5 and 10.0 (Table 3). It was observed that some protease activities were more frequently associated with one T. cruzi group than with the other. Thus, at pH 5.5, 60, 120, and 220 kDa bands were 7, 2.3, and 2 times more frequently found in TCII than in TCI lineage (data not shown). On the other hand, three TCI isolates presented a 90 kDa protease activity at pH 10.0, which was not observed in any TCII isolate. Indeed, in the case of TCII the largest active protease showed a molecular mass of 70 kDa. A number of TCI isolates exhibited four active bands at pH 10.0, whereas TCII isolates expressed a maximum of three active bands (Table 3). Interestingly, only a few isolates demonstrated high-molecular-weight proteases. Specifically, a 200 kDa molecule was detected in a single TCI and two TCII isolates, whereas a 220 kDa protease was expressed by two TCI and four TCII isolates, respectively, at pH 5.5. It was also observed that three TCI samples demonstrated no detectable protease activity at either pH 5.5 or 10.0 (Table 3).

Phenetic analysis of sylvatic T. cruzi protease activity profiles

The heterogeneity among TCI and TCII isolates' protease patterns were analyzed phenetically. Dendrograms generated from global protease analyses showed that the TCI and TCII groups did not appear as discrete groups based on either simple match typing (SSm) (Fig. 2) or Jaccard coefficient (not shown). As an example, strains isolated from Leontopithecus rosalia in Atlantic Rainforest environments formed one cluster at an SSm value of 0.68 with strains having a degree of similarity ranging from more than 90% to almost 75% (TCII GLT493, 600, 291, 672, 481c, 778, 564). Other strains collected from the same environment and animal species were placed in other clusters having less than 60% similarity. On the other hand, higher levels of similarity in protease activity were shared by some TCI and TCII strains, for example, TCI 645 isolated from Didelphis aurita (Atlantic Coastal Rainforest) and TCII GLT745 from L. rosalia (Atlantic Coastal Rainforest), and TCI GS18 and TCII GLT741b (Saguinus bicolor bicolor, Amazon and L. rosalia, Atlantic Coastal Rainforest, respectively) at approximately SSm 0.84. In the same dendrogram, total similarity was found among TCI Thy and TCII GLT633 from Pantanal and Atlantic Forest environments.

FIG. 2.

Phenetic analysis (unweighted pair group method analysis-based dendrogram) of TCI and TCII isolates' protease activities. Phenetic analyses were based on active protease band presence and/or absence at both pH values in TCI and TCII field isolates. Construction is based on simple match typing coefficient.

Even when pH 5.5 and 10.0 active proteases were separately analyzed based on Jaccard coefficient, no separation between the major T. cruzi groups was demonstrated (data not shown), indicating the notable molecular polymorphism among T. cruzi wild isolates.

Discussion

Variation in both the incidence and severity of Chagas disease related to T. cruzi population heterogeneity has been puzzling researchers since the initial description of the disease by Carlos Chagas and Emmanuel Dias (reviewed by Pessoa 1960). The dichotomy into TCI and TCII groups provided evidence of an association between lineages to parasite ecoepidemiological characteristics, with TCI mainly observed in wild mammals and TCII usually found in humans (Devera et al. 2003). However, field observations along different Brazilian biomes do not show the same correlation, where TCII as well as TCI isolates may be collected from a diverse range of sylvatic hosts (Lisboa et al. 2007). Moreover, TCI samples are isolated from humans with Chagas disease associated with oral transmission in Amazonia (Marcili et al. 2009).

Wild T. cruzi isolates maintain their biological features closer to those shown under natural conditions than do laboratory strains. In a previous study, proteases were proposed as good markers for separating TCI and TCII groups based on an analysis of a total of 16 wild isolates (Fampa et al. 2008). In our investigation, we utilized an additional 49, out of 95, field isolates that had formerly been shown to generate variable mice infection patterns, that is, patent, intermittent, or subpatent with variable mortality rates (Lisboa et al. 2007). Electrophoretic analysis using gelatin as substrate demonstrated that the 49 isolates exhibited variable protease activities at both pH 5.5 and 10.0 in terms of both the number of bands and protease molecular weights. These patterns were shown to be independent of the previously established mice infection patterns. Yet, some parallels could be delineated when the current data were compared with those of Fampa et al. (2008). First, there was more heterogeneity in protease activity at pH 5.5 than at pH 10.0. Second, some bands appeared to be more frequently associated with one phylogenetic group than with the other. Third, TCII represented a more a diverse group, given that the majority of TCII isolates were collected from the same host L. rosalia and biome, that is, Atlantic Rainforest (Table 2). Interestingly, the TCII group exhibited protease heterogeneity, which was as marked as that recorded for the TCI isolates, which were more diverse in terms of hosts and biomes of origin. Based on isoenzymes, RAPD (Radom Amplification of Polymorphic DNA), and polymerase chain reaction–restriction fragment length polymorphism analyses (Brisse et al. 2000, Rozas et al. 2008), TCII group was elected as a more diverse group, being partitioned into five sublineages (IIa–e) or discrete typing units. Nonetheless, TCI can also be subdivided by other criteria (O'Connor et al. 2007, Brito et al. 2008, Spotorno et al. 2008).

In this work, we also observed a broader size range of active proteases in cellular extracts at both acid and alkaline pH than has been previously described (Campetella et al. 1990, Lowdes et al. 1996, Cuevas et al. 2003, Fampa et al. 2008). A number of isolates present low-molecular-weight proteases, around 30 kDa, at both pH 5.5 and 10.0. Conversely, very few isolates produced high-molecular-mass proteases, 200–220 kDa. It remains to be determined whether these larger proteases represented monomeric molecules or oligomeric associations of smaller ones. Finally, three out of four TCI isolates that had been collected from coati (Nasua nasua from Pantanal) demonstrated no detectable protease activity at either pH 5.5 or 10.0. Those isolates are clearly capable of survival in the wild and can infect their hosts, which suggests they possess alternative mechanisms to compensate for the absence of gelatinolytic proteases, or maybe their enzymes do not renature properly after being submitted to SDS-polyacrylamide gel electrophoresis denaturating conditions.

A limited number of studies have focused on differential gene expression of factors involved in parasite biology within the TCI and TCII groups. A correlation between the expression of a sialidase homolog gene (Risso et al. 2004), parasite surface glycoproteins involved in mammalian cell invasion (Ruiz et al. 1998), trypomastigote small surface antigen (Di Noia et al. 2002), ectophosphatase activities (Dutra et al. 2006), as well as Tc52, an immunoregulatory parasitic protein (Mathieu-Daudé et al. 2007), revealed a dimorphism that matches the TCI and TCII dichotomy. Finally, our group showed that TCI and TCII groups activate complement system differentially, with the TCII isolates being more resistant to complement system-mediated lysis (Cestari et al. 2008).

Wild isolates represent a largely unexplored group of T. cruzi populations that should be investigated to better understand parasite transmission cycles and identify possible relationships with distinct mammalian hosts. Consequently, methods for prevention of acute infection outbreaks and more efficient strategies against different manifestations of Chagas disease might be developed. The differential combination of protease activities may, in part, account for some of the divergent biological behavior observed among T. cruzi populations.

Footnotes

Acknowledgments

This study was supported by Conselho Nacional de Desenvolvimento Científico e Tecnológico, Fundação Universitária José Bonifácio, and Fundação Carlos Chagas Filho de Amparo à Pesquisa do Estado do Rio de Janeiro. The authors thank Dr. Douglas McIntosh for English review of the article, and Carlos Ardé Ruiz for his technical assistance with parasite study.

Disclosure Statement

No competing financial interests exist.

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