Stage-regulated expression of cruzipain, the major cysteine protease of Trypanosoma cruzi is independent of the level of RNA

doi:10.1016/0166-6851(95)02545-6

Molecular and Biochemical Parasitology

Volume 76, Issues 1–2, February–March 1996, Pages 91-103

https://doi.org/10.1016/0166-6851(95)02545-6 Get rights and content

Abstract

The genes that encode cruzipain, the major cysteine protease of Trypanosoma cruzi are known to be arranged in tandem arrays. To gain a detailed insight into how these arrays are organised at the chromosomal level we have isolated clones from a cosmid library constructed with DNA from the X10.6 strain. In this strain we found that cruzipain is encoded by two allelic clusters composed of approximately 14 and 23 tandemly repeated genes which are located on homologous chromosomes of 650 and 670 kb. With the exception of the 3′-proximal genes, the cruzipain genes were all of identical or very similar sequence. An unusual feature of the 3′-proximal genes is that they lack the sequences that encode the 130 amino acid carboxyl terminal extension which is characteristic of cruzipain. Both gene clusters are situated in a similar chromosomal environment and are flanked by sequences which have the potential to form Z-DNA. In other eukaryotes, these motifs have been associated with recombinational hotspots and have been demonstrated to enhance gene conversion. The cruzipain genes are transcribed to produce a 1.8-kb transcript which is present at the same steady-state level in each of the parasite life cycle stages. However, protein levels and activity are 4–5-times higher in the insect epimastigote stage than in the trypomastigote and amastigote stages. By implication developmental regulation of cruzipain expression occurs predominantly at the translational and/or post-translational levels.

References (44)

A.C.M. Murta et al.
Structural and functional identification of $Gp57 51$ antigen of Trypanosoma cruzi as a cysteine protease
Mol. Biochem. Parasitol.
(1990)
O. Campetella et al.
The major cysteine proteinase (cruzipain) of Trypanosoma cruzi is encoded by multiple polymorphic tandemly organised genes located on different chromosomes
Mol. Biochem. Parasitol.
(1992)
A.E. Eakin et al.
The sequence, organisation and expression of the major cysteine protease (cruzain) from Trypanosoma cruzi
J. Biol. Chem.
(1992)
J.C. Mottram et al.
A cysteine proteinase cDNA from Trypanosoma brucei predicts an enzyme with an unusual C-terminal extension
FEBS Lett.
(1989)
A.E. Souza et al.
Characterisation of a multicopy gene for a major stage-specific proteinase of Leishmania mexicana
FEBS Lett.
(1992)
Y.M. Traub-Cseko et al.
Identification of two distinct cysteine proteinase genes of Leishmania pifanoi axenic amastigotes using the polymerase chain reaction
Mol. Biochem. Parasitol.
(1993)
A.E. Eakin et al.
Production of crystallizable cruzain, the major cysteine protease from Trypanosoma cruzi
J. Biol. Chem.
(1993)
M.N.L. Meirelles et al.
Inhibitors of the major cysteinyl proteinase ( $gp57 51$ ) impair host cell invasion and arrest intracellular development of Trypanosoma cruzi in vitro
Mol. Biochem. Parasitol.
(1992)
J.M. Kelly et al.
An approach to functional complementation by introduction of large DNA fragments into Trypanosoma cruzi and Leishmania donovani using a cosmid shuttle vector
Mol. Biochem. Parasitol.
(1994)
R.P. Bishop et al.
Chromosome size polymorphisms of Leishmania donovani
Mol. Biochem. Parasitol.
(1987)

J.J. Cazzulo et al.

Some kinetic properties of a cysteine proteinase (cruzipain) from Trypanosoma cruzi

Biochim. Biophys. Acta

(1990)

J.H. McKerrow et al.

The cysteine protease of Trypanosoma cruzi as a model for antiparasite drug design

Parasitol. Today

(1995)

S.M. Le Blancq et al.

Evidence for gene conversion between the phosphoglycerate kinase genes of Trypanosoma brucei

J. Mol. Biol.

(1988)

P.D. Smith et al.

Z-DNA forming sequences at a putative duplication site in the human annexi-VI encoding gene

Gene

(1994)

S.V. Graham

Mechanisms of stage-regulated gene expression in Kinetoplastida

Parasitol. Today

(1995)

M. Gale et al.

Translational control mediates the developmental regulation of the Trypanosoma brucei NrK protein

J. Biol. Chem.

(1994)

L. Aslund et al.

The C-terminal extension of the major cysteine proteinase (cruzipain) from Trypanosoma cruzi

Mol. Biochem. Parasitol.

(1991)

G. Harth et al.

Peptide-fluromethyl ketones arrest intracellular replication and intercellular transmission of Trypanosoma cruzi

Mol. Biochem. Parasitol.

(1993)

A.P.C.de A. Lima et al.

Identification of new cysteine protease isoforms in Trypanosoma cruzi

Mol. Biochem. Parasitol.

(1994)

G. Kendall et al.

Trypanosoma cruzi glycosomal glyceraldehyde 3-phosphate dehydrogenase does not conform to the ‘hotspot’ topogenic signal model

EMBO J.

(1990)

M.A. Miles

Culturing and biological cloning of Trypanosoma cruzi

J.M. Kelly

Isolation of DNA and RNA from Leishmania

Cited by (64)

The Trypanosoma cruzi genome; conserved core genes and extremely variable surface molecule families
2011, Research in Microbiology
Citation Excerpt :
It is clear that the repetitive nature of many housekeeping genes is conserved between strains and that there are mechanisms to maintain sequence similarity between repeat copies within tandem arrays. The latter phenomenon is found, for example, for cruzipain, a cysteine protease, that is present as two arrays of more than 20 1.8 kb copies on a homologous pair of chromosomes (Tomas and Kelly, 1996; Arner et al., 2007). In the CL Brener strain, the gene copies within each homologous array are close to identical, while there is 3% polymorphism between the homologs (observed in our laboratory by clone sequencing).
The protozoan parasite Trypanosoma cruzi is an important but neglected pathogen that causes chagas disease, which affects millions of people, mainly in latin America. The population structure and epidemiology of the parasite are complex, with much variability among strains. The genome sequence of a reference strain, CL Brener, was published in 2005, and the availability of this sequence has both revealed the complexity of the parasite genome and greatly facilitated research into parasite biology and pathogenesis, by making the sequences of more than 8000 core genes available. The T. cruzi genome is highly repetitive, which has resulted in inaccuracies in the genome sequence, and attempts have been made to provide a deeper analysis of repeated genes as a complement to the genome sequence. The genome was found to be organized in stable core regions containing housekeeping and other genes, surrounded by highly repetitive, often sub-telomeric highly variable regions containing multiple members of large families of surface molecule genes. Comparative sequencing of T. cruzi strains has been initiated and the results show that the core gene content of the parasite is highly conserved, but that much sequence variability, 3–4% difference at the DNA level on average between strains in coding regions, is present. The additional genomes will improve the understanding of parasite biology and epidemiology.
Aspects of Trypanosoma cruzi Stage Differentiation
2011, Advances in Parasitology
Citation Excerpt :
The cruzipain genes are transcribed as a 1.8-kb transcript which is constitutively expressed throughout the parasite life cycle. Interestingly, the protein abundance and consequently the enzymatic activity are four to five times higher in the insect epimastigote stage than in the trypomastigote and amastigote stages (Hanke et al., 1996). Accordingly, it has been suggested that the developmental regulation of cruzipain expression is likely to be mostly translational and/or post-translational (Tomas and Kelly, 1996).
Trypanosoma cruzi alternates between different morphological and functional types during its life cycle. Since the discovery of this parasite at the beginning of the twentieth century, efforts have been made to determine the basis of its pathogenesis in the course of Chagas disease and its biochemical constituents. There has also been work to develop tools and strategies for prophylaxis of the important disease caused by these parasites which affects millions of people in Latin America. The identification of axenic conditions allowing T. cruzi growth and differentiation has led to the identification and characterization of stage-specific antigens as well as a better characterization of the biological properties and biochemical particularities of each individual developmental stage. The recent availability of genomic data should pave the way to new progress in our knowledge of the biology and pathogenesis of T. cruzi. This review addresses the differentiation and major stage-specific antigens of T. cruzi and attempts to describe the complexity of the parasite and of the disease it causes.
Proteomic analysis of two Trypanosoma cruzi zymodeme 3 strains
2010, Experimental Parasitology
Two Trypanosoma cruzi Z3 strains, designated as 3663 and 4167, were previously isolated from insect vectors captured in the Brazilian Amazon region. These strains exhibited different infection patterns in Vero, C6/36, RAW 264.7 and HEp-2 cell lineages, in which 3663 trypomastigote form was much less infective than 4167 ones. A proteomic approach was applied to investigate the differences in the global patterns of protein expression in these two Z3 strains. Two-dimensional (2D) protein maps were generated and certain spots were identified by mass spectrometry (MS). Our analyses revealed a significant difference in the expression profile of different proteins between strains 3663 and 4167. Among them, cruzipain, an important regulator of infectivity. This data was corroborated by flow cytometry analysis using anti-cruzipain antibody. This difference could contribute to the infectivity profiles observed for each strain by in vitro assay using different cell lines.
Leishmanicidal Activity and Immobilization of dermaseptin 01 antimicrobial peptides in ultrathin films for nanomedicine applications
2009, Nanomedicine: Nanotechnology, Biology, and Medicine
Citation Excerpt :
The pH of the medium was 7.2, and in some cases it was adjusted to 5.5 with 1 M HCl. Incubation was carried out at either 26°C or 32°C, which are the commonly used temperatures for the routine maintenance of L. chagasi promastigotes.26 The effect of different reagents on L. chagasi promastigotes was assessed by a method similar to that of Savoia et al. (2002)22 and Brand et al. (2006).4
Antimicrobial peptides (AMPs) are essential for the innate immune system of eukaryotes, imparting protection against pathogens and their proliferation in host organisms. The recent interest in AMPs as active materials in bionanostructures is due to the properties shown by these biological molecules, such as the presence of an α-helix structure and distribution of positive charges along the chain. In this study the antimicrobial peptide dermaseptin 01 (DS 01), from the skin secretion of Phyllomedusa hypochondrialis frogs was immobilized in nanostructured layered films in conjunction with nickel tetrasulfonated phthalocyanines. The leishmanicidal activity of DS 01 was confirmed using kinetic essays, in which DS 01 promoted death of all metacyclic promastigote cells in 45 minutes. Surprisingly, the immobilized DS 01 molecules displayed electroactivity, as revealed by electrochemical experiments, in which an oxidation peak at about 0.61 V was observed for a DS 01 monolayer deposited on top of a conductive electrode. Such electroactivity was used to investigate the sensing abilities of the nanostructured films toward Leishmania. We observed an increase in the oxidation current as a function of number of Leishmania cells in the electrolytic solution at concentrations down to 10³ cells/mL. The latter is indicative that the use of AMPs immobilized in electroactive nanostructured films may be of interest for applications in the pharmaceutical industry and diagnosis.
The recent interest in Antimicrobial peptides (AMPs) as active materials in bionanostructures is due to the properties shown by these biological molecules. Leishmanicidal activity of a particular AMP is demonstrated in this paper.
Kinetoplastid papain-like cysteine peptidases
2009, Molecular and Biochemical Parasitology
Cysteine peptidases are important for growth and survival of kinetoplastid parasites. The best characterised are those homologous to mammalian cathepsins B and L. To address a somewhat confusing terminology, we introduce a unifying nomenclature for kinetoplastid CATB and CATL peptidases. We review their evolutionary relatedness, genomic organisation, developmental expression, subcellular location and physiological functions. In addition, the applications of kinetoplastid CATB and CATL enzymes as vaccine candidates, diagnostic markers and drug targets are discussed.
Metacyclogenesis as the Starting Point of Chagas Disease
2024, International Journal of Molecular Sciences

View all citing articles on Scopus

^☆: Nucleotide sequence data reported in this paper are available in the GenBank database under the accession numbers U41444, U41454 and U41511.

View full text

Regular paperStage-regulated expression of cruzipain, the major cysteine protease of Trypanosoma cruzi is independent of the level of RNA☆

Abstract

Mol. Biochem. Parasitol.

Mol. Biochem. Parasitol.

J. Biol. Chem.

FEBS Lett.

FEBS Lett.

Mol. Biochem. Parasitol.

J. Biol. Chem.

Mol. Biochem. Parasitol.

Mol. Biochem. Parasitol.

Mol. Biochem. Parasitol.

Biochim. Biophys. Acta

Parasitol. Today

J. Mol. Biol.

Gene

Parasitol. Today

J. Biol. Chem.

The C-terminal extension of the major cysteine proteinase (cruzipain) from Trypanosoma cruzi

Mol. Biochem. Parasitol.

Peptide-fluromethyl ketones arrest intracellular replication and intercellular transmission of Trypanosoma cruzi

Mol. Biochem. Parasitol.

Identification of new cysteine protease isoforms in Trypanosoma cruzi

Mol. Biochem. Parasitol.

Trypanosoma cruzi glycosomal glyceraldehyde 3-phosphate dehydrogenase does not conform to the ‘hotspot’ topogenic signal model

EMBO J.

Culturing and biological cloning of Trypanosoma cruzi

Isolation of DNA and RNA from Leishmania

Regular paper
Stage-regulated expression of cruzipain, the major cysteine protease of Trypanosoma cruzi is independent of the level of RNA☆