Departamento de Patología Experimental, Centro de Investigación y de Estudios Avanzados del IPN (CINVESTAV-IPN), AP 14-740, Mexico City, 07000, Mexico
Correspondence
Rossana Arroyo
rarroyo{at}mail.cinvestav.mx
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ABSTRACT |
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The GenBank/EMBL/DDBJ accession numbers for the tvcp12 gene sequences reported in this article are AY371180 and AY463697.
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INTRODUCTION |
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Conventionally, proteinases are assigned to clans and families depending on a number of characteristics, including sequence similarity, presence of inserted peptide loops and biochemical specificity to small peptide substrates, as well as susceptibility to proteinase inhibitors (Sajid & McKerrow, 2002). CPs of parasitic organisms are grouped mainly into clans CA and CD (Barrett, 1994
; McKerrow et al., 1999
) and those of clan CA are distributed mostly into families C1 and C2.
Proteinases from a variety of protozoan parasites have been characterized at the molecular and cellular levels, and the many roles that proteinases play in these organisms are starting to be understood. Central roles have been proposed for proteases in diverse processes such as host cell invasion and exit, encystation, excystation, catabolism of host proteins, differentiation, cell cycle progression, cytoadherence and both stimulation and evasion of the host immune responses (Klemba & Goldberg, 2002; Sajid & McKerrow, 2002
).
Many CPs play important roles in the virulence of parasites (García-Rivera et al., 1999; Mottram et al., 1996
). In T. vaginalis, some CPs are involved in cytoadherence (Arroyo & Alderete, 1989
, 1995
; Mendoza-López et al., 2000
), haemolysis (Dailey et al., 1990
), cytotoxicity (Álvarez-Sánchez et al., 2000
), disruption of the host cell membrane skeleton (Fiori et al., 1997
), degradation of the secretory leukocyte protease inhibitor (Draper et al., 1998
) and trichomonal immune evasion mechanisms by degradation of human immunoglobulins (Provenzano & Alderete, 1995
). Interestingly, the expression, proteolytic activity and surface localization of certain trichomonad CPs are modulated by the presence of iron in the medium (Alderete et al., 1995
; C. R. León-Sicairos, J. León-Félix & R. Arroyo, unpublished results).
Iron is an essential nutrient for growth, metabolism and expression of virulence factors for many pathogens (Gorrell, 1985; Griffith, 1985
; Wilson & Britigan, 1998
). In trichomonads, iron is acquired from the host via highly specific receptor-mediated mechanisms (Peterson & Alderete, 1984
; Lehker et al., 1990
; Lehker & Alderete, 1992
). In addition, iron controls the expression of hydrogenosomal proteins such as pyruvate : ferredoxin oxidoreductase and ferredoxin (Gorrell, 1985
), and virulence genes, such as those encoding some of the T. vaginalis adhesin proteins (Lehker et al., 1991
; Arroyo et al., 1995
; Engbring et al., 1996
; Alderete et al., 1998
; García et al., 2003
). Tsai et al. (2002)
identified an iron-responsive promoter in the ap65-1 gene which could be involved in the transcriptional regulation of some of these genes. Iron also mediates T. vaginalis resistance to complement lysis due to degradation of the C3 on the trichomonal surface (Alderete et al., 1995
) and negatively regulates the expression of a fibronectin-like gene, flp-1, reported recently (Crouch & Alderete, 2001
).
Here we report the identification and analysis of a novel tvcp12 complete gene that encodes a new papain-like CP of T. vaginalis and is negatively regulated by iron.
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METHODS |
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Cloning of tvcp12 and sequencing.
Previously, we obtained a 3035 bp DNA fragment (T. vaginalis locus 1.2.1.1) by hybridization of a genomic library (Espinosa et al., 2001) with a conserved region of papain-like CPs as a probe. This genomic clone contains a gene fragment and a complete gene separated by 1386 bp. One of the sequences (765 bp) corresponded to a fragment of a CP-encoding gene that we have called tvcp12 (León-Sicairos et al., 2003
). We amplified by PCR this 765 bp fragment using the sense primer CP5-1.2.1.1 (5'-CAGCTCTCAATGAACCATC-3'; nucleotide positions 184202) and the antisense primer CP3-1.2.1.1 (5'-TTATTAAACCTGTGGGACG-3'; nucleotide positions 933948) (Fig. 1b
), corresponding to the ends of the tvcp12 fragment present in the genomic clone. The PCR product was cloned into the vector pCRII-TOPO (Invitrogen). The 5' and 3' ends of the tvcp12 gene were obtained by using the rapid amplification of cDNA ends (RACE) system (Invitrogen), according to the manufacturer's instructions. Briefly, for the synthesis of cDNA, 1 µg total RNA was first dephosphorylated, the cap structure was removed from the mRNA, the oligo-RNA was ligated into the cap-less RNA, and reverse transcription of the trichomonad mRNA was performed. To obtain the missing 5' end of the tvcp12 gene, we used 1 µl cDNA, the GeneRacer 5' nested primer (5'-GGACACTGACATGGACTGAAGGAGTA-3') from the kit and the antisense primer RACE 5'-1.2.1.1 (5'-GGAGTAAAGGTTGCCGTGGTTCTT-3'), designed based on the known gene sequence at nucleotide positions 415438 (Fig. 1b
). To amplify the 3' end of the tvcp12 gene, a PCR was performed with 2 µl cDNA using as a sense primer RACE 3'-1.2.1.1 (5'-CAGGCGATTTCCAGGTTACACAG-3'; nucleotide positions 625647) (Fig. 1b
) and as an antisense primer GeneRacer 3' (5'-GCTGTCAACGATACGCTACGTAACG-3') from the kit. DNA products amplified by PCR were cloned into the vector pCRIV-TOPO (Invitrogen) and both strands were sequenced independently by using the dideoxy chain-termination method (Sanger et al., 1977
) in an AB1377 Applied Biosystems Automatic Sequencer (UNAM, Department of Cell Physiology, Mexico City, Mexico). The DNA sequences were analysed by using the BLAST (http://www.ncbi.nlm.nih.gov/blast/), EXPASY (http://us.expasy.org) and WORKBENCH (http://workbench.sdsc.edu) search engine tools.
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Total RNA was prepared using TRIzol reagent. The poly(A)+ RNA was obtained by oligo(dT) cellulose chromatography. For Northern blot analysis, the mRNA samples were separated on 1 % (w/v) formaldehyde agarose gels and transferred to Zetaprobe nylon membranes using 20x SSC. The filters were pre-hybridized, hybridized and washed as described for Southern blot assays. Each lane on the gel was loaded with 3·4 µg of mRNA as quantified by absorbance at 260 nm (Ausubel et al., 1999).
RT-PCR assays.
These were done using the Superscript RNase H Reverse Transcriptase kit (Stratagene) according to the manufacturer's instructions. Total RNA was reverse-transcribed using AMV reverse transcriptase and the oligo(dT) primer. Then, a fragment of 270 bp of the tvcp12 gene was amplified by PCR using the sense primer PROCP 1.2.1.1 (5'-GATTTCAACTTGCTTCCGGCATT-3'; nucleotide positions 830) and the antisense primer RACE 5'-12 (5'-CTTGACTGTTTGGCCCTTGGAAA-3'; nucleotide positions 254276) (Fig. 1b). A 112 bp fragment of the T. vaginalis
-tubulin gene was amplified by PCR using primer BTUB9 (5'-CATTGATAACGAAGCTCTTTACGAT-3') and primer BTUB2 (5'-GCATGTTGTGCCGGACATAACCAT-3'), as an internal control.
2-D SDS-polyacrylamide gels co-polymerized with gelatin.
Parasite proteinases were analysed by 2-D SDS-PAGE under reducing conditions. The polyacrylamide gels were co-polymerized with 0·2 % gelatin as a substrate (substrate gels) as described previously. After electrophoresis, proteinases were renatured with 2·5 % Triton X-100 and activated with 100 mM sodium acetate buffer pH 4·5 for 18 h at 37 °C. Gels were Coomassie blue-stained and de-stained for analysis (Álvarez-Sánchez et al., 2000).
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RESULTS |
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All T. vaginalis CPs characterized to date have a similar structure, with the most important motifs typical of cathepsin L-like CPs located in the same position (Fig. 2a). The signal peptide was not detected in the amino acid sequence of TvCP12. However, this sequence is present in TvCP2 and TvCP3 but not in TvCP1 and TvCP25, while the amino terminus of TvCP4 is unknown (Fig. 2a
). The ERFNIN-like [Ex3Rx2(Ile/Val)Fx2Nx3Ix3N] motif, which is present in all CPs analysed so far, is located at amino acid residues 3756 in TvCP12, whereas the potential or known start site of the mature TvCP12 is located at residue 99. All CP sequences, except that of TvCP3, have the three residues that form the catalytic triad (Cys-25, His-159 and Asn-175) typical of the papain family located at amino acids 124, 263 and 282. Also, all CPs except TvCP3 have the six conserved cysteine residues (Cys-22/Cys-63, Cys-56/Cys-95 and Cys-153/Cys-200, papain numbering system) that form three disulfide bonds to stabilize the tertiary structure (Fig. 2a
). These Cys residues are located at amino acids 121, 155, 162, 197, 256 and 304 in TvCP12 (Fig. 2a
).
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tvcp12 is a single copy gene transcribed as a 1·3 kb mRNA
To investigate the copy number of the tvcp12 gene in the T. vaginalis genome, we performed Southern blot assays using EcoRI-, NotI- and XbaI-digested genomic DNA and the PCR-amplified 765 bp fragment as a probe. In all cases, a single DNA band was observed (Fig. 3a) suggesting that a single copy of the tvcp12 gene is present in the T. vaginalis genome, as is the case for the tvcp1, tvcp2 and tvcp3 genes (Mallinson et al., 1994
). By Northern blot experiments using the poly(A)+ RNA and the 765 bp probe, we detected a single transcript band of 1·3 kb (Fig. 3b
). The length of this mRNA is consistent with the length of the complete tvcp12 gene sequence, of 948 bp.
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Proteolytic activity of the 30 kDa region is modulated by iron
Lastly, we investigated whether the proteolytic activity of mature CPs with a predicted molecular mass of 30 kDa, which may be the molecular mass of the mature TvCP12 proteinase, was differentially modulated in parasites grown in low and high concentrations of iron. We ran 2-D substrate gels (zymograms) using extracts from T. vaginalis cultured in both iron conditions (Fig. 5
). Differences in the proteolytic activity patterns of the trichomonad's 2535 kDa region were observed among the zymograms of parasite extracts from the different iron concentrations. A total of seven spots with proteolytic activity was observed in this region in the pI range from 4·5 to 6·0. Spots 1, 3, 4, 5 and 6 were observed in the extracts from parasites grown in low and high iron concentrations. Interestingly, spots 2 and 7 were only observed in extracts from parasites grown in low iron medium (Fig. 5
, L), whereas spot 5 activity was more visible in parasites grown in high iron medium (Fig. 5
, H). These results suggest that one of these spots observed only in low concentrations of iron may correspond to TvCP12, but more experiments are necessary to confirm this.
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DISCUSSION |
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The phylogenetic data suggest that TvCP12 appears to be part of a new cluster of CPs that shows high sequence divergence from the two other groups. Explanation for this early divergence will be found once the biochemical and functional characterization of this CP are completed. In addition, these should be considered preliminary phylogenetic data for the T. vaginalis CPs, since only a few of the multiple CP-encoding genes of this parasite have been cloned. The ongoing T. vaginalis genome project could help to confirm or modify this phylogenetic analysis. The pattern of the phylogenetic tree is similar to that of the 20 Entamoeba histolytica CP-encoding genes, which are grouped in two distinct clades suggesting that these amoeba CP-encoding genes evolved from two separate ancestors at a very early stage of parasite evolution (Bruchhaus et al., 2003).
As in other genes, the 5' UTR of tvcp12 is only 21 nt long, but tvcp12 does not have a typical Inr consensus sequence located up to 20 nt upstream of the initiation codon for T. vaginalis genes (Liston & Johnson, 1999). Since no typical TATA box has been described, we do not yet know how this gene is transcribed. However, we know that tvcp12 is expressed as a 1·3 kb mRNA in low iron conditions. In addition, RT-PCR assays evidenced that it is expressed when there is a low concentration of iron in the medium, whereas in high iron concentrations the transcript amount is very low.
The tvcp12 gene is iron-regulated; this is not surprising, because it has been established that different iron concentrations in the medium induce expression of different genes (Lehker & Alderete, 1992). The flp1 and flp2 genes of T. vaginalis, which encode proteins with homology to fibronectin, are induced under low iron growth conditions (Crouch & Alderete, 2001
), whereas ap65 and the ap51 and ap33 genes are overexpressed in high iron concentration media (Arroyo et al., 1995
; Engbring et al., 1996
; Alderete et al., 1998
).
The stability of the mRNA could be influenced by the presence of destabilizing elements such as the AUREs and also due to the length of the poly(A) tail. In mammals, if an AURE from the 3' UTR of an unstable mRNA, for example, one encoding granulocyte macrophage colony stimulating factor (GM-CSF), is placed within the 3' UTR of the -globin stable mRNA, the chimeric transcript decays with a half-life of less than 30 min (Caput et al., 1986
).
-Globin mRNA lacking the AURE is stable for well over 2 h in the same transfected cells. In T. vaginalis, mRNAs have been detected that contain AUREs such as two of the transcripts for the adhesin AP65 genes (Engbring et al., 1996
) and the flp-1 and the flp-2 genes with homology to fibronectin (Crouch & Alderete, 2001
) that could be important for destabilization of these mRNAs. However, studies need to be done to demonstrate if the AUREs found in the tvcp12 mRNAs from two different iron conditions are functioning as destabilizing elements (work in progress).
The stability of the mRNA due to variations in the length of the poly(A) tail could partially explain the differences observed in the transcript of the tvcp12 gene in the two iron conditions. The poly(A) tail length is important in mRNA stability in many genes studied such as the EhPgp1 gene involved in the MDR phenotype of E. histolytica (López-Camarillo et al., 2003). This could also be a regulatory mechanism involved in the low amount of transcript detected in T. vaginalis grown in high iron. These data suggest that iron concentrations during growth are affecting the tvcp12 transcript stability possibly through the size of the poly(A) tail.
The UAAA sequence in the 3' UTR of T. vaginalis mRNAs was proposed as the polyadenylation signal necessary but not sufficient to direct the polyadenylation of transcripts. This signal needs to be positioned at the proper distance between 11 and 30 nt from the cleavage site (AAAUUU) followed by a U-rich motif (Espinosa et al., 2002). We found this putative signal and the cleavage sequence in the genomic sequence of the tvcp12 gene (León-Sicairos et al., 2003
) and our data from the cDNA sequence in Fig. 4(e)
strongly suggest that this could be working as the putative polyadenylation signal of the tvcp12 mRNA.
Lastly, assuming that the pro-region is cleaved in all six cathepsin L-like T. vaginalis CPs (Mallinson et al., 1994; León-Sicairos et al., 2003
) as in other cathepsin L CPs, these proteinases would have a molecular size of between 23 and 30 kDa. However, within this size range only three spots with proteolytic activity have been observed previously in trichomonad extracts (Neale & Alderete, 1990
; Mendoza-López et al., 2000
). These data suggest that not all of the CP-encoding genes identified from this region are expressed in the parasite nor are they activated under the same environmental conditions, and processed in the same way. In addition, other proteinases with narrow specificity, able to degrade certain substrates, could exist in T. vaginalis.
After analysing the proteolytic activity under low and high iron concentrations, we were able to detect up to seven spots with different isoelectric points in the 2535 kDa region; whether these spots belong to the activity of all cathepsin L-like CPs awaits to be determined. However, the size and the down-regulation by iron of tvcp12 suggest that the TvCP12 protein may be one of the papain-like proteolytic spots that are only present in low iron conditions, such as spots 2 and 7.
It is noteworthy that proteinases with the ability to degrade specific substrates such as spectrin (Fiori et al., 1997), human immunoglobulins (Provenzano & Alderete, 1995
) and the secretory leukocyte protease inhibitor (Draper et al., 1998
) have been found in T. vaginalis. Recently, we have also found two legumain-like CPs in the 2535 kDa region with pI 6·3 and 6·5 that do not belong to the papain-like CP family in clan CA (Sajid & McKerrow, 2002
). These legumains appear to be part of a new subfamily of CPs within the family C13 of clan CD (León-Felix et al., 2004
) that has high substrate specificity (Sajid & McKerrow, 2002
).
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ACKNOWLEDGEMENTS |
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Received 20 November 2003;
revised 20 January 2004;
accepted 22 January 2004.