Differential expression of cysteine proteases in developmental stages of the parasitic ciliate Ichthyophthirius multifiliis.
An expressed sequence tag database of the freshwater fish parasite, Ichthyophthirius multifiliis (Ciliophora) was analyzed to seek for proteases potentially involved in the invasion and degradation of host tissues during infection. The translation of the database revealed two cathepsin L cysteine proteases (Icp1 and Icp2) of the C1A peptidase subfamily. The analysis of Icp1 and Icp2 sequences suggested that both proteases would be synthesized as preproproteins, with a mature domain of 27.9 and 22.8 kDa, respectively. Their expression level was determined in the trophont parasitic stage, in the tomont reproductive stage, and in the theront infective stage by real-time RT-PCR. ICP1 and ICP2 were significantly upregulated in trophont and theront stages in comparison with the tomont stage. Mature peptides of Icp1 and Icp2 were identified in crude extracts of I. multifiliis trophonts by LC-MS/MS. Zymograms showed three to seven activity bands at the optimum pH of cathepsin L cysteine proteases. Two bands displaying cysteine protease activity were identified by inhibition with E-64. They represented the major proteolytic activity of the trophont stage at pH 5-7, suggesting that cysteine proteases play an important role in the infection process.
Jousson
Olivier
O
Dipartimento di Patologia Animale, Profilassi e Igiene degli Alimenti, University of Pisa, Italy. oliver.jousson@vet.unipi.it
Di Bello
Domenica
D
Donadio
Elena
E
Felicioli
Antonio
A
Pretti
Carlo
C
D000595:Amino Acid Sequence; D000818:Animals; D002853:Chromatography, Liquid; D003546:Cysteine Endopeptidases / Q000235:genetics / Q000378:metabolism* / Q000502:physiology; D004591:Electrophoresis, Polyacrylamide Gel; D020224:Expressed Sequence Tags; D005786:Gene Expression Regulation; D015723:Gene Library; D016803:Hymenostomatida / Q000201:enzymology* / Q000254:growth & development / Q000472:pathogenicity; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D015800:Protozoan Proteins / Q000235:genetics / Q000378:metabolism* / Q000502:physiology; D020133:Reverse Transcriptase Polymerase Chain Reaction; D016415:Sequence Alignment; D020539:Sequence Analysis, Protein
D015800:Protozoan Proteins; D003546:Cysteine Endopeptidases