Trypsin-like serine proteases in Lutzomyia longipalpis--expression, activity and possible modulation by Leishmania infantum chagasi.
BACKGROUND
Midgut enzymatic activity is one of the obstacles that Leishmania must surpass to succeed in establishing infection. Trypsins are abundant digestive enzymes in most insects. We have previously described two trypsin cDNAs of L. longipalpis: one (Lltryp1) with a bloodmeal induced transcription pattern, the other (Lltryp2) with a constitutive transcription pattern. We have now characterized the expression and activity of trypsin-like proteases of Lutzomyia longipalpis, the main vector of visceral leishmaniasis in Brazil.


RESULTS
In order to study trypsin expression profiles we produced antibodies against peptides specific for Lltryp1 and Lltryp2. The anti-Lltryp1-peptide antibody revealed a band of 28 kDa between 6 and 48 hours. The anti-Lltryp2 peptide antibody did not evidence any band. When proteinaceous substrates (gelatin, hemoglobin, casein or albumin) were co-polymerized in polyacrylamide gels, insect midguts obtained at 12 hours after feeding showed a unique proteolytic pattern for each substrate. All activity bands were strongly inhibited by TLCK, benzamidine and 4-amino-benzamidine, indicating that they are trypsin-like proteases. The trypsin-like activity was also measured in vitro at different time points after ingestion of blood or blood containing Leishmania infantum chagasi, using the chromogenic substrate BArhoNA. L. longipalpis females fed on blood infected with L. i. chagasi had lower levels of trypsin activity after 12 and 48 hours than non-infected insects, suggesting that the parasite may have a role in this modulation.


CONCLUSIONS
Trypsins are important and abundant digestive enzymes in L. longipalpis. Protein production and enzymatic activity followed previously identified gene expression of a blood modulated trypsin gene. A decrease of enzymatic activity upon the parasite infection, previously detected mostly in Old World vectors, was detected for the first time in the natural vector-parasite pair L. longipalpis-L. i. chagasi.
Telleria
Erich Loza
EL
Laboratório de Biologia Molecular de Parasitas e Vetores, Instituto Oswaldo Cruz, Fiocruz, Rio de Janeiro, Rio de Janeiro, Brazil.
de Araújo
Adriana Pereira Oliveira
AP
Secundino
Nágila Francinete
NF
d'Avila-Levy
Claudia Masini
CM
Traub-Csekö
Yara Maria
YM
D000595:Amino Acid Sequence; D000818:Animals; D015153:Blotting, Western; D057075:Enzyme Assays; D005780:Gelatin / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D018314:Leishmania infantum / Q000502:physiology*; D008969:Molecular Sequence Data; D010455:Peptides / Q000276:immunology; D011576:Psychodidae / Q000201:enzymology* / Q000469:parasitology*; D016415:Sequence Alignment; D012697:Serine Endopeptidases / Q000737:chemistry / Q000378:metabolism*; D012996:Solutions; D013379:Substrate Specificity
D010455:Peptides; D012996:Solutions; C114880:trypsin-like serine protease; D005780:Gelatin; D012697:Serine Endopeptidases