Characterization of tripsyns in Lutzomyia longipalpis: main vector of leishmaniose visceral in Brazil.
Visceral Leishmaniasis is a major public health problem. In the New World it is caused by Leishmania infantum chagasi, and in Brazil Lutzomyia longipalpis is the main vector. Insect midgut enzymatic activity during blood digestion is one of the main obstacles which L. i. chagasi must surpass to succeed in establishing infection. Trypsins are the main proteases secreted in this insect gut, and transcription may vary along the blooddigestion process. We are interested in studying molecular aspects of blood feeding, to characterizing specific moleculesthat might have a significant role in parasite-vector interaction, which might be used as potential targets for newstrategies in the fight against the spreading of leishmaniasis. We have isolated and fully sequenced two trypsincDNAs (Lltrip1 and Lltrip2) from an expression library of blood-fed L. longipalpis midgut and their sequencesare similar to other insect trypsins. Specific primers were designed in order to be used in RT-PCR to investigatetrypsin differential expression. Our experiments using semi-quantitative RT-PCR showed that Lltrip1 transcription has a peak at approximately 12 hours after blood ingestion, and disappears after 48 hours, resembling bloodmeal inducedtrypsins from other insects. Lltrip2 has a high transcription in non-fed female and in male insects, much likely tobe a constitutive enzime. Nevertheless its transcription decreases after blood ingestion. High transcription isalso observed in females after 96 hours, when digestive process is finalized and there is no more blood in the gut.We are presently studying trypsin immuno-localization in the insect gut, using an anti-trypsin antibody produced by our group, and our preliminary results indicate that the enzyme distributes through the cytoplasm. We have also used this antibody to determine the appearance of the enzyme over time, through western blots. A band of the expected size is detectable between 6 and 48 hours after bloodmeal and after that, it is no longer detected. Preliminary enzyme activity assays sugests that L. longipalpis adults infected with L. i. chagasi at 48 hours after infective meal, have lower trypsin activity than non-infected bloodfed insects at the same time.
Advisor:Yara Maria Traub-Cseko
School:Faculdades Oswaldo Cruz
Source Type:Master's Thesis
Keywords:Psychodidae Leishmaniasis Visceral Trypsin Insects Polymerase Chain Reaction
Date of Publication:03/23/2007