ABSTRACT
Sandflies (Diptera: Psychodidae) are important disease vectors of parasites of the genus Leishmania, as well as bacteria and viruses. Following studies of the midgut transcriptome of Phlebotomus papatasi, the principal vector of Leishmania major, two non-classical Kazal-type serine proteinase inhibitors were identified (PpKzl1 and PpKzl2). Analyses of expression profiles indicated that PpKzl1 and PpKzl2 transcripts are both regulated by blood-feeding in the midgut of P. papatasi and are also expressed in males, larva and pupa. We expressed a recombinant PpKzl2 in a mammalian expression system (CHO-S free style cells) that was applied to in vitro studies to assess serine proteinase inhibition. Recombinant PpKzl2 inhibited α-chymotrypsin to 9.4% residual activity and also inhibited α-thrombin and trypsin to 33.5% and 63.9% residual activity, suggesting that native PpKzl2 is an active serine proteinase inhibitor and likely involved in regulating digestive enzymes in the midgut. Early stages of Leishmania are susceptible to killing by digestive proteinases in the sandfly midgut. Thus, characterising serine proteinase inhibitors may provide new targets and strategies to prevent transmission of Leishmania.
Subject(s)
Animals , Female , Male , Gastrointestinal Tract/enzymology , Insect Vectors/parasitology , Phlebotomus/enzymology , Serine Proteinase Inhibitors/isolation & purification , CHO Cells , Cricetulus , Chymotrypsin/metabolism , Diptera/genetics , Gene Expression , Leishmaniasis/prevention & control , Life Cycle Stages/genetics , Psychodidae/parasitology , Regression Analysis , Reverse Transcriptase Polymerase Chain Reaction , Recombinant Proteins/isolation & purification , Recombinant Proteins/metabolism , Sequence Analysis , Serine Proteinase Inhibitors/genetics , Serine Proteinase Inhibitors/metabolism , Thrombin/metabolism , Trypsin/metabolismABSTRACT
Changes associated with blood and sugar meals digestion in the sandfly, Phlebotomus langeroni were characterized. Different types of sugars [sucrose, glucose, melibiose, cellobiose, lactose, starch, fig fruits, honey dew and a mixture of sources and sugar sources] were used for the sandfly feeding. Activities of glycosidases and proteases in the sandfly guts after blood and sugar meals were determined using the endpoint method. The results showed that glycosidases [alpha- glycosidase, beta-glycosidase, alpha-galactosidase and beta galactosidase] are present in the sandfly midguts. No activities of the glycosidases [alpha-mannosidase and alpha-amylase] were detected in the sandfly gut. Proteases [trypsin and aminopeptidase] showed activities in the sandfly midguts. It was concluded that the midgut glycosidase may play an important role in the vector-parasite interaction. Trypsin and aminopeptidase induction after a blood meal is controlled by a secretogogues mechanism which indirectly influences the outcome of the Leishmania parasite infection