Kazal-type serine proteinase inhibitors in the midgut of Phlebotomus papatasi

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.

Characterization and expression profiling of serine protease inhibitors in the diamondback moth, Plutella xylostella (Lepidoptera: Plutellidae)

“…The most effective and direct mechanism is to inactivate proteases by protease inhibitors [ 7 , 8 ]. Serine protease inhibitors (SPIs) have been widely studied with documented roles in digestion, metamorphosis and development, as well as in immune responses [ 10 – 16 ]. SPIs can be divided into three distinct types: serpins, canonical inhibitors, and non-canonical inhibitors, based on their mechanisms of action [ 7 , 9 ].…”

“…For example, two Kunitz-type inhibitors from the hemolymph of M. sexta functioned as inhibitors of serine proteases, including trypsin, chymotrypsin and plasmin [ 44 ]. A Kazal-type inhibitor in the midgut of Rhodnius prolixus (Hemiptera: Reduviidae) may be involved in the interaction between microbiota and Trypanosoma cruzi [ 45 ]; two non-classical Kazal-type serine proteinase inhibitors (PpKzl1 and PpKzl2) were identified in Phlebotomus papatasi (Diptera: Psychodidae), and PpKzl2 has been described as an active serine proteinase inhibitor that is possibly involved in regulating digestive enzymes in the midgut [ 10 ]. However, only three P. xylostella SPIs (serpins 2, 4 and 5) have been reported.…”

Are Fireworms Venomous? Evidence for the Convergent Evolution of Toxin Homologs in Three Species of Fireworms (Annelida, Amphinomidae)

“…Kazal-type inhibitors are serine protease inhibitors characterized by the presence of one or more Kazal domains, 40–60 amino acid long domains with a conserved structure of six cysteine residues forming three disulfide bridges ( Laskowski and Kato 1980 ; Rimphanitchayakit and Tassanakajon 2010 ). Kazal-type inhibitors act on a variety of serine proteases including thrombin, trypsin, factor XIIa, subtilisin A, elastase, chymotrypsin, and plasmin ( Sigle and Ramalho-Ortigão 2013 ) and have been reported from the venoms of snakes and bats ( Francischetti et al. 2013 ; Low et al.…”

Identification of Novel Toxin Genes from the Stinging Nettle Caterpillar Parasa lepida (Cramer, 1799): Insights into the Evolution of Lepidoptera Toxins

“…The Kazal-type protease inhibitor is a serpin containing one or more Kazal domains. These proteins inhibit serine protease, e.g., thrombin, trypsin, factor XIIa, subtilisin A, elastase, chymotrypsin, and plasmin [ 84 ]. We identified 14 Kazal-type protease inhibitors from P .…”