Apolipophorin III is a substrate for protease IV fromPseudomonas aeruginosa

Abstract

Our results demonstrated that Pseudomonas aeruginosa serine protease IV degraded apolipophorin III from the haemolymph of Galleria mellonella larvae. ApoLp-III protein was degraded in a stepwise manner. Four intermediate forms of 15, 13.3, 11.9 and 9.5 kDa were detected after 30 min digestion while only one of 5.6 kDa was released after 1-h incubation time. N-terminal amino acid sequence analysis of 5.6 kDa peptide revealed that it was released from apoLp-III after cleavage between lysine 70 and 71. ApoLp-III degradation by protease IV was inhibited by 1 mM TLCK but not 1 mM EDTA, additionally demonstrating that digestion was catalysed by a serine protease. Our data also indicated apoLp-III degradation in vivo during P. aeruginosa infection of G. mellonella larvae.

Changes in Galleria mellonella apolipophorin III level during Pseudomonas aeruginosa infection

“…Our previous report has shown that apoLp-III was degraded in vitro by extracellular serine protease IV isolated from an entomopathogenic strain of P. aeruginosa (Andrejko et al, 2005). In the light of these results, the question arises whether apoLp-III is degraded in vivo during P. aeruginosa infection.…”

“…The reaction was stopped by sample buffer addition and samples were stored at À20°C. The hemolymph extract contained proteins and peptides of Mr below 30 kDa was isolated from the hemocyte-free hemolymph by the acidic/methanol extraction using method adapted from Schoofs et al (1990) as previously described (Andrejko et al, 2005). Protease IV was isolated from P. aeruginosa growth medium using ionexchange chromatography as previously described (Andrejko et al, 2005).…”

“…G. mellonella caterpillars were used as a model host to study the role of the type III secretion system in P. aeruginosa pathogenesis (Miyata et al, 2003). Recently, studies performed in our laboratory indicated that P. aeruginosa serine protease IV degraded apolipophorin-III (apoLp-III) from hemolymph of G. mellonella larvae in vitro (Andrejko et al, 2005).…”

Changes in Galleria mellonella lysozyme level and activity during Pseudomonas aeruginosa infection

“…We showed that apoLp-III G. mellonella was effectively digested both in vitro (Andrejko et al 2005) and in vivo (Andrejko et al 2007) during P. aeruginosa infection. The results prompted us to test whether lysozyme, one of the main components of insect humoral immunity, was degraded in vivo during P. aeruginosa infection.…”

“…Given that, we carried out the studies which indicated that in vitro proteinase IV degrades apoLp-III (apolipophorin III, a multifunctional protein involved in insect immunity) from G. mellonella. On the other hand, lysozyme, despite potential cleavage sites for proteinase IV, was not degraded in vitro (Andrejko et al 2005).…”