Cloning and characterization of Photobacterium damselae ssp. piscicida phospholipase: an enzyme that shows haemolytic activity

Abstract

A phospholipase gene of Photobacterium damselae ssp. piscicida (ppp) was cloned from a genomic library and its nucleotide sequence was determined. The open reading frame consisted of 1218 bp encoding a protein of 405 amino acids with a predicted molecular mass of 46 kDa. The PPP had identities (53-55%) with phospholipase and haemolysin of Vibrio spp., while it showed low identities (23-26%) with glycerophospholipid cholesterol acyltransferase of Aeromonas spp. A recombinant PPP (rPPP) with a His tag at the C-terminus expressed in Escherichia coli and purified showed phospholipase activity. The rPPP also showed lecithin-dependent haemolytic activity against mammalian erythrocytes and direct haemolytic activity against fish erythrocytes. The culture supernatant of wild-type P. damselae ssp. piscicida showed phospholipase activity, while that of a PPP gene knockout mutant did not.

Biochemical Characterization of a Phospholipase A2 from Photobacterium damselae subsp. piscicida

“…Two PL activities with different molecular masses were confi rmed in ECP of Phdp (Hu, 2005). The one with the lower mass was confi rmed as a phospholipase C (Hsu et al, unpublished results), while the one with the higher mass studied here was found similar to that reported by Naka et al (2007). These authors reported the hemolytic activity of a PL from the ECP of Phdp, but did not characterize this PL further.…”

“…BAB85814). Therefore, the correspon ding gene sequence of the PL from Phdp (Naka et al, 2007) was cloned and expressed in Escherichia coli. The recombinant PL was purifi ed, characterized, and found to be a toxin of the bacterium in cobia (Rachycentron canadum).…”

Chromosome-Encoded Hemolysin, Phospholipase, and Collagenase in Plasmidless Isolates of Photobacterium damselae subsp. damselae Contribute to Virulence for Fish

“…The activity of certain bacterial hemolysins is known to be enhanced after supplementation with exogenous lecithin (22,23). We found that the addition of lecithin to sheep erythrocytes caused a slight increase in the translucency of the hemolytic halo, whereas the presence of lecithin clearly enhanced the hemolysis of fish erythrocytes, not only in terms of halo translucency but also in terms of its diameter (Fig.…”

Photobacterium damselae subsp. damselae, an Emerging Pathogen Affecting New Cultured Marine Fish Species in Southern Spain

“…The haemolytic activity in Vibrionaceae can be related to extracellular enzymatic activities, such as phospholipases in V. parahaemolyticus, V. mimicus, V. harveyi and P. damselae subsp. piscicida (Shinoda et al, 1991;Lee et al, 2002;Zhong et al, 2006;Naka et al, 2007) and phospholipase D in P. damselae subsp. damselae (Kreger et al, 1987), or to the direct action of haemolysins that provoke the pore-structure formation on the erythrocyte membrane (Iida & Honda, 1997;Zhang & Austin, 2005).…”