Lysophospholipase L2 of Vibrio cholerae O1 affects cholera toxin production.

The implication in cholera toxin (CT) production of the newly identified gene, lypA, that encodes the lysophospholipase L2 of Vibrio cholerae, was investigated. Introduction of lypA into the V. cholerae O1 mutant (NF404), which has a Tn5-insertion in lypA and has lost CT as well as haemolysin production, restored the lysophospholipase activity and CT production but not the haemolytic activity. Inactivation of the lypA gene of the wild-type strain by chromosomal integration of a plasmid containing a portion of the lypA gene decreased the lysophospholipase L2 activity and the production of CT but not the haemolytic activity. Furthermore, constructed mutants of E1 Tor-biotype and Classical-biotype strains which have a defective lypA failed to produce CT and exhibited decreased enterotoxicity in the ligated rabbit ileal loop test. These results suggest that lypA is possibly required for the expression of CT and may play a role in pathogenicity of V. cholerae.

Genetic analysis of the chromosomal region encoding lysophospholipase L2 of Vibrio cholerae O1.

From the Tn5-inserted mutant library of Vibrio cholerae O1, we found a mutant, NF404, which lost the production of both hemolysin and cholera toxin (CT) even though the Tn5-insertion site was out side from the structural genes for hemolysin and CT. Cloning and sequencing analysis of the homologous region from the wild-type strain, revealed that the sequence spanning the coding region of an ORF1 nominated as lypA, encoding a 39.5 kDa protein. Deduced amino acid sequence of the lypA gene had 37.6% identity to the lysophospholipase L2 (EC 3.1.1.5) of Escherichia coli. In the downstream of lypA, a second open reading frame (ORF2) encoding an unknown protein with molecular weight of 19.9 kDa was found. Assaying the lysophospholipase L2 activity in the cell extract of E.coli harboring lypA in an expression vector showed clear increase in the enzymatic activity.