A gene (pldA) encoding a 35.0-kDa protein with significant homology to the Escherichia coli outer membrane phospholipase was identified upstream of an operon encoding an enterochelin transport system in Campylobacter coli. The results of this study suggest that this gene encodes an outer membrane phospholipase A in C. coli. First, expression of the pldA gene product in a PldA-deficient mutant of E. coli led to the restoration of phospholipase A activity. The recombinant product also partitioned to the outer membrane, suggesting that it may be similarly located in C. coli. Second, heterologous overexpression in E. coli, followed by in vitro folding and purification of C. coli PldA, resulted in pure protein which displayed calcium-dependent lysophospholipase and phospholipase A activities in vitro. Finally, mutants of C. coli in which the pldA gene had been inactivated by allelic exchange were deficient in phospholipase A activity. Phospholipases are associated with lysis of erythrocytes by a number of bacterial pathogens. The pldA mutant was shown to have a reduced hemolytic activity compared to the wild-type strain, suggesting a role for the phospholipase A in the lysis of erythrocytes by C. coli. Since hemolysins are intimately associated with the disease-causing potential of a number of bacterial pathogens, it is likely that the phospholipase A plays some role in Campylobacter virulence. The thermophilic Campylobacter species, particularly Campylobacter jejuni and Campylobacter coli, are established as the most frequently isolated bacteria that cause acute diarrheal disease in humans (11, 27). In view of the incidence of Campylobacter infection, however, there still remains a general absence of information about the mechanisms involved in the pathogenesis of infection (36). Once ingested, the bacteria are thought to colonize the intestinal mucosa, with both flagella and their corkscrew shape enabling them to penetrate this viscous environment. The importance of flagella in colonization of the mucosa has been demonstrated in studies using aflagellate mutants (6, 43). Campylobacters are believed to adhere to and invade intestinal epithelial cells, and adhesion to cell lines has been demonstrated in vitro (18, 41). Although C. jejuni and C. coli have been reported to produce a variety of toxins (12, 24, 30), definitive reports have not yet appeared and the involvement of these proteins in the pathogenesis of infection remains controversial (13, 28). Hemolysins are intimately associated with the virulence of a number of bacterial pathogens (20), and there is increasing evidence that campylobacters may produce one or more hemolysins (17, 23, 29). The modes of action of hemolysins are diverse, but there are several studies which correlate the ability of bacteria to lyse erythrocytes with phospholipase activity (1, 25). These enzymes cleave membrane phospholipids into lysophospholipids, lysophosphatides, and free fatty acids, leading to membrane lysis. In this study, we report the genetic characterization of a gene (p...

In the outer membrane phospholipase A (OMPLA) of Escherichia coli, Ser144 has previously been identified by chemical modification as the active site serine residue. In a specific OMPLA-negative mutant strain, the pldA gene coding for OMPLA was shown to differ from the wild-type gene by a single point mutation, resulting in the substitution of Ser152 by phenylalanine. The role in catalysis of these two serine residues in OMPLA was investigated by site-directed mutagenesis. Ser144 and Ser152 were replaced one at the time by either alanine, valine, phenylalanine, threonine, or cysteine. Ser152 was furthermore replaced by asparagine. Replacement of Ser144 by cysteine resulted in 1% residual activity, whereas the other substitutions at this position yielded virtually inactive enzymes. Substitution of Ser 152 by threonine or asparagine resulted in 40% and 2% residual activity respectively, whereas all other substitutions at this position resulted in the loss of enzymatic activity. We propose that Ser144 is the nucleophile in catalysis, and that Ser152 is involved in hydrogen bonding either to the catalytic triad or in the oxyanion hole.