Molecular analysis of hemolytic and phospholipase C activities of Pseudomonas cepacia

Abstract

By using a gene-specific fragment from the hemolytic phospholipase C (PLC) gene of Pseudomonas aeruginosa as a probe and data from Southern hybridizations under reduced stringency conditions, we cloned a 4.2-kb restriction fragment from a beta-hemolytic Pseudomonas cepacia strain which expressed hemolytic and PLC activities in Escherichia coli under the control of the lac promoter. It was found, by using a T7 phage promoter-directed expression system, that this DNA fragment carries at least two genes. One gene which shares significant DNA homology with both PLC genes from P. aeruginosa encodes a 72-kDa protein, while the other gene encodes a 22-kDa protein. When both genes on the 4.2-kb fragment were expressed from the T7 promoter in the same cell, hemolytic and PLC activities could be detected in the cell lysate. In contrast, when each individual gene was expressed in different cells or when lysates containing the translated products of each separate gene were mixed, neither hemolytic activity nor PLC activity could be detected. Clinical and environmental isolates of P. cepacia were examined for beta-hemolytic activity, PLC activity, sphingomyelinase activity, and reactivity in Southern hybridizations with a probe from P. cepacia which is specific for the larger gene which encodes the 72-kDa protein. There were considerable differences in the ability of the different strains to express hemolytic and PLC activities, and the results of Southern DNA-DNA hybridizations of the genomic DNAs of these strains revealed considerable differences in the probe-reactive fragments between highand medium-stringency conditions as well as remarkable variation in size and number of probe-reactive fragments among different strains. Analysis of the genomic DNAs from hemolytic and nonhemolytic variants of an individual strain (PC-69) by agarose gel electrophoresis, Southern hybridization, and transverse alternating pulsed field gel electrophoresis suggests that the conversion of the hemolytic phenotype to the nonhemolytic phenotype is associated with either the loss of a large plasmid (>200 kb) or a large deletion of the chromosome of P. cepacia PC-69.

Burkholderia cenocepacia, B. multivorans, B. ambifaria and B. vietnamiensis isolates from cystic fibrosis patients have different profiles of exoenzyme production

“…In our study, only B. vietnamiensis isolates were shown to be hemolytic. The low percentage of clinical BCC isolates exhibiting the hemolytic activity described in this report correlates well with earlier findings of Nakazava et al (15) and Vasil et al (30). In contrast, our data differ from those of Bevivino et al (3), which found that almost all isolates (environmental and clinical) belonging to B. ambifaria were hemolytic and that the percentage of hemolytic environmental B. cenocepacia isolates was markedly higher than the percentage of clinical isolates.…”

Virulence factors of Burkholderia cepacia

“…However, all haemolytic strains produce detectable lecithinase activity, and strains of B. cepacia, whether haemolytic or non-haemolytic, appear to produce detectable amounts of extracellular PLC activity. In contrast to the consistent patterns observed in the PLC gene of P. aeruginosa there is hypervariability in genetic organization of the PLC gene of B. cepacia [37]. The variable manner in which a B. cepacia PLC specific gene probe hybridizes with restricted B. cepacia DNA, the variability in expression of haemolytic and PLC activities of different strains, and the association of DNA arrangements with conversion of an Hly + to an Hly-variant may be related to the relatively large number of distinct insertion sequences (IS) reported for B. cepacia (> 25) [37,38]: in contrast these elements have yet to be discovered in P. aeruginosa.…”

“…were haemolytic when erythrocytes from various animals were tested [37]. Unlike the PLC activity of P. aeruginosa the PLC activity in B. cepacia does not correlate with haemolytic activity [29,37].…”

“…were haemolytic when erythrocytes from various animals were tested [37]. Unlike the PLC activity of P. aeruginosa the PLC activity in B. cepacia does not correlate with haemolytic activity [29,37]. However, all haemolytic strains produce detectable lecithinase activity, and strains of B. cepacia, whether haemolytic or non-haemolytic, appear to produce detectable amounts of extracellular PLC activity.…”

“…B. cepacia produces a heat-labile haemolysin which has both phospholipase C and sphingomyelinase activities [37]. Haemolytic and phospholipase C (lecithinase) expression in B. cepacia appears to be a complex phenomena.…”