In Pseudomonas aeruginosa, many exoproduct virulence determinants are regulated via a hierarchical quorum-sensing cascade involving the transcriptional regulators LasR and RhlR and their cognate activators, N-(3-oxododecanoyl)-L-homoserine lactone (3O-C12-HSL) and N-butanoyl-L-homoserine lactone (C4-HSL). In this paper, we demonstrate that the cytotoxic lectins PA-IL and PA-IIL are regulated via quorum sensing. Using immunoblot analysis, the production of both lectins was found to be directly dependent on the rhl locus while, in a lasR mutant, the onset of lectin synthesis was delayed but not abolished. The PA-IL structural gene, lecA, was cloned and sequenced. Transcript analysis indicated a monocistronic organization with a transcriptional start site 70 bp upstream of the lecA translational start codon. A lux box-type element together with RpoS ( S ) consensus sequences was identified upstream of the putative promoter region. In Escherichia coli, expression of a lecA::lux reporter fusion was activated by RhlR/C4-HSL, but not by LasR/3O-C12-HSL, confirming direct regulation by RhlR/C4-HSL. Similarly, in P. aeruginosa PAO1, the expression of a chromosomal lecA::lux fusion was enhanced but not advanced by the addition of exogenous C4-HSL but not 3O-C12-HSL. Furthermore, mutation of rpoS abolished lectin synthesis in P. aeruginosa, demonstrating that both RpoS and RhlR/ C4-HSL are required. Although the C4-HSL-dependent expression of the lecA::lux reporter in E. coli could be inhibited by the presence of 3O-C12-HSL, this did not occur in P. aeruginosa. This suggests that, in the homologous genetic background, 3O-C12-HSL does not function as a posttranslational regulator of the RhlR/ C4-HSL-dependent activation of lecA expression.

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Pseudomonas aeruginosa infection is preceded by selective adhesion of the bacteria to the host target cells via diverse adhesins, including lectins. This step enables maximal damage to the target host cells by the bacterially secreted injurious toxins and enzymes. The production of both lectins and many of the virulence factors is positively controlled by transcription activators including signaling autoinducers (N-acyl-L-homoserine lactones). We show in this communication that erythromycin at subminimal growth inhibitory concentrations simultaneously suppresses the production of P. aeruginosa hemagglutinins (including lectins), protease, hemolysin and homoserine lactone autoinducers. The antibiotic-treated bacteria also show reduced virulence to mice, endorsing clinical observations that indicate the efficiency of low-dose erythromycin treatment of persistent drug-resistant P. aeruginosa infections.

The localization of the Pseudomonas aeruginosa lectins (PA-I and PA-II) was studied using methods of osmotic shock, freezing and thawing and spheroplast formation. Very slight release of the two lectins occurred when P. aeruginosa was exposed to magnesium-osmotic shock or was frozen and thawed. Under these conditions, release of the periplasmic 5'-nucleotidase occurred, whereas no release of cytoplasmic glucose-6-phosphate dehydrogenase activity was detected. Formation of spheroplasts from P. aeruginosa by gradual removal of the bacterial envelopes revealed low lectin activity in the treatment fluids. Osmotic shock treatment of the lysozyme treated mureinoplasts resulted in low release of glucose-6-phosphate dehydrogenase and the two lectins (10-13%) and a considerable activity (38.4%) of 5'-nucleotidase. The presence of the lectins on the outer and the cytoplasmic membranes enabled intact cells and spheroplasts of P. aeruginosa to agglutinate papain-treated human erythrocytes. These results indicate that the two lectins are located mainly in the cytoplasm with small fractions on the cytoplasmic and outer membranes and in the periplasmic space.

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