Identification of the principal T lymphocyte-stimulating antigens of Pseudomonas aeruginosa.

To aid in understanding the role of cellular immunity in limiting Pseudomonas aeruginosa infections, we have identified some of the principal antigens of the organism that are recognized by human T cells. Clones of T cells were selected in such a manner that they would provide information not only about the identity of Pseudomonas antigens, but also the T cell repertoires of immune donors. Most clones were found to be specific for Pseudomonas alkaline protease (AP). Such clones could be physically isolated by selecting with crude Pseudomonas antigens or purified AP. In either case, their fine specificities were the same when tested against a panel of Pseudomonas antigens. The conclusion that AP is the principal immunogen for many donors was confirmed by measuring the absolute frequencies of proliferating T cells committed to AP and all other Pseudomonas antigens. Frequencies of AP-specific clones (1.5-2.7 X 10(-5] were comparable to those from the same donors that were specific for all secreted Pseudomonas antigens (1.3-6.0 X 10(-5]. These results provide a model system for studying human T cell-mediated immunity to bacteria by identifying discrete antigens and measuring the repertoire diversities of cells responding to them.

Expression of Pseudomonas aeruginosa virulence genes requires cell-to-cell communication.

Pseudomonas aeruginosa is an opportunistic human pathogen that causes a variety of infections in immunocompromised hosts and individuals with cystic fibrosis. Expression of elastase, one of the virulence factors produced by this organism, requires the transcriptional activator LasR. Experiments with gene fusions show that gene lasl is essential for high expression of elastase. The lasl gene is involved in the synthesis of a diffusible molecule termed Pseudomonas autoinducer (PAI). PAI provides P. aeruginosa with a means of cell-to-cell communication that is required for the expression of virulence genes and may provide a target for therapeutic approaches.

The involvement of cell-to-cell signals in the development of a bacterial biofilm.

Bacteria in nature often exist as sessile communities called biofilms. These communities develop structures that are morphologically and physiologically differentiated from free-living bacteria. A cell-to-cell signal is involved in the development of Pseudomonas aeruginosa biofilms. A specific signaling mutant, a lasI mutant, forms flat, undifferentiated biofilms that unlike wild-type biofilms are sensitive to the biocide sodium dodecyl sulfate. Mutant biofilms appeared normal when grown in the presence of a synthetic signal molecule. The involvement of an intercellular signal molecule in the development of P. aeruginosa biofilms suggests possible targets to control biofilm growth on catheters, in cystic fibrosis, and in other environments where P. aeruginosa biofilms are a persistent problem.

Identification of functional epitopes of Pseudomonas aeruginosa exotoxin A using synthetic peptides and subclone products.

The structure-function relationship of P. aeruginosa exotoxin A (ETA) was examined using synthetic peptides and genetically engineered ETA deletion mutants. Antibodies directed against synthetic peptides have allowed the identification of three ETA epitopes, two within domain I and one within the last 33 amino acids of domain III. In addition two distinct neutralizing determinants have been identified by antibodies directed against subclone products. One was associated with the amino-terminal half of ETA, the proposed receptor binding region. The second was associated with the carboxy-terminal half of ETA, a region previously not associated with receptor-binding. The amino-terminal subclone also offers potential as an ETA vaccine, since it produces a stable, non-enzymatically active product, effective in inducing ETA neutralizing antibodies. Data derived from these studies were used in a re-evaluation of structure-function relationships between ETA and diphtheria toxin.

Elastase assays.

Two methods of P. aeruginosa elastase purification are described: Method 1 involves concentration of sample supernatants, followed by DEAE-Sepharose liquid chromatography, whereas Method 2 involves initial fractionations followed by molecular sieving and hydrophobic interaction high-performance liquid chromatography. The choice of methods depends on the available equipment and supplies. The methods of assaying elastase activity described as useful for a variety of applications. The elastin-nutrient agar plate method is a qualitative assay to determine the presence of elastase activity produced by a given culture or colony. Use of the quantitative elastin-Congo red assay is appropriate for determining elastase activities of mid-to-high elastase-producing cultures. For more sensitive determinations of P. aeruginosa elastase activity, use of the fluorogenic substrate is advisable.

Quorum sensing, gene expression, and Pseudomonas biofilms.

Quorum sensing has been shown to be important for the development of a normal P. aeruginosa biofilm, and it follows that other microorganisms may employ a similar mechanism in the development of mature biofilms. To methods for detecting the presence of AI activity in biofilms are presented that employ an AI-responsive reporter strain harboring a lacZ fusion. Method 1 involves detection of AI activity in crude biofilms, whereas Method 2 employs an AI purification procedure. By using multiple indicator strains activated by AIs various acyl chain lengths, a wide range of AI molecules can be detected. Chromosomal knockout mutants are extremely useful for examining the contribution of a given gene to a specific phenotype. For quorum-sensing gene expression studies, mutants deficient in the production of AI offer more versatility than R-protein mutants. The main advantage of the AI mutants is that they can be complemented by either the AI synthase gene or the AI itself. Complementation with the AI circumvents having to grow the cells in the presence of antibiotics and allows experimental parameters such as AI concentration and time of addition to be manipulated easily. Finally, three reporter systems suitable for monitoring gene expression in P. aeruginosa biofilms are summarized in T Table II. The choice of reporter fusion depends mainly on whether in vivo analysis is required, whether temporal gene expression is to be examined, and the availability of equipment. In the case of P. aeruginosa, expression of quorum-sensing genes can be monitored either directly, by examining fusions of the R genes or AI synthase genes, or indirectly, by analyzing expression of genes controlled by these quorum-sensing systems.

Pathogenesis of corneal damage from pseudomonas exotoxin A.

Pseudomonas aeruginosa exotoxin A was injected into rabbit corneas. Death of epithelial, endothelial, and stromal cells resulted, and necrosis of the cornea followed. Control eyes with exotoxin neutralized by specific antitoxin showed minimal damage. A dose-response pattern was evident. Antitoxin neutralization of pseudomonas exotoxin A in corneal ulcers may have possible therapeutic implications.

Assessment of elastase as a Pseudomonas aeruginosa virulence factor in experimental lung infection in mink.

Anaesthetized mink were inoculated intratracheally with an elastase-producing Pseudomonas aeruginosa strain (PAO1) and two mutants derived from PAO1 with defective elastase formation (strains PAO1-E64 and PAO1-las-16). Survival times were prolonged in mink infected with the mutants, and microscopic examination of lungs showed that the elastase-positive wild type strain produced more pronounced tissue damage and haemorrhages than did the elastase-defective mutant strains. The strains PAO1 and PAO1-las-16 were also compared to three strains isolated from natural infection in mink which differed in elastase production. The mink strains with high or moderate elastase production produced more severe lung damage and were associated with a higher mortality than the other strains tested. The results indicate that P. aeruginosa may enhance the virulence of the bacterium in lung infections.

Genetic approaches to study Pseudomonas aeruginosa protein antigens.

Pseudomonas aeruginosa produces a large number of extracellular products which may play a role in pathogenesis. We have used genetic techniques to elucidate the relative contribution of these proteins to virulence, and as a method of producing safe toxoids. A mutant has been isolated which produces an immunologically reactive nontoxic form of toxin A, the most toxic extracellular protein produced by P. aeruginosa. Although there are difficulties in production of sufficient quantities of this CRM toxoid, these are likely to be solved by further genetic manipulation. Protection studies with toxin A antibody and studies of mutants deficient in toxin A have confirmed that toxin A plays a role in pathogenesis while clearly showing that toxin A alone cannot totally account for the virulence of P. aeruginosa. Studies of mutants specifically altered in three other products, exoenzyme S, and the two major proteases of P. aeruginosa, elastase and alkaline protease, have clarified the contribution of these products to virulence. Demonstration by genetic studies that exoenzyme S was a major factor in the virulence for one P. aeruginosa strain allowed us to correctly predict that antibody to this product would be protective against infection with that strain.

Effect of anaerobiosis and nitrate on gene expression in Pseudomonas aeruginosa.

DNA microarrays were used to examine the transcriptional response of Pseudomonas aeruginosa to anaerobiosis and nitrate. In response to anaerobic growth, 691 transcripts were differentially expressed. Comparisons of P. aeruginosa grown aerobically in the presence or the absence of nitrate showed differential expression of greater than 900 transcripts.

Cloning and characterization of the Pseudomonas aeruginosa lasR gene, a transcriptional activator of elastase expression.

We report the discovery of the lasR gene, which positively regulates elastase expression in Pseudomonas aeruginosa PAO1. The lasR gene was cloned by its ability to restore a positive elastase phenotype in strain PA103, a strain which possesses the elastase structural gene (lasB) but fails to synthesize the enzyme. Nucleotide sequence analysis revealed an open reading frame of 716 nucleotides encoding a protein of approximately 27 kDa. A labeled LasR protein of 27 kDa was detected in Escherichia coli by using a T7 RNA polymerase expression system. A chromosomal deletion mutant of the lasR gene was constructed in PAO1 by gene replacement. This mutant (PAO-R1) is devoid of elastolytic activity and elastase antigen. The deduced amino acid sequence of LasR is 27% homologous to the positive activator LuxR of Vibrio fischeri and the suspected activator 28K-UvrC of E. coli. Northern (RNA) analysis of total cellular RNA from PAO1, PAO-R1, and PAO-R1 containing the lasR gene on a multicopy plasmid (pMG1.7) revealed that a functional lasR gene is required for transcription of the elastase structural gene (lasB).

Molecular characterization and nucleotide sequence of the Pseudomonas aeruginosa elastase structural gene.

The elastase structural gene (lasB) from Pseudomonas aeruginosa PAO1 has been previously cloned on an 8-kilobase (kb) DNA fragment. The lasB gene, cloned in both orientations in pUC18, produced elastase in Escherichia coli, indicating that its promoter and translation initiation sites are functional in E. coli. Deletion analysis further defined the location of the lasB gene to a 3.0-kb EcoRI-KpnI fragment (pRB1803). Elastase prepared from E. coli TB1 (pRB1803) corresponded in molecular weight to mature P. aeruginosa extracellular elastase (33,000). The lasB gene directed the synthesis of 54- and 50-kilodalton (kDa) proteins in a bacterial cell-free transcription-translation system. The 33-, 50-, and 54-kDa proteins reacted with elastase-specific antiserum. To further characterize the lasB gene, the nucleotide sequence of the 3.0-kb EcoRI-KpnI fragment was determined. This DNA fragment contained a 1,491-base-pair open reading frame encoding 498 amino acids, corresponding to a predicted molecular weight of 53,600. The deduced amino acid sequence contained a putative signal sequence followed by a large polypeptide which preceded the mature 33-kDa elastase protein. Three zinc ligands and an active site were predicted for the mature elastase on the basis of its amino acid sequence homology with Bacillus thermoproteolyticus thermolysin.

Codon usage in Pseudomonas aeruginosa.

We have generated a codon usage table for Pseudomonas aeruginosa. Codon usage in P. aeruginosa is extremely biased. In contrast to E. coli and yeast, P. aeruginosa preferentially uses those codons within a synonymous codon group with the strongest predicted codon-anticodon interaction. We were unable to correlate a particular codon usage pattern with predicted levels of mRNA expressivity. The choice of a third base reflects the high guanine plus cytosine content of the P. aeruginosa genome (67.2%) and cytosine is the preferred nucleotide for the third codon position.

Determination of the amino acid change responsible for the nontoxic, cross-reactive exotoxin A protein (CRM 66) of Pseudomonas aeruginosa PAO-PR1.

Analysis of purified exotoxin A from parental Pseudomonas aeruginosa PAO1 and mutant strain PAO-PR1, which produces enzymatically inactive exotoxin A (CRM 66), revealed that CRM 66 lost 90% of parental enzymatic activity. Nucleotide sequence analysis of cloned exotoxin A genes showed a single amino acid substitution in CRM 66. Position 426 in the mature protein of parental (PAO1) exotoxin A is histidine, whereas in CRM 66, it is tyrosine.

Production of elastase and other exoproducts by environmental isolates of Pseudomonas aeruginosa.

Pseudomonas aeruginosa isolates from environmental sources and bacteremic patients were compared for their levels of elastolytic activity. No significant differences were found. The incidence of production of toxin A, phospholipase C, alkaline protease, and elastase among the environmental strains was also as high as that previously reported for clinical isolates.

Purification and characterization of an extracellular protease of Legionella pneumophila.

An extracellular proteolytic enzyme of Legionella pneumophila was purified by sequential batch separation with DEAE-cellulose, hydrophobic interaction chromatography with octyl-Sepharose, and ion-exchange chromatography with DEAE-Bio-Gel A (Bio-Rad Laboratories, Richmond, Calif.). The resulting protease preparation was determined to be homogeneous by polyacrylamide gel electrophoresis in the presence and absence of sodium dodecyl sulfate. Although free of contaminating proteins, the purified protease separated into two antigenically indistinguishable proteins both of which possessed proteolytic activity. The apparent masses of the proteins were 38 and 40 kilodaltons (kDa) as determined by polyacrylamide gel electrophoresis in sodium dodecyl sulfate, whereas gel filtration chromatography revealed a single mass of 34 kDa. Immunoblot analysis indicated that the 38-kDa protein probably originated from the 40-kDa protein during purification. The isoelectric points of the two protease species were 4.20 and 4.42. Enzyme activity, which was optimum between pH 5.5 and 7.5, was inhibited by various metal chelators; however, no effect was observed after treatment with phenylmethylsulfonyl fluoride, chymostatin, trypsin inhibitor, or dithiothreitol. Enzyme activity inhibited by metal chelators was restored upon the addition of various metal ions, including Zn2+, Fe2+, Mn2+, Cu2+, and Fe3+, but was not restored by Mg2+ or Ca2+. Atomic absorption analysis of the purified protease revealed a single gram-atom of zinc per mole of enzyme. Our findings indicate that the L. pneumophila protease resembles neutral zinc-containing metalloproteases similar to those found in other bacterial species.

Cloning and expression of a common Legionella outer membrane antigen in Escherichia coli.

A genomic library of Legionella pneumophila was constructed by inserting L. pneumophila knoxville-1 strain (LPK-1) chromosome fragments into cosmid vector pHC79. Screening of the library with antibodies directed against a major outer membrane protein/lipopolysaccharide complex from LPK-1 resulted in the identification of six clones that reacted with the antiserum. Western blot analysis indicated that a 19,000 dalton (19 kDa) component was the reactive antigen in all of the clones. Western blot analysis of outer membranes from L. pneumophila serogroups and other Legionella species revealed that the cloned 19 kDa antigen was common to all serogroups and all but one of the five other Legionella species examined. One of the 19 kDa expressing clones was used as an immunoabsorbent to recover antibody to the 19 kDa antigen thus confirming the surface localization of this L. pneumophila antigen in E. coli.