Human monoclonal antibodies to Pseudomonas aeruginosa alginate that protect against infection by both mucoid and nonmucoid strains

Two fully human mAbs specific for epitopes dependent on intact carboxylate groups on the C6 carbon of the mannuronic acid components of Pseudomonas aeruginosa alginate were found to promote phagocytic killing of both mucoid and nonmucoid strains as well as protection against both types of strains in a mouse model of acute pneumonia. The specificity of the mAbs for alginate was determined by ELISA and killing assays. Some strains of P. aeruginosa did not make detectable alginate in vitro, but in vivo protection against lethal pneumonia was obtained and shown to be due to rapid induction of expression of alginate in the murine lung. No protection against strains genetically unable to make alginate was achieved. These mAbs have potential to be passive therapeutic reagents for all strains of P. aeruginosa and the results document that alginate is a target for the proper type of protective Ab even when expressed at low levels on phenotypically nonmucoid strains.     

Microarray analysis and functional characterization of the nitrosative stress response in nonmucoid and mucoid Pseudomonas aeruginosa

The type strain of Pseudomonas aeruginosa, PAO1, showed great upregulation of many nitrosative defense genes upon treatment with S-nitrosoglutathione, while the mucoid strain PAO578II showed no further upregulation above its constitutive upregulation of nor and fhp. NO* consumption however, showed that both strains mount functional, protein synthesis-dependent NO*-consumptive responses.     

Alginate synthesis by mucoid strains of Pseudomonas aeruginosa PAO

Summary Alginic acid production by Pseudomonas aeruginosa PAO strains was studied in yeast extract/2% (w/v) gluconate medium. In all of the five strains studied, synthesis of the alginic acid was shown to occur in the stationary phase of growth. Each strain produced similar amounts of alginic acid at both 30° C and 37° C. However the amount of alginic acid varied from 7.5–11.5 gl^–1 depending upon the strain. The alginic acid was isolated, purified and its chemical composition determined. All strains produced a polysaccharide rich in polymannuronic acid which contained only polymannuronic acid blocks, polyguluronic acid blocks appeared to be absent. The amount of O-acetylation varied considerably from 2.3–14.7%. Analysis of the chain length distribution by poly-acrylamide gel electropheresis indicated that a homogeneous size of polymer was synthesised when compared to a high viscosity algal sample.     

Use of a gene replacement cosmid vector for cloning alginate conversion genes from mucoid and nonmucoid Pseudomonas aeruginosa strains: algS controls expression of algT.

Pseudomonas aeruginosa can convert to a mucoid colony morphology by a genetic mechanism called alginate conversion; this results in the production of copious amounts of the exopolysaccharide alginate. The mucoid phenotype of P. aeruginosa is commonly associated with its ability to cause chronic pulmonary tract infections in patients with cystic fibrosis. In this study we isolated the cis-acting locus involved in alginate conversion, called algS, from both mucoid and nonmucoid isogenic strains. We then examined the role of algS in the control of algT, a trans-active gene required for alginate production in P. aeruginosa. We used a new cosmid cloning vector, called pEMR2, that permitted both the cloning of large DNA fragments and their subsequent gene replacement in P. aeruginosa. To verify the predicted properties of this vector, we isolated and tested a pEMR2 hisI+ clone. Using cloned algS-containing DNA and a method for gene replacement, we constructed isogenic strains of P. aeruginosa that had Tn501 adjacent to algS on the chromosome. Two pEMR2 clone banks containing genomic fragments from isogenic algS(On) (exhibiting the alginate production phenotype) and algS(Off) (exhibiting the non-alginate production phenotype) strains were constructed, and Tn501 served as an adjacent marker to select for clones containing the respective algS allele. The pEMR2 algS(On) and pEMR2 algS(Off) clones were shown to contain the indicated algS allele by gene replacement with the chromosome of strains that carried the opposite allele. To test whether algS controls the expression of the adjacent algT gene, we constructed a pLAFR1 algS(Off)T clone and showed it to be unable to complement an algT::Tn501 mutation in trans. In contrast, a pLAFR1 algS(On)T clone did complement algT::Tn501 in trans. Thus, algS appears to control the activation of algT expression, bringing about alginate conversion.     

Characterization of mucoid Pseudomonas aeruginosa strains isolated from technical water systems

The occurrence of mucoid Pseudomonas aeruginosa strains was investigated in water samples and surface material from non-clinical aquatic environments. Ten of 81 environmental isolates displayed a mucoid colony type after incubation at 36°C for 24 h on Pseudomonas Isolation Agar. The mucoid strains obtained exclusively from surfaces of technical water systems were characterized in terms of medium-dependent expression of mucoid colonial phenotype, exoenzyme profile, pigment production and O-antigen type. The mucoid strains secreted substantially higher quantities of carbohydrate and uronic acid-containing material compared to non-mucoid environmental isolates. Major slime components of the mucoid strains were identified as O-acetylated alginates that contained higher proportions of mannuronate than guluronate monomer residues and were composed of blocks of poly-mannuronate and poly-mannuronate/guluronate, whereas blocks of poly-guluronate were absent. The results suggest that surfaces in aquatic environments may represent a natural habitat for mucoid (i.e. alginate-overproducing) strains of Ps. aeruginosa with properties similar to clinical mucoid strains.     

Isolation and characterization of an alginase from mucoid strains of Pseudomonas aeruginosa.

Strains of Pseudomonas aeruginosa which produce an alginate-like slime polysaccharide were shown to also synthesize an intracellular enzyme which can degrade these polysaccharides and the seaweed alginic acids. The enzyme acts as an eliminase introducing delta 4,5 unsaturation into the uronic acid moiety. It appears to be a polymannuronide lyase which degrades the polysaccharides, depending on their uronic acid composition, to a series of oligosaccharides, the smallest of which is a disaccharide. L-Guluronic acid linkages are not split. The Pseudomonas alginase resembles other bacterial alginases and enzymes from molluscs but differs in some important properties, such as extent of degradation and linkage preference. Nonmucoid forms of the organism produce detectable but much lower amounts of enzyme.     

Siderophore synthesis by mucoid Pseudomonas aeruginosa strains isolated from cystic fibrosis patients.

Nonmucoid Pseudomonas aeruginosa responds to iron deprivation by synthesizing the siderophores pyochelin and pyoverdine. When grown in iron-deficient medium, six mucoid P. aeruginosa strains isolated from cystic fibrosis patients synthesized copious amounts of the exopolysaccharide alginate. A procedure that eliminated the interference of alginate was developed so that siderophores could be extracted from the growth medium. All six isolates were then noted to produce both pyoverdine and pyochelin. This report thus confirms that mucoid P. aeruginosa, like its nonmucoid counterparts, elicits the siderophores commonly cited as those of the microbe.     

Examination of the bactericidal and opsonic activity of normal human serum for a mucoid and nonmucoid strain ofPseudomonas aeruginosa

The bactericidal and opsonic activity of fresh human serum (FHS) for a mucoid strain ofPseudomonas aeruginosa, 144M, and its spontaneous nonmucoid revertant, 144NM, was examined. Strain 144M was sensitive to the bactericidal activity of FHS, but strain 144NM was not. This bactericidal activity was due to the combined interaction of IgG and IgM with complement, activated through both pathways. Neither 144M nor 144NM was ingested by human polymorphonuclear leukocytes (PMNL) without FHS. Whereas maximal phagocytosis of 144M required only 5% FHS, comparable ingestion of 144NM required 25% FHS. Maximal phagocytosis of either 144M or 144NM required IgG, IgM, and complement. However, 144M required a heat-sensitive opsonic IgG, whereas 144NM required a heat-resistant IgG. Using selective absorption techniques, the targets for bactericidal and opsonic immunoglobulins on 144M and 144NM appeared to be different, suggesting that the variant 144NM had one or more altered, absent, or inaccessible cell surface components that account for differences in response to FHS and PMNL.     

Alterations in electrocardiographic parameters induced by acute infection with Pseudomonas aeruginosa wild-type and mucoid strains in mice

Pseudomonas aeruginosa (PA) is a pathogen in patients with cystic fibrosis (CF) and burn wound infections. Few studies have investigated the role of PA in electrocardiogram parameters (ECGs) with acute infection. We hypothesized that PA induces adverse ECGs effects in mice. After inoculation with wild-type strain PA14, the mucoid CF isolate FRD1 (mucA22+), which produces extensive alginate and has a natural mucoid phenotype, and the PA14 algD^? strain that cannot produce alginate, were evaluated by computerized electrocardiographic. ECG conduction changes were present in the P wave, the R-R interval and heart frequency in all PA strains and the PR interval were significantly prolonged with PA infection with PA14 algD^? and FRD1, compared to the control (P < 0.05), indicating slowed atrial and atrial–ventricular conduction. We conclude that PA produces bradycardia and demonstrated dramatic decreases in other ECGs associated with exposure to wild-type and mutant from PA. Furthermore, the mechanisms by which PA affects cardiac conduction remain uncertain.     

Identification of a slime exopolysaccharide depolymerase in mucoid strains ofPseudomonas aeruginosa

Strains ofPseudomonas aeruginosa recovered from pulmonary infections in cystic fibrosis (CF) patients are often mucoid in appearance owing to the secretion of a viscous slime exopolysaccharide (EPS). Unlike most mucoid isolates, strains WcM#2, P10, and P11 produce mucoid colonies after 24 h of incubation at 37°C, which become nonmucoid upon further incubation; this suggests the presence of a slime-degrading enzyme or depolymerase. Using both qualitative and quantitative assays, the presence of a slime EPS depolymerase was confirmed in each of these three strains as well as in four of four additional mucoid strains. Depolymerase activity was lower but still detectable in four of four nonmucoid strains. Enzyme preparations from strains WcM#2, P10, and P11 were active on most, but not all, slime EPS preparations fromP. aeruginosa strains, as well as sodium alginate; greater activity was observed on substrates after deacetylation. Comparisons are made between the enzyme described in this study and previous reports of slime EPS depolymerase in mucoid strains ofP. aeruginosa.