Characterization and expression of the laminin gamma3 chain: a novel, non-basement membrane-associated, laminin chain.

Laminins are heterotrimeric molecules composed of an alpha, a beta, and a gamma chain; they have broad functional roles in development and in stabilizing epithelial structures. Here, we identified a novel laminin, composed of known alpha and beta chains but containing a novel gamma chain, gamma3. We have cloned gene encoding this chain, LAMC3, which maps to chromosome 9 at q31-34. Protein and cDNA analyses demonstrate that gamma3 contains all the expected domains of a gamma chain, including two consensus glycosylation sites and a putative nidogen-binding site. This suggests that gamma3-containing laminins are likely to exist in a stable matrix. Studies of the tissue distribution of gamma3 chain show that it is broadly expressed in: skin, heart, lung, and the reproductive tracts. In skin, gamma3 protein is seen within the basement membrane of the dermal-epidermal junction at points of nerve penetration. The gamma3 chain is also a prominent element of the apical surface of ciliated epithelial cells of: lung, oviduct, epididymis, ductus deferens, and seminiferous tubules. The distribution of gamma3-containing laminins on the apical surfaces of a variety of epithelial tissues is novel and suggests that they are not found within ultrastructurally defined basement membranes. It seems likely that these apical laminins are important in the morphogenesis and structural stability of the ciliated processes of these cells.

Identification of a gene essential for O-acetylation of the Staphylococcus aureus type 5 capsular polysaccharide.

The Staphylococcus aureus serotype 5 capsular polysaccharide (CP5) has a trisaccharide repeating unit of (-->4)-3-O-Ac-beta-D-ManNAcAp-(1-->4)-alpha-L-FucNAcp-(1-->3 )-beta-D-FucNAcp-(1-->). Tn918 mutagenesis of strain Reynolds yielded a mutant that produced wild-type levels of O-deacetylated CP5. The site and orientation of the single transposon insertion in mutant JL232 were determined by analysis of Southern blots and amplification of DNA flanking the transposon. DNA sequencing revealed that Tn918 was inserted within an open reading frame of 627 bp. The predicted amino acid sequence encodes a protein of approximately 26 kDa with homology to members of the NodL-LacA-CysE family of bacterial acetyltransferases. Southern blot analysis showed that genes similar to cap5H were present only in strains of S. aureus belonging to capsular serotypes 2, 4 and 5. In an in vitro assay, the parental strain was more resistant to opsonophagocytic killing than the mutant strain. In a mouse model of staphylococcal infection, the parental strain was able to seed the bloodstream from the peritoneal cavity and colonize the kidneys more efficiently than the O-deacetylated mutant. When cap5H was provided to the mutant in trans, it fully restored CP5 O-acetylation. The virulence of the complemented mutant strain closely approximated that of the parental strain.

Vfr controls quorum sensing in Pseudomonas aeruginosa.

Pseudomonas aeruginosa controls several genes in a cell density-dependent manner through a phenomenon termed quorum sensing. The transcriptional activator protein of the las quorum-sensing system is encoded for by the lasR gene, which is at the top of a quorum-sensing hierarchy. The activation of LasR as a transcriptional activator induces the expression of multiple genes that code for factors important for virulence, and rhlR, which encodes the transcriptional activator protein of the P. aeruginosa rhl quorum-sensing system. Elucidating the method of lasR regulation is crucial to understanding P. aeruginosa quorum sensing. In this report, we present studies on the transcriptional control of lasR. We identified two distinct transcriptional start sites for lasR that were located 201 bp (transcript T1) and 231 bp (transcript T2) upstream from the lasR start of translation. With the use of transcriptional lasRp-lacZ fusions, we showed that in P. aeruginosa, lasR expression is cell density dependent. This gene was expressed at a basal level until it was induced during the second half of log-phase growth, with expression becoming maximal during stationary-phase growth. We also showed that lasR expression was regulated through the cyclic AMP receptor protein (CRP)-binding consensus sequence in its promoter region. Our results from P. aeruginosa mutant studies and gel retardation assays indicated that this regulation was mediated by Vfr, a homolog of the Escherichia coli CRP.

Genetic analysis of type 5 capsular polysaccharide expression by Staphylococcus aureus.

Capsules are produced by over 90% of Staphylococcus aureus strains, and approximately 25% of clinical isolates express type 5 capsular polysaccharide (CP5). We mutagenized the type 5 strain Reynolds with Tn918 to target genes involved in CP5 expression. From a capsule-deficient mutant, we cloned into a cosmid vector an approximately 26-kb EcoRI fragment containing the transposon insertion. In the absence of tetracycline selection, Tn918 was spontaneously excised, thereby resulting in a plasmid containing 9.4 kb of S. aureus DNA flanking the Tn918 insertion site. The 9.4-kb DNA fragment was used to screen a cosmid library prepared from the wild-type strain. Positive colonies were identified by colony hybridization, and a restriction map of one clone (pJCL19 with an approximately 34-kb insert) carrying the putative capsule gene region was constructed. Fragments of pJCL19 were used to probe genomic DNA digests from S. aureus strains of different capsular serotypes. Fragments on the ends of the cloned DNA hybridized to fragments of similar sizes in most of the strains examined. Blots hybridized to two fragments flanking the central region of the cloned DNA showed restriction fragment length polymorphism. A centrally located DNA fragment hybridized only to DNA from capsular types 2, 4, and 5. DNA from pJCL19 was subcloned to a shuttle vector for complementation studies. A 6.2-kb EcoRI-ClaI fragment complemented CP5 expression in a capsule-negative mutant derived by mutagenesis with ethyl methanesulfonate. These experiments provide the necessary groundwork for identifying genes involved in CP5 expression by S. aureus.

Staphylococcus aureus microcapsule expression attenuates bacterial virulence in a rat model of experimental endocarditis.

The virulence of the Staphylococcus aureus strains that differed only in capsule expression was compared in a rat model of catheter-induced experimental endocarditis. The ID50 of all the strains was low (less than 3 x 10(3) cfu of S. aureus), suggesting that this model may be more sensitive than other animal models to differences in bacterial virulence. Compared with the wild-type strains that expressed type 5 or type 8 capsular polysaccharides, mutant strains devoid of capsule had significantly lower ID50 values. In contrast, a mutant that produced scant amounts of the type 5 polysaccharide had an ID50 similar to that of the parental type 5 isolate. As the bacterial inoculum was increased, each of the S. aureus strains reached final concentrations of 10(10)-10(11) cfu/g of vegetation; however, the nonencapsulated mutants colonized the left-sided vegetations at lower inocula than did the wild-type strains. This study indicates that microcapsule expression attenuates bacterial virulence in a rat model of catheter-induced endocarditis.

Virulence of Staphylococcus aureus mutants altered in type 5 capsule production.

Most clinical isolates of Staphylococcus aureus produce microcapsules (uronic acid-containing extracellular polysaccharides) that are detectable by serologic methods but are not visible by negative staining. Among the 11 reported serotypes, capsule types 5 and 8 comprise approximately 75% of all isolates. Transposon mutagenesis was performed on S. aureus to create mutants altered in capsule expression. Tn918 was introduced into the capsule type 5 strain Reynolds by filter mating, and a capsule-deficient transconjugate, JL236, was isolated. The wild-type strain was transformed with JL236 chromosomal DNA to confirm that transfer of the appropriate-size chromosomal fragment containing Tn918 generated a capsule-deficient transformant. Strain Reynolds was mutagenized with ethyl methanesulfonate to obtain a capsule-negative mutant (strain JL240). Capsular phenotypes were determined by colony immunoblots, antibody adsorption experiments, and transmission electron microscopy. The virulences of the parental and mutant strains in mice were compared. The 50% lethal doses for strains Reynolds, JL236, and JL240 were similar (10(8.59), 10(8.98), and 10(8.93) CFU, respectively). Animals injected intraperitoneally with either wild-type or mutant strains had comparable levels of bacteremia at 3 and 24 h after challenge. Quantitative cultures of blood and kidneys from animals challenged intravenously with sublethal doses of the S. aureus strains also showed no differences in bacterial clearance or renal abscess formation. These studies indicate that the type 5 S. aureus microcapsule does not promote bacterial virulence in the animal models tested.

Expression patterns of genes encoding elastase and controlling mucoidy: co-ordinate regulation of two virulence factors in Pseudomonas aeruginosa isolates from cystic fibrosis.

Transcriptional patterns of lasB and algD were compared in isogenic mucoid and non-mucoid Pseudomonas aeruginosa isolates from cystic fibrosis patients. The lasB gene encodes elastase, a major proteolytic enzyme secreted by P. aeruginosa, while algD is required for the synthesis of alginate, an exopolysaccharide frequently overproduced by strains infecting cystic fibrosis patients. A possible coregulation at the transcriptional level of these major virulence determinants was analysed. The lasB and algD genes showed inverse levels of promoter activity. The lasB promoter was active in non-mucoid cells and inactive in mucoid cells (in four out of five tested pairs), while the algD promoter was active in mucoid cells and silent in non-mucoid cells in all cases. When PAO568, a model strain for the analysis of control of the alginate system, was grown under conditions promoting mucoidy, the algD promoter was activated, whereas lasB mRNA could not be detected. This effect was reversed when the cells were grown in a medium suppressing mucoidy. Insertional inactivation of algR, a member of the signal-transduction systems regulating algD transcription, although abolishing algD expression and rendering cells non-mucoid, did not alter the nature of the induction and repression patterns of lasB seen in the parental strain PAO568. These results suggest that the lasB gene and the alginate system are co-ordinately regulated at a level parallel to or above the algR gene.

Increased levels of IgG subclasses specific for Pseudomonas aeruginosa exoenzyme and polysaccharide antigens in chronically infected patients with cystic fibrosis.

IgG subclass levels to Pseudomonas aeruginosa alginate, alkaline proteinase, elastase and exotoxin A in sera of healthy adults, non-infected and infected cystic fibrosis patients were investigated by enzyme linked immunosorbent assay. Whereas healthy adults and non-infected cystic fibrosis patients revealed mostly negative IgG subclass levels to the four antigens, infected cystic fibrosis patients had significantly elevated IgG1, IgG2, IgG3 and IgG4 levels to both the protein antigens as well as the polysaccharide antigen. The study does not support previous findings of an impaired natural IgG2 response to polysaccharide antigen. The study does not support previous findings of an impaired natural IgG2 response to polysaccharide in chronically infected cystic fibrosis patients.

Long-term tobramycin aerosol therapy in cystic fibrosis.

The long-term efficacy and safety of aminoglycoside aerosol therapy for Pseudomonas aeruginosa colonization/infection in cystic fibrosis has not been fully investigated. In the present study, 14 patients with cystic fibrosis, ages 8-19 years (mean: 13.3 years), received tobramycin aerosol therapy for a mean duration of 20 months. Eighty milligrams of a tobramycin solution were inhaled twice daily after physiotherapy via a jet nebulizer. After 1 year, weight for height increased significantly by 2.9% of the predicted normal, and the Kraemer clinical score increased by 2.1 points (P less than 0.05). The frequency of hospital admissions decreased from 2.0 to 1.3 per patient, respectively, during the years before and after the study onset. The antibody response to P. aeruginosa elastase, exotoxin A, and alkaline phosphatase showed a reduction in serum titers against one or more enzymes in eight patients. The best long-term results after 12-38 months of treatment were obtained in moderately ill children. No evidence of ototoxicity or renal damage was observed. Although intermittent bacterial resistance occurred in five patients after 10-21 months of tobramycin inhalation, this was not associated with clinical deterioration. The study demonstrates the safety and clinical efficacy of long-term tobramycin aerosol therapy. Double-blind studies with larger patient cohorts are required to determine the value of aminoglycoside inhalation as an adjunct to the established therapeutic regimens.

Staphylococcus aureus capsular types and antibody response to lung infection in patients with cystic fibrosis.

Chronic respiratory tract infections caused by Staphylococcus aureus are common in patients with cystic fibrosis (CF). Recently, it was shown in a few CF patients that S. aureus isolates produce capsular polysaccharides (CPs). However, it is not known whether this is a common feature and whether an immune response to CPs in CF is detectable. Therefore, we examined 170 S. aureus isolates from CF patients and healthy individuals for production of CP types 5 and 8 by using monoclonal antibodies. We found that CP-producing staphylococcal isolates were randomly distributed among CF patients and healthy carriers. Eighty-five percent of all isolates produced CPs, 77% of which were type 8. Examination of one sputum sample by an immunofluorescence technique suggested that production of CPs is not an in vitro phenomenon. S. aureus isolates from various sites of a single person often yielded more than one CP type. A random distribution of S. aureus strains with CP type 5 or 8 from the skin and respiratory tracts of patients and from the skin of healthy individuals was found. Antibody response to CP types 5 and 8, measured by enzyme-linked immunosorbent assay, was not elevated in CF patients with chronic S. aureus lung infection in comparison with healthy carriers. On the contrary, in CF patients the ratios of antibodies to CP 8 were significantly lower (P less than 0.005; alpha = 0.025). The ratios of antibodies to CP types did not change when monitored longitudinally over several months. This study suggests that the production of CPs is a universal property of S. aureus and that infected CF patients do not have elevated ratios of antibodies to these antigens.

Immunologic aspects of cystic fibrosis.

Bacterial infections determine life expectancy in the hereditary disease cystic fibrosis (CF). The dominant pathogens are Staphylococcus aureus and Pseudomonas aeruginosa, which persist in the patient's respiratory tract. Current explanations of the chronicity of the infections in the apparently immunocompetent host are based on defective opsonophagocytosis. This may be caused by (1) bacterial exopolysaccharide production, leading to cryptic infection types; (2) cleavage of immunoglobulin, complement, and surface receptors on immunocompetent cells by host proteases; and (3) a change from opsonic to nonopsonic antibody isotypes. Continuous antigenic stimulation of the immune system leads to local immune complex formation and a high chronic hypersensitivity reaction as well as to temporary immune unresponsiveness. Progressive tissue damage caused by lysosomal enzymes and oxygen radicals from polymorphonuclear leukocytes is thought to be ultimately responsible for respiratory failure and death in CF. Besides antibiotic treatment, anti-inflammatory therapy is therefore currently considered beneficial.