Protection against mucoid Pseudomonas aeruginosa in rodent models of endobronchial infections.

Chronic endobronchial infection with mucoid Pseudomonas aeruginosa accounts for much of the morbidity and mortality in patients with cystic fibrosis (CF). Reduced morbidity is observed when infection is absent. Clinical investigations have implicated opsonizing antibody specific for the mucoid exopolysaccharide (MEP) surrounding these bacteria as a potential immunologic protective mechanism, whereas nonopsonizing antibody to MEP is not protective. Mice and rats immunized with doses of MEP that elicited opsonizing antibody had reduced levels of infection compared with nonimmune controls after intratracheal challenge with mucoid P. aeruginosa enmeshed in agar beads. Doses of MEP that elicited nonopsonizing antibody were not protective. Parallel experiments in which passive transfer of polyclonal and monoclonal opsonizing and nonopsonizing antibody were used yielded similar results. These data indicate that MEP-specific opsonizing antibody can protect against chronic P. aeruginosa infection in this model of disease.

Role of mutant CFTR in hypersusceptibility of cystic fibrosis patients to lung infections.

Cystic fibrosis (CF) patients are hypersusceptible to chronic Pseudomonas aeruginosa lung infections. Cultured human airway epithelial cells expressing the delta F508 allele of the cystic fibrosis transmembrane conductance regulator (CFTR) were defective in uptake of P. aeruginosa compared with cells expressing the wild-type allele. Pseudomonas aeruginosa lipopolysaccharide (LPS)-core oligosaccharide was identified as the bacterial ligand for epithelial cell ingestion; exogenous oligosaccharide inhibited bacterial ingestion in a neonatal mouse model, resulting in increased amounts of bacteria in the lungs. CFTR may contribute to a host-defense mechanism that is important for clearance of P. aeruginosa from the respiratory tract.

Broadly protective vaccine for Staphylococcus aureus based on an in vivo-expressed antigen.

Vaccines based on preferential expression of bacterial antigens during human infection have not been described. Staphylococcus aureus synthesized poly-N-succinyl beta-1-6 glucosamine (PNSG) as a surface polysaccharide during human and animal infection, but few strains expressed PNSG in vitro. All S. aureus strains examined carried genes for PNSG synthesis. Immunization protected mice against kidney infections and death from strains that produced little PNSG in vitro. Nonimmune infected animals made antibody to PNSG, but serial in vitro cultures of kidney isolates yielded mostly cells that did not produce PNSG. PNSG is a candidate for use in a vaccine to protect against S. aureus infection.

Safety and immunogenicity of high molecular weight polysaccharide vaccine from immunotype 1 Pseudomonas aeruginosa.

The safety and immunogenicity of a high molecular weight polysaccharide from immunotype 1 Pseudomonas aeruginosa were tested in a dose response fashion in adult volunteers. The vaccine lacked toxicity and pyrogenicity for experimental animals. Doses of 50, 75, 150, or 250 microgram were given to groups of individuals as a single dose subcutaneous injection. Doses of 150 and 250 microgram were associated with a significant rise in binding and opsonic antibody at 2 wk postimmunization. Titers remained unchanged for up to 6 mo. The vaccine was almost devoid of toxicity, eliciting no more than a slightly sore and tender arm at the site of injection. High molecular weight polysaccharide antigen appears to induce a good immune response following vaccination that is effective in mediating opsonophagocytic killing of live P. aeruginosa organisms.

Structural analysis and immunogenicity of Pseudomonas aeruginosa immunotype 2 high molecular weight polysaccharide.

We analyzed high molecular weight polysaccharide (PS) from the Fisher immunotype 2 (IT-2) strain of Pseudomonas aeruginosa for molecular composition and structure, then determined its immunogenicity in healthy adults. The PS was composed of 2-acetamido-2,6-dideoxygalactose (N-acetyl fucosamine) and glucose in a molar ratio of 2:1. Structural analysis by carbon-13 and proton nuclear magnetic resonance confirmed that the high molecular weight PS was structurally identical to that of the O-specific side chain of the lipopolysaccharide. PS differed from this material in molecular size. Immunization of 19 adult volunteers with doses of 50-100 micrograms of PS resulted in significant rises (P less than 0.04-P less than 0.0001) in binding antibody levels and killing antibody titers 2 and 4 wk postimmunization. The only reaction to the vaccine was localized tenderness at the immunization site. Analysis of the immunoglobulin isotype response to the vaccine showed a rise in specific serum IgG and IgA antibodies. Heterologous responses to other P. aeruginosa PS antigens were not seen. The antibody levels attained by vaccination were comparable with those in acute-phase serum samples of patients who survived sepsis with IT-2 P. aeruginosa and were significantly higher (P less than 0.03) than specific antibody levels in bacteremic patients who died. These results confirm that PS is a high molecular weight, immunogenic form of the P. aeruginosa IT-2 serotype antigen, eliciting levels of type-specific antibody comparable with those seen among patients surviving an episode of P. aeruginosa sepsis.

T lymphocyte-mediated protection against Pseudomonas aeruginosa infection in granulocytopenic mice.

BALB/c mice immunized with Pseudomonas aeruginosa immunotype 1 polysaccharide develop protective T cell immunity to bacterial challenge. In vitro, T cells from immunized mice kill P. aeruginosa by production of a bactericidal lymphokine. The present study demonstrates that adoptive transfer of T cells from immunized BALB/c mice to granulocytopenic mice resulted in 97% survival on challenge with P. aeruginosa, compared with 17% survival with adoptive transfer of T cells from nonimmune BALB/c mice. This protection is specifically elicited by reexposure to the original immunizing antigen; adoptive recipients cannot withstand challenge with immunotype 3 P. aeruginosa. However, the adoptive recipients do survive simultaneous infection with both P. aeruginosa immunotypes 1 and 3. Adoptive transfer of T cells from the congenic CB.20 mice, which are unable to kill P. aeruginosa in vitro, provides only 20% protection to granulocytopenic mice. These studies indicate that transfer of specific immune T lymphocytes can significantly enhance the resistance to P. aeruginosa infection in granulocytopenic mice.

Immune complexes from immunized mice and infected cystic fibrosis patients mediate murine and human T cell killing of hybridomas producing protective, opsonic antibody to Pseudomonas aeruginosa.

We examined the basis for the absence in cystic fibrosis (CF) patients of opsonic antibodies to the mucoid exopolysaccharide (MEP) antigen surrounding Pseudomonas aeruginosa that infect these patients. Opsonic antibodies to MEP are found in sera of the minority of CF patients that remain noncolonized into the second to fourth decades of life and protect rodents from chronic P. aeruginosa endobronchial infections. High titers of nonopsonic antibodies to MEP are found in P. aeruginosa-infected CF patients. Immunization of mice with doses of MEP that provoke only nonopsonic antibodies elicited CD3+, CD8+, T cell receptor alpha beta receptor+, major histocompatibility complex-unrestricted cytotoxic lymphocytes specific for hybridoma cells producing opsonic but not nonopsonic antibodies. Cytotoxicity was dependent on immune complexes on the surface of the T cells. Normal murine T cells could be activated by concanavalin A and sensitized with immune complexes for cytotoxic killing of hybridoma targets. CF patients infected with P. aeruginosa had serum immune complexes that sensitized concanavalin A-activated human T cells to kill murine hybridoma cells producing opsonic but not nonopsonic antibody. These results could explain the absence in infected CF patients of MEP-specific opsonins, an occurrence that accompanies the persistence of this infectious state.

Role of de-N-acetylase PgaB from Aggregatibacter actinomycetemcomitans in exopolysaccharide export in biofilm mode of growth.

Aggregatibacter actinomycetemcomitans, a Gram-negative bacterium, is the causative agent of localized aggressive periodontitis. Attachment to a biotic surface is a critical first step in the A. actinomycetemcomitans infection process for which exopolysaccharides have been shown to be essential. In addition, the pga operon, containing genes encoding for biosynthetic proteins for poly-N-acetyl glucosamine (PNAG), plays a key role in A. actinomycetemcomitans virulence, as a mutant strain lacking the pga operon induces significantly less bone resorption. Among the genes in the pga operon, pgaB codes for a de-N-acetylase that is responsible for the deacetylation of the PNAG exopolysaccharide. Here we report the role of PgaB in regulation of virulence genes using a markerless, scarless deletion mutant targeting the coding region of the N-terminal catalytic domain of PgaB. The results demonstrate that the N-terminal, catalytic domain of PgaB is crucial for exopolysaccharide export.

[The study of the reaction of terminated oligomerization in the synthesis of oligo-(beta1-6)-glucosamines].

The applicability of terminated oligomerization to the synthesis of oligo-(beta1-6)-glycosamines, fragments of the intercellular polysaccharide adhesin of staphylococci, was studied. The reactions of terminated oligomerization were carried out with mono-, di-, and trisaccharide monomers and N-protected aminopropanol; and spacered mono- and disaccharides as terminating molecules were also attempted. The primary formation of cyclic products of monomer intramolecular glycosylation was observed in almost all the reactions. Only the experiments with the monomer based on the disaccharide bromide under the conditions of the Helferich reaction led to reduced yields (30%) of the cyclic products. However, even in this case, the desired terminated oligosaccharides were generated in approximately 10% yield and mainly were the products of single glycosylation of the terminator by the monomer. These experiments allow the conclusion that, under the examined conditions, the reaction of terminated oligomerization could not result in the synthesis of oligoglucosamines with a high molecular mass.

Theranostic body fluid cleansing: rationally designed magnetic particles enable capturing and detection of bacterial pathogens.

We report on theoretical and experimental considerations on bacteria capturing and enrichment via magnetic separation enabling integrated diagnosis and treatment of blood stream infections. We show optimization of carrier-pathogen interactions based on a mathematical model followed by an experimental proof-of-concept study along with investigations on the process safety.

The role of the CFTR in susceptibility to Pseudomonas aeruginosa infections in cystic fibrosis.

Recent molecular and cellular studies have shed new light on the basis for the susceptibility of cystic fibrosis (CF) patients to Pseudomonas aeruginosa infection. Changes in airway liquid composition and/or viscosity, enhanced bacterial binding to mucin and epithelial cell receptors, increased innate inflammation owing to disruptions in lipid metabolism and a role for the CFTR protein in bacterial ingestion and clearance have all been postulated. The high P. aeruginosa infection rate in CF patients can potentially be explained by the specificity of the interaction between the CFTR and P. aeruginosa.

Establishment of Pseudomonas aeruginosa infection: lessons from a versatile opportunist.

Pseudomonas aeruginosa is an ubiquitous pathogen capable of infecting virtually all tissues. A large variety of virulence factors contribute to its importance in burn wounds, lung infection and eye infection. Prominent factors include pili, flagella, lipopolysaccharide, proteases, quorum sensing, exotoxin A and exoenzymes secreted by the type III secretion system.

Extended contact lens wear enhances Pseudomonas aeruginosa adherence to human corneal epithelium.

Extended wear of soft contact lenses is associated with an increased risk of Pseudomonas aeruginosa infection of the cornea. To assess the role of bacterial adherence in the pathogenesis of these infections, superficial corneal epithelial cells and leukocytes from ten patients who use extended-wear soft lenses and ten control eyes were compared for their propensity to attach P. aeruginosa in vitro. Cells were washed from the cornea by saline irrigation, incubated with a 10-ml solution containing 10(7) colony-forming units/ml of bacteria at 35 degrees C for 30 min, collected on a filter, and prepared using a modified acridine orange staining method. Fluorescence microscopy showed bacterial adherence to corneal epithelial cells, leukocytes, and ocular mucus. The mean number of bacteria adhering to epithelial cells was 2.6 for control eyes and 6.6 for the lens-wearing eyes (P = 0.002). The percentage of epithelial cells attaching greater than or equal to four bacteria was higher for lens-wearing eyes than control eyes (57.4% versus 26.0%, P = 0.0005). There was no significant difference between contact lens-wearing eyes and control eyes in the number of leukocytes collected or in the number of bacteria attached to these cells. These results show that P. aeruginosa adherence to epithelial cells is enhanced in those who use extended-wear soft contact lenses, and this may contribute to the increased incidence of P. aeruginosa keratitis for this population.

Prophylactic and therapeutic efficacy of immunoglobulin G antibodies to Pseudomonas aeruginosa lipopolysaccharide against murine experimental corneal infection.

PURPOSE: To evaluate the efficacy of lipopolysaccharide (LPS)-specific antibodies administered prophylactically or therapeutically to protect against corneal challenge with Pseudomonas aeruginosa. METHODS: The prophylactic efficacy of active immunization with purified P. aeruginosa LPS was evaluated in a murine corneal-scratch model of P. aeruginosa keratitis. The same model was used to evaluate both the prophylactic and the therapeutic efficacy of systemic passive transfer of variable region-identical, isotype-switched, LPS-specific, murine immunoglobulin M (IgM) and immunoglobulin G (IgG) monoclonal antibodies (mAbs). The mAbs were injected intraperitoneally at various times either before or after corneal challenge and the corneal response was monitored macroscopically. In addition, immune rabbit sera were used to evaluate the efficacy of treatment. RESULTS: Active immunization with homologous, but not heterologous, LPS before challenge reduced the severity of corneal disease and protected challenged mice against permanent corneal damage. Passive transfer of the LPS-specific IgM mAb 1F6 before challenge did not prevent corneal damage at any dose tested and had no effect on the course of disease. However, results of dose-response studies of the passive transfer of a variable region-identical IgG2b mAb, 2H3, before challenge indicated a 50% protective dose of 11.8 micrograms. When mAb 2H3 was administered at a dose of 50 micrograms before challenge and the challenge inoculum was increased, all mice were protected from corneal damage up to a challenge inoculum of 2.2 x 10(8) CFU/eye. When given 2 or 4 hours after corneal challenge with P. aeruginosa strain 6294 (which invades corneal epithelial cells during infection) but not when given at 8 or 24 hours, 50 micrograms of mAb 2H3 conferred significant protection (P < 0.05). The maximal interval after challenge during which this antibody could be administered and still protect 50% of mice was calculated by probit analysis to be 9.4 hours. Administration of homologous LPS-specific rabbit antiserum to mice at various times after challenge with P. aeruginosa strain 6206 (which is cytotoxic to corneal epithelial cells and does not remain in these cells during infection) resulted in significant protection when administered 4 or 8 hours after infection. Although probit analysis could not be performed with the available data, 50% of mice were completely protected when the antiserum was given up to 24 hours after challenge. CONCLUSIONS: In an experimental model of P. aeruginosa keratitis, systematically delivered IgG antibodies directed against the O-side-chain antigens of P. aeruginosa, LPS conferred protection against severe corneal damage when administered both prophylactically and therapeutically.

Lipopolysaccharide outer core is a ligand for corneal cell binding and ingestion of Pseudomonas aeruginosa.

PURPOSE: Pseudomonas aeruginosa has been observed to be adherent to and inside epithelial cells during experimental corneal infection. The authors identified bacterial ligands involved in adherence and entry of P. aeruginosa into corneal epithelial cells. METHODS: In vitro gentamicin survival assays were used to determine the intracellular survival of a panel of P. aeruginosa mutants. Strains (10(6) to 10(7) colony-forming units) were added to primary cultures of rabbit corneal epithelial cells (approximately 10(5)/well) for 3 hours, nonadherent bacteria were washed away, and extracellular bacteria were killed with gentamicin. The antibiotic was then washed away, and epithelial cells were lysed with 0.5% Triton X-100 to release internalized bacteria. Bacterial association (sum of bound and internalized bacteria) was measured by the omission of gentamicin. Similar assays were carried out with whole mouse eyes in situ. RESULTS: A lipopolysaccharide core with an exposed terminal glucose residue was found to be necessary for maximal association and entry of P. aeruginosa into corneal cells. Bacterial pili and flagella were not involved. Mutants of P. aeruginosa strains that do not produce an LPS core with a terminal glucose residue had a significantly lower level of association with (approximately 50%) and ingestion by ( > 90%, P < 0.01) corneal cells than did strains with this characteristic. Complementation of the LPS productions defect by plasmid-borne DNA returned association and ingestion to near parental levels. Lipopolysaccharides and delipidated oligosaccharides with a terminal glucose residue in the core inhibited bacterial association and entry into corneal cells. Experiments using P. aeruginosa LPS mutants and corneal cells on whole mouse eyes confirmed the role of the LPS core in cellular entry. CONCLUSIONS: Corneal epithelial cells bind and internalized P. aeruginosa by the exposed LPS core.

Vaccine potential of poly-1-6 beta-D-N-succinylglucosamine, an immunoprotective surface polysaccharide of Staphylococcus aureus and Staphylococcus epidermidis.

Staphylococcus aureus and S. epidermidis are among the most common causes of nosocomial infection, and S. aureus is also of major concern to human health due to its occurrence in community-acquired infections. These staphylococcal species are also major pathogens for domesticated animals. We have previously identified poly-N-succinyl beta-1-6 glucosamine (PNSG) as the chemical form of the S. epidermidis capsular polysaccharide/adhesin (PS/A) which mediates adherence of coagulase-negative staphylococci (CoNS) to biomaterials, serves as the capsule for strains of CoNS that express PS/A, and is a target for protective antibodies. We have recently found that PNSG is made by S. aureus as well, where it is an environmentally regulated, in vivo-expressed surface polysaccharide and similarly serves as a target for protective immunity. Only a minority of fresh human clinical isolates of S. aureus elaborate PNSG in vitro but most could be induced to do so under specific in vitro growth conditions. However, by immunofluorescence microscopy, S. aureus cells in infected human sputa and lung elaborated PNSG. The ica genes, previously shown to encode proteins in CoNS that synthesize PNSG, were found by PCR in all S. aureus strains examined, and immunogenic and protective PNSG could be isolated from S. aureus. Active and passive immunization of mice with PNSG protected them against metastatic kidney infections after intravenous inoculation with eight phenotypically PNSG-negative S. aureus. Isolates recovered from kidneys expressed PNSG, but expression was lost with in vitro culture. Strong antibody responses to PNSG were elicited in S. aureus infected mice, and a PNSG-capsule was observed by electron microscopy on isolates directly plated from infected kidneys. PNSG represents a previously unidentified surface polysaccharide of S. aureus that is elaborated during human and animal infection and is a prominent target for protective antibodies.

Structure of an antigenic teichoic acid shared by clinical isolates of Enterococcus faecalis and vancomycin-resistant Enterococcus faecium.

A shared antigenic teichoic acid, previously found to be a surface capsule-like polysaccharide, was isolated from clinical isolates of Enterococcus faecalis and vancomycin-resistant E. faecium. It was composed of glucose, glycerol, and phosphate as determined by chemical and GC-MS analysis. The repeating-unit structure was elucidated by a series of 1H, 13C, and 31P NMR spectroscopy to be the following: [formula: see text]

Protection from Staphylococcus aureus mastitis associated with poly-N-acetyl beta-1,6 glucosamine specific antibody production using biofilm-embedded bacteria.

Staphylococcus aureus vaccines based on bacterins surrounded by slime, surface polysaccharides coupled to protein carriers and polysaccharides embedded in liposomes administered together with non-biofilm bacterins confer protection against mastitis. However, it remains unknown whether protective antibodies are directed to slime-associated known exopolysaccharides and could be produced in the absence of bacterin immunizations. Here, a sheep mastitis vaccination study was carried out using bacterins, crude bacterial extracts or a purified exopolysaccharide from biofilm bacteria delivered in different vehicles. This polysaccharide reacted specifically with antibodies to poly-N-acetyl-beta-1,6-glucosamine (PNAG) and not with antibodies to other capsular antigens or bacterial components. Following intra-mammary challenge with biofilm-producing bacteria, antibody production against the polysaccharide, milk bacterial counts and mastitis lesions were determined. Bacterins from strong biofilm-producing bacteria triggered the highest production of antibodies to PNAG and conferred the highest protection against infection and mastitis, compared with weak biofilm-producing bacteria and non-cellular inocula. Thus, bacterins from strong biofilm bacteria, rather than purified polysaccharide, are proposed as a cost-efficient vaccination against S. aureus ruminant mastitis.