Correlation of Pseudomonas aeruginosa virulence factors from clinical and environmental isolates with pathogenicity in the neutropenic mouse.

The potential pathogenicity of a microorganism is a major concern for Health Canada evaluators, who will be processing new biotechnology products under the Canadian Environmental Protection Act. Potential pathogenicity is generally predicted by the results of animal pathogenicity studies. In an attempt to define surrogate data for an animal model, this study was initiated. Pseudomonas aeruginosa isolates from clinical and environmental sources were screened for their pilus type, serotype, lipopolysaccharide type, ability to evade host responses, and production of toxin A, exoenzyme S, elastase, phospholipase C, and total protease. The 50% lethal dose (LD50) of the same isolates was determined in the neutropenic mouse model of infection. An attempted correlation was drawn between each (or combinations) of the virulence determinants and the LD50. Stepwise linear regression showed that the presence of high levels of exoenzyme S in association with elastase or phospholipase C, or to a minor extent toxin A, was correlated with low numbers of bacteria required to elicit an LD50. No correlation between any of the other factors examined and virulence was detected. The data suggest that an in vitro high level of exoenzyme S production could be used as surrogate information for neutropenic mouse modelling; however, the levels of all of the extracellular enzymes should be considered when making such an assessment.

Pilin-based anti-Pseudomonas vaccines: latest developments and perspectives.

Among the several adhesins produced by Pseudomonas aeruginosa (Pa), the type-4 pilus promotes the majority of the adherence capability of the bacterium to epithelial cells and it is a major virulence factor in an AB.Y/SnJ mouse infection model. Vaccines targeting the disulfide loop (DSL) adherence binding domain of the pilin protein should therefore provide an effective protection against initial colonization and infection with Pa. To selectively elicit adherence blocking antibodies, the pilin DSL domain was chosen as peptide antigen for the construction of recombinant protein and live vaccines. While synthetic peptide-carrier protein conjugates provided some strain-specific protection, chimeric proteins with N- or C-terminally fused pilin DSL peptides did not engender protective IgG titers mice. Integral fusions of the pilin DSL peptide with the minor coat protein of filamentous phage or surface exposed regions of an outer membrane protein resulted in a display of the peptide on the surface of the phage particles and bacterial cells respectively. However, in immunization studies neither of these live vaccines were effective immunogens. The paracrystalline S-layer of Caulobacter crescentus combines several advantages of an effective antigen surface display system. Recombinant S-layer proteins with singlecopy insertions of a pilin peptide did not engender significant IgG titers, whereas multiple tandem insertions of the same peptide increased the serum IgG response in mice a thousand times. Multiple insertions of DSL peptides from different frequent pilin prototypes may be an interesting alternative for a recombinant cross-protective anti-Pseudomonas vaccine.

Anti-adhesin antibodies that recognize a receptor-binding motif (adhesintope) inhibit pilus/fimbrial-mediated adherence of Pseudomonas aeruginosa and Candida albicans to asialo-GM1 receptors and human buccal epithelial cell surface receptors.

Pseudomonas aeruginosa and Candida albicans were reported to adhere to the glycosphingolipid asialo-GM1 by means of pili and fimbriae, respectively. These diverse adhesins have been previously reported to have an immunologically conserved antigenic epitope and the role of this cross-reactive epitope in adherence to asialo-GM1 was investigated in this study. Both the unbiotinylated PAK pilus and fimbrial adhesins inhibited biotinylated pili from P. aeruginosa PAK and biotinylated C. albicans fimbriae binding to asialo-GM1 and receptors present on human buccal epithelial cells (BECs), which suggested that the same receptor sites were recognized by the two adhesins. Monoclonal antibodies PK99H and Fm16 raised against the P. aeruginosa PAK pili and C. albicans fimbriae, respectively, recognized a conserved epitope present on the two adhesins. Both Fm16 and PK99H blocked fimbriae binding to asialo-GM1 and BEC receptors and also inhibited P. aeruginosa and C. albicans whole cell binding to BECs. These data suggested that the conserved epitope confers receptor-binding properties to the adhesins, demonstrated that (i) asialo-GM1-like receptors present on epithelial cell surfaces are utilized by the pilus and fimbrial adhesins and (ii) the binding to these glycoreceptors is mediated by a conserved epitope that has receptor-binding properties.

Use of synthetic peptides to confirm that the Pseudomonas aeruginosa PAK pilus adhesin and the Candida albicans fimbrial adhesin possess a homologous receptor-binding domain.

Pseudomonas aeruginosa PAK pili and Candida albicans fimbriae are adhesins present on the microbial cell surfaces which mediate binding to epithelial cell-surface receptors. The receptor-binding domain (adhesintope) of the PAK pilus adhesin has been shown previously to reside in the carboxy-terminal disulphide-bonded region of P. aeruginosa pilin (PAK128-144). The delineation of the C. albicans fimbrial adhesintope was investigated in these studies using synthetic peptides which correspond to the whole (PAK128-144) or part of (PAK134-140) adhesintope of the PAK pilus and their respective anti-peptide antisera and biotinylated PAK pili (Bt-PAK pili), fimbriae (Bt-fimbriae), P. aeruginosa whole cells (Bt-P. aeruginosa) and C. albicans whole cells (Bt-C. albicans). The results from these studies confirmed that a structurally conserved motif akin to the PAK(128-144) peptide sequence is present in C. albicans fimbrial adhesin and that the seven-amino-acid residue PAK(134-140) sequence plays an important role in forming the adhesintope for both P. aeruginosa PAK pilus and C. albicans fimbrial adhesins.

Structure-function analysis of the adherence-binding domain on the pilin of Pseudomonas aeruginosa strains PAK and KB7.

The pili of Pseudomonas aeruginosa mediate bacterial binding to human epithelial cell surfaces. We have previously shown that a 17-residue synthetic peptide, KCTSDQDEQFIPKGCSK, corresponding to the C-terminal sequence of the PAK pilin protein (residues 128-144) contains the adherence binding domain. Another pilin strain, KB7, has been cloned and sequenced [Paranchych et al. (1990) in Pseudomonas Biotransformations, Pathogenesis and Evolving Biotechnology, pp 343-351, American Society for Microbiology, Washington, DC]. The C-terminal 17-residue sequence of the KB7 pilin is SCATTVDAKFRPNGCTD, which is semiconserved as compared to the PAK sequence. In this study, the interactions between the A549 human lung carcinoma cells and the two P. aeruginosa pilin strains were elucidated using a single alanine replacement analysis on the C-terminal 17-residue synthetic peptide of the pilins. The ability of these peptide analogs to inhibit the binding of the biotinylated PAK pili to A549 cells was assessed. Six PAK amino acid side chains (Ser131, Gln136, Ile138, Pro139, Gly141, and Lys144) and nine KB7 side chains (Ala130, Thr131, Thr132, Val133, Asp134, Ala135, Lys136, Arg138, and Pro139) were found to be important in mediating the pilus adhesin binding to A549 cells. In addition, a flexible peptide analog with both cysteine residues replaced by alanine failed to inhibit the binding of PAK pili to A549 cells. This suggests that the interactions between the pilin ligand and the A549 cell surface receptors are dependent on the conformation mediated by the disulfide bridge (Cys129 and Cys142). The residues considered to contribute to bacterial adherence are referred to as the "adhesintope". Four PAK and three KB7 side chains were located in a structurally more rigid region of the disulfide-bridged peptide as revealed by two-dimensional NMR studies [McInnes et al. (1993) Biochemistry 32, 13432-13440]. The structural aspects of the pilin-receptor interactions related to the mapped adhesintope sequences are discussed. The dissimilarities between the PAK and KB7 adhesintopes may suggest that compensatory mutations could occur among different pilin strains so as to allow the pilin adhesins to interact with the same receptor.

Studies on multiple Pseudomonas aeruginosa isolates from individual burn patients by RFLP, O antigen serotyping and antibiogram analysis.

Multiple isolates of Pseud. aeruginosa from individual burn patients were tested for antibiotic susceptibility-resistance patterns (antibiogram), O serotype lipopolysaccharide and chromosomal DNA restriction fragment length polymorphisms (RFLP) using a PAK pilin gene probe. Some patients were colonized by isolates identical by all three analytical procedures whereas other patients were found where multiple isolates were identical on the basis of serotype and antibiogram analysis, but different on the basis of RFLP analysis. Examples were found where multiple isolates from an individual patient appeared to be identical on the basis of serotyping and RFLP data, but different on the basis of antibiogram. Strains refractory to O serotyping could be characterized by RFLP type. These results indicate that RFLP analysis provides a valuable addition to routine serotyping and antibiogram studies on Pseud. aeruginosa isolates and that significant numbers of burn patients become co-colonized/co-infected with phenotypically diverse strains of this organism.

Studies on the binding of integration host factor (IHF) and TraM to the origin of transfer of the IncFV plasmid pED208.

The origin of transfer (oriT) of the IncFV plasmid pED208 contains a region with three binding sites for both the plasmid-encoded TraM protein and the integration host factor (IHF) of Escherichia coli, a sequence-specific DNA-binding protein. One region, containing overlapping TraM and IHF binding sites, could be interpreted as containing two binding sites for each protein. Using gel retardation assays, an affinity constant for IHF binding to the three main sites was estimated in the presence and absence of 0.1 M potassium glutamate, which increased the avidity of IHF binding to the weaker sites by two orders of magnitude. DNase I protection analyses and electron microscopy were used to determine the affinity of IHF for oriT-containing DNA in the presence and absence of TraM. The binding of IHF and TraM was found to be non-cooperative by the two techniques employed. Electron microscopy also demonstrated that IHF bent the oriT region in a manner consistent with its previously determined mode of action, while TraM had no discernible effect on the appearance of the DNA. This suggested that IHF and TraM interact with a 295 bp sequence in the oriT region and organize it into a higher order structure that may have a role in the initiation of DNA transfer and control of traM expression.

Development of an anti-adhesive vaccine for Pseudomonas aeruginosa targeting the C-terminal region of the pilin structural protein.

This study describes the development of passive and active vaccines directed at the Pseudomonas aeruginosa pilus adhesin. Passive immunization studies were carried out with P. aeruginosa strain K pilus-specific (PK3B, PK99H) and cross-reactive (PAK-13) monoclonal antibodies (MAbs). When A.BY/SnJ mice were passively immunized with a pilus-specific MAb (PK99H), which inhibited pilus-mediated adherence to respiratory epithelial cells, mice challenged with 5 x LD 50 of P. aeruginosa were completely protected while mice were not protected when animals were passively immunized with a pilus specific MAb (PK3B), which did not inhibit pilus adherence to epithilial cells. MAb PAK-13 was found to cross-react with the C-terminal portion of pili of different strains of P. aeruginosa. When mice were passively immunized with MAb PAK-13, subsequent challenge with KB7 (3 x LD50), PAO (8 x LD50) and PAK (3 x LD50) strains of P. aeruginosa resulted in a 70%, 60% and 90% protection of the mice, respectively. MAb PK99H has been previously shown to recognize a linear antigenic epitope consisting of the sequence DEQFIPK. This epitopic peptide was conjugated to protein carriers using different coupling strategies. Use of an appropriate adjuvant and the correct conjugation strategy were critical for raising high affinity antipeptide antisera. In a comparison of Freund's, alum, and Adjuvax, as adjuvants for a peptide-tetanus toxoid conjugate vaccine, highest titers for the synthetic peptide component of the conjugate were obtained with Adjuvax, while highest titers for the carrier protein components were obtained with Freund's. Of the four peptide-conjugates used in this study, only the C-terminal conjugated peptide failed to produce antibodies that bind to native antigen and did not protect mice in active immunization experiments (no survivors at 80 h in the mouse infection model). Conformationally restricted peptide conjugates in which the peptide was conjugated to the carrier at both ends provided better protection in mice challenged with lethal doses of P. aeruginosa than either N- or C-terminal linked peptide-conjugates. The pilus adhesin plays a critical role in P. aeruginosa pathogenesis and this is an excellent vaccine target for either active or passive immunization strategies.

Use of synthetic peptides in characterization of microbial adhesins.

The characterization of microbial adhesins is greatly facilitated by the use of synthetic peptides. Synthetic peptides can be used to identify specific antigenic epitopes, to delineate receptor-binding domain of adhesins, and to facilitate the characterization of the adhesin, and they allow for a direct examination of structure-binding relationships.

Adherence of Pseudomonas aeruginosa and Candida albicans to glycosphingolipid (Asialo-GM1) receptors is achieved by a conserved receptor-binding domain present on their adhesins.

Pseudomonas aeruginosa, a gram-negative bacterium, and Candida albicans, a dimorphic yeast, are evolutionarily distant microorganisms which can utilize filamentous structures termed pili and fimbriae, respectively, to mediate adherence to glycosphingolipids (asialoganglioside-GM1) receptors. The mechanism of adherence to glycosphingolipid receptors was investigated in these studies. By using monoclonal antibodies (MAbs) against purified pili of P. aeruginosa PAK (PK99H) and monospecific anti-peptide antibodies against the PAK pilin peptides [anti-PAK(128-144) and anti-PAK(134-140)], we demonstrated that these antibodies agglutinated C. albicans whole cells and cross-reacted with C. albicans fimbriae in immunoblots. A control MAb, PKL1, and anti-PAK(75-84) peptide antibodies failed to agglutinate C. albicans whole cells or cross-react with the fimbrial proteins. Conversely, the anti-C. albicans fimbrial MAb Fm16, but not Fm34, agglutinated P. aeruginosa PAK whole cells and Western blots (immunoblots). The interactions between PK99H and Fm16 and their respective homologous antigens were competitively inhibited by heterologous antigens; this demonstrated that the interactions between the antibodies and the heterologous antigens, i.e., PK99H with C. albicans fimbriae and Fm16 with P. aeruginosa pili, were highly specific and suggested that both adhesins share a common antigenic determinant. The immunological cross-reactivity between Fm16 and P. aeruginosa PAK pilin is localized onto the PAK(134-140) region as shown by a competitive enzyme-linked immunosorbent assay. The PAK(134-140) region of PAK pilin contains the epitope recognized by PK99H and also constitutes part of the receptor-binding domain of the pilus adhesin. Thus, the results from these studies suggest that common cell surface receptors are recognized by the P. aeruginosa and C. albicans adhesins because of a conserved receptor-binding domain on the adhesins.

Alteration of the pilin adhesin of Pseudomonas aeruginosa PAO results in normal pilus biogenesis but a loss of adherence to human pneumocyte cells and decreased virulence in mice.

The disulfide loop domain of Pseudomonas aeruginosa PAO pilin was altered by insertion of a chloramphenicol acetyltransferase gene into the pilin gene so that the C-terminal nine amino acids were replaced with 11 new amino acids. The altered pilin gene was transferred into wild-type PAO by recombination, where it did not affect normal piliation as observed by transmission electron microscopy or change of sensitivity to f116, PO4, B9, and Pf1 pilus-specific bacteriophages. However, the binding to human pneumocyte A549 cells was markedly reduced when tested in an in vitro binding assay (2 to 6 bacteria bound per A549 cell for the mutant bacteria compared with 50 bacteria per A549 cell for the wild-type bacteria). Additionally, when susceptible A.BY/SnJ mice were challenged with wild-type P. aeruginosa PAO and with P. aeruginosa PAO-MP (altered pilin gene), a 50% lethal dose of 3 x 10(6) bacteria per mouse was observed for PAO-MP compared with 7 x 10(4) bacteria per mouse for PAO. Approximately 90 of the adherence capability of P. aeruginosa PAO is seemingly attributable to the C-terminal disulfide loop adherence domain of pili. The pilus adherence function contributes significantly to the virulence of P. aeruginosa PAO in the A.BY/SnJ mouse infection model.

Partial characterization of a Candida albicans fimbrial adhesin.

Candida albicans is the primary etiologic agent of candidiasis, a disease that can vary from superficial mucosal lesions to life-threatening systemic or disseminated diseases. Strains of C. albicans have been reported to possess long, thin filamentous protein cell surface appendages termed fimbriae (R.B. Gardiner, M. Canton, and A. W. Day, Bot. Gaz. 143:534-541, 1982). These fimbriae were isolated, purified, and partially characterized. The major structural subunit of the fimbriae is a glycoprotein which consists of 80 to 85% carbohydrate (consisting primarily of D-mannose) and 10 to 15% protein. The molecular weight of the glycosylated fimbrial subunit is approximately 66,000, while unglycosylated protein has an approximate molecular weight of 8,644. The fimbriae function as adhesins mediating C. albicans binding to human buccal epithelial cells. Amino acid analysis of the purified fimbrial subunit indicates that the fimbrial subunit is composed of 50% hydrophobic amino acid residues. The N terminus of the fimbrial subunit is blocked to N-terminal sequencing.

Fimbria-mediated adherence of Candida albicans to glycosphingolipid receptors on human buccal epithelial cells.

Candida albicans is an opportunist fungal pathogen that has the ability to adhere to host cell surface receptors via a number of adhesins. Yu et al. (L. Yu, K. K. Lee, K. Ens, P. C. Doig, M. R. Carpenter, W. Staddon, R. S. Hodges, W. Paranchych, and R. T. Irvin, Infect. Immun. 62:2834-2842, 1994) described the purification and initial characterization of a fimbrial adhesin from C. albicans. In this paper, we show that C. albicans fimbriae also bind to asialo-GM1 [gangliotetraosylceramide: beta Gal(1-3)beta GalNAc(1-4) beta Gal(1-4)beta Glc(1-1)Cer] immobilized on microtiter plates in a saturable and concentration-dependent manner. C. albicans fimbrial binding to exfoliated human buccal epithelial cells (BECs) was inhibited by asialo-GM1 in in vitro binding assays. The fimbriae interact with the glycosphingolipid receptors via the carbohydrate portion of the receptors, since fimbriae were observed to bind to synthetic beta GalNAc(1-4)beta Gal-protein conjugates and the disaccharide was able to inhibit binding of fimbriae to BECs in in vitro binding assays. We conclude from these results that the C. albicans yeast form expresses a fimbrial adhesin that binds to glycosphingolipids displayed on the surface of human BECs.

The binding of Pseudomonas aeruginosa pili to glycosphingolipids is a tip-associated event involving the C-terminal region of the structural pilin subunit.

Pili are one of the adhesins of Pseudomonas aeruginosa that mediate adherence to epithelial cell-surface receptors. The pili of P. aeruginosa strains PAK and PAO were examined and found to bind gangliotetraosyl ceramide (asialo-GM1) and, to a lesser extend, II3N-acetylneuraminosylgangliotetraosyl ceramide (GM1) in solid-phase binding assays. Asialo-GM1, but not GM1, inhibited both PAK and PAO pili binding to immobilized asialo-GM1 on the microtitre plate. PAO pili competitively inhibited PAK pili binding to asialo-GM1, suggesting the presence of a structurally similar receptor-binding domain in both pilus types. The interaction between asialo-GM1 and pili occurs at the pilus tip as asialo-GM1 coated colloidal gold only decorates the tip of purified pili. Three sets of evidence suggest that the C-terminal disulphide-bonded region of the Pseudomonas pilin is exposed at the tip of the pilus: (i) immunocytochemical studies indicate that P. aeruginosa pili have a basal-tip structural differentiation where the monoclonal antibody (mAb) PK3B recognizes an antigenic epitope displayed only on the basal ends of pili (produced by shearing) while the mAb PK99H, whose antigenic epitope resides in residues 134-140 (Wong et al., 1992), binds only to the tip of PAK pili; (ii) synthetic peptides, PAK(128-144)ox-OH and PAO(128-144)ox-OH, which correspond to the C-terminal disulphide-bonded region of Pseudomonas pilin are able to bind to asialo-GM1 and inhibit the binding of pili to the glycolipid; (iii) PK99H was shown to block PAK pilus binding to asialo-GM1.(ABSTRACT TRUNCATED AT 250 WORDS)

The pili of Pseudomonas aeruginosa strains PAK and PAO bind specifically to the carbohydrate sequence beta GalNAc(1-4)beta Gal found in glycosphingolipids asialo-GM1 and asialo-GM2.

Pseudomonas aeruginosa employs pili to mediate adherence to epithelial cell surfaces. The pilus adhesin of P. aeruginosa strains PAK and PAO has been shown to bind to the glycolipid asialo-GM1 (Lee et al., 1994--accompanying article). PAK and PAO pili were examined for their abilities to bind to the synthetic beta GalNAc(1-4)beta Gal (a minimal structural carbohydrate receptor sequence of asialo-GM1 and asialo-GM2 proposed by Krivan et al., 1988a) using solid-phase binding assays. Both pili specifically bound to beta GalNAc(1-4)beta Gal. The binding of beta GalNAc(1-4)beta Gal-Biotin to the immobilized PAK and PAO pili was inhibited by corresponding free pili. The receptor binding domain of the PAK pilus resides in the C-terminal disulphide-looped region (residues 128-144) of the pilin structural subunit (Irvin et al., 1989). Biotinylated synthetic peptides corresponding the C-terminal residues 128-144 of P. aeruginosa PAK and PAO pilin molecules were shown to bind to the beta GalNAc(1-4)beta Gal-(bovine serum albumin (BSA)). The binding of biotinylated peptides to beta GalNAc(1-4)beta GAL-BSA was inhibited by PAK pili, Ac-KCTSDQDEQFIPKGCSK-OH (AcPAK(128-144)ox-OH) and Ac-ACKSTQDPMFTPKGCDN-OH (AcPAO(128-144)ox-OH) peptides. (In these peptides Ac denotes N alpha-acetylation of the N-terminus, -OH means a peptide with a free alpha-carboxyl group at the C-terminus and the 'ox' denotes the oxidation of the sulphhydryl groups of Cys-129 and Cys-142.) Both acetylated peptides were also able to inhibit the binding of beta GalNAc(1-4)beta Gal-biotin to the corresponding BSA-Peptide(128-144)ox-OH conjugates.(ABSTRACT TRUNCATED AT 250 WORDS)

Immune recognition of polar pili from Pseudomonas aeruginosa O.

The B- and T-cell antigenic sites on type O pili from Pseudomonas aeruginosa were determined by using an antipilus antibody competition assay and PAO-immune T-cell blasts in proliferation studies. The citraconylated tryptic digest III region (residues 54 to 120) was determined to be an immunodominant site for both T and B cells on the pilin molecule.

Mutations in the fifth-position glutamate in Pseudomonas aeruginosa pilin affect the transmethylation of the N-terminal phenylalanine.

The pili of Pseudomonas aeruginosa are composed of 15-kDa pilin monomers that are synthesized in the cytoplasm and assembled in the membrane. Processing occurs between the synthesis and assembly steps. The propilin is cleaved by a unique leader peptidase encoded by pilD, which is adjacent to the pilin structural gene pilA. This generates an N-terminal phenylalanine that is subsequently methylated by an as yet uncharacterized transmethylase. The pili of P. aeruginosa belong to the type IV class of pilins, which share a highly conserved N-terminal region 35 amino acids in length, containing a short leader of 6 or 7 amino acids. Two site-specific mutants in the N-terminal region of the mature pilin were constructed. Reestablishing the fifth-position glutamate in a four amino acid deletion mutant (amino acids 4-7) restored the leader peptidase cleavage but not the methylation. A mutation of the fifth-position glutamate to alanine decreased the degree of methylation of the N-terminal phenylalanine. Pili were not assembled by these mutants as assessed by electron microscopy and sensitivity to pilus-specific bacteriophage. Methylation may be required for recognition of the pilin by the assembly machinery and is not residue specific. The fifth-position glutamate appears to play an important role in transmethylase recognition of the pilin subunit.

Localization of the virulence-associated genes pilA, pilR, rpoN, fliA, fliC, ent, and fbp on the physical map of Pseudomonas aeruginosa PAO1 by pulsed-field electrophoresis.

Seven virulence-associated genes have been placed on a genomic map of Pseudomonas aeruginosa PAO1, using pulsed-field electrophoresis, on the basis of the previous physical maps of Romling et al. (U. Romling, M. Duchene, D. Essar, D. Galloway, C. Guidi-Rontani, D. Hill, A. Lazdunski, R. Miller, K. Schleifer, D. Smith, H. Toschka, and B. Tummler, J. Bacteriol. 174:327-330, 1992; U. Romling, D. Grothues, W. Bautsch, and B. Tummler, EMBO J. 8:4081-4089, 1989) and Ratnaningsih et al. (E. Ratnaningsih, S. Dharmsthiti, V. Krishnapillai, A. Morgan, M. Sinclair, and B. W. Holloway, J. Gen. Microbiol. 136:2351-2357, 1990). The new locations for the outer membrane enterobactin iron-siderophore receptor ent gene (41 to 42 min) and the fliA gene (59 to 61 min), which encodes a minor sigma factor of RNA polymerase, are given. The pilA (the pilin structural gene), pilR (a pilin regulatory gene), and rpoN (encoding another minor sigma factor of RNA polymerase) genes map together at 71 to 75 min, locations correcting the previously reported values (V. Shortridge, M. Pato, A. Vasil, and M. Vasil, Infect. Immun. 59:3596-3603, 1990). The fbp gene (28 to 29 min), which encodes an outer membrane ferripyochelin-binding protein of low molecular weight, and the fliC gene (64 to 66 min), the flagellin structural gene, were determined to lie in the previously reported locations.

Low-molecular-mass human salivary mucin, MG2: structure and binding of Pseudomonas aeruginosa.

Low-molecular-mass human salivary mucin, MG2, was isolated from human submandibular-sublingual saliva (HSMSL) employing citraconylation, gel filtration, and ion-exchange chromatography. Following proteolysis with trypsin, two glycopeptides were purified. The higher molecular weight glycopeptide was highly glycosylated with O-linked units. The lower molecular weight glycopeptide was less glycosylated and contained most of the N-linked units. Interaction between components of HSMSL and pili of Pseudomonas aeruginosa was examined by an overlay binding assay. Pili were found to bind to MG2. Preliminary studies indicated that the binding may involve a protein to protein interaction.

Epidemiology of infections with Pseudomonas aeruginosa in burn patients: the role of hydrotherapy.

Pseudomonas aeruginosa remains a cause of serious wound infection and mortality in burn patients. By means of restriction fragment length polymorphism analysis and a DNA probe for the pilin gene of Pseudomonas, a lethal strain of nosocomial P. aeruginosa was identified as the cause of an outbreak of wound infections among burn patients. Environmental surveys suggested an association of the outbreak with hydrotherapy provided to many patients in a common facility. In a trial of burn wound care without hydrotherapy, overall mortality was reduced significantly, mortality associated with pseudomonas sepsis was eliminated, and the strain of P. aeruginosa associated with earlier mortality was eradicated. Moreover, fewer nosocomial pseudomonas infections, lower levels of pseudomonas resistance to aminoglycoside antibiotics, significantly fewer pseudomonas infections of skin graft donor sites, and later appearance of Pseodomonas species in burn patients were found during the period when hydrotherapy was not used.