Pseudomonas aeruginosa bacteriophage phi PLS27-lipopolysaccharide interactions

We investigated the phi PLS27 receptor in Pseudomonas aeruginosa strain PAO lipopolysaccharide (LPS) by analyzing a resistant mutant. This mutant, which was designated AK1282, had the most defective LPS yet reported for a P. aeruginosa rough mutant; this LPS contained only lipid A, 2-keto-3-deoxyoctonate, heptose, and alanine as major components. In addition, this LPS lacked galactosamine, which is present in the inner core of the LPS of other rough mutants. The loss of galactosamine but only a small decrease in the alanine content indicated that the core of strain PAO LPS differed from the core structure which has been suggested for the LPS of other well-characterized P. aeruginosa strains. Our analysis also indicated that galactosamine residues may be crucial for phi PLS27 receptor activity of the LPS. Electrodialysis of LPS and conversion to salt forms (sodium or triethylamine) influenced the phage-inactivating capacity of the LPS, as did the medium in which the inactivation occurred; experiments performed in 1/10-strength broth resulted in much lower PhI50 (concentration of LPS causing a 50% decrease in the titer of phage during 1 h of incubation at 37 degrees C) values than experiments performed in regular-strength broth. Sonication of the LPS also increased the phage-inactivating capacities of the LPS preparations.     

Pseudomonas aeruginosa lipopolysaccharide: evidence that the O side chains and common antigens are on the same molecule.

We investigated whether Pseudomonas aeruginosa produces two distinct lipopolysaccharides (LPS) containing either serologically variable O side chains or a neutral polysaccharide common antigen, designated A bands, that reacts with monoclonal antibody (MAb) E87. Immunoprecipitation of LPS and free O side chains with O-side-chain-specific antibodies or MAb E87 resulted in coprecipitation of both polysaccharides when antibody of either specificity was employed. Chromatography of LPS and free O side chains in a disaggregating deoxycholate buffer indicated the two polysaccharide antigens cochromatograph when eluates were analyzed by sensitive and specific enzyme-linked immunosorbent assay inhibitions. The LPS from a mutant of strain PAO1 that lacks polymerized O side chains but retains the common antigen eluted in fractions containing smaller LPS molecules, indicating the necessity of polymerized O side chains for elution in early fractions containing large LPS monomers. A phosphomannomutase mutant of P. aeruginosa PAO1 makes a rough LPS lacking both O side chains and common antigen but still produces a small (< 6-kDa) common antigen component detectable in cell lysates. Introduction of the cloned pmm gene into this strain restored production of a smooth LPS expressing large MAb E87-reactive common antigen. Destruction with NaOH of O side chains on recombinant LPS molecules eluting early from the molecular sieve column resulted in a shift of the MAb E87-reactive antigen to the late-eluting fractions. These results indicate that on most P. aeruginosa LPS molecules, O side chains and neutral polysaccharide common antigens are both present.     

Isolation and characterization of a bacteriophage specific for the lipopolysaccharide of rough derivatives of Pseudomonas aeruginosa strain PAO

A lipopolysaccharide (LPS)-defective (rough) mutant of Pseudomonas aeruginosa PAO was isolated by selection for resistance to the LPS-specific phage E79. The LPS of this mutant, AK-1012, lacked the O-antigenic side chain-specific amino sugar fucosamine as well as the core-specific sugars glucose and rhamnose. Using this strain, we isolated and characterized a phage, phi PLS27, which is specifically inactivated upon incubation with LPS extracted from rough mutants of P. aeruginosa PAO. phi PLS27 was found to be a Bradley type C phage and was very similar to coliphage T7 in a number of properties, including size, buoyant density, mass, and the number of structural proteins.     

Structural characterization of the outer core and the O-chain linkage region of lipopolysaccharide from Pseudomonas aeruginosa serotype O5.

The point of attachment of the O-chain in the outer core region of Pseudomonas aeruginosa serotype O5 lipopolysaccharide (LPS) was determined following a detailed analysis of the extended core oligosaccharide, containing one trisaccharide O-chain repeating unit, present in both the wild-type strain PAO1 and O-chain deficient mutant strains AK1401 and PAO-rfc. The structure of the extended core oligosaccharide was determined by various mass spectrometric methods as well as one-dimensional and two-dimensional NMR spectroscopy. Furthermore, the one-dimensional analogues of NOESY and TOCSY experiments were applied to confirm the structure of the outer core region in the O-chain polysaccharide. In both the extended core oligosaccharide and the core of the smooth LPS, a loss of one of the beta-glucosyl residues and the translocation of the alpha-rhamnosyl residue, followed by the attachment of the first O-chain repeating unit was observed. This process is complicated and could involve two distinct rhamnosyltransferases, one with alpha-1, 6-linkage specificity and another with alpha-1,3-linkage specificity. It is also plausible that an alpha-1,3 rhamnosyltransferase facilitates the addition of the 'new' alpha-rhamnosyl residue that will act as a receptor for the attachment of the single O-antigen repeating unit in the LPS of the semi-rough mutant. The 2-amino-2-deoxy-fucosyl residue of the first O-chain repeating unit directly attached to the core was found to have a beta-anomeric configuration instead of an alpha configuration, characteristic for this residue as a component of the O-chain polysaccharide. The results of this study provide the first example of the mechanistic implications of the structure of the outer core region in a fully assembled O-chain containing LPS, differing from the O-chain deficient rough LPS.     

Evidence that WapB is a 1,2-glucosyltransferase of Pseudomonas aeruginosa involved in Lipopolysaccharide outer core biosynthesis

Pseudomonas aeruginosa is an important opportunistic pathogen infecting debilitated individuals. One of the major virulence factors expressed by P. aeruginosa is lipopolysaccharide (LPS), which is composed of lipid A, core oligosaccharide (OS), and O-antigen polysaccharide. The core OS is divided into inner and outer regions. Although the structure of the outer core OS has been elucidated, the functions and mechanisms of the glycosyltransferases involved in core OS biogenesis are currently unknown. Here, we show that a previously uncharacterized gene, pa1014, is involved in outer core biosynthesis, and we propose to rename this gene wapB. We constructed a chromosomal mutant, wapB::Gm, in a PAO1 (O5 serotype) strain background. Characterization of the LPS from the mutant by Western immunoblotting showed a lack of reactivity to PAO1 outer core-specific monoclonal antibody (MAb) 5c-101. The chemical structure of the core OS of the wapB mutant was elucidated using nuclear magnetic resonance spectroscopy and mass spectrometry techniques and revealed that the core OS of the wapB mutant lacked the terminal β-1,2-linked-d-glucose residue. Complementation of the mutant with wapB in trans restored the core structure to one that is identical to that of the wild type. Eleven of the 20 P. aeruginosa International Antigenic Typing Scheme (IATS) serotypes produce LPSs that lack the terminal d-glucose residue (Glc(IV)). Interestingly, expressing wapB in each of these 11 serotypes modifies each of their outer core OS structures, which became reactive to MAb 5c-101 in Western immunoblotting, suggesting the presence of a terminal d-glucose in these core OS structures. Our results strongly suggested that wapB encodes a 1,2-glucosyltransferase.     

Characterization of polyagglutinating and surface antigens in Pseudomonas aeruginosa

Mutants of Pseudomonas aeruginosa PAC1R (serotype O:3) which were resistant to bacteriophage D were isolated and shown to react with O:5d, O:9 and O:13 antisera as well as O:3. Antisera to the parent strain and to the three polyagglutinating (PA) mutants also showed cross-reactions. The mutants differed from the parent strain in their lipopolysaccharide (LPS) composition. The LPS from two of the three mutants yielded high molecular weight polysaccharide fractions. Although the high molecular weight fraction from one of the mutants contained the amino sugars and other components characteristic of the O:3 serotype strains, its mobility on Sephadex G75 was different from that of the parent strain. The high molecular weight material from the second mutant lacked the O-antigenic determinants but these were present in a semi-rough LPS fraction. The third mutant appeared rough and completely lacked the O-antigenic components. These three mutants were compared with the parent strain and with a non-agglutinating LPS-defective mutant which lacked both O-antigenic side chains and all neutral sugars in the outer core. Agglutination with absorbed sera and haemagglutination using purified LPS and ELISA detection suggested that wall components other than LPS were responsible for some of the cross-reactions observed. The components responsible were detected after SDS-PAGE of crude outer membrane fractions by a combination of Coomassie blue and silver-staining and antigenic components were detected by immunoelectrophoresis and ELISA-linked immunoblotting of the gels. The main antigenic determinants detected by antiserum to the parent strain were in the high molecular weight O-polysaccharide fractions and in the semirough fractions of the LPS, with some activity due to the H protein of the outer membrane. O:5d antisera reacted with unidentified high molecular weight polysaccharide fractions. Cross-reactions with the O:9 antiserum appeared to be due mainly to the F porin and, to a lesser extent, to the G and E proteins of the outer membrane. O:13 antiserum reacted with high molecular weight polysaccharide fractions but also with the rough core and F and H protein. Cross-reactivity of the other three mutant antisera could largely be interpreted in terms of the outer membrane components exposed in each strain. One reacted strongly with the F porin and the rough core, while the others reacted with a number of protein and LPS-derived fractions.(ABSTRACT TRUNCATED AT 400 WORDS)     

Identification of the cell wall receptor for bacteriophage E79 in Pseudomonas aeruginosa strain PAO.

Bacteriophage E79 was shown to interact with the lipopolysaccharide (LPS) of Pseudomonas aeruginosa strain PAO. LPS isolated from an E79-sensitive, smooth strain inactivated the phage, exhibiting a Phl50 value (concentration of LPS that caused a 50% decrease in the titer of phage during 1 h of incubation at 37 degrees C) of 0.04 microgram/ml, whereas the LPS isolated from a rough mutant derived from the wild type showed no neutralizing activity towards E79. EDTA and sodium deoxycholate were demonstrated to abolish the neutralizing capacity of the smooth LPS. One E79 receptor site was shown to be equivalent to 10(-16) g of LPS.     

Molecular cloning of genes involved with expression of A-band lipopolysaccharide, an antigenically conserved form, in Pseudomonas aeruginosa.

Most strains of Pseudomonas aeruginosa can express two chemically and immunologically distinct types of lipopolysaccharide (LPS), an antigenically conserved form called A band and the serotype-specific form called B band. To study the molecular controls regulating expression of the A-band LPS antigen, we have cloned the genes involved with A-band LPS expression. Strain AK1401, a phage-resistant mutant of PAO1 which was shown previously to produce only A-band LPS and not the O-antigen-containing B-band LPS, was mutagenized by using ethyl methanesulfonate to generate an A-band-deficient mutant called rd7513. A cosmid clone bank of P. aeruginosa PAO1 whole genomic DNA was constructed in Escherichia coli. The gene bank was mobilized en masse into strain rd7513, and detection of complementation of synthesis of A band was done by screening transconjugants in a colony immunoblot assay with the A-band-specific monoclonal antibody N1F10. One recombinant cosmid, pFV3, complemented synthesis of A-band polysaccharide in rd7513. Silver-stained polyacrylamide gel and Western immunoblot analyses of LPS extracted from the transconjugant rd7513(pFV3) showed that the A band produced had a higher molecular weight than the A band of AK1401. Analysis of the plasmid pFV3 showed that it contained a chromosomal insert of 27 kb. Two subclones of pFV3, namely, pFV35 and pFV36, containing chromosomal inserts of 5.3 and 4.2 kb, respectively, also complemented A-band expression in rd7513. The LPS banding profile of rd7513(pFV35) was similar to that of AK1401, while the LPS profile of rd7513(pFV36) more closely resembled that of rd7513(pFV3). pFV3 complemented A-band expression in five of the six P. aeruginosa O serotypes which lack A band as well as in rough strain AK44 but failed to complement A-band expression in core mutants AK1012 and AK1282, suggesting that pFV3 contains genes for A-band expression and that synthesis of a complete core region in isogenic mutant strains is required for A-band synthesis.     

The Pseudomonas aeruginosa algC gene encodes phosphoglucomutase, required for the synthesis of a complete lipopolysaccharide core.

We have constructed strains of Pseudomonas aeruginosa with mutations in the algC gene, previously shown to encode the enzyme phosphomannomutase. The algC mutants of a serotype O5 strain (PAO1) and a serotype O3 strain (PAC1R) did not express lipopolysaccharide (LPS) O side chains or the A-band (common antigen) polysaccharide. The migration of LPS from the algC mutant strains in Tricine-sodium dodecyl sulfate-polyacrylamide gels was similar to that of LPS from a PAO1 LPS-rough mutant, strain AK1012, and from a PAC1R LPS-rough mutant, PAC605, each previously shown to be deficient in the incorporation of glucose onto the LPS core (K. F. Jarrell and A. M. Kropinski, J. Virol. 40:411-420, 1981, and P. S. N. Rowe and P. M. Meadow, Eur. J. Biochem. 132:329-337, 1983). We show that, as expected, the algC mutant strains had no detectable phosphomannomutase activity and that neither algC strain had detectable phosphoglucomutase (PGM) activity. To confirm that the PGM activity was encoded by the algC gene, we transferred the cloned, intact P. aeruginosa algC gene to a pgm mutant of Escherichia coli and observed complementation of the pgm phenotype. Our finding that the algC gene product has PGM activity and that strains with mutations in this gene produce a truncated LPS core suggests that the synthesis of glucose 1-phosphate is necessary in the biosynthesis of the P. aeruginosa LPS core. The data presented here thus demonstrate that the algC gene is required for the synthesis of a complete LPS core in two strains with different LPS core and O side chain structures.     

Lipopolysaccharide-Defective Mutants of the Wilt Pathogen Pseudomonas solanacearum

Lipopolysaccharide (LPS)-defective mutants of Pseudomonas solanacearum were used to test the hypothesis that differences in LPS structure are associated with the ability or inability of different strains to induce a hypersensitive response (HR) in tobacco. To obtain these mutants, LPS-specific bacteriophage of P. solanacearum were isolated and used to select phage-resistant mutants of the virulent, non-HR-inducing strain K60. The LPS of 24 of these mutants was purified and compared with that of K60 and its HR-inducing variant, B1. Upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis, LPS from K60 and other smooth strains separated into many evenly spaced bands that migrated slowly, whereas LPS from B1 and most phage-resistant strains separated into one to three bands that migrated rapidly. Carbohydrate analysis showed that the LPS of the phage-resistant strains lacked O-antigen sugars (rhamnose, xylose, and N-acetylglucosamine) and could be grouped into (i) those that had all core sugars (rhamnose, glucose, heptose, and 2-keto-3-deoxyoctonate), (ii) those that had no core rhamnose, and (iii) those that lacked all core sugars except for 2-keto-3-deoxyoctonate. The LPS composition of 10 of the rough, phage-resistant mutants was similar to that of the HR-inducing strain, B1, yet none of them induced the HR. Only 2 of 13 mutant strains tested caused wilting of tobacco, and these had rough LPS but produced large amounts of extracellular polysaccharide, unlike most LPS-defective mutants. The evidence did not support the hypothesis that the initial interaction between rough LPS and tobacco cell walls is the determining factor in HR initiation.     

Structure of a highly phosphorylated lipopolysaccharide core in the Delta algC mutants derived from Pseudomonas aeruginosa wild-type strains PAO1 (serogroup O5) and PAC1R (serogroup O3)

Lipopolysaccharides (LPS) were isolated from rough-type mutant strains of Pseudomonas aeruginosa (Delta algC) derived from wild-type strains PAO1 (serogroup O5) and PAC1R (serogroup O3). Structural studies of the LPS core region with a special focus on the phosphorylation pattern were performed by 2D NMR spectroscopy, including a 1H,(31)P HMQC-TOCSY experiment, MALDI-TOF MS, and Fourier-transform ion cyclotron resonance ESIMS using the capillary skimmer dissociation technique. Both LPS were found to contain two residues each of 3-deoxy-D-manno-oct-2-ulosonic acid (Kdo) and L-glycero-D-manno-heptose (Hep), one residue of N-(L-alanyl)-D-galactosamine and one O-carbamoyl group (Cm) on the distal Hep residue. The following structures of a tetrasaccharide trisphosphate from strain PAC1R Delta algC and that carrying an additional ethanolamine phosphate group (PEtN) from strain PAO1 Delta algC were elucidated: [carbohydrate structre: see text] where R=P in PAC1R Delta algC and PPEtN in PAO1 Delta algC. To our knowledge, in this work the presence of ethanolamine diphosphate is unambiguously confirmed and its position established for the first time in the LPS core of a rough-type strain of P. aeruginosa. In addition, the structure of the complete LPS core of wild-type strain P. aeruginosa PAO1 was reinvestigated and the position of the phosphorylation sites was revised.     

The isolation and characterization of lipopolysaccharide-defective mutants of Pseudomonas aeruginosa PAC1.

Mutants with defective lipopolysaccharides (LPSs) were isolated from Pseudomonas aeruginosa PACIR (Habs serogroup 3) by selection for resistance to aeruginocin from P. aeruginosa PI6 Carbenicillin-sensitive mutants were isolated from P. aeruginosa PACI but not all had defective LPSs. Rough colonial morphology and resistance to bacteriophage II9X appeared to be independent of LPS composition. The LPSs from five mutants were analysed and compared with that of the parent strain. Separation of partially-degraded polysaccharides from LPS from PACI on Sephadex G75 yielded two different high molecular weight fractions and a phosphorylated low molecular weight fraction (L). The mutant LPSs lacked most or all of the high molecular weight fractions but retained some low molecular weight material. That from PACI and two of the mutants was separated by elution from Biogel P6 into two fractions. One, L2, was the core polysaccharide while the other, LI, contained short antigenic side-chains attached to the core like the semi-rough (SR) LPSs of the Enterobacteriaceae. The two mutants which gave the LI fraction with Habs 3 and PACI antisera as did the parent strain. The other three mutants were unreactive and their LPSs contained core components only. One appeared to have a complete core while the other two lacked rhamnose and rhammose plus glucose respectively. Thus there may be four types of LPS in PACI: one contains unsubstituted core polysaccharide and yields L2 on acid hydrolysis, another has short antigenic side-chains of the SR type and yields the LI fraction, while the two high molecular weight fractions are derived from core polysaccharides with different side-chains.     

Functional characterization of MigA and WapR: putative rhamnosyltransferases involved in outer core oligosaccharide biosynthesis of Pseudomonas aeruginosa

Pseudomonas aeruginosa lipopolysaccharide (LPS) contains two glycoforms of core oligosaccharide (OS); one form is capped with O antigen through an alpha-1,3-linked L-rhamnose (L-Rha), while the other is uncapped and contains an alpha-1,6-linked L-Rha. Two genes in strain PAO1, wapR (PA5000) and migA (PA0705), encode putative glycosyltransferases associated with core biosynthesis. We propose that WapR and MigA are the rhamnosyltransferases responsible for the two linkages of L-Rha to the core. Knockout mutants with mutations in both genes were generated. The wapR mutant produced LPS lacking O antigen, and addition of wapR in trans complemented this defect. The migA mutant produced LPS with a truncated outer core and showed no reactivity to outer core-specific monoclonal antibody (MAb) 5C101. Complementation of this mutant with migA restored reactivity of the LPS to MAb 5C101. Interestingly, LPS from the complemented migA strain was not reactive to MAb 18-19 (specific for the core-plus-one O repeat). This was due to overexpression of MigA in the complemented strain that caused an increase in the proportion of the uncapped core OS, thereby decreasing the amount of the core-plus-one O repeat, indicating that MigA has a regulatory role. The structures of LPS from both mutants were elucidated using nuclear magnetic resonance spectroscopy and mass spectrometry. The capped core of the wapR mutant was found to be truncated and lacked alpha-1,3-L-Rha. In contrast, uncapped core OS from the migA mutant lacked alpha-1,6-L-Rha. These results provide evidence that WapR is the alpha-1,3-rhamnosyltransferase, while MigA is the alpha-1,6-rhamnosyltransferase.     

Functional characterization of WaaL, a ligase associated with linking O-antigen polysaccharide to the core of Pseudomonas aeruginosa lipopolysaccharide

The O antigen of Pseudomonas aeruginosa B-band lipopolysaccharide is synthesized by assembling O-antigen-repeat units at the cytoplasmic face of the inner membrane by nonprocessive glycosyltransferases, followed by polymerization on the periplasmic face. The completed chains are covalently attached to lipid A core by the O-antigen ligase, WaaL. In P. aeruginosa the process of ligating these O-antigen molecules to lipid A core is not clearly defined, and an O-antigen ligase has not been identified until this study. Using the sequence of waaL from Salmonella enterica as a template in a BLAST search, a putative waaL gene was identified in the P. aeruginosa genome. The candidate gene was amplified and cloned, and a chromosomal knockout of PAO1 waaL was generated. Lipopolysaccharide (LPS) from this mutant is devoid of B-band O-polysaccharides and semirough (SR-LPS, or core-plus-one O-antigen). The mutant PAO1waaL is also deficient in the production of A-band polysaccharide, a homopolymer of D-rhamnose. Complementation of the mutant with pPAJL4 containing waaL restored the production of both A-band and B-band O antigens as well as SR-LPS, indicating that the knockout was nonpolar and waaL is required for the attachment of O-antigen repeat units to the core. Mutation of waaL in PAO1 and PA14, respectively, could be complemented with waaL from either strain to restore wild-type LPS production. The waaL mutation also drastically affected the swimming and twitching motilities of the bacteria. These results demonstrate that waaL in P. aeruginosa encodes a functional O-antigen ligase that is important for cell wall integrity and motility of the bacteria.     

Heterogeneity of lipopolysaccharides from Pseudomonas aeruginosa: analysis of lipopolysaccharide chain length.

Lipopolysaccharide (LPS) from smooth strains of Pseudomonas aeruginosa 503, PAZ1, PAO1715, PAO1716, and Z61 was fractionated by gel filtration chromatography. LPS samples from the first four strains, all PAO1 derivatives, separated into three major size populations, whereas LPS from strain Z61, a Pac K799/WT mutant strain, separated into two size populations. When column fractions were applied to sodium dodecyl sulfate-polyacrylamide gels in their order of elution, molecules of decreasing size were resolved, and the ladder of molecules with different-length O antigens formed a diagonal across the gel. The LPS from the PAO1 derivatives contained two distinct sets of bands, distinguished on the gels as two sets of diagonals. The set of bands with the faster mobility, the B bands, was found in column fractions comprising the three major amino sugar-containing peaks. In the sample from strain 503, a fourth minor peak which contained B bands was resolved. The slower-moving set of bands, the A bands, were recovered in a minor peak. LPS from strain Z61 contained only one set of bands, with the higher-molecular-weight molecules eluting from the column in a volume similar to that of the B bands of the PAO1 strains. Analysis of the fractions of LPS from all strains indicated that less than 8% of the LPS molecules had a long, attached O antigen. Analysis of the peak that contained mainly A bands indicated a lack of reactive amino sugar and phosphate, although heptose and 2-keto-3-deoxyoctulosonic acid were detected. Reaction of isolated fractions with monoclonal antibody specific for the PAO1 O-antigen side chain indicated that only the B bands from the PAO1 strains were antigenically reactive. The bands from strain Z61 showed no reactivity. The data suggest that the A and B bands from the PAO1 strains are antigenically distinct. We propose that PAO1 strains synthesize two types of molecules that are antigenically different.     

Characterisation of the katA gene encoding a catalase and evidence for at least a second catalase activity in Staphylococcus xylosus,bacteria used in food fermentation

Insertional inactivation of wbpM in Pseudomonas aeruginosa serogroup O11 strain PA103 resulted in mutants exhibiting three distinct lipopolysaccharide (LPS) phenotypes. One mutant, PA103 wbpM-C, had a truncated LPS core and lacked O antigen. These defects were not complemented by the cloned wbpM gene, suggesting a secondary mutation was present. When the wild-type galU gene was introduced into strain PA103 wbpM-C containing the cloned wbpM gene, both LPS defects were corrected. Construction of galU mutants in P. aeruginosa serogroups O11, O5, O6 and O17 strains led to truncation of the LPS core, indicating the involvement of GalU in P. aeruginosa LPS core synthesis.     

Bacteriophage attachment sites, serological specificity, and chemical composition of the lipopolysaccharides of semirough and rough mutants of Salmonella typhimurium

Extracted lipopolysaccharides (LPS) from one smooth, one semirough, and five rough mutants of Salmonella typhimurium LT2 or LT7, for which the chemical structure of the polysaccharide chain had been elucidated by using methylation analysis, were characterized with passive hemagglutination inhibition and phage inactivation experiments. Each addition of a sugar residue to a LPS from chemotype Rc was reflected in changed serological reactivity and phage-inhibiting activity of a collection of bacteriophages of the isolated LPS. Thus, certain criteria can be established for a classification of rough mutants of S. typhimurium. The observation that the serological RII specificity corresponds to a completed common core polysaccharide was verified. The serological RI specificity was found in LPS with terminal d-galactose I residues. One of the mutants, SL733, yielded a LPS which cross-reacted with anti-O5 factor serum although the polysaccharide was virtually free from contaminating O-specific material. The O5 reactivity was destroyed by alkaline treatment of SL733 LPS. The smooth- and rough-specific Felix O-l (FO) and the rough-specific 6SR and Br2 phages were shown to have their receptors in the LPS. There was a good correlation between the adsorption rate constant to whole cells and the phage inhibiting activity of isolated LPS suggesting that the LPS exert the major influence on the attachment of these phages to the bacteria. The polysaccharide structures in the LPS necessary for attachment of the 6SR and Br2 phages were defined. It was found that measuring the phage-inhibiting properties of isolated LPS as PhI(50) (LPS concentration required to inactivate 50% of the phages under defined conditions) was a more sensitive method for a characterization of the LPS than the serological and chemical assays used.     

Epithelial cell contact-induced alterations in Salmonella enterica serovar Typhi lipopolysaccharide are critical for bacterial internalization

The invasion of Pseudomonas aeruginosa and Salmonella enterica serovar Typhi into epithelial cells depends on the cystic fibrosis transmembrane conductance regulator (CFTR) protein as an epithelial receptor. In the case of P. aeruginosa , the bacterial ligand for CFTR is the outer core oligosaccharide portion of the lipopolysaccharide (LPS). To determine whether serovar Typhi LPS is also a bacterial ligand mediating internalization, we used both P. aeruginosa and serovar Typhi LPS as a competitive inhibitor of serovar Typhi invasion into the epithelial cell line T84. P. aeruginosa LPS containing a complete core efficiently inhibited serovar Typhi invasion. However, neither killed wild-type Typhi cells nor purified LPS were effective inhibitors. LPS from mutant Typhi strains defective in O side-chain synthesis, but with an apparently normal core, was capable of inhibiting invasion, but LPS obtained from a deeper rough mutant strain with alterations in fast-migrating core oligosaccharide failed to inhibit invasion. Lastly, exposure of wild-type serovar Typhi to T84 cultures before heat killing resulted in a structural alteration in its LPS that allowed the heat-killed cells to inhibit invasion of wild-type serovar Typhi. These data indicate that the serovar Typhi LPS core, like the P. aeruginosa LPS core, is a ligand mediating internalization of bacteria by epithelial cells, and that exposure of this ligand on wild-type Typhi is induced by the bacteria's interaction with host cells.     

Immunochemistry of the surface carbohydrate antigens of Bacteroides fragilis and definition of a common antigen

The components extracted by aqueous phenol from whole cells of Bacteroides fragilis were analysed by SDS-PAGE and immunoblotting and shown to consist of a series of strain-specific, cross-reactive and common antigens. Regularly-spaced ladder patterns on silver-stained gels indicated that in most strains the LPS was present as a predominantly smooth type, but with chain lengths of varying molecular mass, ranging within each particular strain from essentially rough forms to long chain-length smooth forms. The rough form of the LPS at the gel front possessed an antigen common to most of the strains investigated. Another antigen, which migrated behind the rough LPS on SDS gels, was common to all strains of the species. The smooth LPS forms and the other high molecular mass components were strain-specific antigens. Previously published methods are not capable of producing pure LPS or capsular polysaccharide for this organism.     

Identification of the lipopolysaccharide core region as the receptor site for a cytotoxin-converting phage, phi CTX, of Pseudomonas aeruginosa.

A temperate phage, phi CTX, is a cytotoxin-converting phage of Pseudomonas aeruginosa. In this study, we characterized the lipopolysaccharide (LPS) structures of phi CTX-resistant mutants derived from phi CTX-sensitive strains. phi CTX infectivity was neutralized by LPS preparations derived from sensitive strains but not by those from resistant strains. phi CTX-resistant mutants had lower-molecular-weight rough (R)-type LPS than the parental strains and lacked the reactivity of some anti-LPS core monoclonal antibodies. Some LPS core components were lacking or significantly decreased in the resistant mutants. These results suggested that a receptor site of the cytotoxin-converting phage phi CTX was the LPS core region and that especially L-rhamnose and D-glucose residues in the outer core were involved in phage binding. The host range of phi CTX was nearly O-serotype dependent, probably because of the diversity of the LPS core structure among P. aeruginosa strains. phi CTX bound to most strains of Homma serotypes A, G, and I but not to strains of serotypes B and E. Furthermore, we found that a genetic locus specifying phi CTX sensitivity (and consequently participating in the biosynthesis of part of the LPS core) existed in or near the locus participating in the determination of O-serotype specificity (somA), which has been mapped between leu-10 and eda-9001. phi CTX, as well as anti-LPS core monoclonal antibodies, will be a good tool for structural characterization of the P. aeruginosa LPS core region.