Protection against fatal Pseudomonas aeruginosa sepsis by immunization with smooth and rough lipopolysaccharides.

The protective capacity of various native and mutant lipopolysaccharide antigens against fatal Pseudomonas aeruginosa burn wound sepsis was evaluated. Immunization with O-polysaccharide-deficient lipopolysaccharides derived from Escherichia coli J5 or Salmonella typhi Ty 21a afforded substantial protection against only one of five Pseudomonas aeruginosa challenge strains of various serotypes. Immunization with both lipopolysaccharide antigens evoked antibody of the immunoglobulin G class which recognized lipopolysaccharide isolated from the challenge strain against which protection was noted. This was not seen for the remaining four challenge strains. Attempts to demonstrate cross-serotype protection using O-antigen-deficient and core-deficient Pseudomonas aeruginosa lipopolysaccharide antigens was, for the most part, unsuccessful. In contrast, high levels of protection against all six serotypes of challenge strains were seen following immunization with homologous lipopolysaccharide.

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)

Simian liver alcohol dehydrogenase: isolation and characterization of isoenzymes from Macaca mulatta.

Like human liver alcohol dehydrogenase, that of Macaca mulatta can be purified and separated into anodic and cathodic pyrazole-insensitive and cathodic pyrazole-sensitive enzyme forms. Their inhibition by 4-methylpyrazole and their substrate specificities are analogous to those observed for the corresponding isoenzymes of human liver. However, on the basis of data available so far, the physiochemical and compositional characteristics, i.e., molecular weight, zinc content, and dimeric structure, of all simian alcohol dehydrogenase forms are virtually identical with those of other mammalian alcohol dehydrogenases studied up to now. Zinc is essential for their enzymatic function, as demonstrated by inhibition with chelating agents.

Biosynthesis of the core part of the lipopolysaccharide of Pseudomonas aeruginosa.

The incorporation of rhamnose and glucose into the core part of the lipopolysaccharide (LPS) of Pseudomonas aeruginosa was studied using enzyme preparations from strain PAC1R and LPS-defective mutants derived from it. Crude membrane preparations from the LPS-defective mutant PAC556 transferred rhamnose from dTDP-L-[14C]rhamnose to material insoluble in trichloracetic acid. The preparations contained both transferase enzyme and acceptor, the former being destroyed by heating. Between 60 and 70% of the radioactive rhamnose transferred to the membranes was extractable by aqueous phenol and non-diffusible. The material extracted did not move in any of the chromatography solvents tested and contained rhamnose as the sole radioactive component. Soluble dTDP-L-rhamnose-LPS rhamnosyltransferase was obtained from the parent strain PAC1R by ammonium sulphate precipitation of a 105000 g supernatant fraction from broken bacteria. It was most active at pH 8 with 5 mM-MgCl2 and required heat-treated membranes of PAC556 as acceptor. This mutant, whose LPS lacks both O-antigenic side-chains and rhamnose in the core, was shown to lack either the epimerase or the NADP-dependent oxidoreductase used to synthesize dTDPrhamnose. After preincubation with soluble transferase and UDPglucose, heated membranes of mutant strains PAC611, PAC612 and PAC605 could also act as acceptors for rhamnose. These mutants all lacked some or all of the glucose as well as the rhamnose from the core of their LPS and the experiments thus provided confirmation that rhamnose was the terminal hexose of the core in P. aeruginosa PAC1.

Morphology and morphogenesis of cerebral ring hemorrhages in anemic monkeys.

Cerebral ring hemorrhages were observed in four species of monkeys with various forms of anemia. The lesions resembled those described in humans with a variety of diseases, including pernicious anemia. The pale centers of most lesions were surrounded by rings of erythrocytes. Also observed were petechiae, pale foci without hemorrhage, and glial nodules. Ultrastructural study revealed that the pale cores were foci of degenerating neurites associated with infiltration of fibrin into the finest interstitial spaces. Leakage of firbinogen and erythrocytes apparently occurred from capillary sized vessels. A few of these were thrombosed, some were normal and some had only small fibrin thrombi in apparent apposition to damaged endothelial tight junctions. These findings suggest that a primary effect of anemia and other insults associated with ring hemorrhages is damage to tight junctions. Consequent fibrinous exudation clogs the interstitium and results in neuritic degeneration.

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.

Heterogeneity of the lipopolysaccharide from Pseudomonas aeruginosa.

Lipopolysaccharide isolated from pseudomonas aeruginosa PAC1 and its phage-resistant mutant was degraded by mild acid hydrolysis into lipid A and three major polysaccharide-containing fractions which were separated on Sephadex G-75. The low-molecular-weight fraction contained glucose, rhamnose, heptose, galactosamine, alanine and phosphate. The higher-molecular-weight fractions consisted mainly of glucose, rhamnose and glucosamine together with amino compounds. Alkaline degradation of the lipopolysaccharide produced at least four different species each of which contained a low-molecular-weight polysaccharide similar if not identical to that produced by acid hydrolysis. Under certain growth conditions an abnormal lipopolysaccharide was produced which was defective in the low-molecular-weight polysaccharide and contained mainly high-molecular-weight material. Strains of different serotype yielded lipopolysaccharides which also exhibited heterogeneity but contained a low-molecular-weight polysaccharide similar to that obtained from strain PAC1 and PAC1R. It is suggested that each strain of P. aeruginosa may produce several lipopolysaccharides each containing a polysaccharide common to all. The relative proportions of the various lipopolysaccharides may be changed by growth conditions.