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ABSTRACTBrucella abortusrough lipopolysaccharide (LPS) mutants were obtained by transposon insertion into two wbkgenes (wbkA[putative glycosyltransferase; formerly rfbU] and per[perosamine synthetase]), into manB(pmm[phosphomannomutase; formerly rfbK]), and into an unassigned gene. Consistent with gene-predicted roles, electrophoretic analysis, 2-keto-3-manno-d-octulosonate measurements, and immunoblots with monoclonal antibodies to O-polysaccharide, outer and inner core epitopes showed no O-polysaccharide expression and no LPS core defects in the wbkmutants. The rough LPS of manBmutant lacked the outer core epitope and the gene was designated manBcoreto distinguish it from the wbk manBO-Ag. The fourth gene (provisionally designated wa**) coded for a putative glycosyltransferase involved in inner core synthesis, but the mutant kept the outer core epitope. Differences in phage and polymyxin sensitivity, exposure or expression of outer membrane protein, core and lipid A epitopes, and lipid A acylation demonstrated that small changes in LPS core caused significant differences in B. abortusouter membrane topology. In mice, the mutants showed different degrees of attenuation and induced antibodies to rough LPS and outer membrane proteins. Core-defective mutants and strain RB51 were ineffective vaccines against B. abortusin mice. The mutants perand wbkAinduced protection but less than the standard smooth vaccine S19, and controls suggested that anti O-polysaccharide antibodies accounted largely for the difference. Whereas no core-defective mutant was effective against B. ovis, S19, RB51, and the wbkAand permutants afforded similar levels of protection. These results suggest that rough Brucellavaccines should carry a complete core for maximal effectiveness.

ABSTRACTPathogenic biotypes of Yersinia enterocolitica(serotypes O:3, O:8, O:9, and O:13), but not environmental biotypes (serotypes O:5, O:6, O:7,8, and O:7,8,13,19), increased their permeability to hydrophobic probes when they were grown at pH 5.5 or in EGTA-supplemented (Ca2+-restricted) media at 37°C. A similar observation was also made when representative strains of serotypes O:8 and O:5 were tested after brief contact with human monocytes. The increase in permeability was independent of the virulence plasmid. The role of lipopolysaccharide (LPS) in this phenomenon was examined by using Y. enterocoliticaserotype O:8. LPS aggregates of bacteria grown in acidic or EGTA-supplemented broth took up more N-phenylnaphthylamine than LPS aggregates of bacteria grown in standard broth and also showed a marked increase in acyl chain fluidity which correlated with permeability, as determined by measurements obtained in the presence of hydrophobic dyes. No significant changes in O-antigen polymerization were observed, but lipid A acylation changed depending on the growth conditions. In standard medium at 37°C, there were hexa-, penta-, and tetraacyl lipid A forms, and the pentaacyl form was dominant. The amount of tetraacyl lipid A increased in EGTA-supplemented and acidic media, and hexaacyl lipid A almost disappeared under the latter conditions. Our results suggest that pathogenic Y. enterocoliticastrains modulate lipid A acylation coordinately with expression of virulence proteins, thus reducing LPS packing and increasing outer membrane permeability. The changes in permeability, LPS acyl chain fluidity, and lipid A acylation in pathogenic Y. enterocoliticastrains approximate the characteristics in Yersinia pseudotuberculosisand Yersinia pestisand suggest that there is a common outer membrane pattern associated with pathogenicity.

ABSTRACTThe outer membrane (OM) of the intracellular parasiteBrucella abortusis permeable to hydrophobic probes and resistant to destabilization by polycationic peptides and EDTA. The significance of these unusual properties was investigated in a comparative study with the opportunistic pathogens of the genusOchrobactrum, the closest known Brucellarelative. Ochrobactrumspp. OMs were impermeable to hydrophobic probes and sensitive to polymyxin B but resistant to EDTA. These properties were traced to lipopolysaccharide (LPS) because (i) insertion of B. abortusLPS, but not of Escherichia coliLPS, into OchrobactrumOM increased its permeability; (ii) permeability and polymyxin B binding measured with LPS aggregates paralleled the results with live bacteria; and (iii) the predicted intermediate results were obtained with B. abortus-Ochrobactrum anthropiand E. coli-O. anthropiLPS hybrid aggregates. Although Ochrobactrumwas sensitive to polymyxin, self-promoted uptake and bacterial lysis occurred without OM morphological changes, suggesting an unusual OM structural rigidity.Ochrobactrumand B. abortusLPSs showed no differences in phosphate, qualitative fatty acid composition, or acyl chain fluidity. However, OchrobactrumLPS, but not B. abortusLPS, contained galacturonic acid. B. abortusand Ochrobactrumsmooth LPS aggregates had similar size and zeta potential (−12 to −15 mV). Upon saturation with polymyxin, zeta potential became positive (1 mV) for Ochrobactrumsmooth LPS while remaining negative (−5 mV) for B. abortussmooth LPS, suggesting hindered access to inner targets. These results show that although Ochrobactrumand Brucellashare a basic OM pattern, subtle modifications in LPS core cause markedly different OM properties, possibly reflecting the adaptive evolution ofB. abortusto pathogenicity.