Envelope proteins in Salmonella minnesota mutants.

Envelope proteins of one smooth (S) strain and seven rough (R) mutants of Salmonella minnesota were examined by sodium dodecyl sulphate-polyacrylamide gel electrophoresis. All strains gave similar band patterns although some consistent differences were detected. A major polypeptide band at 54k, which coincided with the flagellar component, was more prominent in S, Ra and Rb than in the Rc, Rd and Re chemotypes. The latter strains, however, showed more prominent bands at 48, 19 and 18k. The stage of growth at which the cultures were harvested was also found to affect the band patterns, particularly in the 54 and 40k regions. A closer examination of S, Ra and Re strains suggested that the levels of the major 40 and 37k bands were slightly reduced in Re. It is concluded that the progressive loss of lipopolysaccharide components which occurs from the S chemotype through various degrees of roughness to Re is accompanied by a change in the envelope protein composition, apparently between Rb and Rc.     

Interaction of lipopolysaccharide with detergents and its possible role in the detergent resistance of the outer membrane of Gram-negative bacteria.

In the presence of MgCl2, amounts of detergents which disrupted phospholipid vesicles caused lipopolysaccharide I from Proteus mirabilis to aggregate and form vesicular, membrane-like structures. Vesicle formation with P. mirabilis lipopolysaccharide II containing longer O-polysaccharide chains was extremely poor. Lipopolysaccharides of Salmonella minnesota R mutants (chemotypes Ra, Rc and Re) displayed a growing tendency for vesicle formation with increasing deficiency of the R core polysaccharide. Lipopolysaccharides of chemotypes Rc and Re produced vesicles even in the absence of MgCl2 and detergent. Spherical aggregates consisting of P. mirabilis lipopolysaccharide I MgCl2 and detergent were unable to either entrap or retain [14C]-sucrose, [3H=inulin or [3H]dextran. On the other hand, S. minnesota R mutant lipopolysaccharides of chemotypes Rc and Re could entrap all three saccharides and retain them for at least short periods of time. Leakage of [3H]-inulin out of re-lipopolysaccharide vesicles was greatly retarded by addition of MgCl2 to the vesicle system. Incorporation of P. mirabilis lipopolysaccharide I or S. minnesota Rc lipopolysaccharide into phospholipid vesicles protected these model membranes from disruption by detergent. This suggested a similar protective function of lipopolysaccharide in the outer membrane of enteric bacteria against the action of surfactants occurring in their normal intestinal habitat.     

The sensitivity of smooth and rough mutants ofSalmonella typhimurium to bactericidal and bacteriolytic action of serum,lysozyme and to phagocytosis

The sensitivity of smooth and rough mutants ofSalmonella typhimurium belonging to different chemotypes to bactericidal and bacteriolytic action of antibodies, complement and lysozyme was investigated. By the action of specific antibodies and complement the majority of strains was killed and in the presence of sufficient amount of lysozyme even lysed. Sera of newborn piglet which do not contain antibodies however, were able to kill regularly only strains of lower chemotypes (Rc and Rb). Smooth strains were usually resistant to the action of piglet serum whereas strains of Ra chemotype were killed only by sera of some piglets. Using the liver clearance method a significant correlation between the lipopolysaccharide chemotype of the particular strain and the degree of phagocytosis was found.     

Delayed hypersensitivity in murine salmonellosis: specificity of footpad reaction in mice infected with rough mutants of Salmonella typhimurium

Delayed type (footpad) hypersensitivity (DTH) in BALB/c mice immunized with rough mutant strains of Salmonella typhimurium LT2 was examined. Injection of live organisms of an Rb mutant TV148 strain induced DTH in mice, while injection of the heat-killed organisms did not. The mice immunized with live organisms of the Ra, Rb, Rc, Rd, and Re mutant strains showed positive footpad reactions to the heat-killed cell antigen of LT2 (wild type) strain. The mice immunized with the Rb mutant strain also showed positive footpad swellings in response to heat-killed cell antigens of S. paratyphi A, S. paratyphi B, S. typhi, S. enteritidis, and S. cholerae-suis. Furthermore, positive reactions to antigens of Escherichia coli and Shigella flexneri were seen in the TV148-immunized mice, but the mice did not respond to heat-killed organisms of Pseudomonas aeruginosa or Staphylococcus aureus. The cross-reactive footpad reaction to E. coli could be transferred adoptively with T cells prepared from the spleens of TV148-immunized mice into syngeneic recipients. These results suggest that the cross-reactive DTH antigen(s) is widely distributed among related organisms such as Shigella and Escherichia.     

The disaccharide L-alpha-D-heptose1-->7-L-alpha-D-heptose1-->of the inner core domain of Salmonella lipopolysaccharide is accessible to antibody and is the epitope of a broadly reactive monoclonal antibody.

We generated a panel of mAb containing at least one specificity against each of the known chemotypes of the Salmonella LPS core domain and used them to investigate the accessibility of core determinants in smooth LPS. Most of the mAb were reactive with at the most three chemotypes of the core as determined by enzyme immunoassay and failed to bind smooth LPS or any of the complete cores of E. coli. One mAb, MASC1-MM3 (MM3), reacted with six different Salmonella core chemotypes, the R2 core of Escherichia coli and a variety of smooth LPS. This mAb reacted equally well with live and heat-killed bacteria. It bound to 123 of 126 clinical isolates of Salmonella and 11 of 73 E. coli strains in a dot-immunoblot assay. Typical ladder-like patterns of bands were observed after immunoblotting of this mAb against electrophoretically resolved smooth LPS from the five major serogroups of Salmonella species (A, B, C1, D1, and E). MM3 had no reactivity with BSA conjugates of O-Ag polysaccharides from the above serogroups confirming specificity for a core epitope. Polysaccharides derived from or synthetic saccharides representative of the various chemotypes of Salmonella LPS core were tested as competitive inhibitors of the binding of MM3 to LPS. The results led to a conclusion that MM3 recognizes the structure, L-alpha-D-Heptose1-->7-L-alpha-D-Heptose1-->disaccharide present as a branch in the Ra, Rb1, Rb2, Rb3 and Rc but lacking in the Rd1, Rd2, and Re chemotypes of the Salmonella LPS core. This disaccharide seems free and accessible on the basis of the previously calculated conformations of the Salmonella (Ra) and E. coli complete cores (R1, R2, R3, R4, and K12). It therefore defines or contains an epitope within the inner core subdomain of Salmonella LPS that is accessible to antibody in long-chained LPS and in intact bacteria with complete LPS.     

Staining of O-specific polysaccharide chains of lipopolysaccharides with ruthenium red

S-form lipopolysaccharides (LPS) from Klebsiella strain LEN-1 (O3: K1-) and from Salmonella minnesota strain 1114 were positively stained with ruthenium red, whereas R-form LPS from Klebsiella strain LEN-111 (O3-: K1-) and Ra, Rb1, RcP+, Rd1P-, and Re LPS from the respective mutant strains of S. minnesota were not or only faintly stained by such treatment. From these results it was concluded that ruthenium red stains the O-specific polysaccharide chains of LPS. The appearance of stained preparations of S-form LPS suggested that the material responsible for this positive staining corresponded to the surface projections which were seen by the negative staining technique as attached to the ribbon-like structures and spherules of the LPS.     

Suppressive effects of serum on the LPS-induced production of nitric oxide and TNF-alpha by a macrophage-like cell line, WEHI-3, are dependent on the structure of polysaccharide chains in LPS

The effect of serum on LPS-induced activation of a murine macrophage-like cell line, WEHI-3, was examined. Foetal calf serum strongly inhibited the production of nitric oxide (NO) and TNF-alpha by LPS-stimulated WEHI-3 cells, while it enhanced the production of both by other macrophage-like cell lines, J774.1 and BAM3, on treatment with LPS. This suppressive effect of serum on WEHI-3 cells was most remarkable when the cells were stimulated with rough-chemotype LPS, Ra LPS, Rc LPS and Rd2 LPS. Foetal calf serum also inhibited TNF-alpha production by the same cells stimulated with high concentrations of smooth-form LPS (S LPS; > 1000 ng/mL). Serum-mediated suppression was also observed for expression of the TNF-alpha gene in Rc LPS-stimulated WEHI-3 cells. This suppressive effect of FCS was most remarkable during the 1-2 h before the addition of LPS, but it was not observed when FCS was added at 1 h after the addition of LPS, suggesting dependence on the time of FCS addition to LPS-stimulated cells. No significant difference was observed in the expression of CD14 on WEHI-3 cells cultured in the presence and absence of serum, suggesting that CD14 is not involved in the serum-mediated suppression of these LPS-responses. On the contrary, FCS showed enhancing effects on the production of NO and TNF-alpha by WEHI-3 cells stimulated with low concentrations (< 100 ng/mL) of S LPS and rough mutant Salmonella minnesota Re LPS. These results suggest that the ability of FCS to suppress LPS-induced activation of WEHI-3 cells in mainly dependent on the structure of polysaccharide chains and also on the concentration of LPS employed.     

Relationship between immune system and gram-negative bacteria: monocyte chemotaxis induced by supernatants from human peripheral blood OKT8+ lymphocytes stimulated with smooth and rough Salmonella strains

It has been recently reported that smooth (S) Salmonella typhimurium LT-2 and rough (R) mutants, S. minnesota R345 (Rb) and R595 (Re), spontaneously adhere to human peripheral blood mononuclear cells (PBMC). The binding is confined to T cells and is mediated by the lipopolysaccharide (LPS) moiety of the bacteria outer membrane. In this study, we have investigated the monocyte chemotactic responsiveness induced by supernatants recovered from human PBMC stimulated with either S or R Salmonella strains. All supernatants were able to trigger monocyte chemotaxis, even if to a different degree according to the bacterial strain used. These data were further supported by experiments which showed that stimulation of PBMC by purified homologous LPS led to the release of a chemotactic lymphokine (CLK) for human monocytes. Moreover, this CLK seems to be released by T cells, and in particular OKT8+ cells, since OKT8- PBMC failed to produce CLK.     

Bacteriophage receptor development and synthesis of O-specific side chains after addition of D-galactose to the uridine diphosphate-galactose-4-epimeraseless mutant Salmonella typhimurium LT2-M1

The formation of complete cell wall core lipopolysaccharide (LPS) and O-antigenic side chains after addition of d-galactose to the uridine diphosphate-galactose-4-epimeraseless mutant, Salmonella typhimurium LT2-M1, has been studied by (i) determination of adsorption rates of smooth and rough specific bacteriophages, (ii) passive hemagglutination inhibition, and (iii) qualitative and quantitative determination of the polysaccharide composition and structure. A rapid synthesis of the complete core LPS and O side chains occurred in bacteria in the log phase and the early stationary phase. Phage C21, which attaches to unsubstituted Rc structures, was adsorbed by the bacteria for only 10 min after the addition of d-galactose. Unsubstituted Rc structures, however, could still be detected after 160 min by immunological and chemical assays. Attachment of the P22 phage, which requires O-specific side chains with more than one repeating unit for adsorption, was demonstrated 10 min after the addition of d-galactose. Attachment of the Felix O-1 phage, which requires a complete core, was observed between 20 and 80 min after the addition of d-galactose. The rough specific phages 6SR and Br2 did not adsorb to the bacteria at any time after the addition of d-galactose. By passive hemagglutination inhibition, the presence of O-specific structures could be demonstrated after 10 min. No antigenic activity of the Ra and Rb structures was observed in the LPS preparations isolated at any time after the addition of d-galactose. Methylation analysis of LPS preparations isolated at 10 and 160 min after the addition of d-galactose showed that the O-specific side chains contained an average of 11 and 15 repeating units, respectively. In the 10-min sample, every 25th "Rc structure" carried a side chain, compared to every 3rd residue in the 160-min sample.     

Monoclonal antibodies to Salmonella lipopolysaccharide: anti-O-polysaccharide antibodies protect C3H mice against challenge with virulent Salmonella typhimurium

The present investigation reports the production of monoclonal antibodies to antigenic determinants of the O-polysaccharide of Salmonella typhimurium lipopolysaccharide (LPS), and assesses the effectiveness of these antibodies in protecting C3H mice against the lethal effects of Salmonella infection. Hybridomas were generated by fusing spleen cells from (BALB/c X A/J)F1 (CAF1) mice hyperimmunized by i.v. injection with acetone-killed S. typhimurium SR-11 with X63-Ag8.653 murine myeloma cells. Hybridomas producing antibodies reactive with S. typhimurium SR-11 whole cells were subcloned, and seven of the resulting clones as well as one previously described clone were selected for use in the studies reported here. Monoclonal antibodies from these eight clones were of the IgG1 (1), IgG3 (6), or IgM (1) isotype and were specific for the O-polysaccharide region of Salmonella LPS, reacting with LPS from smooth S. typhimurium SR-11 and LT-2, but not with LPS from rough S. minnesota R60 (Ra), R345 (Rb), or R595 (Re). The effectiveness of each monoclonal antibody in protecting C3H/HeN and C3H/HeJ mice against the lethal effects of Salmonella infection was evaluated by comparing the median length of survival of groups of mice given antibody by i.p. injection before i.p. challenge with virulent S. typhimurium SR-11 to that of animals that received no antibody. Three out of eight monoclonal anti-O-polysaccharide antibodies, ST-1 (IgM), 10-5-47 (IgG3), and 10-5-6 (IgG3), provided significant (p less than 0.01) protection to C3H/HeN mice challenged with approximately 10(4) LD100 of Salmonella. Only antibodies ST-1 and 10-5-6, however, extended the median length of survival of C3H/HeJ mice beyond that of infected controls. Mouse antiserum prepared against S. typhimurium SR-11 was equally protective in C3H/HeJ mice. In an attempt to understand the contribution of antibody specificity to the relative differences in the protective capacities of the monoclonal antibodies, their reactivities with several Salmonella reference strains of different classical serotypes were examined. Although some differences in reactivity against the different strains were apparent, this approach was not adequate for defining the fine specificity of these monoclonal antibodies. The results of this study provide evidence that monoclonal antibodies with specificity to the O-polysaccharide region of Salmonella LPS can protect C3H mice against challenge with the homologous bacterial strain.     

Chemical and ultrastructural comparison of endotoxins extracted from Salmonella minnesota wild type and R mutants

Endotoxic lipopolysaccharide and glycolipids ( RGl ) extracted from Salmonella minnesota wild type and R mutant cells ( chemotypes Ra, Rb, Rc, Rd1, and Rd2 ), respectively, with hot phenol-water (PW) and phenol-chloroform-petroleum ether (PCP) were analyzed chemically and electron microscopically. All RGl extracted with PW ( RGl -PW) contained excess amounts of phosphate, O-ester linked fatty acids and neutral sugars, while all RGl extracted with PCP ( RGl -PCP) contained excess amounts of free amino groups and fatty acids, in addition to the RGl constituents. Polyamine (cadaverine), phosphoethanolamine, and an unidentified amino compound were contained in RGl -PCP as free amino groups. When stained with uranyl formate, the ultrastructure of RGl -PW showed a spherical form (onion-like form), whereas the micrographs of RGl -PCP showed a filamentous structure, regardless of strain differences. On the other hand, the micrographs of RGl -PW represented spherical and doughnut-shaped forms, and the micrographs of RGl -PCP showed filamentous or stick forms, when stained with uranyl acetate. Thus, it is suggested that the ultrastructures of RGl were dominated by the solvent systems used for extraction, and not by the strains used here.     

Crystallization of R-form lipopolysaccharides from Salmonella minnesota and Escherichia coli.

Salmonella minnesota Re and Ra lipopolysaccharides (LPSs) and Escherichia coli K-12 LPS formed three-dimensional crystals, either hexagonal plates (preferential growth along the a axis) or solid columns (preferential growth along the c axis), when they were precipitated by the addition of 2 volumes of 95% ethanol containing 375 mM MgCl2 and incubated in 70% ethanol containing 250 mM MgCl2 at 4 degrees C for 10 days. Analyses of crystals suggested that they consist of hexagonal lattices with the a axis (a side of the lozenge as a unit cell on the basal plane) of 0.462 nm for all these three kinds of LPSs and the c axes (perpendicular to the basal plane) of 5.85, 8.47, and 8.75 nm for S. minnesota Re and Ra LPSs and E. coli K-12 LPS, respectively, and that hydrocarbon chains of the lipid A portion play the leading part in crystallization, whereas the hydrophilic part of the lipid A (the disaccharide backbone) and R core exhibit a disordered structure or are in a random orientation. The phenomenon of doubling of the a axis to 0.924 nm was observed with crystals of S. minnesota Re LPS when they were incubated in 70% ethanol for an additional 180 days, but not with crystals of S. minnesota Ra LPS or E. coli K-12 LPS. S. minnesota S-form LPS possessing the O-antigen-specific polysaccharide and S. minnesota free lipid A obtained by acid hydrolysis of Re LPS did not crystallize under the same experimental conditions.     

Relationship between Mg2(+)-induced hexagonal assembly of R-form lipopolysaccharides and chemical structure of their R-cores

The relationship between formation of the Mg2(+)-induced hexagonal lattice structure by R-form lipopolysaccharides (LPS) and chemical structure of their R-cores was investigated using different kinds of R-form LPS from a series of mutants of Salmonella minnesota or S. typhimurium. The optimal experimental condition for formation of the hexagonal lattice structure was to suspend LPS preparations, from which cationic material was removed by electrodialysis, in 50 mM tris (hydroxymethyl) aminomethane buffer at pH 8.5 containing 10 mM MgCl2. Under this experimental condition, Rb1 LPS formed the hexagonal lattice structure with the lattice constant of 14.0 +/- 0.2 nm. Ra LPS, which possesses the full length of R-core, also formed the hexagonal lattice structure but its lattice constant was larger (18.1 +/- 0.2 nm) than that of Rb1 LPS (the lattice structure by Ra LPS was looser than that by Rb1 LPS). All the other R-form LPS preparations tested, RcP+, PcP-, Rd1P-, and Re LPS, whose R-cores are shorter than that of Rb1 LPS, did not form the hexagonal lattice structure, but formed membranous structures showing various shapes which consisted of multiple bilayer structures. Failure to form the hexagonal lattice structure was the common feature of these kinds of R-form LPS irrespective of temperature at which the LPS suspensions in 10 mM MgCl2-50 mM Tris buffer were incubated. From the results of the present study it was concluded that capability of R-form LPS to form the hexagonal lattice structure has a close correlation with the chemical structure of their R-cores.     

Porins and lipopolysaccharide of Escherichia coli ATCC 25922 and isogenic rough mutants

Abstract The lipopolysaccharide and porin profile of Escherichia coli ATCC 25922, a smooth strain commonly used in antibiotic susceptibility testing, and five isogenic rough mutants was examined. The lipopolysaccharide of the parent strain had the characteristic ladder pattern on polyacrylamide gels, while that of the mutants appeared similar to chemotypes Ra and Rc of Salmonella typhimurium with some changes in chemical composition. Of the porins, OmpC appeared markedly reduced in the parent strain while OmpF appeared markedly reduced in the mutants. In addition, a new outer-membrane protein of size intermediate to that of OmpC and OmpF was detected in all mutants. Neither parent nor mutants were susceptible to the LPS core-specific P1 phage or the porin-specific PA2 and K20 phages.     

Structural investigations on the inner core region of lipopolysaccharides from Salmonella minnesota rough mutants

The structure of the inner core region (L-glycero-D-mannoheptose/2-keto-3-deoxy-D-mannooctulosonic acid region) of lipopolysaccharides from Salmonella minnesota rough mutants was investigated. Using conventional methods (neutral sugar analysis, Smith degradation and methylation analysis) combined with gas chromatography/mass spectrometry (GC/MS) of higher oligosaccharides (up to tetrasaccharide), the linkages of the core sugars of lipopolysaccharides from S. minnesota rough mutants, strains R4 (Rd2P-), R7 (Rd1P-) and R5 (RcP-) were determined as: (formula see text) with R representing H in R4, L-glycero-D-mannoheptopyranosyl in R7, and D-glucopyranosyl-(1----3)-L-glycero-D-mannoheptopyranosyl in R5, respectively. In addition, it is shown that heterogeneity within the neutral sugar part of these lipopolysaccharides is low.     

High-molecular-weight components in lipopolysaccharides of Salmonella typhimurium, Salmonella minnesota, and Escherichia coli.

Lipopolysaccharide from smooth strains of Salmonella typhimurium, Salmonella minnesota, and Escherichia coli O111:B4, O55:B5, and O127:B8 was fractionated by gel filtration chromatography. All lipopolysaccharide samples separated into three major populations. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the fractions from S. typhimurium and S. minnesota indicated that the three peaks were made up of molecules with average O-antigen lengths of (i) 70 or more repeat units, (ii) 30 and 20 repeats units in the samples from S. typhimurium and S. minnesota, respectively, and (iii) 1 repeat unit. In contrast to the Salmonella samples, peak 1 from the E. coli samples was not detected on polyacrylamide gels and lacked detectable phosphate. This high-molecular-weight material had a sugar composition similar to that of O-antigen and was tentatively identified as capsular polysaccharide. Peaks 2 and 3 of the E. coli samples were analogous to those of the Salmonella isolates, containing lipopolysaccharide molecules with averages of 18 and 1 O-antigen repeat units, respectively. These lipopolysaccharide molecules did not completely dissociate during electrophoresis, and multimers were detected as distinct, anomalous, slow-migrating bands. Increasing the concentration of sodium dodecyl sulfate in the gels resulted in the dissociation of these multimers.     

The role of lipopolysaccharide structure in the recipient cell during plasmid-mediated bacterial conjugation

The role of the lipopolysaccharide (LPS) structure in the recipient cell during bacterial conjugation was studied using a series of well-defined LPS mutations in Salmonella minnesota. The plasmids Flac (IncFI) and R1drd19 (IncFII) transferred at a high frequency to the smooth S218 parent strain and the rough LPS mutants. However, R64drd1 1 (IncI alpha) transferred poorly to the LPS mutants compared with transfer to the smooth LPS parent strain. The decrease in R64drd1 1 transfer frequency correlated with the extent of the defect in LPS structure, suggesting that intact LPS on the recipient is a major requirement for R64drd1 1 mating. Transfer of the P-group plasmid, RK2, was not affected by changes in LPS structure. These results show that plasmids use different cell surface structures during conjugation, and that LPS is particularly important for R64drd1 1 transfer.     

Estimation of mean molecular weight of Enterobacteriaceae lipopolysaccharides using gas chromatography for determination of fatty acids

In the paper, we propose a method for estimation of the mean molecular weight of lipopolysaccharide, which is important for accuracy of endotoxin activity investigation. In our study, it was assumed that lipid A portion in Enterobacterial lipopolysaccharide is substituted by four 3-hydroxytetradecanoic acid residues. Lipopolysaccharides of S, Ra, Rc and Re chemotypes being laboratory preparations as well as purchased from Sigma were investigated. Fatty acids were determined by of gas chromatography as methyl esters according to the procedure described by Wollenweber and Rietschel. Mean molecular weight was calculated by the formula: MMW = [formula: see text]. A high agreement between the estimated and the theoretical molecular weight values was demonstrated in the case of Salmonella minnesota R595 (Re) LPS preparation. As expected, LPS heterogeneity increase together with enlargement of polysaccharide chain length which is visible in electrophoregrams also. Except for LPS mean molecular weight estimation, the method allows its detection in various preparations and samples, distinguishing of R and S LPS forms as well as the determination of mean length of O-specific chain in lipopolysaccharides which structures are known.     

Relationship between immune system and gram-negative bacteria. IV. T lymphocytes from Lpsd mice possess binding site(s) for Rb Salmonella

We have previously shown that Salmonella minnesota R345 (Rb) spontaneously binds to 50 to 55% of human peripheral blood mononuclear cells (PBMC). In the present study, we have compared Rb cytoadherence to lymphoid cells from various tissues of lipopolysaccharide (LPS) hyporesponsive (Lpsd) and LPS responsive (Lpsn) mouse strains. A higher number of spleen cells from Lpsd mice (C3H/HeJ and C57BL/10ScN) bound Rb bacteria (22 to 30%) than cells from Lpsn mice (4 to 9%). Rb bound mainly to T cells, and cytoadherence occurred in both Lyt-1+ and Lyt-2+ T cell subsets. By contrast, purified splenic B cells from Lpsd and Lpsn mice gave less than 4% Rb cytoadherence. In both mouse strains, cytoadherence was mediated by the homologous LPS structure, because purified Rb-LPS blocked Rb Salmonella binding to T cells. On the other hand, smooth Salmonella typhimurium LT-2 LPS (S-LPS) and Salmonella R595 (Re) LPS (Re-LPS), which contain mainly lipid A, were without effect on Rb binding. Increased Rb binding was seen with T cells from Peyer's patches (PP), mesenteric lymph nodes (MLN), and peripheral blood than from spleen of C3H/HeN (Lpsn) mice; however, greater cytoadherence was always seen with T cells of these tissues from C3H/HeJ mice. Interestingly, treatment of whole spleen or purified T cells from C3H/HeN mice with neuraminidase enhanced cytoadherence to levels seen with C3H/HeJ cells. The observed Rb binding to PP, MLN, and PBMC cells in both mouse strains suggests that gut microbial environment may play an important role in Rb cytoadherence. This is also supported by the evidence that when spleen cells of germfree and conventional mice were tested for Rb binding, higher cytoadherence was observed in conventional mice only. Taken together, these results indicate that T cells of Lpsd mice express binding site(s) for Salmonella, whereas Lpsn mice have T cells with these structure(s) in a cryptic configuration.     

Sensitivity of Rough Gram-negative Bacteria to the Bactericidal Action of Serum

Three Salmonella minnesota rough mutants were found to be more sensitive to the bactericidal action of antibody and complement than was the parent smooth strain. The antibodies involved were shown to be against components which are all present in the smooth parent strain and are not identical with the lipopolysaccharides isolated by the phenol-water extraction procedure. The lipopolysaccharide of the smooth strain was shown to confer resistance by blocking access of antibody to the sensitive antigens.