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In an experimental urinary tract infection of the mouse, colonization of the urinary bladder by isogenic strains of Salmonella enterica serovar Typhimurium was found to depend on the motility of the bacteria. Strains were obtained by genetic recombination between a highly motile O-6,7 and a poorly motile O-4,5,12 strain. The O antigen did not interfere with the colonization, whereas motility did; flagellated and motile O-6,7 and O-4,5,12 bacteria colonized the bladder equally well.

We used crossed immunoelectrophoresis to study detergent-solubilized components of the outer membrane of Salmonella typhimurium under nondenaturing conditions. The antisera used were raised against nondenatured outer membrane preparations. Lipopolysaccharide and lipoprotein were identified easily as discrete precipitates when they were solubilized with Triton X-100. However, solubilization of the porins with Triton X-100 resulted in a complex precipitate pattern, indicating incomplete dissociation of protein-protein interactions. A clear-cut pattern was obtained when the porins were first solubilized and denatured with hot sodium dodecyl sulfate, followed by removal of the sodium dodecyl sulfate and renaturation in the presence of Triton X-100. Our findings suggested that crossed immunoelectrophoresis can be used to study the antigenicity of nondenatured porins and the antibody responses to them.

Porin (outer membrane protein) preparations extracted from a rough (Rb2) mutant of Salmonella typhimurium proved to be good immunogens in mice and rabbits. The antibody response achieved was measured by using enzyme-linked immunosorbent assay techniques. High titers of both antiporin and antilipopolysaccharide were detected in both species. The rabbit antiserum raised against the porins and the porin preparations themselves had a highly significant protective capacity against intraperitoneal Salmonella infection of mice. Absorption of the rabbit antiporin serum with lipopolysaccharide immunosorbent did not change its protective capacity in a passive immunization experiment, suggesting that the antiporin antibody preparations were the active components.

Mutants resistant to bacteriophages (P221 and PH105 or PH51) were isolated from a rfa strain of Salmonella typhimurium. They were found deficient in separate 33,000- to 36,000-dalton band proteins (major band proteins). Double mutants derived from both types of mutants were deficient in both of the bands. The growth behavior of all the mutants was normal. The outer membrane of the mutants appeared to be more wrinkled than normal and formed vesicles in many of the mutants. In freeze-fractured cells, changes were seen in the outer membrane (particleless patches in the concave fracture face, the particles themselves being smaller than normal). These changes were more marked in the double mutants.

A crude major outer membrane (porin) preparation, obtained from a rough strain of Salmonella typhimurium earlier shown to be protective both in active and passive immunization of mice against challenge with smooth S. typhimurium, was further purified. Removal of the main impurities, lipopolysaccharide (LPS) and lipoprotein, was accompanied by loss of protective capacity in passive immunization experiments. Reconstitution with rough LPS restored the protective capacity. Protection was, however, concluded not to be due to anti-LPS, because a large fraction of the anti-LPS antibodies could be removed from the protective rabbit antiserum with an LPS immunosorbent without loss of protection.

The role of several outer membrane components as virulence factors is well established. We have now isolated spontaneous mutants and conjugational hybrids of smooth mouse virulent Salmonella typhimurium deficient in two major outer-membrane proteins. The lack of the 34,000- and/or 36,000-dalton proteins was confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Mutants and recombinants were then tested for their mouse virulence by intraperitoneal inoculation and were found to be as virulent as the parent strain.

The antibodies responsible for the protective effect of sera obtained by immunizing rabbits with the major outer membrane protein porin of Salmonella typhimurium complexed with LPS (Kuusi et al., Infect. Immun., 1981; 34:328-332) were found to be directed to the O antigen. They were effective in very small concentration which probably accounted for our failure to identify them before or now by in vitro methods. Although both the porin and the LPS in the complexes used for immunization were isolated from rough (R) form organisms devoid of O antigen by all usually applied criteria, the antigen contained a small amount of smooth form LPS, most probably derived from a slight leakiness of the rfa mutation responsible for the R character. The small amount of contaminating O antigen was apparently rendered highly immunogenic by complexation with the outer membrane protein.

Outbred mice were vaccinated with various artificial Salmonella vaccines and subsequently challenged intraperitoneally with graded doses of virulent Salmonella typhimurium. The Salmonella vaccines used were: (i) octasaccharide, obtained by hydrolysis of the O-antigenic polysaccharide chain of S. typhimurium strain SH 4809 with phage P22-associated endo-rhamnosidase and covalently linked to either diphtheria toxin or edestine; (ii) purified outer membrane proteins (porins) from S. typhimurium; and (iii) octasaccharide covalently linked to porins. All vaccines induced significant protection against experimental infection of mice with S. typhimurium. However, vaccination with the octasaccharide-porin conjugate resulted in better protection than that obtained by vaccination with octasaccharide or porin vaccines separately. Rabbit antibodies raised against the different vaccines were also passively administered intravenously to mice. Such mice were protected against challenge with virulent S. typhimurium by antibodies specific for the S. typhimurium O-antigen or for the porins. Thus, active immunization with more than one surface component of Salmonella bacteria improved the efficacy of the vaccine. The data from the passive immunization experiments also emphasized the role of humoral immunity for protection against S. typhimurium infection.

Salmonella typhimurium LT2 lines, if phenotypically rough, are fully sensitive to bacteriocin 4-59, produced by Salmonella canastel strain SL1712. Bacteriocin-resistant mutants fell into three classes. Those resistant to phage ES18 and to albomycin proved to be mutants of class chr (equivalent to tonB of Escherichia coli); these mutants still adsorb the bacteriocin and so are classified as tolerant. Another class of (incompletely) tolerant mutants was resistant to phage PH51; their envelope fractions lacked the band corresponding to outer membrane protein 34K, known to serve for adsorption of phage PH51. A third class of mutants, which did not adsorb the bacteriocin, was unaltered in sensitivity to phages. Their envelopes lacked the 33K band, indicating absence of the outer membrane protein 33K, considered to correspond to outer membrane protein II* of E. coli, which in that species is determined at locus ompA (formerly tolG or con). Phage P22 HT105/1 cotransduced the 33K S. typhimurium gene (to be called ompA, to accord with E. coli usage) with pyrD+ at about 30% frequency when the donor allele was ompA+ or one ompA, but at only 3 to 11% when the donor allele was another ompA. When the donor carried either of two long deletions of the put (proline utilization) operon, phage P22 HT105/1 cotransduced put (and ompA+) with pyrD+ at low frequency. The cotransduction data indicate that ompA of S. typhimurium is located between pyrD and put, nearer the former. This corresponds to the map position of ompA in E. coli K-12.