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Lipopolysaccharide (LPS) is the major component of the outer leaflet of the outer membrane of gram-negative bacteria. LPS has been described as having three structural and functional domains: the lipid A, core, and O antigen domains. The biological and toxic activities associated with LPS lie within the lipid A domain. The genetic basis for lipid A biosynthesis has largely been determined (15), allowing the construction of defined mutations that result in bacteria synthesizing altered lipid A structures. Previously, mutations in lipid A biosynthesis genes resulted in conditional lethality, but recently mutations in msbB (renamed waaN) in Escherichia coli and Salmonella enterica serovar Typhimurium which do not affect bacterial growth have been described (11, 12, 16). htrB (renamed waaM) mutants have also been generated which are conditional for growth at less than 32°C for survival (17). The waaM and waaN genes are responsible for the late acylation reactions that complete lipid A biosynthesis. Loss of these acyl chains from lipid A reduces the ability of the molecule to induce release of cytokines and other mediators of the immune response (7, 9, 12, 13, 16).

In mice, mutation of waaM in serovar Typhimurium resulted in reduced virulence and reduced growth in vivo, probably in large part due to the temperature sensitivity of these mutants (9). Mutation of waaN led to a very different phenotype. These mutants were able to grow at the same rate as the wild-type bacteria in murine livers and spleens following intravenous inoculation but reached higher numbers (approximately 10⁹ CFU per organ) than those typically associated with death during infection with wild-type serovar Typhimurium. Only a small proportion of the mice died, and the surviving mice eventually cleared the infection (12). The levels of proinflammatory cytokines and nitric oxide production were considerably lower during the course of infection with the waaN mutant than with the wild-type bacteria. This suggests that death in the mouse typhoid model of infection is dependent on high levels of cytokine release in response to lipid A.

In order to assess the role of lipid A in other Salmonella infection systems, Everest et al. (3) tested a waaN mutant in a rabbit ligated ileal loop model for enteropathogenesis and found that it showed no difference compared with wild-type serovar Typhimurium. This result was surprising in that the mutant is reduced in its ability to induce cytokines, which have been implicated in the induction of enteropathogenic responses (1, 2, 14). We have therefore reevaluated the role of lipid A in Salmonella enteropathogenesis by testing the waaN mutant in the bovine ligated ileal loop model. The results from this model using defined isogenic bacterial mutants correlate well with the severity of enteritis in orally inoculated calves (18, 20, 21).

Effect of mutation of waaN on induction of enteropathogenic responses by serovar Typhimurium. Bacteria were incubated in bovine midileal loops for 12 h, during which time polymorphonuclear leukocytes (PMNs) from each calf were isolated, labeled with ¹¹¹In, and reinjected. The surgical procedure is described in detail elsewhere (18). After 12 h, the secretory response (volume of fluid within a loop/length of loop in milliliters per centimeter) and the  emission of PMNs in the test loops compared with the negative control loops (PMN influx ratio) were recorded. The bacterial strains used, serovar Typhimurium C5 and its derivative waaN mutant and S. enterica serovar Dublin SD2229 and its derivative sipB mutant, have been described previously (12, 22). The inocula were incubated overnight in Luria-Bertani (LB) broth at 25°C and 100 rpm, subcultured approximately 1:3 into fresh LB broth, and incubated for 2 h at 37°C and 130 rpm. The optical density of the subcultures was adjusted by the addition of LB broth as required. The mean inoculum ± standard error of the mean (SEM) was 9.4 ± 0.08 log₁₀ CFU per loop, and the mean secretory response ± SEM in the negative control loops (inoculated with sterile LB broth) was 0.02 ± 0.02 ml cm¹.

View larger version (24K): [in this window] [in a new window]   FIG. 1.   Induction of enteropathogenic responses by serovar Dublin SD2229, serovar Typhimurium C5, and derivative mutants in bovine ligated ileal loops. (a) Secretory response. (b) Inflammatory response. Each bar represents the mean from three ligated loops and is presented with the SEM. WT, wild type.

The waaN mutant is impaired in secretion of proteins required for invasion and enteropathogenesis. Induction of enteropathogenic responses by serovars Typhimurium and Dublin requires the appropriate secretion and translocation of type III secretion system-1 (TTSS-1) Salmonella invasion proteins (Sips) and Salmonella outer proteins (Sops) (4, 10, 21). The effect of the waaN mutation on the secretion of proteins by serovar Typhimurium was assessed. Bacterial cultures were prepared by incubation overnight in LB broth at 25°C and 100 rpm, subculture 1:10 into fresh LB broth, and incubation for 4 h at 37°C and 130 rpm. There were no differences in the optical densities of the cultures (1.106 and 1.109 at 600 nm) or the number of viable bacteria (2.5 × 10⁹ and 4.0 × 10⁹ CFU ml¹) between the wild-type and waaN mutant, respectively, in a representative experiment. The culture supernatant was obtained by centrifugation at 10,000 × g for 10 min at 4°C and filtration with 0.45-µm-pore-size disposable filters. Proteins present in the supernatant were precipitated by the addition of trichloroacetic acid, separated on a sodium dodecyl sulfate-12% polyacrylamide gel, and stained with Coomassie brilliant blue as described previously (22). Several proteins were present in larger amounts in wild-type serovar Typhimurium C5 than in the waaN mutant (Fig. 2) in each of three separate experiments. Two of the most prominent of these proteins had molecular sizes similar to those reported for SipA (87 kDa) and SipC (42 kDa) (8, 21). We have previously demonstrated that a 42-kDa secreted protein from serovar Typhimurium is recognized by an anti-SipC monoclonal antibody (21).

View larger version (52K): [in this window] [in a new window]   FIG. 2.   Secretion of proteins by serovar Typhimurium C5 wild-type and its isogenic waaN mutant analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The sizes of molecular weight markers are indicated on the left. The gel was scanned with a Kodak DCS420 digital camera, and the contrast of the image was adjusted by using Adobe Photoshop 3.0.

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