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Salmonella enterica serovar Typhimurium, a gram-negative bacterial species, is a facultative intracellular pathogen which infects its hosts through the oral route (25). Human diseases caused by Salmonella serotypes include gastroenteritis, bacteremia, and typhoid fever (11). Most infections occur as a result of ingestion of undercooked eggs or contaminated food (meats and dairy products) or water (1, 11, 22). Each year in the United States two to four million cases of gastroenteritis are caused by Salmonella bacteria (23), along with a few hundred cases of typhoid fever (3). According to the World Health Organization, Salmonella is probably the most common cause of diarrhea globally (11), and at least 12 million cases of typhoid fever are reported each year, with a mortality rate of 10 to 12% (7).

Like other gram-negative bacteria, S. typhimurium has an outer membrane surrounding the periplasmic space. The outer membrane contains numerous proteins, referred to as OMPs. A subset of these, called porins, form water-filled channels across the outer membrane to facilitate the transport of small hydrophilic molecules (16). S. typhimurium expresses three porins when grown under normal conditions (Lennox broth at 37°C): OmpD (34 kDa), OmpF (35 kDa), and OmpC (36 kDa) (12, 15, 21). OmpD is found in S. typhimurium but is absent from other gram-negative bacteria, including Escherichia coli. OmpD is homologous with the NmpC and Lc porins in E. coli K-12 (21), both of which (NmpC and Lc) can only be expressed in E. coli K-12 mutants which lack normal outer membrane proteins (18). Little is known about the OmpD porin, apart from the genomic location of the ompD gene and the immunochemical and topological structure of the porin itself (20, 21).

Dorman and colleagues (6) showed that mutations in some porin-associated genes affect the virulence of S. typhimurium in BALB/c mice. Specifically, a mutation in the ompR gene, which encodes a positive regulator of porin gene expression, has a dramatic effect on virulence, increasing the 50% lethal dose (LD₅₀) by more than three log units compared to that of a wild-type strain. In the same study, Dorman et al. characterized the effect of mutations in the ompC, ompF, and ompD genes. They report that strains containing ompF or ompC mutations were as virulent as their wild-type parent, while a strain containing an ompD mutation showed a slight reduction in virulence (23-fold increase in LD₅₀ between the wild type and the ompD mutant). Interestingly, OmpR regulates the expression of the genes coding for porins OmpC and OmpF; but it does not seem to regulate expression of ompD (6).

In a subsequent study, Chatfield et al. (2) showed that a mutant lacking both the OmpF and OmpC porins is attenuated, displaying an oral LD₅₀ that is three log units greater than that of the wild-type parent. This result explains, in part, the attenuation of ompR mutants. However, because ompR mutants display higher oral and intravenous LD₅₀s than the ompF ompC double mutant, it is likely that there are other genes regulated by OmpR which encode proteins involved in virulence.

Traditional programs to design live, attenuated oral vaccines against Salmonella have concentrated on using mutations in the bacterial biochemical pathways or using mutations in global regulators (19). Eliminating global regulators can render a strain avirulent and immunogenic. Inactivating some of the genes regulated by a global regulator should account for some of the avirulence and immunogenicity seen in strains containing a mutation in the global regulator. The ompD gene is regulated by adenylate cyclase and the cyclic AMP regulatory protein (CRP) (16). Strains of S. typhimurium which have cya and crp mutations are avirulent (4). Our goal was to determine if a mutation in ompD could account for some of the avirulence of the cya and crp mutants. In this study, an ompD mutation was transduced into virulent S. typhimurium SL1344 and UK-1. The resultant transductants were compared with their wild-type parents with respect to their abilities to adhere to and invade cells in culture and to colonize tissues and cause disease in BALB/c mice. In contrast to the results of a previous study (6), our results show that ompD mutants are not attenuated, eliminating the possibility that nonexpression of OmpD contributes to the avirulence of cya and crp mutants.

Bacterial strains, media, and phenotypic screens. The bacterial strains used in this study are listed in Table 1. To generate strains specifically for this project, standard P22HTint transductions were performed. Strains were constructed by transducing the ompD::Tn10 mutation from strain BRD455 (6) into virulent S. typhimurium SL1344 strain 3339 (9) and UK-1 strain 3761 (5) to yield strains 8201 and 8202, respectively. Transductants were purified, and the presence of the ompD mutation was verified by examining outer membrane fractions from 8201 and 8202 by protein electrophoresis as described below.

Membrane isolation and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis. Bacterial cells were grown in Lennox broth overnight and then sedimented by centrifugation at 5,000 rpm (Sorvall SS-34 rotor in a Sorvall RC5C centrifuge) at 4°C for 10 min and resuspended in phosphate-buffered saline at pH 7.4. Cells were lysed with a French press at 2,000 psi. After the cellular debris was removed by centrifugation at 6,000 rpm (Sorvall SS-34 rotor in a Sorvall RC5C centrifuge) at 4°C for 10 min, the outer membrane proteins were specifically selected by ultracentrifugation at 36,000 rpm (Sorvall SW41Ti rotor in a Sorvall OTD65B ultracentrifuge) at 4°C for 1 h. The pellet containing both cytoplasmic and outer membrane proteins was resuspended in phosphate-buffered saline containing 0.5% Sarkosyl (sodium lauryl sarcosinate) (Sigma, St. Louis, Mo.), followed by another round of ultracentrifugation at 36,000 rpm at 4°C for 1 h to precipitate the outer membrane fractions. Tris-glycine SDS-polyacrylamide gels (10% acrylamide and 1.5 M Tris [pH 8.8] for the slab gel; 5% acrylamide and 1.0 M Tris [pH 6.8] for the stacking gel) were used to separate the outer membrane proteins. The gels were stained with 0.25% Coomassie blue stain and destained with a solution of 10% glacial acetic acid and 30% methanol.

Virulence assays. The abilities of S. typhimurium mutants to adhere to and invade Intestine-407 (Int-407) cells (10) were analyzed by using a protocol based on a method developed by Galán and Curtiss (8), as described previously (24).

Verification of the ompD mutants. As described above, the ompD::Tn10 mutation was transduced into S. typhimurium SL1344 strain 3339 and S. typhimurium UK-1 strain 3761 to obtain 8201 and 8202, respectively. SDS-PAGE analysis of outer membrane fractions from 8201 and 8202 was performed to verify the absence of the protein band corresponding to OmpD. The results confirm that both 8201 and 8202 strains did not express OmpD (Fig. 1).

View larger version (49K): [in this window] [in a new window]   FIG. 1.   Separation of outer membrane fractions of six strains of S. typhimurium by SDS-10% PAGE. Lane 1, wild-type SL1344 strain 3339; lane 2, ompC mutant BRD454; lane 3, ompD mutant BRD455; lane 4, ompD mutant SL1344 strain 8201; lane 5, wild-type UK-1 strain 3761; lane 6, ompD mutant UK-1 strain 8202.

Adherence and invasion capabilities of the ompD S. typhimurium mutants. Dorman and colleagues (6) reported that a mutation in ompD had only a small effect on the virulence of S. typhimurium SL1344 when BALB/c mice were inoculated perorally. Consequently, the abilities of ompD mutants to adhere to and invade intestinal epithelial cells were compared with those of the wild type. The effect of the ompD mutation was examined in both the virulent SL1344 and UK-1 strain backgrounds. As shown in Table 2, in both SL1344 and UK-1 backgrounds, the ompD mutants show no significant difference from the wild type in either adherence to or invasion of Int-407 cells. This result indicates that the OmpD porin is not involved in either the adherence or invasiveness of S. typhimurium.

Determination of the virulence of wild-type and ompD strains of S. typhimurium with BALB/c mice. As our results showed no effect of the ompD mutation on adherence or invasion, we decided to assay virulence in an animal model. The LD₅₀s of the ompD mutants and their corresponding wild-type parents were determined. This was done to verify the original observation made by Dorman et al. (6) that ompD mutants were somewhat attenuated. Concurrently, we performed colonization studies to determine at which point in the infection process these mutants may be blocked.

Discussion. Dorman and colleagues (6) have reported that an ompD mutant of S. typhimurium SL1344 is less virulent than the wild type, with an oral LD₅₀ about 23-fold higher. LD₅₀s determined in this study show that there is no difference in virulence between ompD mutants of S. typhimurium SL1344 or UK-1 and their corresponding wild-type parents. In support of this conclusion, our colonization data show that there is no significant difference between the abilities of two different wild-type S. typhimurium strains and their respective ompD mutants to colonize or reach the Peyer's patches, intestinal wall, intestinal contents, spleen, or liver. Furthermore, adherence and invasion assays performed with cultured intestinal epithelial cells showed no significant difference between the wild type and ompD mutants of either S. typhimurium UK-1 or SL1344 in their abilities to adhere to or invade host cells.