Salmonella species are known to initiate infection of mammalian hosts by penetrating the intestinal epithelium of the small bowel. These bacteria preferentially interact with Peyer's patches which are collections of lymphoid follicles making up the gut-associated lymphoid tissue. We infected murine ligated intestinal loops with invasive and noninvasive Salmonella typhimurium strains for 30, 60, 120, and 180 min and examined the infected tissue by transmission electron microscopy. Within 30 min, we found that invasive S. typhimurium exclusively entered M cells found within the follicle-associated epithelium (FAE) of the Peyer's patches. Initially, interactions between invasive bacteria and enterocytes adjacent to the M cells were not found. Invasion of M cells was associated with the ability of the bacteria to invade tissue culture cells. S. typhimurium mutants, which were noninvasive for tissue culture cells, could not be found in ligated loops associated with M cells or enterocytes after incubations of 30, 60, 120, or 180 min. At 60 min, internalized invasive S. typhimurium were cytotoxic for the M cells. Destruction of an M cell formed a gap in the FAE which allowed organisms to invade enterocytes adjacent to the dead cell. Later in the infection process (120 and 180 min), the presence of bacteria beneath the FAE correlated with changes in the cytoarchitecture of the lymphoid follicle. In addition, replicating Salmonella began to enter both the apical and basolateral surfaces of enterocytes adjacent to infected M cells.

Salmonella typhimurium penetrate intestinal epithelial cells during infection. In vitro studies reveal that the availability of oxygen during bacterial growth decreases their capacity to adhere to and enter cultured epithelial cells. To identify S. typhimurium genes involved in epithelial cell entry, mutants were selected that entered HEp-2 cells when grown under repressing, aerobic culture conditions. Two types of transposons were used to generate bacterial mutations--transposons that disrupt genes (Tn10 and Tn5) and one transposon (Tn5B50) that, in addition to disrupting genes, can cause constitutive expression of genes from the neo promoter at one end of the transposon. Three classes of mutations were found that increased the ability of aerobically grown S. typhimurium to enter HEp-2 cells. One class of mutations disrupts the che operons and results in a nonchemotactic phenotype. The second class of mutations revealed that defects in rho, which encodes an essential transcription termination factor, result in hyperinvasiveness. The third class of mutations was obtained only from mutagenesis with Tn5B50, suggesting that their increased invasiveness is due to constitutive expression of a gene(s) from the exogenous neo promoter. Analysis of this third class of mutations identified a S. typhimurium locus hil (hyperinvasion locus), which is essential for bacterial entry into epithelial cells. The results suggest that hil encodes an invasion factor or an activator of invasion factor expression. hil maps between srl and mutS near minute 59.5 of the S. typhimurium chromosome, a region adjacent to other loci that have been identified as required for S. typhimurium invasiveness and virulence.

Recent work has demonstrated that Salmonella enterica serovar Typhimurium forms biofilms on HEp-2 tissue culture cells in a type 1 fimbria-dependent manner. To investigate how biofilm growth of HEp-2 tissue culture cells affects gene expression in Salmonella , we compared global gene expression during planktonic growth and biofilm growth. Microarray results indicated that the transcription of ∼100 genes was substantially altered by growth in a biofilm. These genes encode proteins with a wide range of functions, including antibiotic resistance, central metabolism, conjugation, intracellular survival, membrane transport, regulation, and fimbrial biosynthesis. The identification of five fimbrial gene clusters was of particular interest, as we have demonstrated that type 1 fimbriae are required for biofilm formation on HEp-2 cells and murine intestinal epithelium. Mutations in each of these fimbriae were constructed in S. enterica serovar Typhimurium strain BJ2710, and the mutants were found to have various biofilm phenotypes on plastic, HEp-2 cells, and chicken intestinal tissue. The pef and csg mutants were defective for biofilm formation on each of the three surfaces tested, while the lpf mutant exhibited a complete loss of the ability to form a biofilm on chicken intestinal tissue but only an intermediate loss of the ability to form a biofilm on tissue culture cells and plastic surfaces. The bcf mutant displayed increased biofilm formation on both HEp-2 cells and chicken intestinal epithelium, while the sth mutant had no detectable biofilm defects. In all instances, the mutants could be restored to a wild-type phenotype by a plasmid carrying the functional genes. This is the first work to identify the genomic responses of Salmonella to biofilm formation on host cells, and this work highlights the importance of fimbriae in adhering to and adapting to a eukaryotic cell surface. An understanding of these interactions is likely to provide new insights for intervention strategies in Salmonella colonization and infection.

Invasive enteric bacteria must pass through the intestinal epithelium in order to establish infection. It is becoming clear that a common target for intestinal mucosa penetration is the specialized epithelial cell of Peyer’s patches, the M cell. In order to gain a better understanding of how bacteria interact with M cells, we have compared the interactions ofSalmonella typhimurium,Listeria monocytogenes, andShigella flexneriwith M cells by using a murine ligated-loop model. Our results indicate thatS. typhimuriumpossesses a highly efficient mechanism for M cell entry that targets and destroys these cells, whileL. monocytogenesandS. flexneriappear to be internalized into M cells in a less disruptive fashion. Early uptake ofListeriaorShigellainto M cells appeared to lead to the death of some cells, as evidenced by the appearance of holes in the intestinal epithelium. At later time points, the follicle-associated epithelium of animals infected with these bacteria displayed extensive destruction. These data indicate that enteric pathogens use different strategies to interact with M cells and initiate infection of a host.

Recently, we demonstrated thatSalmonella entericaserovar Typhimurium can form biofilm on HEp-2 cells in a type 1 fimbria-dependent manner. Previous work onSalmonellaexopolysaccharide (EPS) in biofilm indicated that the EPS composition can vary based upon the substratum on which the bacterial biofilm forms. We have investigated the role of genes important in the production of colanic acid and cellulose, common components of EPS. A mutation in the colanic acid biosynthetic gene,wcaM, was introduced intoS. entericaserovar Typhimurium strain BJ2710 and was found to disrupt biofilm formation on HEp-2 cells and chicken intestinal tissue, although biofilm formation on a plastic surface was unaffected. Complementation of thewcaMmutant with the functional gene restored the biofilm phenotype observed in the parent strain. A mutation in the putative cellulose biosynthetic gene,yhjN, was found to disrupt biofilm formation on HEp-2 cells and chicken intestinal epithelium, as well as on a plastic surface. Our data indicate thatSalmonellaattachment to, and growth on, eukaryotic cells represent complex interactions that are facilitated by species of EPS.

Conjugate vaccines are among the most effective methods for preventing bacterial infections. However, existing manufacturing approaches limit access to conjugate vaccines due to centralized production and cold chain distribution requirements. To address these limitations, we developed a modular technology for in vitro conjugate vaccine expression (iVAX) in portable, freeze-dried lysates from detoxified, nonpathogenic Escherichia coli. Upon rehydration, iVAX reactions synthesize clinically relevant doses of conjugate vaccines against diverse bacterial pathogens in 1 hour. We show that iVAX-synthesized vaccines against Francisella tularensis subsp. tularensis (type A) strain Schu S4 protected mice from lethal intranasal F. tularensis challenge. The iVAX platform promises to accelerate development of new conjugate vaccines with increased access through refrigeration-independent distribution and portable production.