We have demonstrated that the IL-8–releasing activity of EAEC 042 can be attributed to a single protein moiety that has a sequence very similar to that of a flagellin from S. dysenteriae. Furthermore, we have shown that flagella from 042 and several other EAEC isolates are extremely potent in releasing IL-8, and that aflagellar 042 loses its IL-8–releasing activity. Finally, EAEC flagellin expressed as a His-tagged fusion protein possesses the same activity. These findings show that the EAEC IL-8–releasing factor is in fact a flagellar protein that interacts with epithelial cells, leading to IL-8 secretion.
Interestingly, whereas flagella have been implicated in the production of inflammation by several other gram-negative pathogens, this activity has not previously been attributed to a single gene product. Pseudomonas aeruginosa
aflagellar mutants demonstrate reduced pathogenicity in the mouse pneumonia model, and purified flagella can themselves produce pulmonary inflammation (12
). In addition, P. aeruginosa
isolates with altered flagellar expression demonstrate reduced ability to release IL-8 from A549 pulmonary epithelial cells (13
). Helicobacter pylori
isolates with natural polymorphisms in flagellar genes induce different amounts of IL-8 release from MKN45 gastric epithelial cells, and IL-8 release correlates with motility (14
). Purified phase I Salmonella
flagellin and Salmonella fliC
expressed in E. coli
each cause release of TNF-α from a human promonocytic cell line (15
). Finally, Salmonella typhi
flagella impair antigen uptake and presentation by human macrophages (16
), and induce a cytokine cascade in these cells (17
Whereas most E. coli
express flagella and are motile, the closely related Shigella
species were until recently thought to be devoid of flagella and constitutively nonmotile. However, Tominaga et al. demonstrated that S. flexneri
and S. sonnei
both possess genes homologous to fliC
that can be expressed in E. coli
to produce flagella (18
). A corresponding sequence for S. dysenteriae
was submitted to GenBank. However, this putative protein has not been described in a publication, nor has any function been ascribed to it.
Flagellar operons are present in all four Shigella
subgroups, but all contain disrupting elements that prevent coordinated expression of intact flagella (19
). Nevertheless, Girón demonstrated that under carefully controlled conditions, Shigella
can in fact be induced to express flagella, but in very low numbers (one flagellum per 300–1,000 organisms), and that they do exhibit some motility in very soft (0.175%) agar (21
). The high degree of homology between the flagellin from EAEC 042 and that from S. dysenteriae
and S. flexneri
raises the intriguing hypothesis that expression of flagellin in vivo may contribute to the severe inflammation characteristic of shigellosis.
A comparison of the predicted amino acid sequence of FliC-EAEC with that of other flagellins reveals substantial homology at the NH2
and COOH termini. This is consistent with the model of flagellin structure proposed by Vonderviszt et al. (22
). In monomeric form, flagellin consists of disordered NH2
- and COOH-terminal regions and three organized domains, labeled G1, G2, and G3. The G3 domain, formed by the NH2
- and COOH-subterminal regions of the molecule, forms a compact structure and is highly conserved among many E. coli
); it presumably is required for proper assembly of the molecule. In contrast, the G1 and G2 domains, which are formed by the middle of the molecule, are hypervariable and presumed to contain the H-antigenic epitopes. The divergence in G1 and G2 domains has been proposed to be driven by antigenic diversity (as a way to avert host response); our work suggests that these domains may confer important and heretofore unrecognized pathogenic phenotypes.
The expression of proinflammatory flagella by EAEC may be an important and novel pathogenic characteristic of these organisms. The phenotype of aggregative adherence, which is pathognomonic of EAEC, is one of several virulence traits identified in these organisms, but is not associated with inflammation. Along with aggregative adherence fimbriae, the large AA plasmid in some isolates carries the genes for Pet, a 104-kDa serine protease enterotoxin and cytotoxin; EAST-1, a small ST-like polypeptide enterotoxin; and several cryptic loci with homology to virulence genes of other bacterial species. None of these genes is required for IL-8 release; plasmid-cured 042 maintains its activity (8
). Virulence traits present on the EAEC 042 chromosome include pic mucinase, which is encoded on the antisense strand in an overlapping configuration with Shigella
enterotoxin 1 (24
), and putative genes for yersiniabactin (25
) and a putative invasion protein, Tia (26
). Although these genes may confer pathogenic phenotypes in vivo, there is no evidence that they are involved in producing inflammation.
Proinflammatory flagella could play an important role in EAEC pathogenesis by inducing epithelial IL-8 production and recruiting neutrophils to the epithelial surface. In other intestinal infections, neutrophil transmigration has been shown to cause fluid secretion and epithelial barrier disruption (27
). This could enhance the effects of other EAEC toxins such as Pet or EAST-1, although whether this occurs in vivo remains to be seen. The presence of IL-8–releasing flagella in diverse EAEC isolates with heterogeneity in other pathogenic genotypes suggests that these flagella may be pathogenic independent of other EAEC toxins. Further studies will be needed to determine the range of effects of proinflammatory flagella on the intestinal epithelium. These findings would in turn lead to a better understanding of how EAEC and other intestinal bacteria produce persistent diarrhea, intestinal inflammation, growth impairment, and other potentially unrecognized manifestations of early childhood infection.