We have previously shown that most EAEC strains in our collection harbor a partially conserved virulence plasmid (designated pAA), which typically encodes the transcriptional activator AggR. Here, we show that aggR is part of a highly conserved two-gene cluster, which also encodes a novel low–molecular weight secreted protein that mediates dispersal of EAEC on the colonic mucosa. In recognition of this phenotype, we have herein designated this gene aap (anti-aggregation protein). We now further suggest the more descriptive name “dispersin” for the aap gene product. Our data suggest that dispersin is secreted to the environment, but that it remains noncovalently associated with the bacterial cell surface.
Several roles for dispersin in adherence and colonization are plausible. All enteric pathogens must adhere to the epithelial surface. But at the same time, it may be beneficial to negatively modulate adherence to permit dispersal across the epithelial surface. Knutton et al. have shown that the bundle-forming pilus of enteropathogenic E. Coli
(EPEC) mediates both aggregation and dispersal, which occur sequentially (27
). In this way, EPEC can adhere and multiply early in the pathogenetic sequence, thereby establishing a foothold on the epithelium, but the bacteria then disperse individual progeny, which are free to establish new foci of infection. This paradigm is intuitively beneficial and therefore may be a property of many other mucosal pathogens. Indeed, Benitez et al. have suggested that the Hap mucinase of Vibrio cholerae
may serve as an enzymatic “detachase,” mutation of which results in increased density of bacterial colonization but also attenuation of virulence (28
We also hypothesized that dispersin may play a role in penetration of the intestinal mucous blanket, expecting that large aggregates would be retarded in translocation through a viscous gel. Indeed, using a purified mucin gel column assay, we showed that the dispersin mutant penetrated more slowly than the wild-type parent. The human colonic mucosa is covered with a continuous mucous gel approaching 1 mm in thickness (29
), which provides a formidable barrier against colonic pathogens. Single bacteria may be more adept at swimming through this layer; alternatively, biophysical characteristics conferred by the dispersin protein coat could promote mucous layer penetration. Whatever their precise roles, Aap, Hap, and some component of bundle-forming pili are apparently the first examples of bacterial “dispersins,” which may facilitate efficient colonization via bacterial dispersal. Thorough elucidation of the roles of these dispersins in vivo has not yet been accomplished, because of the limited availability of good whole-animal models, but is clearly worthy of investigation.
We can propose one potential mechanism by which dispersin may promote dispersal. AAF fimbriae are highly hydrophobic (10
), and this hydrophobicity would favor strong autoagglutination in an aqueous environment. The predicted amino sequence of dispersin is more hydrophilic than that of AAF/II, and thus the presence of dispersin on the surface of the bacterial cell may ameliorate surface hydrophobicity. Scanning electron microscopy data are consistent with this hypothesis. Absence of dispersin results in striking alteration in the morphology of the AAF fimbriae. In the absence of dispersin, the fimbriae cling to the surface of the bacterial cell, although their number and diameter do not appear changed. This would imply that the surface of the outer membrane under the dispersin coat is largely hydrophobic; such hydrophobicity could be mediated by outer membrane proteins, or perhaps by other hydrophobic surface structures previously described in EAEC (30
Notably, despite the fact that aap is coregulated with AAF adhesins and that dispersin has C-terminal characteristics consistent with secretion by the usher-chaperone pathway, we found that mutants in either the usher or the chaperone secreted normal levels of dispersin. Thus, the mechanism by which dispersin is translocated across the outer membrane is as yet unknown. We found it interesting that AAF mutants bound less dispersin protein in the presence of Triton X-100. Immunofluorescence microscopy experiments suggest that some dispersin is bound within AAF-mediated aggregates (not shown), providing one possible explanation for this observation.
The requirement for AggR in aap
expression is highly significant. AggR has been shown to be required for expression of AAF biogenesis genes (31
), yet there are many strains that carry aggR
and do not have an identified AAF adhesin (6
). Our data thus suggest that AggR acts as a more versatile regulator of virulence genes in EAEC. Indeed, recent data from our laboratory suggest that AggR activates additional unlinked genes on the pAA plasmid and the EAEC chromosome (J. Nishi et al., unpublished results; and E. Dudley et al., unpublished results). Additional characterization of the AggR regulon and its role in EAEC pathogenesis is underway. Coexpression of AAF and dispersin in a common AggR regulon is consistent with an interactive role of the two factors in colonization, but it is interesting that the bacterium would use the same regulator to express both an adhesin and a negative modulator of adhesion.
EAEC is an emerging pathogen, but vaccine development is in its earliest stages. Several obstacles must be overcome before a candidate vaccine can be constructed. Chief among these obstacles is the antigenic heterogeneity of EAEC strains. In addition, given that EAEC infection is a serious health concern predominantly in developing countries, there is little economic incentive toward the development of a vaccine. Therefore, the best approach to EAEC vaccine development would be to identify a highly conserved, highly prevalent, surface-exposed and immunogenic protein that could be expressed in an attenuated vector vaccine. The ideal vector would be one engineered to provide protection against another, perhaps more serious threat (such as shigellosis or typhoid fever). Dispersin fulfills many of these criteria, and experiments are underway to test its feasibility as an EAEC immunogen.