Heat-Resistant Agglutinin 1 Is an Accessory Enteroaggregative Escherichia coli Colonization Factor

Enteroaggregative Escherichia coli (EAEC) is an important cause of acute and persistent diarrhea. The defining stacked brick adherence pattern of Peruvian EAEC isolate 042 has previously been attributed to aggregative adherence fimbriae II (AAF/II), which confer aggregative adherence on laboratory E. coli strains. EAEC strains also show exceptional autoaggregation and biofilm formation, other phenotypes that have hitherto been ascribed to AAF/II. We report that EAEC 042 carries the heat-resistant agglutinin (hra1) gene, also known as hek, which encodes an outer membrane protein. Like AAF/II, the cloned EAEC 042 hra1 gene product is sufficient to confer autoaggregation, biofilm formation, and aggregative adherence on nonadherent and nonpathogenic laboratory E. coli strains. However, an 042 hra1 deletion mutant is not deficient in these phenotypes compared to the wild type. EAEC strain 042 produces a classic honeycomb or stacked brick pattern of adherence to epithelial cells. Unlike wild-type 042, the hra1 mutant typically does not form a tidy stacked brick pattern on HEp-2 cells in culture, which is definitive for EAEC. Moreover, the hra1 mutant is significantly impaired in the Caenorhabditis elegans slow kill colonization model. Our data suggest that the exceptional colonization of strain 042 is due to multiple factors and that Hra1 is an accessory EAEC colonization factor.Enteroaggregative Escherichia coli (EAEC) was originally identified as the etiologic agent of persistent diarrhea in developing countries but is gaining increasing prominence for its role in a wider spectrum of diarrheal syndromes. EAEC strains have been implicated in acute as well as persistent diarrhea among adults and children (reviewed in references 25 and 40). A recent meta-analysis found that EAEC is significantly associated with disease in every group at high risk for diarrhea, including young children, human immunodeficiency virus-positive individuals, and visitors to developing countries (24). In addition to its association with disease in epidemiological studies in developing countries, EAEC has also been identified as a principal cause of diarrheal disease in Germany, the United Kingdom, and the United States (11, 26, 51).Aggregative adherence is the defining characteristic of EAEC (38). EAEC strains adhere to the intestinal epithelium, and to epithelial cells in culture, in a characteristic two-dimensional “stacked brick” fashion. The pattern features bacteria adhering to the eukaryotic surface, other bacteria, and the solid substratum. Four types of fimbriae have so far been documented as conferring aggregative adherence (4, 14, 17, 37). Two noncontiguous plasmid loci containing the complete complement of genes encoding aggregative adherence fimbriae I (AAF/I) or AAF/II are sufficient to confer aggregative adherence on nonadherent E. coli (14, 49). The plasmid bearing type IV pili found in Serbian EAEC outbreak strain C1096 are also sufficient to confer a weak aggregative adherence phenotype on E. coli K-12 (17). AAF additionally play an essential role in production of a superfluous EAEC-associated biofilm, which could account for the association of these strains with persistent diarrhea in epidemiological studies (46).Some categories of diarrheagenic pathogens have a conserved set of adhesins which allow them to overcome flushing across the intestinal epithelium. Typical enteropathogenic E. coli isolates, for example, all possess bundle-forming pili and the outer membrane adhesin intimin, whereas atypical enteropathogenic E. coli isolates possess intimin but not bundle-forming pili (reviewed in reference 10). EAEC strains, by contrast, are considerably heterogeneous. While many EAEC strains carry genes encoding one of the known aggregative adherence fimbriae, some EAEC do not harbor any known AAF even though they do demonstrate aggregative adherence (4, 7, 13, 14). This, and the presence of multiple adhesins in most mucosal colonizers (53), points to the likelihood of other EAEC adhesins. Imuta et al. recently implicated a TolC secreted factor in adherence (27), and Montiero-Neto et al. (33) described a 58-kDa nonstructural adhesin in O111:H12 EAEC. However, the former factor is only a contributor to aggregative adherence and the latter adhesin is not found in other EAEC. Overall, nonstructural EAEC adhesins have received little attention.The outer membrane protein Tia was originally characterized as an invasin and later shown to confer adhesive properties on enterotoxigenic E. coli (ETEC) (20, 21). Fleckenstein et al. (21) observed that a tia gene probe hybridized to DNA from non-ETEC strains, one of which was EAEC strain 042. As the Southern blot data published by Fleckenstein et al. showed bands of different intensities, as well as size, between ETEC strain {"type":"entrez-nucleotide","attrs":{"text":"H10407","term_id":"875229","term_text":"H10407"}}H10407, which carries tia, and EAEC strain 042, we hypothesized that the probe was recognizing a similar, rather than identical, gene (21).We have determined that EAEC strain 042 harbors a gene encoding the heat-resistant agglutinin 1 (hra1), a hemagglutinin originally reported from an O9:H10:K99 porcine ETEC strain. Hra1 has also been reported from uropathogenic E. coli strains and neonatal meningitis E. coli strain RS218, in which context it is otherwise known as Hek (19, 48). (The hek nomenclature was introduced after hra1, to delineate the form of the gene found in invasive human pathogens from that of a porcine isolate 19].) A role for the outer membrane protein Hra1/Hek in adherence by neonatal meningitis E. coli has recently been defined (19).Although hra1/hek has been reported from multiple pathogens, its role in colonization and virulence has only been conclusively studied in the neonatal meningitis E. coli strain RS218 (19). In this paper, we demonstrate that the EAEC hra1 gene is sufficient to confer colonization-associated phenotypes, including aggregative adherence and biofilm formation, on laboratory E. coli strains. Intriguingly, we find that although it confers these phenotypes on K-12 and is expressed in 042, hra1 is not required for in vitro colonization-associated phenotypes demonstrated by 042. The hra1 gene is, however, essential for the formation of a true stacked brick pattern in EAEC and for optimal in vivo colonization in a Caenorhabditis elegans model.