EAEC pathogenesis

Página 1

The pathogenesis of EAEC is really complex as strains are relatively heterogeneous. The best-studied virulence factor is AggR, the master regulator of EAEC virulence, which controls expression of adherence factors, a dispersin protein, and a large cluster of genes encoded on the EAEC chromosome [41]. EAEC pathogenesis involves three stages: (1) adherence to the intestinal mucosa by aggregative adherence fimbriae (AAF) and adherence factors, (2) increased production of mucus that encrusts EAEC on the surface of enterocytes; and (3) release of toxins and elicitation of an inflammatory response, mucosal toxicity, and intestinal secretion [28, 41].

AggR regulates the expression of a secreted low-molecular weight protein known as dispersin (aap) which has been identified in 80% of EAEC isolates from one laboratory [54]. Dispersin is responsible for mediating an antiaggregation phenotype by inducing changes in the outer membrane polarization of the bacterial cell, which requires an ABC transporter system encoded by the att [55]. In a volunteer challenge study, dispersin has been demonstrated to be highly immunogenic, suggesting that it is a potential vaccine candidate [56]. Unlike dispersin, a separate transporter located in the outer membrane protein (OMP) called TolC, which is encoded by the AatA and has also been associated with the secretion of a yet to be characterized factor that contributes to aggregation [57]. Multidrug resistance efflux pumps including the AcrAB-TolC system have been reported to be associated with the colonization and persistence of bacteria in the host and to have roles in bacterial pathogenicity [58, 59]. One study suggested that strains possessing CVD432 and EAST-1 virulence markers are most commonly associated with chronic diarrhea in children [60]; whereas another study suggested that strains possessing aggR, aap, and astA (which encodes for EAST-1 protein) are most commonly associated with acute diarrhea in adults [61].

EAEC adherence to the intestinal mucosa requires fimbrial structures called aggregative adherence factors (AAF) [62]. Although three fimbriae encoded by the pAA plasmid are aggA (AAF/I), aafA (AAF/II), and agg-3(AAF/III), each EAEC isolate carries only one AAF subtype. aggA is responsible for the aggregative phenotype and human erythrocyte haemagglutination of EAEC [63]. aafA allows EAEC to adhere to the intestinal mucosa [64]. agg-3 functions as an adhesin [65]. Three membrane-associated proteins (MAP), of 18, 20 and 58 kDa, are believed to play an important role in EAEC adherence to and haemagglutination of animal cells [66]. One study has characterized the OMP profiles of EAEC strains from children with diarrhea from Sao Paulo, Brazil, and has observed a heterogeneity in OMP profiles [66]. The binding of EAEC fimbriae to the extracellular matrix proteins fibronectin, laminin, and type IV collagen is an initial step in adherence to the intestinal mucosa [67].

Certain strains carry a high molecular weight plasmid associated with the aggregative adherence on which a number of virulence genes are located [68]. Furthermore, a serine protease involved in the colonization process (Pic) is encoded by the chromosome from strain 042 [30, 69]. Other virulence factors that are believed to be associated with EAEC are 18 and 30 kDa outer membrane adhesins [70, 71]. Recently, two de novo OMP of sizes 41 kDa and 48 kDa demonstrated significant cross reactivity with known adhesins were observed in EAEC at pH 4.0 [72, 73]. However, some EAEC strains with “stacked-brick” aggregative adherence lack AAF, suggesting the presence of other adhesion mechanisms. A novel aggregative adhesion pilin has recently been demonstrated in EAEC strains lacking known AAF. This novel adhesion pilin is encoded by hdaA, also regulated by the AggR regulon [74], is distantly related to the Dr family of adhesins. Undoubtedly, other potential AAF and adherence factors exist, and need to be explored.

In addition, different pathogenicity islands have been identified within the EAEC group, including Shigella shepathogenicity island, containing enterotoxin and mucinase genes [69], and Yersinia high-pathogenicity island, containing the yersiniabactin siderophore gene [75]. pheU pathogenicity island in the chromosome of EAEC is regulated by AggR [76]. A few EAEC carry the haemolysin-pyleonephritis associated pili island associated with extraintestinal E coli and show a cell-detaching-aggregative phenotype on HEp-2 cells [77]. EAEC strains frequently have toxin genes located on the chromosome, where genes involved in iron capture, such as irp2 and fyuA, were also found [78]. A pathogenicity island, the locus for proteolysis activity, is also present in some EAEC [79]. Recently, a novel secretion system, called type VI secretion (T6S) system (T6SS) present on the pheU pathogenicity island with two copies of T6S gene clusters, called sci-1 and sci-2 has been reported in EAEC [80]. sciN is a critical gene necessary for T6S-dependent secretion of the Hcp-like SciD protein and for biofilm formation [80].

The second stage of EAEC pathogenesis involves adherence to the mucosa characterized by the presence of a thick, aggregating biofilm [48]. Animal and in vitro culture studies demonstrate that EAEC survives within the mucus layer, explaining why individuals infected with EAEC, especially children in developing countries with pre-existent malnutrition, may develop mucoid stools, malnutrition, and persistent colonization with prolonged diarrhea. However, the mucus layer and biofilm possibly do not explain malnutrition in affected children. This is because, for the biofilm to impair nutrient absorption, it would have to cover most of the small intestinal mucosa, but there is no evidence that this actually occurs. It is more likely that inflammatory responses or altered intestinal microbiota are primarily responsible. One study has identified biofilm production in 48 of 62 (77%) EAEC strains from Japanese children with diarrhea, using a quantitative biofilm assay, suggesting that this assay may be a useful and convenient screening tool for EAEC [48]. Recently, Pic protease has been reported in intestinal colonization and growth of EAEC [81]. The production of biofilm is also regulated by AggR and requires several genes, including Fis, which codes for a DNA-binding protein involved in growth regulation, and yafK, which codes for a 28-kDa protein [82]. EilA, a HilA-like regulator, and air, encoding the predicted OMP in EAEC chromosome, are associated with biofilm formation [83]. Recently, a gene coding for the 32.8-kDa Shf protein has been localized in one of the three open reading frames between aafC and aatA, and has also been implicated in biofilm formation [84]. However, loss of biofilm formation and diffuse adherence pattern was observed in EAEC at pH 4.0 whereas at pH 7.4, typical aggregative adherence pattern was observed (Figure 1) [72, 73].