New adhesin of enteroaggregative Escherichia coli related to the Afa/Dr/AAF family

Abstract

Enteroaggregative Escherichia coli (EAEC) is an important cause of diarrhea worldwide. We analyzed 17 Danish EAEC strains, isolated in the course of a case control study, for phenotypic and genotypic properties. The strains belonged to at least 14 different serotypes. Using PCR to investigate the prevalence of various putative virulence genes, we found that all but two strains were typical EAEC, as they harbored all or part of the previously described AggR regulon. The majority of the strains harbored genes encoding aggregative adherence fimbriae (AAF). The most common was AAF/I, found in nine strains; eight strains carried no known AAF-related genes. We utilized TnphoA mutagenesis to localize the aggregative adherence (AA) adhesin from one typical EAEC strain, C1010-00, which lacked a known AAF. We identified a TnphoA insertion in a hypothetical Dr-related pilin deposited in GenBank as HdaA. Four additional Danish strains harbored HdaA, and all but one displayed AA to HEp-2 cells. By using PCR primers derived from the pilins and ushers from the three AAF and Hda, we found that 16 of 17 strains exhibited evidence of one of these factors; importantly, the one negative strain also lacked the aggR gene. Cloning of the complete Hda gene cluster and expression in E. coli DH5alpha resulted in AA and complementation of the C1010-00 nonadherent mutant. Four related adhesins have now been found to confer AA in typical EAEC strains; our data suggest that, together, these variants may account for AA in the large majority of strains.

FIG. 1.

Biofilm formation on glass surfaces after 20 h of growth. (A) C1010-00; (B) C1010-00hdaA::phoA; (C) C1010-00hdaA::phoA(pNBO1); (D) DH5α(pNBO1). Scale bars = 50 μm. The inset in panel D shows aggregates of bacteria.

FIG. 2.

The hdaA gene is under the control of AggR. (A) Alkaline phosphatase activity assay. C1010-00hdaA::phoA was complemented with pBADaggR, and the alkaline phosphatase activity was measured under repressing conditions (0.2% glucose) or activating conditions (0.2% arabinose). Shown are the mean absorbances of the results from three independent experiments with error bars representing one standard deviation. (B) RT-PCR for the hdaA transcript. RNA was extracted from EAEC strains C1010-00 (lane 1) and C1010-00aggR::pJP5603 (lane 2) and subjected to reverse transcription and cDNA amplification by PCR for hdaA (top) or the constitutive chloramphenicol acetyltransferase gene (cat; bottom).

FIG. 3.

Annotation of the hda biogenesis gene cluster as determined by nucleotide sequence analysis of the product amplified from strain C1010-00. Gene designations follow from GenBank accession number EU637023. All ORFs encoding >50 predicted amino acids are indicated. The nucleotide positions of the predicted translational start and stop codons are indicated below the line. The inverted triangle indicates the position of the TnphoA insertion into the hdaA gene of C1010-00, which corresponds to nucleotide 378 downstream of the predicted hdaA translational start codon.

FIG. 4.

Quantitation of biofilm formation by Hda-expressing clones. Bacteria were cultivated in DMEM-0.45% glucose for 20 h at 37°C in 24-well dishes. Biofilms were fixed and stained with crystal violet, and then the stains were solubilized and quantitated spectrophotometrically at 470 nm. The bars represent the means of the results from triplicate wells; error bars indicate one standard deviation. 1, C1010-00hdaA::phoA(pNBO1); 2, HB101(pJPN45)(pNBO1); 3, DH5α(pNBO1); 4, C1010-00; 5, HB1010(pJPN45); 6, C1010-00hdaA::phoA; 7, DH5α.

FIG. 5.

Neighbor-joining tree analysis of the predicted pilin protein of the Dr superfamily. Pilin-encoding gene sequences were downloaded from GenBank, translated, and truncated at the sites of proven or predicted signal peptidase cleavage. Derived amino acid sequences were aligned using ClustalW, and a neighbor-joining consensus tree was constructed using Phylip. An unrooted phylogram including weighted branch lengths was plotted using Phylodendron software. Afa variants are derived from urinary tract isolates, DaaE is from diffusely adherent E. coli, Agg and Aaf represent variants from EAEC, and M-agglutinin and AfaE-8 are derived from veterinary isolates.