Analysis of fimbriae from different E.coli isolates causing agglutination of boar sperm cells.

In E. coli, as well as in other pathogenic bacteria, fimbriae allow the adhesion of the bacteria to receptors on the host cell by recognition of specific carbohydrate structures and subsequent colonization of mucosae. Some E. coli are found to produce strong agglutination of sperm cells in humans, but these studies have not been extended to animal models.
Here we show the main aspects in the characterization of Type 1-like fimbriae expressed by E. coli isolates involved in spermagglutination in boars.
Transmission electron microscopy after negative staining of eight randomly collected isolates shows that seven of them express typical Type 1-like appendages and no other hair-like structures could be identified on any of them. All isolates agglutinate boar sperm cells, yeast cells and rabbit erythrocytes, but the inhibition pattern by alpha-D-mannose and methyl-alpha-D-mannopyranoside was found to be slightly different from one isolate to another, especially when compared to that of E. coli strain K514. The latter express exclusively Type 1 fimbriae and were used as control strain in all our studies. Sugars other than those mentioned did not inhibit the agglutinating activity of the eight isolates. One of the E. coli isolates designated FV5506 expresses Type 1-like structures, but shows a different inhibition of agglutination pattern when compared to the other samples. One E. coli isolate named FV5507 did not express any fimbriae but nevertheless agglutinates boar sperm cells, yeast cells and rabbit erythrocytes, pointing to the presence of an adhesin in the outer membrane. The agglutination is also solely inhibited by alpha-D-mannose and methyl-alpha-D-mannopyranoside.
PCR amplification analysis showed that the DNA of E coli FV5506 is not recognized by any of the primers designed to amplify the different genes of the Type 1 cluster. E. coli FV5507 on the other hand express the fimH gene (encoding for the adhesion) but not fimA (encoding for the major subunit). The fimH gene was amplified in all the isolates investigated, except in FV5506. Sequence comparison reveals a high homology amongst the different isolates and also to six E. coli Type 1 sequences deposited in the NCBI protein sequence database. Furthermore, sequence homology is observed to FimH genes from E. coli of bovine, porcine and avian origin. Primers designed to show the presence of other genes of the Type 1 cluster (FimC, FimD and FimG) amplified DNA from our E. coli FV5491, FV5492 and FV5494 isolates. Primers designed for PCR amplification of the whole Type 1 cluster recognized all DNA from our isolates, except E. coli FV5506. DNA from E. coli FV5507 was amplified but the PCR product obtained was smaller, suggesting a deletion of a substantial part of the genome in this isolate.