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Escherichia coli as contaminant of boar semen: role of F1 fimbrial lectins in the spermagglutination phenomena

vrijdag, 22 september, 2006 - 16:00
Campus: Brussels Humanities, Sciences & Engineering campus
Faculteit: Science and Bio-engineering Sciences
L
2.210
Luis Orlando Maroto Martin
doctoraatsverdediging

Escherichia coli is a normal inhabitant of the large intestine of mammals and birds. Some strains however colonize other niches as well and are the causative agents of intestinal and extra-intestinal diseases of man and his livestock. During the last decades, much attention has been paid to E. coli that provoke disease. The research described in this PhD work aimed at identifying and characterizing microorganisms in boar semen that cause agglutination of spermatozoa and to get a better understanding of the underlying mechanisms.

Screening of 115 ejaculates of boars to be used in artificial insemination (AI) revealed that 75% of them were contaminated with E. coli, Proteus spp., Serratia spp., Aerobacter spp., Klebsiella spp., Staphylococcus spp., Streptococcus spp. and Pseudomonas spp., E. coli being the major contaminant. Bacterial contamination of services used in AI was shown to significantly lower the litter size.

Eight E. coli strains, that were isolated from boar semen and that provoked agglutination of sperm cells, were selected for further characterization. All strains agglutinated sperm cells but also yeast cells and rabbit erythrocytes in a mannoside-inhibitable way. Four strains were non-typeable after challenge with a battery of 177 O-antisera, while two strains are serotype O8, one is O105 and one is O146. The latter serotypes are very uncommon in swine but more frequently associated with humans. Most strains were found to be susceptible to most antibiotics, except for streptomycin that is abundantly used in the preparation of the extenders for AI. From pulse field gel electrophoresis it is concluded that the eight strains belong to at least six different lineages.
Seven of the eight strains expressed F1 or F1-like fimbriae, as was revealed by transmission electron microscopy. Attempts to amplify genes that encode the major virulence factors of UPEC (fimbriae: PapG variants, type S and F1C; toxins: CNF1 and Hly; other factors: Afa and Aer) and ETEC (fimbriae: F4, F5, F6, F18 and F41; toxins: LT, STa and STb) were all negative and also the genes for intimin (eae) and capsular protein K1 could not be amplified. Only fimH, the gene that encodes the minor subunit of F1 fimbriae that is responsible for carbohydrate-binding, could be amplified in seven of the eight strains. Taking all these results together, and in view of the fact that the boars did not show any symptoms of disease, it can be hypothesized that the E. coli in the boar semen samples result from contamination introduced during or after semen collection.
Besides fimH, also the genes fimA, fimC, fimD and fimG were amplified, encoding respectively the major fimbrial subunit, the chaperone, the usher and another minor subunit. The strain EcFV5507 that did not express fimbriae but nevertheless agglutinates sperm cells was shown to be devoid of the major subunit FimA forming the shaft of the fimbrial structure. It is supposed that this strain has the adhesin FimH incorporated in the outer membrane.

The agglutination caused by one strain (EcFV5506) is not inhibited by mannose, but only by methyl-╬▒,D-mannopyranoside, though at very high concentration. None of the genes of the F1 operon were amplified in this strain using primers that were able to amplify all genes of the F1 operon in the other seven strains. Protein sequencing of the major subunit confirmed that the fimbriae expressed by the strain EcFV5506 are related to the classical F1 fimbriae and they are referred to as F1-like fimbriae. The major subunit of this strain has a deletion of some 30 amino acids. To the best of our knowledge, this is the first report of a FimA subunit with such an important internal deletion, that nevertheless forms properly folded polypeptides that can successfully build up the shaft of functional fimbriae.

The sequences of FimA, FimC, FimD, FimG and FimH obtained in our study of the sperm agglutinating strains can easily be aligned with sequences availabe from the NCBI Protein Database, though a number of substitutions not known so far were detected.

To unequivocally demonstrate that the sperm agglutination observed is mediated by F1 and only F1 fimbriae, the fimH gene of one strain (EcFV5491) was knocked out by the insertion of the gene that codes for chloramphenicol resistance. The cat gene was inserted in such a way as to replace the complete lectin domain of FimH. Transformants obtained were indeed shown to have completely lost their sperm agglutinating activity.