Strain-specific differences of appearance and numbers of pili-like structures on the surface of C. diphtheriae strains were shown by ultrastructural analyses via atomic force microscopy. Additionally, RNA hybridization and Western blotting experiments revealed distinct differences in the expression patterns of pili subunits for the investigated strains.

To our knowledge, this is the first time that isolate-specific differences in pili formation were characterized. Mandlik and co-workers [13] showed that type AZD2014 mouse III pili length of strain NCTC13129 depends on spaH expression and can be manipulated by deletion or overexpression of spaH. These results are supplemented here by showing that this is a phenomenon which occurs also as natural variation in different C. diphtheriae wild type isolates. Strains ISS4746 and ISS4749 showed the most extended pili structures, an observation which is correlated with high expression of spaA and spaH in these strains, while medium-length pili of DSM43989 are correlated with lack of spaH expression. As mentioned above, it was shown by Ku-0059436 concentration mutant analyses of strain NCTC13129 that expression of spaB and spaC is crucial for adhesion to D562 cells [13]. Natural variations of the spaB and spaC expression patterns observed here indicate that this correlation is not as strict as suggested, since strain ISS4060 shows only low spaB and no spaC expression

but a high adhesion rate, indicating that other

factors are important for adhesion as well and expression of these might differ in various isolates. The lack of any PCR product for spaD, spaE, and spaF and the absence of a SpaD signal in Western blotting experiments suggest that these genes are absent in the investigated strains. All pili-encoding genes of C. diphtheriae are located on pathogenicity islands [20, 21]. Based on the genome sequence of strain NCTC13129, C. diphtheriae possesses 13 of these genomic islands [20, 22] and pili cluster II is located on genomic island CDGI-2, which has a size of 17.5 kb and is located directly adjacent to 36.5 kb pathogenicity island CDGI-1, the tox + corynephage [20]. Data of PCR experiments (not shown) indicate that the pili-encoding genes located on CDGI-2 are missing in all investigated ISS and DSM strains and consequently Acetophenone the genetic repertoire of C. diphtheriae isolates is rather variable. This observation is in agreement with a recent genome survey of C. diphtheriae C7(-) and PW8 strains [23] indicating that 11 of the 13 putative pathogenicity islands of the sequenced reference strain NCTC13129 are absent in the C7(-) strain. The importance of bacterial appendices and surface proteins for host cell contact were also shown recently for a non-fimbrial protein, DIP1281, previously annotated as invasion-associated protein. This protein is a virulence factor involved in cell surface organization, adhesion and internalization in epithelial cells.