| Fimbriae are proteinaceous hair-like structures
that mediate the adhesion of bacteria to host tissue surfaces.
Due to their high hydrophobicity, fimbrial proteins are
considered notoriously difficult to analyze and are not
amenable to standard proteomics techniques such as LC-MS.
As a result, hydrophobic fimbriae have not been well-studied
so far. However, mass spectrometry is the predominant
proteomics technique to characterize proteins, their variants
and post-translational modifications. Therefore, the focus
of our study was the development of adequate LC-MS and
LC-MS/MS techniques that enabled us to characterize fimbriae
of gram negative and gram positive bacteria.
After isolation of the fimbriae, dissociated fimbria
were analyzed directly by LC-MS using a mixture of 5%
hexafluoroisopropanol in acetonitrile as the organic
modifier. In addition, fimbrial subunits were chemically
and/or enzymatically cleaved and analyzed by LC-MS/MS
to characterize different variants. Undissociated fimbria
of gram positive bacteria were analyzed with a combined
chemical and enzymatic cleavage approach. The fragments
were characterized by LC-MS/MS using a QTOF 2 mass spectrometer.
The gram negative model organism studied was enterotoxicogenic
E. coli. A novel LC-ESI-MS technique that uses a 5%
addition of HFIP to the organic phase kept highly hydrophobic
proteins in solution and allowed us to determine the
molecular weight of more than twenty different fimbrial
subunits. E coli fimbriae and closely related variants
have been extensively characterized, mostly through
chemical cleavage or enzymatic digestion.
Fimbriae of the gram positive bacteria Actinomyces
naeslundii contribute to the pathogenesis of caries.
These fimbriae form highly stable structures on the
bacterial surface that are resistant to standard digestion
methods. With a combination of chemical and enzymatic
cleavages, we were able to cover more than 95% of the
503 amino acid that comprise FimP, the major subunit
of A. naeslundii fimbriae and a variety of cross-links
and post-translational modifications. The present investigation
provides the first evidence of post-translational modifications
of A. naeslundii subunits during fimbrial biogenesis.
Taken together, an unexpected number of new subunits
and structural features of fimbrial membrane proteins
was identified. They further our understanding of microbial
infection and the potential for intervention. |
