POSTERS - BLAST X - University of Utah

BLAST X Poster #26
MECHANISM FOR SORTASE LOCALIZATION AND ROLE IN EFFICIENT PILUS ASSEMBLY
IN ENTEROCOCCUS FAECALIS
Kimberly A. Kline*† Andrew L. Kau*, Swaine L. Chen*, Birgitta Henriques-Normark†, Staffan
Normark†, Michael G. Caparon* and Scott J. Hultgren*
*Department of Molecular Microbiology, Washington University School of Medicine, St. Louis,
Missouri 63110; †Department of Microbiology,Tumor and Cell Biology, Karolinska Institutet,
Solna, Sweden
Pathogenic streptococci and enterococci primarily rely on the conserved secretory (Sec)
pathway for the translocation and secretion of virulence factors out of the cell. Since many of
these secreted Gram positive virulence factors are subsequently attached to the bacterial cell
surface via sortase enzymes, we sought to investigate the spatial relationship between
secretion and cell wall attachment in Enterococcus faecalis. We discovered that Sortase A
(SrtA) and Sortase C (SrtC) are co-localized with SecA at single foci in enterococcus. The SrtAprocessed
substrate aggregation substance accumulated in single foci implying a single site of
secretion for these proteins. Furthermore, we show by electron and immunofluorescent
microscopy and immunoblot analysis that in the absence of the pilus polymerizing SrtC, pilin
subunits also accumulate in single foci. Proteins that localized to single foci in E. faecalis were
found to share a positively charged domain flanking a transmembrane helix. Mutation or
deletion of this domain in SrtC abolished both its retention at single foci and its function in
efficient pilus assembly. We conclude that a positively charged domain adjacent to a
transmembrane helix can act as a localization retention signal for the focal
compartmentalization of membrane proteins. Finally, we have examined the localization of the
secretion and sorting machinery and substrates throughout the bacterial cell cycle and present a
model for spatial and temporal organization of the molecular components leading to efficient
pilus biogenesis in E. faecalis.
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