Staphylococcus aureus is a major cause of community- and hospital-acquired diseases.
Infections with antibiotic-resistant MRSA strains are associated with increased mortality. To
identify targets for anti-MRSA therapy, this proposal analyzes the trafficking of surface proteins,
which is essential for S. aureus colonization and invasive disease. Surface proteins are
anchored to the cell wall, a process that requires specific signals and targeting mechanisms.
Sortase A recognizes the LPXTG motif in C-terminal sorting signals, which are cut and
covalently linked to peptidoglycan. Two types of surface protein precursors are distinguished.
Precursors with canonical signal peptides are deposited in the envelope at the cell poles.
Precursors with YSIRKxxxGxxS motif signal peptides traffic to the cross-wall. Confined by
septal membranes during cell division, the cross-wall separates newly divided cells. It
represents the site of de novo peptidoglycan synthesis and is split for completion of the cell
cycle. Here we report that lipoteichoic acid (LTA) is synthesized at septal membranes and
required for septal secretion of YSIRKxxxGxxS precursors. During secretion, the YSIRKx
peptide is removed from the precursor. We screened temperature-sensitive mutants for defects
in YSIRKxxxGxxS precursor secretion and identified variants with mutations in secA, secG and
pepV. secA mutants are defective for the transport of all precursors, polar and septal secretion.
secG and pepV mutants exhibit defects in septal secretion. SecA co-purifies with
YSIRKxxxGxxS precursors and with PepV. We will test the hypothesis that lipid products of LTA
synthesis are critical for the activation of PepV at septal membranes. Although YSIRKxxxGxxS
precursors associate with SecA, we predict that precursor complexes cannot activate SecYEG
translocons unless the YSIRKx peptide is removed. Upon completion of the cross-wall, LtaS,
the catalyst of LTA synthesis, is cut and released while the products of LTA synthesis are
dispersed until the next cell division cycle. The proposal will analyze LtaS variants defective in
LTA synthesis and LtaS cleavage and characterize the contributions of specific lipids and LtaS
regulation for septal secretion. We will characterize YSIRKxxxGxxS precursors bound to SecA
for their ability to activate translocons in the presence of specifc lipids and PepV and study
precursor mutants for their ability to activate SecYEG translocons. Last, we will characterize the
subcellular distribution of PepV, its association with septal lipids, ability to cleave YSIRKxxGxxS
precursors and contribution to S. aureus colonization and pathogenesis.