PB0641 - Alternative cleavage method of LPXTG motif in group A streptococcus by LPXTGase-like protein has a higher selectivity towards aromatic fourth position residues at the sortase A consensus sequence

Background: Group A Streptococcus (GAS) is a human specific gram-positive bacterium responsible for several deadly infections, such as necrotizing fasciitis and streptococcal toxic shock syndrome. Plasminogen-binding group A streptococcal M-like protein (PAM) is the primary protein responsible for binding human plasminogen (hPg) in several Group A streptococcus (GAS) strains and is bound to the cell wall through cleavage and transpeptidation by Sortase A (srtA) interacting with the LPXTG motif.

Aims: Investigating an alternative cleavage site of the LPXTG motif in AP53 GAS cells characteristic of LPXTGase that shows an increased affinity for aromatic residues in the fourth position.

Methods: Peptides with mutations in the fourth position of the LPXTG motif containing FRET partners, Dabcyl and EDANS were synthesized (Dabcyl-LPSXGEAA-EDANS). Tyrosine at X had the lowest affinity for srtA. A mutant strain of AP53 containing PAM protein with a mutated LPSYG was made and tested to determine the levels of PAM within the cell wall and cell membrane. Cell membrane fractions were tested with the peptides to determine cleavage rates and mass spectrometry was performed to determine the cleavage sites for Dabcyl-LPSTGEAA-EDANS and Dabcyl-LPSYGEAA-EDANS.

Results: The LPSYG peptides showed reduced affinity for srtA protein. However, LPSYG-mutated PAM protein was cleaved and transpeptidated at the same level as WT-PAM. Interactions between the peptides and AP53 cell membrane fractions showed that Dabcyl-LPSYGEAA-EDANS was cleaved at rates higher than Dabcyl-LPSTGEAA-EDANS in both wild type and srtA membranes. Mass spectrometry results show an alternative cleavage site for LPSYGEAA.

Conclusion(s): LPSYGEAA was shown to be cleaved at rates far higher than the WT srtA consensus sequence in both the presence and absence of srtA . Mass spectrometry results show that the LPSYGEAA is cleaved at an alternative site C-terminal to the glutamic acid which has been shown to be the cleavage site for another membrane protein in GAS, LPXTGase.