Study On Mechanism Of Efficient Lysis Of Bacterial Cell Wall By Staphylococcus

Staphylococcus aureus, one of the most common human pathogens, causes a wide range of serious infectious diseases, including pneumonia, osteomyelitis, endocarditis, and sepsis. The development of resistance to penicillin, methicillin, and vancomycin has dramatically limited the treatment options for S. aureus infections. There is an urgent need to develop novel antibacterial agents with completely different modes of action.Lysostaphin is a peptidoglycan hydrolase secreted by Staphylococcus simulans. It can specifically lyse Staphylococcus aureus, and is being tested as a novel antibacterial agent. The mature lysostaphin protein (27kDa) contains two domains:an N-terminal catalytic domain (CAT) and a C-terminal bacterial cell wall targeting domain (CWT). The CAT domain cleaves peptide bonds between the third and the fourth glycine residues of pentaglycine cross-bridges, while the CWT domain specifically binds to pentaglycine cross-bridges in cross-linked peptidoglycans of S. aureus. The CAT domain of LytM and CWT domain of ALE-1were structurally determined, but crystallization of the full-length lysostaphin or homologous enzymes has so far been unsuccessful. It remains unclear how the two domains are coordinated together to efficiently cleave bacterial peptidoglycan.In our study, we generated recombinant proteins for lysostaphin and the separated domains, and tested the biochemical activities of the proteins. The lysostaphin CAT domain alone had strong lytic activity against S. aureus in low ionic strength buffer, although the activity was about3-fold lower than that of the full-length lysostaphin. Further analysis showed that the bacteriolytic activity of the CAT domain was dramatically reduced by the presence of50mM NaCl, which had little effect on the full-length lysostaphin. In contrast, the CWT domain alone displayed similar strong binding to S. aureus under150mM NaCl as the full-length lysostaphin protein in our affinity assays using live cells. These results clearly show that CWT-mediated substrate binding is critical for the lytic activity of the CAT domain.Then we employed homology modeling to predict the3D models of the lysostaphin CAT and CWT domains using the crystal structures of the LytM CAT domain (residues185-315)(PDB code:2B13) and the ALE-1CWT domain (residues270-362)(PDB code:1R77) as templates. The two structures share about63%and89%amino-acid sequence similarities respectively with the lysostaphin domains. To predict the structural organization of the CAT and CWT domains in the overall lysostaphin structure, we carried out protein-protein docking using the obtained3D models of the CAT and CWT domains, with the program RosettaDock. Ten thousand docking models were predicted and ranked by the Rosetta energy scores of the CAT-CWT interfaces. The top20models with the lowest Rosetta interface energies were extracted and clustered. However, these docked structures showed the CWT domain distributed all around the CAT domain.Finally, we used hydrogen/deuterium exchange mass spectrometry (H/DX-MS) and site-directed disulfide cross-linking to probe the interface between the lysostaphin catalytic and targeting domains. H/DX-MS mediated comparison of peptides from full-length lysostaphin and the separated domains identified four peptides (amino acids331-339,248-260,454-473, and474-488) of lower solvent accessibility in the full-length protein, suggesting that the protein regions covered by those peptides might be involved in CAT-CWT domain associations. We introduced cysteine-pair substitutions into the two domains and examined the formation of disulfide bonds between those residues. Two cysteine pairs (K332C/T464C and K336C/T464C) engineered within the peptides331-339and454-473could form disulfide bonds, strongly supporting that they could be arranged in close proximity. The cross-linked mutant (K332C/T464C) exhibited a similar binding capacity to S. aureus as the wild-type protein, but significantly lower bacteriolytic activity probably because of restraint in conformation. The diminished activity was further reduced with increasing NaCl concentrations that can cause contractions of bacterial peptidoglycan. The lytic activity, however, could be fully recovered by reducing the disulfide bonds. These results suggest that lysostaphin may require dynamic association of the two domains for coordinating substrate binding and target cleavage on the elastic peptidoglycan.The results from our study will improve our understanding of the bacteriolytic mechanism of lysostaphin. One important goal of our study in the future is to develop site-specific PEGylated lysostaphin as a new antibacterial agent to treat systemic S. aureus infections. The results presented here will help select appropriate PEGylation sites without interfering with the activity of lysostaphin.