Contributions of Net Charge on the PlyC Endolysin CHAP Domain

Abstract

Bacteriophage endolysins, enzymes that degrade the bacterial peptidoglycan (PG), have gained an increasing interest as alternative antimicrobial agents, due to their ability to kill antibiotic resistant pathogens efficiently when applied externally as purified proteins. Typical endolysins derived from bacteriophage that infect Gram-positive hosts consist of an N-terminal enzymatically-active domain (EAD) that cleaves covalent bonds in the PG, and a C-terminal cell-binding domain (CBD) that recognizes specific ligands on the surface of the PG. Although CBDs are usually essential for the EADs to access the PG substrate, some EADs possess activity in the absence of CBDs, and a few even display better activity profiles or an extended host spectrum than the full-length endolysin. A current hypothesis suggests a net positive charge on the EAD enables it to reach the negatively charged bacterial surface via ionic interactions in the absence of a CBD. Here, we used the PlyC CHAP domain as a model EAD to further test the hypothesis. We mutated negatively charged surface amino acids of the CHAP domain that are not involved in structured regions to neutral or positively charged amino acids in order to increase the net charge from -3 to a range from +1 to +7. The seven mutant candidates were successfully expressed and purified as soluble proteins. Contrary to the current hypothesis, none of the mutants were more active than wild-type CHAP. Analysis of electrostatic surface potential implies that the surface charge distribution may affect the activity of a positively charged EAD. Thus, we suggest that while charge should continue to be considered for future engineering efforts, it should not be the sole focus of such engineering efforts.

Characterization of an Endolysin Targeting Clostridioides difficile That Affects Spore Outgrowth

“…The secondary structure of cell surface and non-steric cell wall factors may also play a role in preventing access the cell wall [ 35 , 46 ]. Previous studies reported a correlation between the positive charge on lysin’s catalytic domain and its bactericidal activity in the absence of CBD [ 47 , 48 ]. The net positive charge present in CWH 351—656 could interact more efficiently with the negatively charged bacterial surfaces in the absence of CBD.…”

Electrostatic Interaction with the Bacterial Cell Envelope Tunes the Lytic Activity of Two Novel Peptidoglycan Hydrolases

“…The observation of positive charge on the surface of PG hydrolases and a negative one on bacterial CW has for many years been interpreted as a simple mechanism of providing electrostatic interactions that facilitate the attachment of the enzymes to the bacterial surface ( 4 , 5 ). However, recently published results revealed that this relationship is not as simple and straightforward as was previously assumed ( 9 , 49 ).…”

“…In addition, enhanced lytic activity was reported after increasing positive surface net charge of the CW binding module of the Cpl-7 phage lysozyme ( 4 ). However, an increase in the net surface charge of the PlyC catalytic domain (CHAP) from −3 to +7 did not improve the performance of the enzyme, leading to the conclusion that the surface charge cannot be considered the only mechanism that regulates the interactions between the enzyme and the bacterial CW ( 9 ). The change in charge on the surface of both domains of Lss indicated much more complex relationships between charge effect, lysin electrostatics, bacterial targeting, and lysis efficiency ( 49 ).…”

“…The protein surface charge is an important factor for the interactions between the PG hydrolases and the bacterial CW ( 3 – 5 ). A current hypothesis suggests that the positive net surface charge of PG hydrolases allows them to interact with the negatively charged bacterial ( 5 , 6 ) or eukaryotic cells surfaces ( 7 , 8 ); however, other reports have undermined this theory ( 9 ). This issue has been investigated by many research groups and discussed in several reports but their conclusions have been inconsistent, probably because the proteins analyzed were different in other respects (i.e., distribution of the surface charge, electrostatics of the binding groove) except for the net surface charge or because the studied enzymes were artificially engineered ( 9 ).…”

Two New M23 Peptidoglycan Hydrolases With Distinct Net Charge

“…The results revealed that there was a correlation between enzyme activity and bacterial surface charge. However, recently published results do not support Low's hypothesis (Shang and Nelson, 2019), as an engineered increase in surface charge did not result in increased enzyme activity. These discrepancies may be explained by the presence of other regulatory mechanisms/ elements.…”