An Engineered Multimodular Enzybiotic against Methicillin-Resistant Staphylococcus aureus

Development of multidrug antibiotic resistance in bacteria is a predicament encountered worldwide. Researchers are in a constant hunt to develop effective antimicrobial agents to counter these dreadful pathogenic bacteria. Here we describe a chimerically engineered multimodular enzybiotic to treat a clinical isolate of methicillin-resistant Staphylococcus aureus (S. aureus). The cell wall binding domain of phage ϕ11 endolysin was replaced with a truncated and more potent cell wall binding domain from a completely unrelated protein from a different phage. The engineered enzybiotic showed strong activity against clinically relevant methicillin-resistant Staphylococcus aureus. In spite of a multimodular peptidoglycan cleaving catalytic domain, the engineered enzybiotic could not exhibit its activity against a veterinary isolate of S. aureus. Our studies point out that novel antimicrobial proteins can be genetically engineered. Moreover, the cell wall binding domain of the engineered protein is indispensable for a strong binding and stability of the proteins.

Potential Application of Phage ɸ11 Lytic Proteins in Rapid Detection and Elimination of Staphylococcus aureus

The increasing antibiotic resistance conferred by Staphylococcus aureus to multiple potential antibiotics has become a serious issue of concern and threat to mankind worldwide. In light of this, phage lytic proteins have been reported which show potential antimicrobial activity against pathogenic microorganisms that could be a promising alternative to antibiotics to eradicate the antibiotic resistant problems. This review discusses the various applications of S. aureus phage lytic proteins and the potentiality of aureophage phi 11 endolysin and virion associated peptidoglycan hydrolase (VAPGH) against staphylococcus strains. Phage Phi11 endolysin harbors two enzymatically active domain; cysteine and histidine-dependent amidohydrolase/peptidase (CHAP) and Amidase 2 at the N-terminus and a cell wall binding domain (CBD) SH3 5 at the C-terminus, while virion associated peptidoglycan hydrolase (VAPGH) has two catalytic domains, CHAP and Glucosaminidase (Mannosyl-glycoprotein endo-beta-N-acetylglucosaminidase) at its N-terminal and C-terminal, respectively.

Characterization of an Endolysin Targeting Clostridioides difficile That Affects Spore Outgrowth

Clostridioides difficile is a spore-forming enteric pathogen causing life-threatening diarrhoea and colitis. Microbial disruption caused by antibiotics has been linked with susceptibility to, and transmission and relapse of, C. difficile infection. Therefore, there is an urgent need for novel therapeutics that are effective in preventing C. difficile growth, spore germination, and outgrowth. In recent years bacteriophage-derived endolysins and their derivatives show promise as a novel class of antibacterial agents. In this study, we recombinantly expressed and characterized a cell wall hydrolase (CWH) lysin from C. difficile phage, phiMMP01. The full-length CWH displayed lytic activity against selected C. difficile strains. However, removing the N-terminal cell wall binding domain, creating CWH351—656, resulted in increased and/or an expanded lytic spectrum of activity. C. difficile specificity was retained versus commensal clostridia and other bacterial species. As expected, the putative cell wall binding domain, CWH1—350, was completely inactive. We also observe the effect of CWH351—656 on preventing C. difficile spore outgrowth. Our results suggest that CWH351—656 has therapeutic potential as an antimicrobial agent against C. difficile infection.

Characterization of Endolysin LyJH307 with Antimicrobial Activity against Streptococcus bovis

Streptococcus bovis (S. bovis) is one of the critical initiators of acute acidosis in ruminants. Therefore, we aimed to develop and characterize the endolysin LyJH307, which can lyse ruminal S. bovis. We tested the bactericidal activity of recombinant LyJH307 against S. bovis JB1 under a range of pH, temperature, NaCl, and metal ion concentrations. In silico analyses showed that LyJH307 has a modular design with a distinct, enzymatically active domain of the NLPC/P60 superfamily at the N-terminal and a cell wall binding domain of the Zoocin A target recognition domain (Zoocin A_TRD) superfamily at the C-terminal. The lytic activity of LyJH307 against S. bovis JB1 was the highest at pH 5.5, and relatively higher under acidic, than under alkaline conditions. LyJH307 activity was also the highest at 39 °C, but was maintained between 25°C and 55°C. LyJH307 bactericidal action was retained under 0-500 mM NaCl. While the activity of LyJH307 significantly decreased on treatment with ethylenediaminetetraacetic acid (EDTA), it was only restored with supplementation of 10 mM Ca2+. Analyses of antimicrobial spectra showed that LyJH307 lysed Lancefield groups D (S. bovis group and Enterococcus faecalis) and H (S. sanguinis) bacteria. Thus, LyJH307 might help to prevent acute ruminal acidosis.

Bacteriophage-Derived Endolysins Applied as Potent Biocontrol Agents to Enhance Food Safety

Endolysins, bacteriophage-encoded enzymes, have emerged as antibacterial agents that can be actively applied in food processing systems as food preservatives to control pathogens and ultimately enhance food safety. Endolysins break down bacterial peptidoglycan structures at the terminal step of the phage reproduction cycle to enable phage progeny release. In particular, endolysin treatment is a novel strategy for controlling antibiotic-resistant bacteria, which are a severe and increasingly frequent problem in the food industry. In addition, endolysins can eliminate biofilms on the surfaces of utensils. Furthermore, the cell wall-binding domain of endolysins can be used as a tool for rapidly detecting pathogens. Research to extend the use of endolysins toward Gram-negative bacteria is now being extensively conducted. This review summarizes the trends in endolysin research to date and discusses the future applications of these enzymes as novel food preservation tools in the field of food safety.

LysPBC2, a Novel Endolysin Harboring aBacillus cereusSpore Binding Domain

ABSTRACTTo control the spore-forming human pathogenBacillus cereus, we isolated and characterized a novel endolysin, LysPBC2, from a newly isolatedB. cereusphage, PBC2. Compared to the narrow host range of phage PBC2, LysPBC2 showed very broad lytic activity against allBacillus,Listeria, andClostridiumspecies tested. In addition to a catalytic domain and a cell wall binding domain, LysPBC2 has a spore binding domain (SBD) partially overlapping its catalytic domain, which specifically binds toB. cereusspores but not to vegetative cells ofB. cereus. Both immunogold electron microscopy and a binding assay indicated that the SBD binds the external region of the spore cortex layer. Several amino acid residues required for catalytic or spore binding activity of LysPBC2 were determined by mutagenesis studies. Interestingly, LysPBC2 derivatives with impaired spore binding activity showed an increased lytic activity against vegetative cells ofB. cereuscompared with that of wild-type LysPBC2. Further biochemical studies revealed that these LysPBC2 derivatives have lower thermal stability, suggesting a stabilizing role of SBD in LysPBC2 structure.IMPORTANCEBacteriophages produce highly evolved lytic enzymes, called endolysins, to lyse peptidoglycan and release their progeny from bacterial cells. Due to their potent lytic activity and specificity, the use of endolysins has gained increasing attention as a natural alternative to antibiotics. Since most endolysins from Gram-positive-bacterium-infecting phages have a modular structure, understanding the function of each domain is crucial to make effective endolysin-based therapeutics. Here, we report the functional and biochemical characterization of aBacillus cereusphage endolysin, LysPBC2, which has an unusual spore binding domain and a cell wall binding domain. A single point mutation in the spore binding domain greatly enhanced the lytic activity of endolysin at the cost of reduced thermostability. This work contributes to the understanding of the role of each domain in LysPBC2 and will provide insight for the rational design of efficient antimicrobials or diagnostic tools for controllingB. cereus.