Agents Targeting the Bacterial Cell Wall as Tools to Combat Gram-Positive Pathogens.

The cell wall is an indispensable element of bacterial cells and a long-known target of many antibiotics. Penicillin, the first discovered beta-lactam antibiotic inhibiting the synthesis of cell walls, was successfully used to cure many bacterial infections. Unfortunately, pathogens eventually developed resistance to it. This started an arms race, and while novel beta-lactams, either natural or (semi)synthetic, were discovered, soon upon their application, bacteria were developing resistance. Currently, we are facing the threat of losing the race since more and more multidrug-resistant (MDR) pathogens are emerging. Therefore, there is an urgent need for developing novel approaches to combat MDR bacteria. The cell wall is a reasonable candidate for a target as it differentiates not only bacterial and human cells but also has a specific composition unique to various groups of bacteria. This ensures the safety and specificity of novel antibacterial agents that target this structure. Due to the shortage of low-molecular-weight candidates for novel antibiotics, attention was focused on peptides and proteins that possess antibacterial activity. Here, we describe proteinaceous agents of various origins that target bacterial cell wall, including bacteriocins and phage and bacterial lysins, as alternatives to classic antibiotic candidates for antimicrobial drugs. Moreover, advancements in protein chemistry and engineering currently allow for the production of stable, specific, and effective drugs. Finally, we introduce the concept of selective targeting of dangerous pathogens, exemplified by staphylococci, by agents specifically disrupting their cell walls.

Dual-Functional Implant Based on Gellan-Xanthan Hydrogel with Diopside, BMP-2 and Lysostaphin for Bone Defect Repair and Control of Staphylococcal Infection.

A new dual-functional implant based on gellan-xanthan hydrogel with calcium-magnesium silicate ceramic diopside and recombinant lysostaphin and bone morphogenetic protein 2 (BMP-2)-ray is developed. In this composite, BMP-2 is immobilized on microparticles of diopside while lysostaphin is mixed directly into the hydrogel, providing sustained release of BMP-2 to allow gradual bone formation and rapid release of lysostaphin to eliminate infection immediately after implantation. Introduction of diopside of up to 3% (w/v) has a negligible effect on the mechanical properties of the hydrogel but provides a high sorption capacity for BMP-2. The hydrogels show good biocompatibility and antibacterial activity. Lysostaphin released from the implants over a 3 h period efficiently kills planktonic cells and completely destroys 24 h pre-formed biofilms of Staphylococcus aureus. Furthermore, in vivo experiments in a mouse model of critically-sized cranial defects infected with S. aureus show a complete lack of osteogenesis when implants contain only BMP-2, whereas, in the presence of lysostaphin, complete closure of the defect with newly formed mineralized bone tissue is observed. Thus, the new implantable gellan-xanthan hydrogel with diopside and recombinant lysostaphin and BMP-2 shows both osteogenic and antibacterial properties and represents a promising material for the treatment and/or prevention of osteomyelitis after bone trauma.

ABD-3, the confluence of powerful antibacterial modalities: ABDs delivering and expressing lss, the gene encoding lysostaphin.

In response to the antimicrobial resistance crisis, we have developed a powerful and versatile therapeutic platform, the Antibacterial Drone (ABD) system. The ABD consists of a highly mobile staphylococcal pathogenicity island re-purposed to deliver genes encoding antibacterial proteins. The chromosomally located island is induced by a co-resident helper phage, packaged in phage-like particles, and released in very high numbers upon phage-induced lysis. ABD particles specifically adsorb to bacteria causing an infection and deliver their DNA to these bacteria, where the bactericidal cargo genes are expressed, kill the bacteria, and cure the infection. Here, we report a major advance of the system, incorporation of the gene encoding a secreted, bactericidal, species-specific lytic enzyme, lysostsphin. This ABD not only kills the bacterium that has been attacked by the ABD, but also any surrounding bacteria that are sensitive to the lytic enzyme which is released by secretion and by lysis of the doomed cell. So while the killing field is thus expanded, there are no civilian casualties (bacteria that are insensitive to the ABD and its cargo protein(s) are not inadvertently killed). Without amplifying the number of ABD particles (which are not re-packaged), the expression and release of the cargo gene's product dramatically extend the effective reach of the ABD. A cargo gene that encodes a secreted bactericidal protein also enables the treatment of a mixed bacterial infection in which one of the infecting organisms is insensitive to the ABD delivery system but is sensitive to the ABD's secreted cargo protein.

PLGA nanoparticle-encapsulated lysostaphin for the treatment of Staphylococcus aureus infections.

Staphylococcus aureus possesses the ability to become pathogenic, leading to severe and life-threatening infections. Its methicillin-resistant variant MRSA has garnered high-priority status due to its increased morbidity and associated mortality. This emphasizes the urgency for novel anti-staphylococcal agents. The bacteriocin lysostaphin stands out for its remarkable bactericidal activity against S. aureus, including MRSA, outperforming conventional antibiotics. However, the clinical application of lysostaphin faces challenges, including enzymatic activity loss under physiological conditions and potential immunogenicity. This study introduces a novel approach by encapsulating lysostaphin within polylactic-co-glycolic acid (PLGA) nanoparticles, a biodegradable copolymer known for its biocompatibility and sustained drug release ability. The study assesses the antimicrobial activity of lysostaphin-loaded PLGA nanoparticles against different S. aureus strains, and we also used GFP-expressing S. aureus for facilitating its traceability in planktonic, biofilm, and intracellular infection models. The results showed the significant reduction in bacteria viability both in planktonic and biofilm states. The in vitro intracellular infection model demonstrated the significantly enhanced efficiency of the developed nanoparticles compared to the treatment with the free bacteriocin. This research presents lysostaphin encapsulation within PLGA nanoparticles and offers promising avenues for enhancing lysostaphin's therapeutic efficacy against S. aureus infections.

Alginate Gel Encapsulated with Enzybiotics Cocktail Is Effective against Multispecies Biofilms.

The development of new and effective antibacterials for pharmaceutical or cosmetic skin care that have a low potential for the emergence and expansion of bacterial resistance is of high demand in scientific and applied research. Great hopes are placed on alternative agents such as bactericidal peptidoglycan hydrolases, depolymerases, etc. Enzybiotic-based preparations are being studied for the treatment of various infections and, among others, can be used as topical formulations and dressings with protein-polysaccharide complexes. Here, we investigate the antibiofilm properties of a novel enzybiotic cocktail of phage endolysin LysSi3 and bacteriocin lysostaphin, formulated in the alginate gel matrix and its ability to control the opportunistic skin-colonizing bacteria Staphylococcus aureus, Pseudomonas aeruginosa, and Klebsiella pneumoniae, as well as mixed-species biofilms. Our results propose that the application of SiL-gel affects different components of biofilm extracellular polymeric substances, disrupts the matrix, and eliminates the bacteria embedded in it. This composition is highly effective against biofilms composed of Gram-negative and Gram-positive species and does not possess significant cytotoxic effects. Our data form the basis for the development of antibacterial skin care products with a gentle but effective mode of action.

Efficacy of lysostaphin-coated titanium plates on implant-associated MRSA osteitis in minipigs.

PURPOSE: The growing incidence of implant-associated infections (IAIs) caused by biofilm-forming Staphylococcus aureus in combination with an increasing resistance to antibiotics requires new therapeutic strategies. Lysostaphin has been shown to eliminate this biofilm. Own studies confirm the effectiveness in a murine model. The current study characterizes the effects of lysostaphin-coated plates in an IAI minipig model. METHODS: The femur of 30 minipigs was stabilized with a five-hole plate, a bone defect was created, and in 20 cases methicillin-resistant Staphylococcus aureus was applied. Ten animals served as control group. After 14 days, local debridement, lavage, and plate exchange (seven-hole plate) were performed. Ten of the infected minipigs received an uncoated plate and 10 a lysostaphin-coated plate. On day 84, the minipigs were again lavaged, followed by euthanasia. Bacterial load was quantified by colony-forming units (CFU). Immunological response was determined by neutrophils, as well as interleukins. Fracture healing was assessed radiologically. RESULTS: CFU showed significant difference between infected minipigs with an uncoated plate and minipigs with a lysostaphin-coated plate (p = 0.0411). The infection-related excessive callus formation and calcification was significantly greater in the infected animals with an uncoated plate than in animals with a lysostaphin-coated plate (p = 0.0164/p = 0.0033). The analysis of polymorphonuclear neutrophils and interleukins did not reveal any pioneering findings. CONCLUSION: This study confirms the minipig model for examining IAI. Furthermore, coating of plates using lysostaphin could be a promising tool in the therapeutic strategies of IAI. Future studies should focus on coating technology of implants and on translation into a clinical model.

Efficacy of Lysostaphin functionalized silicon catheter for the prevention of Staphylococcus aureus biofilm.

Staphylococcus aureus readily forms biofilms on tissue and indwelling catheter surfaces. These biofilms are resistant to antibiotics. Consequently, effective prevention and treatment strategies against staphylococcal biofilms are actively being pursued over the past two decades. One of the proposed strategies involve the incorporation of antibiotics and antiseptics into catheters, however, a persistent concern regarding the possible emergence of antimicrobial resistance is associated with these medical devices. In this study, we developed two types of silicone catheters: one with Lysostaphin (Lst) adsorbed onto the surface, and the other with Lst functionalized on the surface. To confirm the presence of Lst protein on the catheter surface, we conducted FTIR-ATR and SEM-EDS analysis. Both catheters exhibited hemocompatibility, biocompatibility, and demonstrated antimicrobial and biofilm prevention activities against both methicillin-sensitive and resistant strains of S. aureus. Furthermore, the silicone catheters that were surface-functionalized with Lst showed substantially better and more persistent anti-biofilm effects when compared to the catheters where Lst was surface-adsorbed, both under in vitro static and flow conditions, as well as in vivo in BALB/c mice. These results indicate that surface-functionalized Lst catheters have the potential to serve as a promising new medical device for preventing S. aureus biofilm infections in humans.

Engineered probiotic Lactobacillus plantarum WCSF I for monitoring and treatment of Staphylococcus aureus infection.

IMPORTANCE: Bacterial infection and the emergence of drug-resistant strains are major problems in clinical treatment. Staphylococcus aureus, which typically infects the skin and blood of animals, is also a potential intestinal pathogen that needs to be addressed by the emergence of a new treatment approach. Probiotic therapy is the most likely alternative to antibiotic therapy to solve the problem of bacterial drug resistance in clinical practice. In this study, the engineered Lactobacillus plantarum can not only sense the signal AIP to detect S. aureus but also kill S. aureus by secreting the lysostaphin enzyme. Our strategy employed an Agr quorum-sensing genetic circuit to simultaneously detect and treat pathogenic bacteria, which provided a theoretical possibility for solving practical clinical bacterial infection cases in the future.

Effect of poly (lactic-co-glycolic acid) polymer nanoparticles loaded with vancomycin against Staphylococcus aureus biofilm.

Staphylococcus aureus is a unique challenge for the healthcare system because it can form biofilms, is resistant to the host's immune system, and is resistant to numerous antimicrobial therapies. The aim of this study was to investigate the effect of poly (lactic-co-glycolic acid) (PLGA) polymer nanoparticles loaded with vancomycin and conjugated with lysostaphin (PLGA-VAN-LYS) on inhibiting S. aureus biofilm formation. Nano drug carriers were produced using the double emulsion evaporation process. we examined the physicochemical characteristics of the nanoparticles, including particle size, polydispersity index (PDI), zeta potential, drug loading (DL), entrapment efficiency (EE), Lysostaphin conjugation efficiency (LCE), and shape. The effect of the nano drug carriers on S. aureus strains was evaluated by determining the minimum inhibitory concentration (MIC), conducting biofilm formation inhibition studies, and performing agar well diffusion tests. The average size, PDI, zeta potential, DL, EE, and LCE of PLGA-VAN-LYS were 320.5 ± 35 nm, 0.270 ± 0.012, -19.5 ± 1.3 mV, 16.75 ± 2.5%, 94.62 ± 2.6%, and 37% respectively. Both the agar well diffusion and MIC tests did not show a distinction between vancomycin and the nano drug carriers after 72 h. However, the results of the biofilm analysis demonstrated that the nano drug carrier had a stronger inhibitory effect on biofilm formation compared to the free drug. The use of this technology for treating hospital infections caused by the Staphylococcus bacteria may have favorable effects on staphylococcal infections, considering the efficacy of the nano medicine carrier developed in this study.

Two Recombinant Bacteriocins, Rhamnosin and Lysostaphin, Show Synergistic Anticancer Activity Against Gemcitabine-Resistant Cholangiocarcinoma Cell Lines.

Cholangiocarcinoma (CCA), a bile duct cancer with a high mortality rate, has a poor prognosis due to its highly invasive and drug-resistant phenotypes. More effective and selective therapies are urgently needed. Bacteriocins are broad-spectrum antimicrobial peptides/proteins produced by bacterial strains to compete with other bacteria. Recent studies have reported that bacteriocins exhibit anticancer properties against various cancer cell lines with minimal toxicity toward normal cells. In this study, two types of recombinant bacteriocins, rhamnosin from probiotic Lacticaseibacillus rhamnosus and lysostaphin from Staphylococcus simulans, were highly produced in Escherichia coli and subsequently purified via immobilized-Ni2+ affinity chromatography. When their anticancer activity was investigated against CCA cell lines, both rhamnosin and lysostaphin were found capable of inhibiting the growth of CCA cell lines in a dose-dependent fashion but were less toxic toward a normal cholangiocyte cell line. Rhamnosin and lysostaphin as single treatments could suppress the growth of gemcitabine-resistant cell lines to the same extent as or more than they suppressed the parental counterparts. A combination of both bacteriocins more strongly inhibited growth and enhanced cell apoptosis in both parental and gemcitabine-resistant cells partly through the increased expression of the proapoptotic genes BAX, and caspase-3, -8, and -9. In conclusion, this is the first report to demonstrate an anticancer property of rhamnosin and lysostaphin. Using these bacteriocins as single agents or in combination would be effective against drug-resistant CCA.

In vivo efficiency of the produced recombinant lysostaphin antimicrobial peptide in treatment of methicillin-resistant Staphylococcus aureus (MRSA) skin infection in a mouse model.

Background and Objectives: Staphylococcus simulans secretes an antimicrobial compound called lysostaphin, which has bactericidal properties. It destroys staphylococci through the hydrolysis of peptidoglycan in the cell wall. Therefore, this unique property indicates the high ability of lysostaphin in the treatment of staphylococcal infections and is considered as an anti-staphylococcal agent. Materials and Methods: Escherichia coli BL21 (DE3) competent cells were transformed with pET32a-lysostaphin clone and induced by isopropyl-ß-D-thio-galactoside (IPTG). The recombinant protein was purified by affinity chromatography. Recombinant lysostaphin -A-based ointment was used for external wound healing in animal model. In vivo activity of ointment was evaluated by clinical evidences and cytological microscopic assessment. Results: Our results showed the recombinant protein was produced exactly. The results of checkerboard tests showed MIC, MBC and antibacterial activity test an acute reduction of cell viability during the use of lysostaphin, and SEM results approved the intense wrecking effects of lysostaphin in combination on bacterial cells. Macroscopic findings and microscopic data showed that the recombinant lysostaphin ointment was effective on excisional wound healing. Conclusion: Our findings proved that the recombinant lysostaphin ointment was effective on wound healing due to Staphylococcus aureus infection.

Conserved loop residues-Tyr270 and Asn372 near the catalytic site of the lysostaphin endopeptidase are essential for staphylolytic activity toward pentaglycine binding and catalysis.

Lysostaphin endopeptidase cleaves pentaglycine cross-bridges found in staphylococcal cell-wall peptidoglycans and proves very effective in combatting methicillin-resistant Staphylococcus aureus. Here, we revealed the functional importance of two loop residues, Tyr270 in loop 1 and Asn372 in loop 4, which are highly conserved among the M23 endopeptidase family and are found close to the Zn2+-coordinating active site. Detailed analyses of the binding groove architecture together with protein-ligand docking showed that these two loop residues potentially interact with the docked ligand-pentaglycine. Ala-substituted mutants (Y270A and N372A) were generated and over-expressed in Escherichia coli as a soluble form at levels comparable to the wild type. A drastic decrease in staphylolytic activity against S. aureus was observed for both mutants, suggesting an essential role of the two loop residues in lysostaphin function. Further substitutions with an uncharged polar Gln side-chain revealed that only the Y270Q mutation caused a dramatic reduction in bioactivity. In silico predicting the effect of binding site mutations revealed that all mutations displayed a large ΔΔGbind value, signifying requirements of the two loop residues for efficient binding to pentaglycine. Additionally, MD simulations revealed that Y270A and Y270Q mutations induced large flexibility of the loop 1 region, showing markedly increased RMSF values. Further structural analysis suggested that Tyr270 conceivably participated in the oxyanion stabilization of the enzyme catalysis. Altogether, our present study disclosed that two highly conserved loop residues, loop 1-Tyr270 and loop 4-Asn372, located near the lysostaphin active site are crucially involved in staphylolytic activity toward binding and catalysis of pentaglycine cross-links.

The choice of chromatographic resin for the purification of recombinant lysostaphin affects its activity.

Lysostaphin is a zinc-dependent endopeptidase that is effective against both antibiotic-sensitive and antibiotic-resistant strains of Staphylococcus aureus. Lysostaphin is typically purified on cation-exchange or metal-chelate affinity resins, and there are data indicating potential influence of the chromatographic resin on the lysostaphin activity. In this study, we systematically investigated the impact of the resin used to purify the recombinant lysostaphin on its activity. To this end, recombinant lysostaphin with an additional histidine tag at the C-terminus was purified using a cation-exchange resin, three types of nickel-chelate resins with different strength of metal ion binding, or a zinc-chelate resin. Lysostaphin samples purified on the cation-exchange resin (WorkBeads 40S), the nickel-chelate resin with a strong nickel ion binding (WorkBeads NiMAC), and the zinc-chelate resin (WorkBeads NTA with immobilized zinc ions) had equal activity. On the contrary, the activity of lysostaphin preparations purified on nickel-chelate resins with medium (WorkBeads Ni-NTA) and relatively weak (WorkBeads Ni-IDA) nickel ion binding was significantly reduced. The decrease in activity can be explained by the interaction of lysostaphin with the nickel ions leached from the resin and is caused by either the exchange of the zinc ion in the lysostaphin active center with a nickel ion from the resin, or binding of an additional ion that inhibits the enzymatic activity. Removal of the metal ions from the active site of lysostaphin and subsequent incorporation of the native zinc ions lead to complete restoration of the activity of the enzyme.

Peptidoglycan-Targeting Staphylolytic Enzyme Lysostaphin as a Novel and Efficient Protease toward Glycine-Rich Flexible Peptide Linkers.

Glycine-rich flexible peptide linkers have been widely adopted in fusion protein engineering; however, they can hardly be cleaved for the separation of fusion partners unless specific protease recognition sites are introduced. Herein, we report the use of the peptidoglycan-targeting staphylolytic enzyme lysostaphin to directly digest the glycine-rich flexible linkers of various lengths including oligoglycine linkers and (G4S)x linkers, without the incorporation of extra amino acids. Using His-MBP-linker-LbCpf1 as a model substrate, we show that both types of linkers could be digested by lysostaphin, and the digestion efficiency improved with increasing linker length. The enzyme LbCpf1 retained full activity after tag removal. We further demonstrated that the proteolytic activity of lysostaphin could be well maintained under different environmental conditions and in the presence of a series of chemical reagents at various concentrations that are frequently used in protein purification and stabilization. In addition, such a digestion strategy could also be applied to remove the SUMO domain linked to LwCas13a via an octaglycine linker. This study extends the applications of lysostaphin beyond an antimicrobial reagent and demonstrates its potential as a novel, efficient, and robust protease for protein engineering.

Engineering active lysostaphin variants that incorporate noncanonical amino acids and characterizing the effects of site-specific PEGylation.

We describe a facile strategy to identify sites for the incorporation of noncanonical amino acids into lysostaphin-an enzyme that degrades the cell wall of Staphylococcus aureus-while retaining stapholytic activity. We used this strategy to generate active variants of lysostaphin incorporating para-azidophenylalanine. The incorporation of this "reactive handle" enabled the orthogonal site-specific modification of the enzyme variants with polyethylene glycol (PEG) using copper-free click cycloaddition. PEGylated lysostaphin variants could retain their stapholytic activity, with the extent of retention depending on the site of modification and the PEG molecular weight. The site-specific modification of lysostaphin could be useful not only for PEGylation to improve biocompatibility but also for the incorporation of the enzyme into hydrogels and other biomaterials and for studies of protein structure and dynamics. Moreover, the approach described herein could be readily applied to identify suitable sites for the incorporation of reactive handles into other proteins of interest.

Antibacterial and Anti-Biofilm Properties of Diopside Powder Loaded with Lysostaphin.

BACKGROUND: Diopside-based ceramic is a perspective biocompatible material with numerous potential applications in the field of bone prosthetics. Implantable devices and materials are often prone to colonization and biofilm formation by pathogens such as Staphylococcus aureus, which in the case of bone grafting leads to osteomyelitis, an infectious bone and bone marrow injury. To lower the risk of bacterial colonization, implanted materials can be impregnated with antimicrobials. In this work, we loaded the antibacterial enzyme lysostaphin on diopside powder and studied the antibacterial and antibiofilm properties of such material to probe the utility of this approach for diopside-based prosthetic materials. METHODS: Diopside powder was synthesized by the solid-state method, lysostaphin was loaded on diopside by adsorption, the release of lysostaphin from diopside was monitored by ELISA, and antibacterial and anti-biofilm activity was assessed by standard microbiological procedures. RESULTS AND CONCLUSIONS: Lysostaphin released from diopside powder showed high antibacterial activity against planktonic bacteria and effectively destroyed 24-h staphylococcal biofilms. Diopside-based materials possess a potential for the development of antibacterial bone grafting materials.

Host Cationic Antimicrobial Molecules Inhibit S. aureus Exotoxin Production.

Innate immune molecules, including antimicrobial peptides (for example, defensins) and lysozyme, function to delay or prevent bacterial infections. These molecules are commonly found on mucosal and skin surfaces. Staphylococcus aureus is a common pathogen and causes millions of infections annually. It is well known that innate immune molecules, such as defensins and lysozyme, either poorly inhibit or do not inhibit the growth of S. aureus. Our current studies show that the α-defensin human neutrophil α-defensin-1 (HNP-1) and lysozyme inhibit exotoxin production, both hemolysins and superantigens, which are required for S. aureus infection. HNP-1 inhibited exotoxin production at concentrations as low as 0.001 µg/mL. Lysozyme inhibited exotoxin production at 0.05 to 0.5 µg/mL. Both HNP-1 and lysozyme functioned through at least one two-component system (SrrA/B). The ß-defensin human ß-defensin 1 (HBD-1) inhibited hemolysin but not superantigen production. The cation chelator S100A8/A9 (calprotectin), compared to EDTA, was tested for the ability to inhibit exotoxin production. EDTA at high concentrations inhibited exotoxin production; these were the same concentrations that interfered with staphylococcal growth. S100A8/A9 at the highest concentration tested (10 µg/mL) had no effect on S. aureus growth but enhanced exotoxin production. Lower concentrations had no effect on growth or exotoxin production. Lysostaphin is regularly used to lyse S. aureus. The lytic concentrations of lysostaphin were the only concentrations that also inhibited growth and exotoxin production. Our studies demonstrate that a major activity of innate defensin peptides and lysozyme is inhibition of staphylococcal exotoxin production but not inhibition of growth. IMPORTANCE Staphylococcus aureus causes large numbers of both relatively benign and serious human infections, which are mediated in large part by the organisms' secreted exotoxins. Since 1921, it has been known that lysozyme and, as shown later in the 1900s, other innate immune peptides, including human neutrophil α-defensin-1 (HNP-1) and human ß-defensin 1 (HBD-1), are either not antistaphylococcal or are only weakly inhibitory to growth. Our study confirms those findings but, importantly, shows that at subgrowth inhibitory concentrations, these positively charged innate immune peptides inhibit exotoxin production, including both hemolysins and the superantigen toxic shock syndrome toxin-1. The data show that the principal activity of innate immune peptides in the host is likely to be inhibition of exotoxin production required for staphylococcal mucosal or skin colonization rather than growth inhibition.

Whole-Genome Analysis of Starmerella bacillaris CC-PT4 against MRSA, a Non-Saccharomyces Yeast Isolated from Grape.

Starmerella bacillaris is often isolated from environments associated with grape and winemaking. S. bacillaris has many beneficial properties, including the ability to improve the flavor of wine, the production of beneficial metabolites, and the ability to biocontrol. S. bacillaris CC-PT4 (CGMCC No. 23573) was isolated from grape and can inhibit methicillin-resistant Staphylococcus aureus and adaptability to harsh environments. In this paper, the whole genome of S. bacillaris CC-PT4 was sequenced and bioinformatics analyses were performed. The S. bacillaris CC-PT4 genome was finally assembled into five scaffolds with a genome size of 9.45 Mb and a GC content of 39.5%. It was predicted that the strain contained 4150 protein-coding genes, of which two genes encoded killer toxin and one gene encoded lysostaphin. It also contains genes encoding F1F0-ATPases, Na(+)/H(+) antiporter, cation/H(+) antiporter, ATP-dependent bile acid permease, major facilitator superfamily (MFS) antiporters, and stress response protein, which help S. bacillaris CC-PT4 adapt to bile, acid, and other stressful environments. Proteins related to flocculation and adhesion have also been identified in the S. bacillaris CC-PT4 genome. Predicted by antiSMASH, two secondary metabolite biosynthesis gene clusters were found, and the synthesized metabolites may have antimicrobial effects. Furthermore, S. bacillaris CC-PT4 carried genes associated with pathogenicity and drug resistance. Overall, the whole genome sequencing and analysis of S. bacillaris CC-PT4 in this study provide valuable information for understanding the biological characteristics and further development of this strain.

One fold, many functions-M23 family of peptidoglycan hydrolases.

Bacterial cell walls are the guards of cell integrity. They are composed of peptidoglycan that provides rigidity to sustain internal turgor and ensures isolation from the external environment. In addition, they harbor the enzymatic machinery to secure cell wall modulations needed throughout the bacterial lifespan. The main players in this process are peptidoglycan hydrolases, a large group of enzymes with diverse specificities and different mechanisms of action. They are commonly, but not exclusively, found in prokaryotes. Although in most cases, these enzymes share the same molecular function, namely peptidoglycan hydrolysis, they are leveraged to perform a variety of physiological roles. A well-investigated family of peptidoglycan hydrolases is M23 peptidases, which display a very conserved fold, but their spectrum of lytic action is broad and includes both Gram- positive and Gram- negative bacteria. In this review, we summarize the structural, biochemical, and functional studies concerning the M23 family of peptidases based on literature and complement this knowledge by performing large-scale analyses of available protein sequences. This review has led us to gain new insight into the role of surface charge in the activity of this group of enzymes. We present relevant conclusions drawn from the analysis of available structures and indicate the main structural features that play a crucial role in specificity determination and mechanisms of latency. Our work systematizes the knowledge of the M23 family enzymes in the context of their unique antimicrobial potential against drug-resistant pathogens and presents possibilities to modulate and engineer their features to develop perfect antibacterial weapons.

Lysostaphin: Engineering and Potentiation toward Better Applications.

Lysostaphin is a potent bacteriolytic enzyme with endopeptidase activity against the common pathogen Staphylococcus aureus. By digesting the pentaglycine crossbridge in the cell wall peptidoglycan of S. aureus including the methicillin-resistant strains, lysostaphin initiates rapid lysis of planktonic and sessile cells (biofilms) and has great potential for use in agriculture, food industries, and pharmaceutical industries. In the past few decades, there have been tremendous efforts in potentiating lysostaphin for better applications in these fields, including engineering of the enzyme for higher potency and lower immunogenicity with longer-lasting effects, formulation and immobilization of the enzyme for higher stability and better durability, and recombinant expression for low-cost industrial production and in situ biocontrol. These achievements are extensively reviewed in this article focusing on applications in disease control, food preservation, surface decontamination, and pathogen detection. In addition, some basic properties of lysostaphin that have been controversial and only elucidated recently are summarized, including the substrate-binding properties, the number of zinc-binding sites, the substrate range, and the cleavage site in the pentaglycine crossbridge. Resistance to lysostaphin is also highlighted with a focus on various mechanisms. This article is concluded with a discussion on the limitations and future perspectives for the actual applications of lysostaphin.