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Lysostaphin is a potent bacteriolytic enzyme with endopeptidase activity against the common pathogen

Multifunctional antimicrobial strategies are urgently needed to treat methicillin-resistant Staphylococcus aureus (MRSA) caused pneumonia due to its increasing resistance, enhanced virulence, and high pathogenicity. Here, we report that lysostaphin, a bacteriolytic enzyme, encapsulated within poly(lactic-co-glycolic acid) microspheres (LyIR@MS) specially treats planktonic MRSA bacteria, mature biofilms, and related pneumonia. Optimized LyIR@MS with suitable diameters could deliver a sufficient amount of lysostaphin to the lung without a decrease in survival rate after intravenous injection. Furthermore, the degradable properties of the carrier make it safe for targeted release of lysostaphin to eliminate MRSA, repressing the expression of virulence genes and improving the sensitivity of biofilms to host neutrophils. In the MRSA pneumonia mouse model, treatment or prophylaxis with LyIR@MS significantly improved survival rate and relieved inflammatory injury without introducing adverse events. These findings suggest the clinical translational potential of LyIR@MS for the treatment of MRSA-infected lung diseases.

Background Antibiotic tolerance allows bacteria to overcome antibiotic treatment transiently and potentially accelerates the emergence of resistance. However, our understanding of antibiotic tolerance at the genetic level during adaptive evolution of Staphylococcus aureus remains incomplete. We sought to identify the mutated genes and verify the role of these genes in the formation of vancomycin tolerance in S. aureus. Methods Vancomycin-susceptible S. aureus strain Newman was used to induce vancomycin-tolerant isolates in vitro by cyclic exposure under a high concentration of vancomycin (20× MIC). WGS and Sanger sequencing were performed to identify the genetic mutations. The function of mutated genes in vancomycin-tolerant isolates were verified by gene complementation. Other phenotypes of vancomycin-tolerant isolates were also determined, including mutation frequency, autolysis, lysostaphin susceptibility, cell wall thickness and cross-tolerance. Results A series of vancomycin-tolerant S. aureus (VTSA) strains were isolated and 18 mutated genes were identified by WGS. Among these genes, pbp4, htrA, stp1, pth and NWMN_1068 were confirmed to play roles in VTSA formation. Mutation of mutL promoted the emergence of VTSA. All VTSA showed no changes in growth phenotype. Instead, they exhibited reduced autolysis, decreased lysostaphin susceptibility and thickened cell walls. In addition, all VTSA strains were cross-tolerant to antibiotics targeting cell wall synthesis but not to quinolones and lipopeptides. Conclusions Our results demonstrate that genetic mutations are responsible for emergence of phenotypic tolerance and formation of vancomycin tolerance may lie in cell wall changes in S. aureus.

Background: Mature lysostaphin (~28-kDa Lss) from Staphylococcus simulans proves effective in killing methicillin-resistant Staphylococcus aureus (MRSA) which is endemic in hospitals worldwide. Lss is Zn2+-dependent endopeptidase, but its bacteriolytic activity could be affected by exogenously added Zn2+. Objective: To gain greater insights into structural and functional impacts of Zn2+and Ni2+on Lss-induced bioactivity. Methods: Lss purified via immobilized metal ion-affinity chromatography was assessed for bioactivity using turbidity reduction assays. Conformational change of metal ion-treated Lss was examined by circular dichroism and intrinsic fluorescence spectroscopy. Co-sedimentation assay was performed to study interactions between Zn2+-treated Lss and S. aureus peptidoglycans. Metal ionbinding prediction and intermolecular docking were used to locate an extraneous Zn2+-binding site. Results: A drastic decrease in Lss bioactivity against S. aureus and MRSA was revealed only when treated with Zn2+, but not Ni2+, albeit no negative effect of diethyldithiocarbamate—Zn2+-chelator on Lss-induced bioactivity. No severe conformational change was observed for Lss incubated with exogenous Zn2+ or Ni2+. Lss pre-treated with Zn2+ efficiently bound to S. aureus cell-wall peptidoglycans, suggesting non-interfering effect of exogenous metal ions on cell-wall targeting (CWT) activity. In silico analysis revealed that exogenous Zn2+, but not Ni2+, preferably interacted with a potential extraneous Zn2+-binding site (His253, Glu318 and His323) placed near the Zn2+-coordinating Lssactive site within the catalytic (CAT) domain. Conclusion: Our present data signify the adverse influence of exogenous Zn2+ ions on Lss-induced staphylolytic activity through the exclusive presence within the CAT domain of an extraneous inhibitory Zn2+-binding site, without affecting the CWT activity.

The bacteriolysin lysostaphin (Lst) and endolysin PlyPH are potent modular lytic enzymes with activity against clinically-relevant Gram-positive Staphylococcus aureus and Bacillus cereus, respectively. Both enzymes possess an N-terminal catalytic domain and C-terminal binding domain, with the latter conferring significant enzyme specificity. Lst and PlyPH show reduced activity in the presence of bacterial growth-supporting conditions, such as complex media. Here, we hypothesize that Lst and PlyPH bind poorly to their targets in growth media, which may influence their use in antimicrobial applications in the food industry, as therapeutics, and for control of microbial communities. To this end, binding of isolated Lst and PlyPH binding domains to target bacteria was quantified in the presence of three increasingly complex media - phosphate buffered saline (PBS), defined growth medium (AAM) and undefined complex medium (TSB) by surface plasmon resonance (SPR) and flow cytometry. Evaluation of binding kinetics by SPR demonstrated that PlyPH binding was particularly sensitive to medium composition, with 8-fold lower association and 3.4-fold lower dissociation rate constants to B. cereus in TSB compared to PBS. Flow cytometry studies indicated a decrease in the binding-dependent fluorescent populations of S. aureus and B. cereus, for lysostaphin binding domain and PlyPH binding domain, respectively, in TSB compared to PBS. Enzyme binding behavior was consistent with the enzymes' catalytic activity in the three media, thereby suggesting that compromised enzyme binding could be responsible for poor activity in more complex growth media.

Staphylococcus aureus, an opportunistic pathogen, causes diverse community and nosocomial-acquired human infections, including folliculitis, impetigo, sepsis, septic arthritis, endocarditis, osteomyelitis, implant-associated biofilm infections and contagious mastitis in cattle. In recent days, both methicillin-sensitive and methicillin-resistant S. aureus infections have increased. Highly effective anti-staphylococcal agents are urgently required. Lysostaphin is a 27 kDa zinc metallo antimicrobial lytic enzyme that is produced by Staphylococcus simulans biovar staphylolyticus and was first discovered in the 1960s. Lysostaphin is highly active against S. aureus strains irrespective of their drug-resistant patterns with a minimum inhibitory concentration of ranges between 0·001 and 0·064 μg ml-1 . Lysostaphin has activity against both dividing and non-dividing S. aureus cells; and can seep through the extracellular matrix to kill the biofilm embedded S. aureus. In spite of having excellent anti-staphylococcal activity, its clinical application is hindered because of its immunogenicity and reduced bio-availability. Extensive research with lysostaphin lead to the development of several engineered lysostaphin derivatives with reduced immunogenicity and increased serum half-life. Therapeutic efficacy of both native and engineered lysostaphin derivatives was studied by several research groups. This review provides an overview of the therapeutic applications of native and engineered lysostaphin derivatives developed to eradicate S. aureus infections.

Intracellular methicillin-resistant Staphylococcus aureus (MRSA) is extremely difficult to remove by common antibiotics, leading to infection recurrence and resistance. Herein we report a novel exosome-based antibiotic delivery platform for eradicating intracellular MRSA, where mannosylated exosome (MExos) is employed as the drug carrier and preferentially taken up by macrophages, delivering lysostaphin (MExoL) and vancomycin (MExoV) to intracellular pathogens. Combination of MExoL and MExoV eradicated intracellular quiescent MRSA. Moreover, MExos rapidly accumulated in mouse liver and spleen, the target organs of intracellular MRSA, after intravenous (IV) administration. Thus, the MExos antibiotic delivery platform is a promising strategy for combating intracellular infection.

Lysostaphin is a glycylglycine endopeptidase, secreted by Staphylococcus simulans, capable of specifically hydrolyzing pentaglycine crosslinks present in the peptidoglycan of the Staphylococcus aureus cell wall. In this paper, we describe the cloning and expression of the lysostaphin enzyme gene in Bacillus subtilis WB600 host using pWB980 expression system. Plasmid pACK1 of S. simulans was extracted using the alkaline lysis method. Lysostaphin gene was isolated by PCR and cloned into pTZ57R/T-Vector, then transformed into Escherichia coli DH5α. The amplified gene fragment and uncloned pWB980 vector were digested using PstI and XbaІ enzymes and purified. The restricted gene fragment was ligated into the pWB980 expression vector by the standard protocols, then the recombinant plasmid was transformed into B. subtilis WB600 using electroporation method. The recombinant protein was evaluated by the SDS-PAGE method and confirmed by western immunoblot. Analysis of the target protein showed a band corresponding to 27-kDa r-lysostaphin. Protein content was estimated 91 mg/L by Bradford assay. The recombinant lysostaphin represented 90% of its maximum activity at 40 °C and displayed good thermostability by keeping about 80% of its maximum activity at 45 °C. Heat residual activity assay of recombinant lysostaphin demonstrated that the enzyme stability was up to 40 °C and showed good stability at 40 °C for 16 h incubation.

The effect of enzymatic and physical modifications of the surface of two different strains from lactic acid bacteria, Lactobacillus rhamnosus (LGG) and Lactobacillus delbruekii subs. lactis ATCC 4797 (LBD), to stabilize medium-chain triglyceride (MCT) oil based Pickering emulsions were investigated. A section of cell wall degrading enzymes, lysozyme from chicken egg white and human, lysostaphin, mutanolysin from Streptomyces globisporus and proteinase k and the hydrophobic protein zein were used for enzymatic and physical surface modifications. Cell surface modifications were characterized by optical microscopy, scanning electron cryo-microscopy (Cryo-SEM), transmission electron microscopy (TEM), microbial adhesion to hexadecane (MATH) test and zeta potential measurements. The modified cell hydrophobicity in terms of MATH values were increased (around four times) by the enzymatic and physical modifications for LBD and LGG compared to the control. Emulsions stabilized by modified bacterial cells showed higher stability in comparison with unmodified samples, especially for the samples modified with chicken egg lysozyme. Confocal microscopy revealed that the modified bacterial cells were absorbed at the interface between oil and water and preventing the oil particles from coalescence. Thus, modified bacterial cells can be used to formulate food-grade stable Pickering emulsions. Such Pickering emulsions can potentially be clean label alternatives to replace the conventional emulsion preparations.

Peptidoglycan-degrading enzymes are a group of proteins intensively studied as novel antibacterials, with some of them having reached pre-clinical and clinical stages of research. Many peptidoglycan-degrading enzymes have modular organization and consist of a catalytic and a cell wall binding domain. This property has been exploited in enzyme engineering efforts, and many new peptidoglycan-degrading enzymes were generated through domain exchange. However, rational combination of domains from different enzymes is still challenging since relative contribution of every domain to the cumulative bacteriolytic activity is not yet clearly understood. In this work, we investigated the influence of ionic strength and pH on the catalytic efficiency and cell binding of peptidoglycan-degrading enzyme lysostaphin and how this influence is reflected in the lysostaphin bacteriolytic activity. Contrary to generally accepted view, lysostaphin domains are not completely independent and their combination within one protein leads to increased bacteriolytic activity with increasing NaCl concentration, despite both catalysis and cell binding being inhibited by NaCl. This effect is likely mediated by changes in conformation of bacterial cell wall peptidoglycan rather than the physical inter-domain interaction. KEY POINTS: • NaCl enhances bacteriolytic activity of lysostaphin but not of its catalytic domain. • Catalytic activity and cell binding of lysostaphin are inhibited by NaCl. • Peptidoglycan conformation likely affects lysostaphin bacteriolytic activity.

Bacterial cell walls represent one of the most prominent targets of antibacterial agents. These agents include natural products (e.g., vancomycin) and proteins stemming from the innate immune system (e.g., peptidoglycan-recognition proteins and lysostaphin). Among bacterial pathogens that infect humans, Staphylococcus aureus (S. aureus) continues to impose a tremendous healthcare burden across the globe. S. aureus has evolved countermeasures that can directly restrict the accessibility of innate immune proteins, effectively protecting itself from threats that target key cell well components. We recently described a novel assay that directly reports on the accessibility of molecules to the peptidoglycan layer within the bacterial cell wall of S. aureus. The assay relies on site-specific chemical remodeling of the peptidoglycan with a biorthogonal handle. Here, we disclose the application of our assay to a screen of a nonredundant transposon mutant library for susceptibility of the peptidoglycan layer with the goal of identifying genes that contribute to the control of cell surface accessibility. We discovered several genes that resulted in higher accessibility levels to the peptidoglycan layer and showed that these genes modulate sensitivity to lysostaphin. These results indicate that this assay platform can be leveraged to gain further insight into the biology of bacterial cell surfaces.

Lytic enzymes are novel antimicrobial agents that degrade bacterial cell walls, resulting in cell rupture and death. We tested one enzyme, the bacteriocin lysostaphin, for treatment of nonhuman primates (Macaca mulatta) with persistent methicillinresistant Staphylococcus aureus (MRSA) infection of their cranial implant margins. The goal of this study was to determine if topical lysostaphin, either alone or as an adjunct therapy, could eliminate MRSA. Lysostaphin had in vitro lytic activity against all 4 previously identified NHP MRSA clones, as well as against 12 MRSA isolates of the same clonal type (MLST ST3862 and spa type t4167) before and after treatment, with no resistance discovered. In an in vivo pilot study, a 2-d application of lysostaphin alone reduced MRSA in the implant margins by 3-logs during treatment of one animal; however, MRSA titers had returned to control levels by 1 wk after treatment. In the main study, all animals (n = 4) received 10 d of systemic antibiotic treatment and both the animals and their environment (cages, equipment, room) underwent 5-d of decontamination. The experimental animals (n = 2) received 5 doses of topical lysostaphin (15 mg, every other day) applied onto their implant margins. Daily cultures showed that MRSA counts decreased significantly (≤ 25 colony-forming units/mL; P < 0.05). However, sampling of the cranial implant margin 7 d after last treatment showed that MRSA counts had returned to control levels. Our study suggests that lysostaphin, coupled with other treatment modalities, can decrease MRSA infection short-term but do not completely eradicate MRSA in the long-term. This reappearance of MRSA may be due to cross-contamination or reinfection from other infected areas, an inability of the treatment to reach all colonized areas, or insufficient dosing or length of treatment. Topical lysostaphin may be more useful clinically for superficial nonimplant associated wounds in which the lytic enzyme has better access to the infected tissue.

Lysostaphin is a protein zinc metalloproteinase, extracted from Staphylococcus simulans, which disrupting peptide layer of S. aureus. In this study, Lysostaphin gene was detected in the S. simulans isolates. The molecular weight of the Lysostaphin gene is 750 bp. We were used the pET-32a(+) plasmid to cloning lysostaphin gene which transformed to competent rubidium chloride E. coli DH5α for producing the lysostaphin protein. The lysostaphin protein from this gene which isolated from S. simulans, then used the expression of used to killed S. aureus, which has the thick layer of wall that is the very difficult bacteria response to treatment. The result was reported succeeded pET-32a (+) plasmid to expressed lysostaphin gene and gave lysostaphin protein with high quality and quantity. As well as the result was appeared the high accuracy of his tag method in protein extraction and purification, and the quality and quantity more than other studies.

The increasing prevalence of multidrug resistant bacteria has warranted the search for new antimicrobial agents as existing antibiotics lose their potency. Among these, bacteriophage therapy, as well as the administration of specific bacteriolysis agents, i.e., lytic enzymes, have emerged as attractive alternatives. Nebulizers offer the possibility for delivering these therapeutics directly to the lung, which is particularly advantageous as a non-invasive and direct route to treat bacterial lung infections. Nevertheless, nebulizers can often result in significant degradation of the bacteriophage or protein, both structurally and functionally, due to the large stresses the aerosolization process imposes on these entities. In this work, we assess the capability of a novel low-cost and portable hybrid surface and bulk acoustic wave platform (HYDRA) to nebulize a Myoviridae bacteriophage (phage K) and lytic enzyme (lysostaphin) that specifically targets Staphylococcus aureus. Besides its efficiency in producing phage or protein-laden aerosols within the 1–5 μ m respirable range for optimum delivery to the lower respiratory tract where lung infections commonly take place, we observe that the HYDRA platform—owing to the efficiency of driving the aerosolization process at relatively low powers and high frequencies (approximately 10 MHz)—does not result in appreciable denaturation of the phages or proteins, such that the loss of antimicrobial activity following nebulization is minimized. Specifically, a low (0.1 log 10 (pfu/ml)) titer loss was obtained with the phages, resulting in a high viable respirable fraction of approximately 90%. Similarly, minimal loss of antimicrobial activity was obtained with lysostaphin upon nebulization wherein its minimum inhibitory concentration (0.5 μ g/ml) remained unaltered as compared with the non-nebulized control. These results therefore demonstrate the potential of the HYDRA nebulization platform as a promising strategy for pulmonary administration of alternative antimicrobial agents to antibiotics for the treatment of lung diseases caused by pathogenic bacteria.

Extraction and purification of ribonucleic acid (RNA) from Gram-positive methicillin resistant Staphylococcus aureus (MRSA) is problematic, because the MRSA has a rigid cell wall that contains lipoteichoic acid and peptidoglycan, thus causing difficulty when utilizing the standard methods. For this reason, the aim of the current study was to improve and modify the method of extraction of RNA from MRSA, with good integrity, purity, low cost, and with saved time of extraction. A fast and an inexpensive method involving the use of acid phenol: chloroform (5: 1 [v/v]) at low pH (4.5), with lysostaphin and Triton X-100 for effective isolation of RNA from the MRSA is developed. As a result of this study, yields of this method presented high concentration of RNA 1175.26 ng/ µl/ 3 ml of bacterial culture broth, with high RNA integration number (RIN). In similar assays such as using; the RNeasy Mini kit, GeneJET RNA purification kit, TRIzol kit and hot phenol: chloroform (1: 1 [v/v]) extraction method, they yielded low concentrations of RNA (92-700 ng/ µl); with lower purity, quantity, and also little integrity, compared to using the current acid phenol chloroform (5: 1 [v/v]) extraction method. In conclusion, this new method for extraction of RNA from MRSA can be used to save time, cost, and provide high quality of RNA.

Here, «host-vector» expression system of Brevibacillus choshinensis was developed and used for producing a recombinant lysostaphin with high-output. The recombinant plasmid pNCMO2/lsf12 was constructed, and its expression in Brevibacillus choshinensis (strain Brevibacillus choshinensis/pNCMO2/lsf12) provided a synthesis of the 27-kDa protein, which was secreted into the culture medium. Its specific staphylolitic activity being 557 U/mg at optimal pH (7.5-8.0) and temperature (50-55 °C) values was comparable with the natural and recombinant analogs. We hope that developed methods of a deep cultivation of the recombinant Brevibacillus choshinensis/pNCMO2/lsf12 strain for a high-yield production (up to 90 mg/L) and a single-stage purification of lysostaphin (up to 90% homogeneity) become the basis for the production of the enzyme on an industrial scale. Brevibacillus choshinensis, ion-exchange chromatography, lysostaphin The work was financially supported by the Grant No. 050 of Rospotrebnadzor «Monitoring of borreliosis pathogens circulation in regions of the Russian Federation and improvement of diagnostic tools for borreliosis»

Mature lysostaphin (mLst) is a glycineglycine endopeptidase, capable of specifically cleaving penta-glycine crosslinker in the peptidoglycan of Staphylococcus aureus cell wall. It is a very effective therapeutic enzyme to kill the multidrug-resistant S. aureus often encountered in hospital acquired infections. Fusing cellulose binding domain (CBD) to mLst significantly reduced the insoluble expression of mLst in E. coli. Employing mLst-cleavable peptides as fusion linkers leaded to an effective self-cleavage expression that CBD and mLst could be completely cleaved off from the fusions during the expression process. The presence of residue linker fragment at N-terminus of the cleaved-off mLst strongly inhibited the cell lytic activity of the recovered recombinant mLst, and only ~ 50% of the wild-type mLst activity could be retained. Intact CBD-Lst fusions were obtained when uncleavable peptide linkers were employed. With CBD at N-terminus of mLst, the intact fusion completely lost its cell lytic activity but the dipeptidase activity still remained. In contrast, approximately 10% cell lytic activity of mLst still could be maintained for the fusion with CBD at C-terminus of mLst. KEY POINTS: • CBD fusion enhanced soluble expression of recombinant lysostaphin. • In vivo self-cleavage of fusion linkers by the expressed lysostaphin fusions. • Self-cleaved lysostaphin fusions retain most of dipeptidase but lose 50% cell lytic activity.

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.

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.

Multifunctional antimicrobial strategies are urgently needed to treat methicillin-resistant

Staphylococcus aureus expresses virulence factors to trigger its internalization into eukaryote cells and to survive inside different subcellular compartments. This paper describes an enzyme protection assay to study the extent of S. aureus internalization and its intracellular survival in adherent non-professional phagocytic cells (NPPCs) as well as the intracellular efficacy of antimicrobial compounds. NPPCs are grown in a multi-well plate until they reach 100% confluence. S. aureus cultures are grown overnight in cell culture medium. The bacterial suspension is diluted according to the number of cells per well to inoculate the cells at a controlled multiplicity of infection. Inoculated cells are incubated for 2 h to allow the bacteria to be internalized by the NPPCs, following which lysostaphin is added to the culture medium to selectively kill extracellular bacteria. Lysostaphin is present in the culture medium for the rest of the experiment. At this point, the infected cells could be incubated with antimicrobial compounds to assess their intracellular activities against S. aureus. Next, the cells are washed three times to remove the drugs, and intracellular S. aureus load is then quantified by culturing on agar plates. Alternatively, for studying staphylococcal virulence factors involved in intracellular survival and cell toxicity, lysostaphin could be inactivated with proteinase K to eliminate the need for washing steps. This tip improves the reliability of the intracellular bacterial load quantification, especially if cells tend to detach from the culture plate when they become heavily infected because of the multiplication of intracellular S. aureus. These protocols can be used with virtually all types of adherent NPPCs and with 3D cell culture models such as organoids.

Bacterial cell walls represent one of the most prominent targets of antibacterial agents. These agents include natural products (e.g., vancomycin) and proteins stemming from the innate immune system (e.g., peptidoglycan-recognition proteins and lysostaphin). Among bacterial pathogens that infect humans,Staphylococcus aureus(S. aureus) continues to impose a tremendous healthcare burden across the globe.S. aureushas evolved countermeasures that can directly restrict the accessibility of innate immune proteins, effectively protecting itself from threats that target key cell well components. We recently described a novel assay that directly reports on the accessibility of molecules to the peptidoglycan layer within the bacterial cell wall ofS. aureus. The assay relies on site-specific chemical remodeling of the peptidoglycan with a biorthogonal handle. Here, we disclose the application of our assay to a screen of a nonredundant transposon mutant library for susceptibility of the peptidoglycan layer with the goal of identifying genes that contribute to the control of cell surface accessibility. We discovered several genes that resulted in higher accessibility levels to the peptidoglycan layer and showed that these genes modulate sensitivity to lysostaphin. These results indicate that this assay platform can be leveraged to gain further insight into the biology of bacterial cell surfaces.Table of Contents Figure

The growing incidence of multidrug-resistant bacterial strains presents a major challenge in modern medicine. Antibiotic resistance is often exhibited by Staphylococcus aureus, which causes severe infections in human and animal hosts and leads to significant economic losses. Antimicrobial agents with enzymatic activity (enzybiotics) and phage therapy represent promising and effective alternatives to classic antibiotics. However, new tools are needed to study phage–bacteria interactions and bacterial lysis with high resolution and in real-time. Here, we introduce a method for studying the lysis of S. aureus at the single-cell level in real-time using atomic force microscopy (AFM) in liquid. We demonstrate the ability of the method to monitor the effect of the enzyme lysostaphin on S. aureus and the lytic action of the Podoviridae phage P68. AFM allowed the topographic and biomechanical properties of individual bacterial cells to be monitored at high resolution over the course of their lysis, under near-physiological conditions. Changes in the stiffness of S. aureus cells during lysis were studied by analyzing force–distance curves to determine Young's modulus. This allowed observing a progressive decline in cellular stiffness corresponding to the disintegration of the cell envelope. The AFM experiments were complemented by surface plasmon resonance (SPR) experiments that provided information on the kinetics of phage-bacterium binding and the subsequent lytic processes. This approach forms the foundation of an innovative framework for studying the lysis of individual bacteria that may facilitate the further development of phage therapy.

Purpose Staphylococcus aureus is the most common persistent pathogen in humans, so development of new formulations to combat pathogen invasion is quite necessary. Methods In the current study, for the first time, the synergistic activity of recombinant lysostaphin and LL-37 peptide was studied against S. aureus. Moreover, different niosomal formulations of the peptide and protein were prepared and analyzed in terms of size, shape, zeta potential, and entrapment efficiency. Also, a long-term antibacterial activity of the best niosomal formulation and free forms was measured against S. aureus in vitro. Results The optimal niosomal formulation was obtained by mixing the surfactants (span60 and tween60; 2:1 w/w), cholesterol, and dicetylphosphate at a ratio of 47:47:6, respectively. They showed uniform spherical shapes with the size of 565 and 325 nm for lysostaphin and LL-37, respectively. This formulation showed high entrapment efficiency for the peptide, protein, and a slow-release profile over time. Release kinetic was best fitted by Higuchi model indicating a diffusion-based release of the drugs. The lysostaphin/LL-37 niosomal formulation synergistically inhibited growth of S. aureus for up to 72 hours. However, the same amounts of free forms of both anti-microbial agents could not hold the anti-microbial effect and growth was seen in the following 72 hours. Cytotoxicity assay specified that lysostaphin/LL-37 niosomal combination had no deleterious effect on normal fibroblast cells at effective antimicrobial concentrations. Conclusion This study indicated that the use of lysostaphin in combination with LL-37, either in niosomal or free forms, synergistically inhibited growth of S. aureus in vitro. In addition, niosomal preparation of antimicrobial agents could provide a long-term protection against bacterial infections.

Lysostaphin is a bacteriolytic enzyme targeting peptidoglycan, the essential component of the bacterial cell envelope. It displays a very potent and specific activity towards staphylococci, including methicillin-resistant Staphylococcus aureus (MRSA). Lysostaphin causes rapid cell lysis and disrupts biofilms, and is therefore a therapeutic agent of choice to eradicate staphylococcal infections. The C-terminal SH3b domain of lysostaphin recognizes peptidoglycans containing a pentaglycine crossbridge and has been proposed to drive the preferential digestion of staphylococcal cell walls. Here, we elucidate the molecular mechanism underpinning recognition of staphylococcal peptidoglycan by the lysostaphin SH3b domain. We show that the pentaglycine crossbridge and the peptide stem are recognized by two independent binding sites located on opposite sides of the SH3b domain, thereby inducing a clustering of SH3b domains. We propose that this unusual binding mechanism allows a synergistic and structurally dynamic recognition of S. aureus peptidoglycan and underpins the potent bacteriolytic activity of this enzyme.

Introduction: Staphylococcus aureus is a source of nosocomial infections and one of the significant concerns in patients with indwelling devices. Lysostaphin is a bacterially produced endopeptidase with a unique activity on S. aureus. Plasma, the fourth state of the material, consists of charged ions, free electrons, and activated neutral species. Biomedical applications of cold plasma are rapidly growing due to its capacity to treat heat-sensitive objects such as polymeric materials and biological samples. It activates surfaces by etching them to stabilize proteins. The direct effect of cold atmospheric plasma on the eradication of microorganisms have been investigated. However, there is no report on immobilizing antibiotic agents. Methods: In this study, the lysostaphin protein was expressed and purified using Ni-NTA column, then the purified enzyme was immobilized on glass surfaces pretreated with cold atmospheric plasma for 150 s, 200 s, and 300 s. The antimicrobial activity of immobilized lysostaphin on S. aureus was approved by in vitro analysis. Results: The 300 s plasma treatment confirmed to be the best time arrangement for more lysostaphin immobilization, shown by Atomic Force Microscopy. Conclusion: Our results showed that passive adsorption to the treated surface does not affect the structure and subsequent antimicrobial function of the recombinant protein compared to the standard proteins.

Accurate and rapid identification of Staphylococcus aureus (S. aureus) is of great significance for controlling the food poisoning and infectious diseases caused by S. aureus. In this study, a novel strategy that combines lysin cell-binding domain (CBD)-based magnetic separation with fluorescence detection was developed for the specific and sensitive quantification of S. aureus in authentic samples. The S. aureus cells were separated from the sample matrix by lysin CBD-functionalized magnetic beads. Following lysis by lysostaphin, intracellular catalase was released from S. aureus cells and detected by a fluorometric system composed of horseradish peroxidase (HRP), hydrogen peroxide (H2O2), and Amplex Red. S. aureus was quantified via the inhibitory effect of the released intracellular catalase on the fluorometric system since the catalase could decompose the H2O2. Optimized conditions afforded a calibration curve for S. aureus ranging from 1.0 × 102 to 1.0 × 107 CFU mL−1. The detection limit was as low as 78 CFU mL−1 in phosphate-buffered saline (PBS), and the total detection process could be completed in less than 50 min. Other bacteria associated with common food-borne and nosocomial infections negligibly interfered with S. aureus detection, except for Staphylococcus epidermidis, which may have slightly interfered. Moreover, the potential of this proposed method for practical applications has been demonstrated by detection assays of sterilized milk and human serum.

Bacteriolytic enzymes (cell lytic enzymes) are promising alternatives to antibiotics especially in killing drug-resistant bacteria. However, some bacteria slowly become resistant to various classes of peptidoglycan hydrolases, for reasons not well studied, in the presence of growth-supporting nutrients, which are prevalent at sites of infection. Here, we show that Staphylococcus aureus, a human and animal pathogen, while susceptible to the potent staphylolytic enzyme lysostaphin (Lst) in buffered saline, is highly resistant in the rich medium tryptic soy broth (TSB). Through a series of biochemical analysis, we identified that the resistance was due to prevention of Lst-cell binding mediated by the wall teichoic acids (WTAs) present on the cell surface. Inhibition or deletion of the gene tarO responsible for the first step of WTA biosynthesis greatly reduced S. aureus resistance to Lst in TSB. To overcome the resistance, we took advantage of the gene regulation potential of CRISPR-dCas9 and demonstrated that downregulation of tarO, tarH, and/or tarG gene expression, the latter two encoding enzymes that anchor WTAs in the outer layer of cell wall peptidoglycan, sensitized S. aureus to Lst and enabled eradication of the bacterium in TSB in 24 hr. As a result, we elucidate a key mechanism of Lst resistance in metabolically active S. aureus and provide a potential approach for treating life-threatening or hard-to-treat infections caused by Gram-positive pathogens.

Background and aim: Methicillin-resistant Staphylococcus aureus (MRSA) is one of the most common causes of surgical infection, and its resistance to numerous conventional antibiotics makes treatment difficult. Although vancomycin is often an effective agent for the initial therapy of MRSA, clinical failure sometimes occurs. Therefore, there is an urgent need to develop better therapies. Here, we prepared some vancomycin-loaded nanoliposomes coupled with anti-staphylococcal protein (lysostaphin) and evaluated their in vitro and in vivo efficacy as a topical MRSA therapy. Methods: Vancomycin was encapsulated in liposomes, and the coupling of lysostaphin with the surface of liposomes was carried out through cyanuric functional groups. The bactericidal efficacies and a full characterization were evaluated. To define different nanoliposomal– bacterium interactions and their bactericidal effect, flow cytometry was employed. Finally, in vivo, the topical antibacterial activity of each formulation was measured against surgical wound MRSA infection in a mouse model. Results: High encapsulation and conjugation efficiency were achieved for all formulations. All the formulations showed a significant reduction in bacterial counts (p

Mastitis is the disease that most affects dairy cattle with losses above US$ 2 billion per year in the United States alone. It frequently presents bacterial origin, with Staphylococcus aureus (S. aureus) standing out as a pathogen challenging to eliminate because of the high resistance to antimicrobials. Antimicrobial therapy often demonstrates failure, with low cure rates, bacterial resistance and bacterial seclusion in the outbreaks of infection as well as leaving its residues in soil, water, and even animal products. Advances in research may provide benefits to animal welfare by increasing cow's resistance to mastitis by inducing mammary gland cells to secrete an antibacterial protein called lysostaphin, which is a potent staphylocolytic enzyme. Over the years, research groups have developed projects aimed at developing particular immunomodulators, as well as transgenic lysostaphin-secreting cows. The focus of this review is to compile studies on the use of lysostaphin and in the therapeutic and prophylactic control of staphylococcal mastitis using genetic engineering and biotechnology as an alternative tool. In the transgenic models of mice and cows, lysostaphin was able to prevent staphylococcal mastitis presenting little effect on the integrity of the mammary gland, animal physiology and milk produced. Further studies should be performed not only related to cases of prevention of staphylococcal mastitis, but also in the treatment and maintenance of the long-term action of lysostaphin. Keywords: Staphylococcus aureus, Biotechnology, rLYS, Animal bioreactors

Purpose. The purpose of the present study was to determine the relatedness of Staphylococcus aureus strains successively isolated over a 7-day period from a single bacteraemic patient undergoing antibiotic treatment with vancomycin. Methods. The S. aureus strains had been isolated and sequenced previously. Antibiotic susceptibility testing, population analysis profiling, and lysostaphin sensitivity and phagocytic killing assays were used to characterize these clonal isolates. Results. The seven isolates (MEH1–MEH7) were determined to belong to a common multilocus sequence type (MLST) and spa type. Within the third and fifth day of vancomycin treatment, mutations were observed in the vraS and rpsU genes, respectively. Population analysis profiles revealed that the initial isolate (MEH1) was vancomycin-susceptible S. aureus (VSSA), while those isolated on day 7 were mostly heteroresistant vancomycin-intermediate S. aureus (hVISA). Supporting these findings, MEH7 was also observed to be slower in growth, to have an increase in cell wall width and to have reduced sensitivity to lysostaphin, all characteristics of VISA and hVISA strains. In addition, MEH7, although phagocytosed at numbers comparable to the initial isolate, MEH1, survived in higher numbers in RAW 264.7 macrophages. Macrophages infected with MEH7 also released more TNF-α and IFN-1β. Conclusion. We report an increasing resistance to vancomycin coupled with daptomycin that occurred within approximately 3 days of receiving vancomycin and steadily increased until the infection was cleared with an alternative antibiotic therapy. This study reiterates the need for rapid, efficient and accurate detection of hVISA and VISA infections, especially in high-bacterial load, metastatic infections like bacteraemia.

Growing antimicrobial resistance and biofilm infection resilience have led researchers to study the potential laying in antimicrobial combinations, including those encompassing enzymes with biofilm disrupting abilities. Therefore, this work set out to evaluate the undergone journey of antimicrobial enzyme combination research and gain insights into its current status and most promising leads. Expert curators annotated and analysed all published experimental data on enzyme-encompassing combinations for two major biofilm-forming pathogens, Pseudomonas aeruginosa and Staphylococcus aureus. This entailed the construction of the first publically accessible online database on antimicrobial enzyme combinations, the Antimicrobial Enzyme Combinations Database (www.ceb.uminho.pt/aecd). Gathered data was also reconstructed as knowledge-networks to help analyse and visualize annotated entities (e.g. enzymes, methods, strains, combination outputs). The database currently holds 122 and 206 annotated combinations for P. aeruginosa and S. aureus, respectively, and their analysis allowed a systematic review of the available evidence on enzyme combinations, reliably illustrating the studies being performed. The most tested enzymes (e.g. lysozyme, DNAse, lysostaphin) were scrutinised and the rationale behind each combination explained. This research area is still growing even though current research gaps/opportunities were identified, such as lack of biofilm testing and of studies on polymicrobial scenarios. Hopefully, this work will shed light on the synergetic potential resting in enzyme combinations and alleviate some of the time and resource-consuming tasks related to enzyme combination research by helping the selection and design of new enzyme related therapeutic options for P. aeruginosa and S. aureus infections., This work was supported by the Portuguese Foundation for Science and Technology (FCT) under the scope of the strategic funding of UID/BIO/04469 unit and COMPETE 2020 [POCI-01-0145-FEDER-006684] and BioTecNorte operation [NORTE-01-0145-FEDER-000004], funded by the European Regional Development Fund under the scope of Norte2020 - Programa Operacional Regional do Norte. The authors also recognise COMPETE2020 and FCT for the project POCI-01-0145-FEDER-029841., info:eu-repo/semantics/publishedVersion

Staphylococcus aureus, among other staphylococcal species, developed multidrug resistance and causes serious health risks that require complex treatments. Therefore, the development of novel and effective strategies to combat these bacteria has been gaining importance. Since Staphylococcus simulans lysostaphin is a peptidoglycan hydrolase effective against staphylococcal species, the enzyme has a significant potential for biotechnological applications. Despite promising results of lysostaphin as a bacteriocin capable of killing staphylococcal pathogens, it is still not widely used in healthcare settings due to its high production cost. In this study, medium engineering techniques were applied to improve the expression yield of recombinant lysostaphin in E. coli. A new effective inducible araBAD promoter system and different mediums were used to enhance lysostaphin production. Our results showed that the composition of autoinduction media enhanced the amount of lysostaphin production 5-fold with the highest level of active lysostaphin at 30 degrees C. The production cost of 1000U of lysostaphin was determined as 4-fold lower than the previously proposed technologies. Therefore, the currently developed bench scale study has a great potential as a large-scale fermentation procedure to produce lysostaphin efficiently.

Introduction: Staphylococcus aureus (S. aureus) infections are associated with increased morbidity, mortality and health-care costs. Persistent nasal carriage of S. aureus found in 10-30% of the general population, constitutes a risk factor for these infections. Nasal decolonization is one of the used strategies to prevent this risk in some situations. Areas covered: Mupirocin nasal ointment has been used for the nasal decolonization and prevention of staphylococcal infections in various settings like surgeries. However, rising rates of resistance to mupirocin require the development of new decolonization agents. In this review, we will discuss mupirocin, its origins, studies that proved its efficacy and the associated resistance, as well as other decolonization agents under investigation. Expert opinion: As some limitations exist to mupirocin use, further research for alternatives is encouraged. Some old approved antiseptics (chlorhexidine, povidone-iodine) or antibiotics (rifampicin, bacitracin) have been investigated for their efficacy in this indication. Other new agents (tea tree oil, retapamulin, LTX-109, XF-73, phages, lysostaphin, squalamine analogues, etc.) are being studied. Some of them are still in preclinical phases, and others have reached clinical trials, but further research is needed. Special interest should be given to single dose decolonization strategies and to molecules that do not select resistant strains.

The increasing spread of antibiotic-resistant microorganisms has led to the necessity of developing alternative antimicrobial treatments. The use of peptidoglycan hydrolases is a promising approach to combat bacterial infections. In our study, we constructed a 2 kb-triple-acting fusion gene (TF) encoding the N-terminal amidase-5 domain of streptococcal LambdaSA2 prophage endolysin (D-glutamine-L-lysin endopeptidase), a mid-protein amidase-2 domain derived from the staphylococcal phage 2638A endolysin (N-acetylmuramoyl-L-alanine amidase) and the mature version (246 residues) of the Staphylococcus simulans Lysostaphin bacteriocin (glycyl-glycine endopeptidase) at the C-terminus. The TF gene was expressed in Nicotiana benthamiana plants using the non-replicating Cowpea mosaic virus (CPMV)-based vector pEAQ-HT and the replicating Alternanthera mosaic virus (AltMV)-based pGD5TGB1L8823-MCS-CP3 vector, and in Escherichia coli using pET expression vectors pET26b+ and pET28a+. The resulting poor expression of this fusion protein in plants prompted the construction of a TF gene codon-optimized for expression in tobacco plants, resulting in an improved codon adaptation index (CAI) from 0.79 (TF gene) to 0.93 (TFnt gene). Incorporation of the TFnt gene into the pEAQ-HT vector, followed by transient expression in N. benthamiana, led to accumulation of TFnt to an approximate level of 0.12 mg/g of fresh leaf weight. Antimicrobial activity of purified plant- and bacterial-produced TFnt proteins was assessed against two strains of Gram-positive Staphylococcus aureus 305 and Newman. The results showed that plant-produced TFnt protein was preferentially active against S. aureus 305, showing 14% of growth inhibition, while the bacterial-produced TFnt revealed significant antimicrobial activity against both strains, showing 68 (IC50 25 µg/ml) and 60% (IC50 71 µg/ml) growth inhibition against S. aureus 305 and Newman, respectively. Although the combination of codon optimization and transient expression using the non-replicating pEAQ-HT expression vector facilitated production of the TFnt protein in plants, the most functionally active antimicrobial protein was obtained using the prokaryotic expression system.

Lysostaphin is widely used in clinical settings against Staphylococcus aureus, but its mutants can abolish its killing activity. The difficulty in studies of mutations in lysostaphin is the shortage of data, which may need many decades to collect, although lysostaphin is so important for clinical therapeutics and drug development. In order not to passively wait for the accumulation of new data, in this study 1) the 23,442 mutations in 1408 proteins from databank were used to determine whether the mutations in lysostaphin follow the general mutation trend obtained from the databank, 2) the amino-acid pair predictability was used to explore the underlined mechanism for lysostaphin mutations, and 3) the amino-acid distribution probability was used to associate the mutation with dysfunction of lysostaphin. The results show that the mutations in lysostaphin follow the general trend of mutations in proteins; the underlined mechanism for mutations in lysostaphin is explainable from a viewpoint of randomness, and a mutation with increased distribution probability would have a larger chance to dysfunction lysostaphin. This study provides useful information for future design of anti-S. aureus drug and enzyme engineering.

Cutibacterium acnes is a pathogenic bacterium that cause inflammatory diseases of the skin and intervertebral discs. The immune activation induced by C. acnes requires multiple cellular responses in the host. Silkworm, an invertebrate, generates melanin by phenoloxidase upon recognizing bacterial or fungal components. Therefore, the melanization reaction can be used as an indicator of innate immune activation. A silkworm infection model was developed for evaluating the virulence of C. acnes, but a system for evaluating the induction of innate immunity by C. acnes using melanization as an indicator has not yet been established. Here we demonstrated that C. acnes rapidly causes melanization of the silkworm hemolymph. On the other hand, Staphylococcus aureus, a gram-positive bacterium identical to C. acnes, does not cause immediate melanization. Even injection of heat-killed C. acnes cells caused melanization of the silkworm hemolymph. DNase, RNase, and protease treatment of the heat-treated C. acnes cells did not decrease the silkworm hemolymph melanization. Treatment with peptidoglycan-degrading enzymes, such as lysostaphin and lysozyme, however, decreased the induction of melanization by the heat-treated C. acnes cells. These findings suggest that silkworm hemolymph melanization may be a useful indicator to evaluate innate immune activation by C. acnes and that C. acnes peptidoglycans are involved in the induction of innate immunity in silkworms.

Drug-resistant bacterial pathogens are a serious threat to global health, and antibacterial lysins are at the forefront of innovative treatments for these life-threatening infections. While lysins' general mechanism of action is well understood, the design principles that might enable engineering of performance-enhanced variants are still being formulated. Here, we report a detailed analysis of molecular determinants underlying the in vivo efficacy of lysostaphin, a canonical anti-MRSA (methicillin-resistant Staphylococcus aureus) lysin. Systematic analysis of bacterial binding, growth inhibition, lysis kinetics, and in vivo therapeutic efficacy revealed that binding affinity, and not inherent catalytic firepower, is the dominant driver of lysostaphin efficacy. This insight enabled electrostatic affinity tuning of lysostaphin to produce a single point mutant that manifested dramatically enhanced processivity and lysis kinetics and trended toward improved in vivo efficacy. More generally, these studies provide important insights into the complex relationships between lysin electrostatics, bacterial targeting, cell lysis efficiency, and in vivo efficacy. The lessons learned may enable engineering of other high-performance antibacterial biocatalysts.

There is an urgent need for novel agents to treat drug-resistant bacterial infections, such as multidrug-resistant Staphylococcus aureus (MRSA). Desirable properties for new antibiotics include high potency, narrow species selectivity, low propensity to elicit new resistance phenotypes, and synergy with standard of care (SOC) chemotherapies. Here, we describe analysis of the anti-MRSA potential exhibited by F12, an innovative anti-MRSA lysin that has been genetically engineered to evade detrimental antidrug immune responses in human patients. F12 possesses high potency and rapid onset of action, it has narrow selectivity against pathogenic Staphylococci, and it manifests synergy with numerous SOC antibiotics. Additionally, resistance to F12 and β-lactam antibiotics appears mutually exclusive, and importantly we provide evidence that F12 re-sensitizes normally resistant MRSA strains to β-lactams both in vitro and in vivo These results suggest that combinations of F12 and SOC antibiotics could be a promising new approach to treating refractory S. aureus infections.

Lysostaphin enzyme is an effective antibacterial agent proven to be promising for the prevention of healthcare-associated infections related to methicillin-resistant Staphylococcus aureus (MRSA). Here, safe, and non-toxic antibacterial hybrid coatings consisting of lysostaphin and waterborne polyurethane (PU) are presented. Lysostaphin was covalently immobilized onto polydopamine functionalized halloysite nanotubes (HNTs) to obtain stable HNT-lysostaphin nanohybrid structures with high effective lysostaphin concentrations, which were then incorporated into PU coatings by a facile spray coating process. Resulting PU/HNT-lysostaphin hybrid coatings presented strong antibacterial activity against S. aureus with a >99 % killing efficiency. The incorporation of lysostaphin into the polymer matrix via HNTs as a carrier enhanced the stability of the enzyme resulting in non-leaching coatings that presented high operational stability over multiple bacterial incubation cycles and high storage stability without any significant loss of enzymatic activity. Furthermore, PU/HNT-lysostaphin coatings demonstrated significant antibiofilm properties and reduced the formation of S. aureus biofilms by 70 % relative to neat PU coatings. The lysostaphin based antibacterial hybrid coatings developed in this study, which can be easily applied to surfaces in healthcare facilities and medical devices offer an effective approach for the prevention of S. aureus associated nosocomial infections.

Methicillin-resistant Staphylococcus aureus sequence type 72 (ST72) is prevalent in South Korea and has shown resistance to multiple antimicrobials. ST72 isolates display different levels of resistance to the antistaphylococcal lysostaphin. Draft genome sequencing of ST72 human isolates exhibiting lysostaphin resistance or susceptibility was performed to better understand the mechanism of lysostaphin resistance using subtractive genomics.

Gaining an insight into the mechanism underlying antimicrobial-resistance development in Staphylococcus aureus is crucial for identifying effective antimicrobials. We isolated S. aureus sequence type 72 from a patient in whom the S. aureus infection was highly resistant to various antibiotics and lysostaphin, but no known resistance mechanisms could explain the mechanism of lysostaphin resistance. Genome-sequencing followed by subtractive and functional genomics revealed that serine hydroxymethyltransferase (glyA or shmT gene) plays a key role in lysostaphin resistance. Serine hydroxymethyltransferase (SHMT) is indispensable for the one-carbon metabolism of serine/glycine interconversion and is linked to folate metabolism. Functional studies revealed the involvement of SHMT in lysostaphin resistance, as ΔshmT was susceptible to the lysostaphin, while complementation of the knockout expressing shmT restored resistance against lysostaphin. In addition, the ΔshmT showed reduced virulence under in vitro (mammalian cell lines infection) and in vivo (wax-worm infection) models. The SHMT inhibitor, serine hydroxymethyltransferase inhibitor 1 (SHIN1), protected the 50% of the wax-worm infected with wild type S. aureus. These results suggest SHMT is relevant to the extreme susceptibility to lysostaphin and the host immune system. Thus, the current study established that SHMT plays a key role in lysostaphin resistance development and in determining the virulence potential of multiple drug-resistant S. aureus.

The high-glucose-induced cytotoxicity in diabetes has been widely recognized. Staphylococcus aureus is the most frequent pathogen isolated from diabetic foot ulcers, but the properties of this bacterium under high glucose conditions remain unclear. S. aureus grew in medium usually forms weak biofilm, and which was significantly increased by addition of glucose. However, extracellular DNA (eDNA), an important component of biofilms, was markedly decreased in presence of 15 mM glucose. The reduced eDNA content was not caused by degradation, because the nuclease activity of biofilm supernatants with glucose was significantly decreased due to the acidic pH of the medium. Under planktonic state, the growth of S. aureus was significantly decreased in the Luria-Bertani (LB) medium supplemented with 25 mM glucose, and the reduced growth of S. aureus by glucose was dose-dependent. Except for glucose, the growth of planktonic S. aureus was also markedly decreased by fructose or sucrose. Amounts of acid metabolites were produced under high glucose conditions, but the survival of planktonic S. aureus was unaffected by these acidic conditions. Cells of S. aureus from the culture medium with glucose had a thinner cell wall and highly resistant to lysostaphin compared with the bacteria cultured in LB medium. mRNA expression of genes encoding pentaglycine bridges, the substrate of lysostaphin, was significantly decreased in S. aureus by glucose. In addition to S. aureus, the growth of Staphylococcus haemolyticus and Staphylococcus epidermidis was also significantly decreased by an excess of glucose, but strains of Enterococcus faecalis, Escherichia coli, and Pseudomonas aeruginosa were unaffected by glucose. In conclusion, the reduced growth of S. aureus under high glucose conditions is due to impairment of the unique cell-wall structure, pentaglycine bridges.

Antibacterial lysins are enzymes that hydrolyze bacterial peptidoglycan, which results in the rapid death of bacterial cells due to osmotic lysis. Lysostaphin is one of the most potent and well-studied lysins active against important nosocomial pathogen Staphylococcus aureus. Similarly to most other lysins, lysostaphin is composed of enzymatic and peptidoglycan-binding domains, and both domains influence its antibacterial activity. It is thus desirable to be able to study the activity of both domains independently. Lysostaphin cleaves pentaglycine cross-bridges within the staphylococcal peptidoglycan. Here, we report the protocol to study the catalytic activity of lysostaphin on the isolated pentaglycine peptide that is based on the chromogenic reaction of peptide amino groups with ninhydrin. Unlike previously reported assays, this protocol does not require in-house chemical synthesis or specialized equipment and can be readily performed in most laboratories. We demonstrate the use of this protocol to study the effect of EDTA treatment on the lysostaphin enzymatic activity. We further used this protocol to determine the catalytic efficiency of lysostaphin on the isolated pentaglycine and compared it to the apparent catalytic efficiency on the whole staphylococcal cells. These results highlight the relative impact of enzymatic and peptidoglycan-binding domains of lysostaphin on its bacteriolytic activity.

Drug-resistant bacterial pathogens are a serious threat to global health, and antibacterial lysins are at the forefront of innovative treatments for these life-threatening infections. While lysins’ general mechanism of action is well understood, the design principles that might enable engineering of performance-enhanced variants are still being formulated. Here, we report a detailed analysis of molecular determinants underlying the in vivo efficacy of lysostaphin, a canonical anti-MRSA (methicillin-resistant Staphylococcus aureus) lysin. Systematic analysis of bacterial binding, growth inhibition, lysis kinetics, and in vivo therapeutic efficacy revealed that binding affinity, and not inherent catalytic firepower, is the dominant driver of lysostaphin efficacy. This insight enabled electrostatic affinity tuning of lysostaphin to produce a single point mutant that manifested dramatically enhanced processivity and lysis kinetics and trended toward improved in vivo efficacy. More generally, these studies provide important insights into the complex relationships between lysin electrostatics, bacterial targeting, cell lysis efficiency, and in vivo efficacy. The lessons learned may enable engineering of other high-performance antibacterial biocatalysts.

Abstract: Staphylococcus aureus is a prominent human pathogen that causes nosocomial and community acquired infections. The accelerating emergence and prevalence of staphylococcal infections have grotesque health consequences which are mostly due to its anomalous capability to acquire drug resistance and scarcity of novel classes of antibacterials. Many combating therapies are centered on primary targets of S. aureus which are cell envelope, ribosomes and nucleic acids. This review describes various chemotherapeutic strategies for combating S. aureus infections including monotherapy, combination drug therapy, phage endolysin therapy, lysostaphins and antibacterial drones. Monotherapy has dwindled in due course of time, but combination therapy, endolysin therapy, lysostaphin and antibacterial drones are emerging alternatives which efficiently conquer the shortcomings of monotherapy. Combinations of more than one antibiotic agents or combination of adjuvant with antibiotics provide a synergistic approach to combat infections causing pathogenic strains. Phage endolysin therapy and lysostaphin are also presented as possible alternatives to conventional antibiotic therapies. Antibacterial Drones go a step further by specifically targeting the virulence genes in bacteria, giving them a certain advantage over existing antibacterial strategies. But the challenge remains on the better understanding of these strategies for executing and implementing them in the health sector. In this day and age, most of the S. aureus strains are resistant to an ample number of antibiotics, so there is an urgent need to overcome such multidrug-resistant strains for the welfare of our community.

Antibiotic resistance and the colonization of resistant bacteria such as Staphylococcus aureus on surfaces, often in the form of biofilms, prolong hospitalization periods and increase mortality, thus is a significant concern for healthcare providers. To prevent biofilm formation, the inadequate concentration of using nanoparticles as antibacterial coating agents is one of the major obstacles. This study aimed to design a hypervalency TiO2 nanocomposite as a reserved base to carry a high amount of active antibacterial agents such as lysostaphin via a biotin-streptavidin-biotin bridge. The utilization of the streptavidin-biotin system could increase the abundance of lysostaphin. Lysostaphin was expressed in Escherichia coli and purified. Both recombinant lysostaphin and titanium oxide nanocomposite were conjugated with biotin and linked to a streptavidin bridge. The kinetics and activity of the enzyme were examined after each step utilizing N-acetylhexaglycine as a substrate. Physical characteristics of nanoparticles containing lysostaphin were determined using AFM, SEM, FTIR, and zeta potential. The results showed changes in size, charge, and morphology of the nanoparticles following the lysostaphin attachment. Also, the stability and kinetics of the active biological enzymes on nanoparticles were reexamined following 8 months of storage. Exploiting this approach, various biotinylated antibacterial agents could be prepared and rapidly immobilized on a nanoparticle as an active net against related infectious agents.

Lytic enzymes are novel antimicrobial agents that degrade bacterial cell walls, resulting in cell rupture and death. We tested one enzyme, the bacteriocin lysostaphin, for treatment of nonhuman primates (Macaca mulatta) with persistent methicillin-resistant Staphylococcus aureus (MRSA) infection of their cranial implant margins. The goal of this study was to determine if topical lysostaphin, either alone or as an adjunct therapy, could eliminate MRSA. Lysostaphin had in vitro lytic activity against all 4 previously identified NHP MRSA clones, as well as against 12 MRSA isolates of the same clonal type (MLST ST3862 and spa type t4167) before and after treatment, with no resistance discovered. In an in vivo pilot study, a 2-d application of lysostaphin alone reduced MRSA in the implant margins by 3-logs during treatment of one animal; however, MRSA titers had returned to control levels by 1 wk after treatment. In the main study, all animals (n = 4) received 10 d of systemic antibiotic treatment and both the animals and their environment (cages, equipment, room) underwent 5-d of decontamination. The experimental animals (n = 2) received 5 doses of topical lysostaphin (15 mg, every other day) applied onto their implant margins. Daily cultures showed that MRSA counts decreased significantly (≤ 25 colony-forming units/mL; P < 0.05). However, sampling of the cranial implant margin 7 d after last treatment showed that MRSA counts had returned to control levels. Our study suggests that lysostaphin, coupled with other treatment modalities, can decrease MRSA infection short-term but do not completely eradicate MRSA in the long-term. This reappearance of MRSA may be due to cross-contamination or reinfection from other infected areas, an inability of the treatment to reach all colonized areas, or insufficient dosing or length of treatment. Topical lysostaphin may be more useful clinically for superficial nonimplant associated wounds in which the lytic enzyme has better access to the infected tissue.

There is an urgent need for novel agents to treat drug-resistant bacterial infections, such as multidrug-resistant Staphylococcus aureus (MRSA). Desirable properties for new antibiotics include high potency, narrow species selectivity, low propensity to elicit new resistance phenotypes, and synergy with standard-of-care (SOC) chemotherapies. Here, we describe analysis of the antibacterial potential exhibited by F12, an innovative anti-MRSA lysin that has been genetically engineered to evade detrimental antidrug immune responses in human patients. F12 possesses high potency and rapid onset of action, it has narrow selectivity against pathogenic staphylococci, and it manifests synergy with numerous SOC antibiotics. Additionally, resistance to F12 and β-lactam antibiotics appears mutually exclusive, and, importantly, we provide evidence that F12 resensitizes normally resistant MRSA strains to β-lactams both in vitro and in vivo. These results suggest that combinations of F12 and SOC antibiotics are a promising new approach to treating refractory S. aureus infections.