Immunomimetic Designer Cells Protect Mice from MRSA Infection.

Many community- and hospital-acquired bacterial infections are caused by antibiotic-resistant pathogens. Methicillin-resistant Staphylococcus aureus (MRSA) predisposes humans to invasive infections that are difficult to eradicate. We designed a closed-loop gene network programming mammalian cells to autonomously detect and eliminate bacterial infections. The genetic circuit contains human Toll-like receptors as the bacterial sensor and a synthetic promoter driving reversible and adjustable expression of lysostaphin, a bacteriolytic enzyme highly lethal to S. aureus. Immunomimetic designer cells harboring this genetic circuit exhibited fast and robust sense-and-destroy kinetics against live staphylococci. When tested in a foreign-body infection model in mice, microencapsulated cell implants prevented planktonic MRSA infection and reduced MRSA biofilm formation by 91%. Notably, this system achieved a 100% cure rate of acute MRSA infections, whereas conventional vancomycin treatment failed. These results suggest that immunomimetic designer cells could offer a therapeutic approach for early detection, prevention, and cure of pathogenic infections in the post-antibiotic era.

Rapid Antibacterial Activity Assessment of Chimeric Lysins.

Various chimeric lysins have been developed as efficacious antibiotics against multidrug-resistant bacteria, but direct comparisons of their antibacterial activities have been difficult due to the preparation of multiple recombinant chimeric lysins. Previously, we reported an Escherichia coli cell-free expression method to better screen chimeric lysins against Staphylococcus aureus, but we still needed to increase the amounts of expressed proteins enough to be able to detect them non-isotopically for quantity comparisons. In this study, we improved the previous cell-free expression system by adding a previously reported artificial T7 terminator and reversing the different nucleotides between the T7 promoter and start codon to those of the T7 phage. The new method increased the expressed amount of chimeric lysins enough for us to detect them using Western blotting. Therefore, the qualitative comparison of activity between different chimeric lysins has become possible via the adjustment of the number of variables between samples without protein purification. We applied this method to select more active chimeric lysins derived from our previously reported chimeric lysin (ALS2). Finally, we compared the antibacterial activities of our selected chimeric lysins with reported chimeric lysins (ClyC and ClyO) and lysostaphin and determined the rank orders of antibacterial activities on different Staphylococcus aureus strains in our experimental conditions.

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.

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.

Antimicrobial peptidase lysostaphin at subinhibitory concentrations modulates staphylococcal adherence, biofilm formation, and toxin production.

BACKGROUND: The ability of antimicrobial agents to affect microbial adherence to eukaryotic cell surfaces is a promising antivirulence strategy for combating the global threat of antimicrobial resistance. Inadequate use of antimicrobials has led to widespread instances of suboptimal antibiotic concentrations around infection sites. Therefore, we aimed to examine the varying effect of an antimicrobial peptidase lysostaphin (APLss) on staphylococcal adherence to host cells, biofilm biomass formation, and toxin production as a probable method for mitigating staphylococcal virulence. RESULTS: Initially, soluble expression in E. coli and subsequent purification by immobilized-Ni2+ affinity chromatography (IMAC) enabled us to successfully produce a large quantity of highly pure ~ 28-kDa His-tagged mature APLss. The purified protein exhibited potent inhibitory effects against both methicillin-sensitive and methicillin-resistant staphylococcal strains, with minimal inhibitory concentrations (MICs) ranging from 1 to 2 µg/mL, and ultrastructural analysis revealed that APLss-induced concentration-specific changes in the morphological architecture of staphylococcal surface membranes. Furthermore, spectrophotometric and fluorescence microscopy revealed that incubating staphylococcal strains with sub-MIC and MIC of APLss significantly inhibited staphylococcal adherence to human vaginal epithelial cells and biofilm biomass formation. Ultimately, transcriptional investigations revealed that APLss inhibited the expression of agrA (quorum sensing effector) and other virulence genes related to toxin synthesis. CONCLUSIONS: Overall, APLss dose-dependently inhibited adhesion to host cell surfaces and staphylococcal-associated virulence factors, warranting further investigation as a potential anti-staphylococcal agent with an antiadhesive mechanism of action using in vivo models of staphylococcal toxic shock syndrome.

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.

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.

Genetic Determinants of Surface Accessibility in Staphylococcus aureus.

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.

Two-site recognition of Staphylococcus aureus peptidoglycan by lysostaphin SH3b.

Lysostaphin is a bacteriolytic enzyme targeting peptidoglycan, the essential component of the bacterial cell envelope. It displays a very potent and specific activity toward staphylococci, including methicillin-resistant Staphylococcus aureus. 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 synergistic and structurally dynamic recognition of S. aureus peptidoglycan and underpins the potent bacteriolytic activity of this enzyme.

Influence of NaCl and pH on lysostaphin catalytic activity, cell binding, and bacteriolytic activity.

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.

Self-cleaved expression of recombinant lysostaphin from its cellulose binding domain fusion.

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.

Electrostatic-Mediated Affinity Tuning of Lysostaphin Accelerates Bacterial Lysis Kinetics and Enhances In Vivo Efficacy.

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.

Deimmunized Lysostaphin Synergizes with Small-Molecule Chemotherapies and Resensitizes Methicillin-Resistant Staphylococcus aureus to ß-Lactam Antibiotics.

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 ß-lactam antibiotics appears mutually exclusive, and, importantly, we provide evidence that F12 resensitizes normally resistant MRSA strains to ß-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.

High-yield production of active recombinant S. simulans lysostaphin expressed in E. coli in a laboratory bioreactor.

Staphylococcus aureus (S. aureus), which has developed multidrug resistance, leads to many healthcare-associated infections resulting in significant medical and economic losses. Therefore, the development of new efficient strategies to deal with these bacteria has been gaining importance. Lysostaphin is a peptidoglycan hydrolase that has considerable potential as a bacteriocin. However, there have been few reported optimization and scale-up studies of the lysostaphin bioproduction process. Our preliminary results have revealed that the composition of auto-induction media at 30 °C increases the produced lysostaphin around 10-fold in shake flasks. In this study, achieving higher yields for recombinant lysostaphin in E. coli at a laboratory scale has been the aim, through the use of auto-induction media. Optimized medium composition and fermentation parameters were transferred to a laboratory-scale bioreactor. The tested conditions improved protein yields up to 184 mg/L in a 3 L stirred bioreactor and the productivity was improved 2-fold in comparison to previously published reports. Furthermore, this study also showed that lysostaphin is an effective bacteriocin on both commercially available and isolated S. aureus strains. These results will contribute to future larger-scale production of lysostaphin via the proposed fermentation conditions.

Therapeutic applications of lysostaphin against Staphylococcus aureus.

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.

Immobilization of recombinant lysostaphin on nanoparticle through biotin-streptavidin conjugation technology as a geometrical progressed confrontation against Staphylococcus aureus infection.

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.

Hydrogel delivery of lysostaphin eliminates orthopedic implant infection by Staphylococcus aureus and supports fracture healing.

Orthopedic implant infections are a significant clinical problem, with current therapies limited to surgical debridement and systemic antibiotic regimens. Lysostaphin is a bacteriolytic enzyme with high antistaphylococcal activity. We engineered a lysostaphin-delivering injectable PEG hydrogel to treat Staphylococcus aureus infections in bone fractures. The injectable hydrogel formulation adheres to exposed tissue and fracture surfaces, ensuring efficient, local delivery of lysostaphin. Lysostaphin encapsulation within this synthetic hydrogel maintained enzyme stability and activity. Lysostaphin-delivering hydrogels exhibited enhanced antibiofilm activity compared with soluble lysostaphin. Lysostaphin-delivering hydrogels eradicated S. aureus infection and outperformed prophylactic antibiotic and soluble lysostaphin therapy in a murine model of femur fracture. Analysis of the local inflammatory response to infections treated with lysostaphin-delivering hydrogels revealed indistinguishable differences in cytokine secretion profiles compared with uninfected fractures, demonstrating clearance of bacteria and associated inflammation. Importantly, infected fractures treated with lysostaphin-delivering hydrogels fully healed by 5 wk with bone formation and mechanical properties equivalent to those of uninfected fractures, whereas fractures treated without the hydrogel carrier were equivalent to untreated infections. Finally, lysostaphin-delivering hydrogels eliminate methicillin-resistant S. aureus infections, supporting this therapy as an alternative to antibiotics. These results indicate that lysostaphin-delivering hydrogels effectively eliminate orthopedic S. aureus infections while simultaneously supporting fracture repair.

Differential Induction of Type I and III Interferons by Staphylococcus aureus.

Staphylococcus aureus is a leading cause of bacterial pneumonia, and we have shown previously that type I interferon (IFN) contributes to the pathogenesis of this disease. In this study, we screened 75 S. aureus strains for their ability to induce type I and III IFN. Both cytokine pathways were differentially stimulated by various S. aureus strains independently of their isolation sites or methicillin resistance profiles. These induction patterns persisted over time, and type I and III IFN generation differentially correlated with tumor necrosis factor alpha production. Investigation of one isolate, strain 126, showed a significant defect in type I IFN induction that persisted over several time points. The lack of induction was not due to differential phagocytosis, subcellular location, or changes in endosomal acidification. A correlation between reduced type I IFN induction levels and decreased autolysis and lysostaphin sensitivity was found between strains. Strain 126 had a decreased rate of autolysis and increased resistance to lysostaphin degradation and host cell-mediated killing. This strain displayed decreased virulence in a murine model of acute pneumonia compared to USA300 (current epidemic strain and commonly used in research) and had reduced capacity to induce multiple cytokines. We observed this isolate to be a vancomycin-intermediate S. aureus (VISA) strain, and reduced Ifnb was observed with a defined mutation in walK that induces a VISA phenotype. Overall, this study demonstrates the heterogeneity of IFN induction by S. aureus and uncovered an interesting property of a VISA strain in its inability to induce type I IFN production.

A functional type II-A CRISPR-Cas system from Listeria enables efficient genome editing of large non-integrating bacteriophage.

CRISPR-Cas systems provide bacteria with adaptive immunity against invading DNA elements including bacteriophages and plasmids. While CRISPR technology has revolutionized eukaryotic genome engineering, its application to prokaryotes and their viruses remains less well established. Here we report the first functional CRISPR-Cas system from the genus Listeria and demonstrate its native role in phage defense. LivCRISPR-1 is a type II-A system from the genome of L. ivanovii subspecies londoniensis that uses a small, 1078 amino acid Cas9 variant and a unique NNACAC protospacer adjacent motif. We transferred LivCRISPR-1 cas9 and trans-activating crRNA into Listeria monocytogenes. Along with crRNA encoding plasmids, this programmable interference system enables efficient cleavage of bacterial DNA and incoming phage genomes. We used LivCRISPR-1 to develop an effective engineering platform for large, non-integrating Listeria phages based on allelic replacement and CRISPR-Cas-mediated counterselection. The broad host-range Listeria phage A511 was engineered to encode and express lysostaphin, a cell wall hydrolase that specifically targets Staphylococcus peptidoglycan. In bacterial co-culture, the armed phages not only killed Listeria hosts but also lysed Staphylococcus cells by enzymatic collateral damage. Simultaneous killing of unrelated bacteria by a single phage demonstrates the potential of CRISPR-Cas-assisted phage engineering, beyond single pathogen control.

Functional Identification of Serine Hydroxymethyltransferase as a Key Gene Involved in Lysostaphin Resistance and Virulence Potential of Staphylococcus aureus Strains.

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.