Assaf Raz

Combination Therapy With Lysin CF-301 and Antibiotic Is Superior to Antibiotic Alone for Treating Methicillin-Resistant Staphylococcus aureus–Induced Murine Bacteremia

Lysins are bacteriophage-derived enzymes that degrade bacterial peptidoglycans. Lysin CF-301 is being developed to treat Staphylococcus aureus because of its potent, specific, and rapid bacteriolytic effects. It also demonstrates activity on drug-resistant strains, has a low resistance profile, eradicates biofilms, and acts synergistically with antibiotics. CF-301 was bacteriolytic against 250 S. aureus strains tested including 120 methicillin-resistant S. aureus (MRSA) isolates. In time-kill studies with 62 strains, CF-301 reduced S. aureus by 3-log10 within 30 minutes compared to 6–12 hours required by antibiotics. In bacteremia, CF-301 increased survival by reducing blood MRSA 100-fold within 1 hour. Combinations of CF-301 with vancomycin or daptomycin synergized in vitro and increased survival significantly in staphylococcal-induced bacteremia compared to treatment with antibiotics alone (P < .0001). Superiority of CF-301 combinations with antibiotics was confirmed in 26 independent bacteremia studies. Combinations including CF-301 and antibiotics represent an attractive alternative to antibiotic monotherapies currently used to treat S. aureus bacteremia.

Sortase A localizes to distinct foci on the Streptococcus pyogenes membrane

Cell wall peptidoglycan-anchored surface proteins are essential virulence factors in many gram-positive bacteria. The attachment of these proteins to the peptidoglycan is achieved through a transpeptidation reaction, whereby sortase cleaves a conserved C-terminal LPXTG motif and covalently attaches the protein to the peptidoglycan precursor lipid II. It is unclear how the sorting reaction is regulated spatially and what part sortase localization plays in determining the distribution of surface proteins. This is mainly the result of inadequate immunofluorescence techniques required to resolve these issues in certain bacterial pathogens. Here we describe the utilization of the phage lysin PlyC to permeabilize the cell wall of Streptococcus pyogenes to antibodies, thereby allowing the localization of sortase A using deconvolution immunofluorescence microscopy. We find that sortase localizes within distinct membranal foci, the majority of which are associated with the division septum and colocalize with areas of active M protein anchoring. Sortase distribution to the new septum begins at a very early stage, culminates during septation, and decays after division is completed. This implies that the sorting reaction is a dynamic, highly regulated process, intimately associated with cell division. The ability to study cytoplasmic and membrane antigens using deconvolution immunofluorescence microscopy will facilitate further study of cellular processes in S. pyogenes.

Use of a Bacteriophage Lysin to Identify a Novel Target for Antimicrobial Development

We identified an essential cell wall biosynthetic enzyme in Bacillus anthracis and an inhibitor thereof to which the organism did not spontaneously evolve measurable resistance. This work is based on the exquisite binding specificity of bacteriophage-encoded cell wall-hydrolytic lysins, which have evolved to recognize critical receptors within the bacterial cell wall. Focusing on the B. anthracis-specific PlyG lysin, we first identified its unique cell wall receptor and cognate biosynthetic pathway. Within this pathway, one biosynthetic enzyme, 2-epimerase, was required for both PlyG receptor expression and bacterial growth. The 2-epimerase was used to design a small-molecule inhibitor, epimerox. Epimerox prevented growth of several Gram-positive pathogens and rescued mice challenged with lethal doses of B. anthracis. Importantly, resistance to epimerox was not detected (<10−11 frequency) in B. anthracis and S. aureus. These results describe the use of phage lysins to identify promising lead molecules with reduced resistance potential for antimicrobial development.

A Highly Active and Negatively Charged Streptococcus pyogenes Lysin with a Rare d-Alanyl-l-Alanine Endopeptidase Activity Protects Mice against Streptococcal Bacteremia

ABSTRACT Bacteriophage endolysins have shown great efficacy in killing Gram-positive bacteria. PlyC, a group C streptococcal phage lysin, represents the most efficient lysin characterized to date, with a remarkably high specificity against different streptococcal species, including the important pathogen Streptococcus pyogenes. However, PlyC is a unique lysin, in terms of both its high activity and structure (two distinct subunits). We sought to discover and characterize a phage lysin active against S. pyogenes with an endolysin architecture distinct from that of PlyC to determine if it relies on the same mechanism of action as PlyC. In this study, we identified and characterized an endolysin, termed PlyPy (phage lysin from S. pyogenes), from a prophage infecting S. pyogenes. By in silico analysis, PlyPy was found to have a molecular mass of 27.8 kDa and a pI of 4.16. It was active against a majority of group A streptococci and displayed high levels of activity as well as binding specificity against group B and C streptococci, while it was less efficient against other streptococcal species. PlyPy showed the highest activity at neutral pH in the presence of calcium and NaCl. Surprisingly, its activity was not affected by the presence of the group A-specific carbohydrate, while the activity of PlyC was partly inhibited. Additionally, PlyPy was active in vivo and could rescue mice from systemic bacteremia. Finally, we developed a novel method to determine the peptidoglycan bond cleaved by lysins and concluded that PlyPy exhibits a rare d-alanyl-l-alanine endopeptidase activity. PlyPy thus represents the first lysin characterized from Streptococcus pyogenes and has a mechanism of action distinct from that of PlyC.

Attenuated neutrophil axis in atopic dermatitis compared to psoriasis reflects TH17 pathway differences between these diseases

Nikhil Dhingra, BSa,b,c, Mayte Suarez-Farinas, PhDa,c, Judilyn Fuentes-Duculan, MDa, Julia K. Gittler, BAa,d, Avner Shemer, MDe, Assaf Raz, PhDf, Vincent A. Fischetti, PhDf, James G. Krueger, MD, PhDa, and Emma Guttman-Yassky, MD, PhDa,g Emma Guttman-Yassky: eguttman@rockefeller.edu aThe Laboratory for Investigative Dermatology, The Rockefeller University, New York, NY bColumbia University College of Physicians & Surgeons, New York, NY cThe Center for Clinical and Translational Science, The Rockefeller University, New York, NY dAlbert Einstein College of Medicine, Bronx, NY eThe Department of Dermatology, Tel-Hashomer, Tel Aviv, Israel fThe Laboratory of Bacterial Pathogenesis and Immunology, The Rockefeller University, New York, NY gThe Department of Dermatology, Icahn School of Medicine at Mount Sinai, New York, NY

Bacteriophage Lysin CF-301, a Potent Antistaphylococcal Biofilm Agent

ABSTRACT Biofilms pose a unique therapeutic challenge because of the antibiotic tolerance of constituent bacteria. Treatments for biofilm-based infections represent a major unmet medical need, requiring novel agents to eradicate mature biofilms. Our objective was to evaluate bacteriophage lysin CF-301 as a new agent to target Staphylococcus aureus biofilms. We used minimum biofilm-eradicating concentration (MBEC) assays on 95 S. aureus strains to obtain a 90% MBEC (MBEC90) value of ≤0.25 μg/ml for CF-301. Mature biofilms of coagulase-negative staphylococci, Streptococcus pyogenes, and Streptococcus agalactiae were also sensitive to disruption, with MBEC90 values ranging from 0.25 to 8 μg/ml. The potency of CF-301 was demonstrated against S. aureus biofilms formed on polystyrene, glass, surgical mesh, and catheters. In catheters, CF-301 removed all biofilm within 1 h and killed all released bacteria by 6 h. Mixed-species biofilms, formed by S. aureus and Staphylococcus epidermidis on several surfaces, were removed by CF-301, as were S. aureus biofilms either enriched for small-colony variants (SCVs) or grown in human synovial fluid. The antibacterial activity of CF-301 was further demonstrated against S. aureus persister cells in exponential-phase and stationary-phase populations. Finally, the antibiofilm activity of CF-301 was greatly improved in combinations with the cell wall hydrolase lysostaphin when tested against a range of S. aureus strains. In all, the data show that CF-301 is highly effective at disrupting biofilms and killing biofilm bacteria, and, as such, it may be an efficient new agent for treating staphylococcal infections with a biofilm component.

Cellular aspects of the distinct M protein and SfbI anchoring pathways in Streptococcus pyogenes

Wall‐anchored surface proteins are critical for the in vivo survival of Streptococcus pyogenes. Cues in the signal sequence direct the membrane translocation of surface proteins: M protein to the septum, and SfbI to the poles. Both proteins are subsequently anchored to the wall by the membrane bound enzyme sortase A. However, the cellular features of these pathways are not fully understood. Here we show that M protein and SfbI are anchored simultaneously throughout the cell cycle. M protein is rapidly anchored at the septum, and in part of the cell cycle, is anchored simultaneously at the mother and daughter septa. Conversely, SfbI accumulates gradually on peripheral peptidoglycan, resulting in a polar distribution. Sortase is not required for translocation of M protein or SfbI at their respective locations. Methicillin‐induced unbalanced peptidoglycan synthesis diminishes surface M protein but not SfbI. Furthermore, overexpression of the division regulator DivIVA also diminishes surface M protein but increases SfbI. These results demonstrate a close connection between the regulation of cell division and protein anchoring. Better understanding of the spatial regulation of surface anchoring may lead to the identification of novel targets for the development of anti‐infective agents, given the importance of surface molecules for pathogenesis.

Propionibacterium acnes biofilm is present in intervertebral discs of patients undergoing microdiscectomy.

Background In previous studies, Propionibacterium acnes was cultured from intervertebral disc tissue of ~25% of patients undergoing microdiscectomy, suggesting a possible link between chronic bacterial infection and disc degeneration. However, given the prominence of P. acnes as a skin commensal, such analyses often struggled to exclude the alternate possibility that these organisms represent perioperative microbiologic contamination. This investigation seeks to validate P. acnes prevalence in resected disc cultures, while providing microscopic evidence of P. acnes biofilm in the intervertebral discs. Methods Specimens from 368 patients undergoing microdiscectomy for disc herniation were divided into several fragments, one being homogenized, subjected to quantitative anaerobic culture, and assessed for bacterial growth, and a second fragment frozen for additional analyses. Colonies were identified by MALDI-TOF mass spectrometry and P. acnes phylotyping was conducted by multiplex PCR. For a sub-set of specimens, bacteria localization within the disc was assessed by microscopy using confocal laser scanning and FISH. Results Bacteria were cultured from 162 discs (44%), including 119 cases (32.3%) with P. acnes. In 89 cases, P. acnes was cultured exclusively; in 30 cases, it was isolated in combination with other bacteria (primarily coagulase-negative Staphylococcus spp.) Among positive specimens, the median P. acnes bacterial burden was 350 CFU/g (12 - ~20,000 CFU/g). Thirty-eight P. acnes isolates were subjected to molecular sub-typing, identifying 4 of 6 defined phylogroups: IA1, IB, IC, and II. Eight culture-positive specimens were evaluated by fluorescence microscopy and revealed P. acnes in situ. Notably, these bacteria demonstrated a biofilm distribution within the disc matrix. P. acnes bacteria were more prevalent in males than females (39% vs. 23%, p = 0.0013). Conclusions This study confirms that P. acnes is prevalent in herniated disc tissue. Moreover, it provides the first visual evidence of P. acnes biofilms within such specimens, consistent with infection rather than microbiologic contamination.

Lysibodies are IgG Fc fusions with lysin binding domains targeting Staphylococcus aureus wall carbohydrates for effective phagocytosis

Significance Antibiotic resistance is an ever-increasing problem; for certain pathogens, few treatment options remain. Vaccines and therapeutic antibodies represent important alternatives to antibiotics, yet despite extensive effort no approved vaccine is available for Staphylococcus aureus. Although wall carbohydrates are ideal immunotherapeutic targets due to their abundance and high level of conservation, their poor immunogenicity compared with conventional protein targets complicates the production of effective antibodies. The approach presented here fuses the high-affinity binding domains from bacteriophage lysins and autolysins that recognize specific cell wall carbohydrate epitopes to IgG Fc, creating effective therapeutic antibodies, or lysibodies. This approach is generalizable, allowing production of antibodies to poorly immunogenic carbohydrate epitopes of many Gram-positive pathogens. Lysibodies thus represent a broad class of anti-infectives. The cell wall of Gram-positive bacteria contains abundant surface-exposed carbohydrate molecules that are highly conserved within and often across species. The potential therapeutic usefulness of high-affinity antibodies to cell wall carbohydrates is unquestioned, however obtaining such antibodies is challenging due to the poor overall immunogenicity of these bacterial targets. Autolysins and phage lysins are peptidoglycan hydrolases, enzymes that have evolved over a billion years to degrade bacterial cell wall. Such wall hydrolases are modular enzymes, composed of discrete domains for high-affinity binding to cell wall carbohydrates and cleavage activity. In this study, we demonstrate that binding domains from autolysins and lysins can be fused to the Fc region of human IgG, creating a fully functional homodimer (or “lysibody”) with high-affinity binding and specificity for carbohydrate determinants on the bacterial surface. Furthermore, we demonstrate that this process is reproducible with three different binding domains specific to methicillin-resistant Staphylococcus aureus (MRSA). Cell-bound lysibodies induced the fixation of complement on the bacterial surface, promoted phagocytosis by macrophages and neutrophils, and protected mice from MRSA infection in two model systems. The lysibody approach could be used to target a range of difficult-to-treat pathogenic bacteria, given that cell wall hydrolases are ubiquitous in nature.

Streptococcus pyogenes Sortase Mutants Are Highly Susceptible to Killing by Host Factors Due to Aberrant Envelope Physiology.

Cell wall anchored virulence factors are critical for infection and colonization of the host by Gram-positive bacteria. Such proteins have an N-terminal leader sequence and a C-terminal sorting signal, composed of an LPXTG motif, a hydrophobic stretch, and a few positively charged amino acids. The sorting signal halts translocation across the membrane, allowing sortase to cleave the LPXTG motif, leading to surface anchoring. Deletion of sortase prevents the anchoring of virulence factors to the wall; the effects on bacterial physiology however, have not been thoroughly characterized. Here we show that deletion of Streptococcus pyogenes sortase A leads to accumulation of sorting intermediates, particularly at the septum, altering cellular morphology and physiology, and compromising membrane integrity. Such cells are highly sensitive to cathelicidin, and are rapidly killed in blood and plasma. These phenomena are not a loss-of-function effect caused by the absence of anchored surface proteins, but specifically result from the accumulation of sorting intermediates. Reduction in the level of sorting intermediates leads to a return of the sortase mutant to normal morphology, while expression of M protein with an altered LPXTG motif in wild type cells leads to toxicity in the host environment, similar to that observed in the sortase mutant. These unanticipated effects suggest that inhibition of sortase by small-molecule inhibitors could similarly lead to the rapid elimination of pathogens from an infected host, making such inhibitors much better anti-bacterial agents than previously believed.