Antibiotics target conserved bacterial cellular pathways or growth functions and therefore cannot selectively kill specific members of a complex microbial population. Here, we develop programmable, sequence-specific antimicrobials using the RNA-guided nuclease Cas91, 2 delivered by a bacteriophage. We show that Cas9 re-programmed to target virulence genes kills virulent, but not avirulent, Staphylococcus aureus. Re-programming the nuclease to target antibiotic resistance genes destroys staphylococcal plasmids that harbor antibiotic resistance genes3, 4 and immunizes avirulent staphylococci to prevent the spread of plasmid-borne resistance genes. We also demonstrate the approach in vivo, showing its efficacy against S. aureus in a mouse skin colonization model. This new technology creates opportunities to manipulate complex bacterial populations in a sequence-specific manner.

The plasmin(ogen) binding property of group A streptococci is incriminated in tissue invasion processes. We have characterized a novel 45-kDa protein displaying strong plasmin(ogen) binding activity from the streptococcal surface. Based on its biochemical properties, we confirmed the identity of this protein as ␣-enolase, a key glycolytic enzyme. Dose-dependent ␣-enolase activity, immune electron microscopy of whole streptococci using specific antibodies, and the opsonic nature of polyclonal and monoclonal antibodies concluded the presence of this protein on the streptococcal surface. We, henceforth, termed the 45-kDa protein, SEN (streptococcal surface enolase). SEN is found ubiquitously on the surface of most streptococcal groups and serotypes and showed significantly greater plasmin(ogen) binding affinity compared with previously reported streptococcal plasminogen binding proteins. Both the C-terminal lysine residue of SEN and a region N-terminal to it play a critical role in plasminogen binding. Results from competitive plasminogen binding inhibition assays and cross-linking studies with intact streptococci indicate that SEN contributes significantly to the overall streptococcal ability to bind plasmin(ogen). Our findings, showing both the protected protease activity of SENbound plasmin and SEN-specific immune responses, provide evidence for an important role of SEN in the disease process and post-streptococcal autoimmune diseases.Streptococcus pyogenes is responsible for a wide variety of human diseases that range from suppurative infections of the throat (pharyngitis), skin (impetigo), and underlying tissues (necrotizing fasciitis), to an often fatal toxic shock syndrome, and the post-streptococcal sequelae, rheumatic fever, and acute glomerulonephritis. Bacterial surface proteins play a major role in these disease processes by exhibiting a wide range of functions. As data have become available, it is clear that most surface proteins found on Gram-positive bacteria, particularly those on group A streptococci, have a great deal of structural similarities (1, 2). Proteins for which the function(s) has been defined have been found to be multifunctional, whereas in others a function has only been attributed to one of two or more domains (2, 3). Thus, the multifunctional characteristics of these surface proteins increase the complexity of the Grampositive surface beyond what has been previously imagined.We recently described one such multifunctional protein, streptococcal surface dehydrogenase (SDH), 1 as a major surface protein on group A streptococci and other streptococcal groups which is structurally and functionally related to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (4). SDH also has an ADP-ribosylating activity (5) and exhibits multiple binding activities to several mammalian proteins such as fibronectin and cytoskeletal proteins (4). A structurally and enzymatically similar streptococcal protein, Plr, was also identified on group A streptococci, based on its ability to bind plasmin (6). SDH, how...

The dormant and durable spore form of Bacillus anthracis is an ideal biological weapon of mass destruction. Once inhaled, spores are transported by alveolar macrophages to lymph nodes surrounding the lungs, where they germinate; subsequent vegetative expansion causes an overwhelming flood of bacteria and toxins into the blood, killing up to 99% of untreated victims. Natural and genetically engineered antibiotic-resistant bacilli amplify the threat of spores being used as weapons, and heighten the need for improved treatments and spore-detection methods after an intentional release. We exploited the inherent binding specificity and lytic action of bacteriophage enzymes called lysins for the rapid detection and killing of B. anthracis. Here we show that the PlyG lysin, isolated from the gamma phage of B. anthracis, specifically kills B. anthracis isolates and other members of the B. anthracis 'cluster' of bacilli in vitro and in vivo. Both vegetative cells and germinating spores are susceptible. The lytic specificity of PlyG was also exploited as part of a rapid method for the identification of B. anthracis. We conclude that PlyG is a tool for the treatment and detection of B. anthracis.

SummaryThe surface of streptococci presents an array of different proteins, each designed to perform a specific function. In an attempt to understand the early events in group A streptococci infection, we have identified and purified a major surface protein from group A type 6 streptococci that has both an enzymatic activity and a binding capacity for a variety of proteins. Mass spectrometric analysis of the purified molecule revealed a monomer of 35.8 kD. Molecular sieve chromatography and sodium dodecyl sulfate (SDS)-gel dectrophoresis suggest that the native conformation of the protein is likely to be a tetramer of 156 kD. NH2-terminal amino acid sequence analysis revealed 83% homology in the first 18 residues and about 56% in the first 39 residues with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) of eukaryotic or bacterial origin. This streptococcal surface GAPDH (SDH) exhibits a dose-dependent dehydrogenase activity on glyceraldehyde-3-phosphate in the presence of fl-nicotinamide adenine dinucleotide both in its pure form and on the streptococcal surface. Its sensitivity to trypsin on whole organism and its inability to be removed with 2 M NaCI or 2% SDS support its surface location and tight attachment to the streptococcal cell. Affinity-purified antibodies to SDH detected the presence of this protein on the surface of all M serotypes of group A streptococcal tested. Purified SDH was found to bind to fibronectin, lysozyme, as well as the cytoskeletal proteins myosin and actin. The binding activity to myosin was found to be localized to the globular heavy meromyosin domain. SDH did not bind to streptococcal M protein, tropomyosin, or the coiled-coil domain of myosin. The multiple binding capacity of the SDH in conjunction with its GAPDH activity may play a role in the colonization, internalization, and the subsequent proliferation of group A streptococci.

For 70 years antibiotics have saved countless lives and enabled the development of modern medicine, but it is becoming clear that the success of antibiotics may have only been temporary and we now anticipate a long-term, generational and perhaps never-ending challenge to find new therapies to combat antibiotic-resistant bacteria. As the search for new conventional antibiotics has become less productive and there are no clear strategies to improve success, a broader approach to address bacterial infection is needed. This review of potential alternatives to antibiotics (A2As) was commissioned by the Wellcome Trust, jointly funded by the Department of Health, and involved scientists and physicians from academia and industry. For the purpose of this review, A2As were defined as non-compound approaches (that is, products other than classical antibacterial agents) that target bacteria or approaches that target the host. In addition, the review was limited to agents that had potential to be administered orally, by inhalation or by injection for treatment of systemic/invasive infection. Within these criteria, the review has identified 19 A2A approaches now being actively progressed. The feasibility and potential clinical impact of each approach was considered. The most advanced approaches (and the only ones likely to deliver new treatments by 2025) are antibodies, probiotics, and vaccines now in Phase II and Phase III trials. These new agents will target infections caused by P. aeruginosa, C. difficile and S. aureus. However, other than probiotics for C. difficile, this first wave will likely best serve as adjunctive or preventive therapies. This suggests that conventional antibiotics will still be needed. The economics of pathogen-specific therapies must improve to encourage innovation, and greater investment into A2As with broad-spectrum activity (e.g. antimicrobial-, host defense-and, anti-biofilm peptides) is needed. Increased funding, estimated at >£1.5 bn over 10 years is required to validate and then develop these A2As. Investment needs to be partnered with translational expertise and targeted to support the validation of these approaches at Clinical Phase II proof of concept. Such an approach could transform our understanding of A2As as effective new therapies and should provide the catalyst required for both active engagement and investment by the pharma/biotech industry. Only a sustained, concerted and coordinated international effort will provide the solutions needed for the next decade.

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M protein is a major virulence determinant for the group A streptococcus by virtue of its ability to allow the organism to resist phagocytosis. Common in eucaryotes, the fibrillar coiled-coil design for the M molecule may prove to be a common motif for surface proteins in gram-positive organisms. This type of structure offers the organism several distinct advantages, ranging from antigenic variation to multiple functional domains. The close resemblance of this molecular design to that of certain mammalian proteins could help explain on a molecular level the formation of epitopes responsible for serological cross-reactions between microbial and mammalian proteins. Many of the approaches described in the elucidation of the M-protein structure may be applied for characterizing similar molecules in other microbial systems.

Nasopharyngeal carriage is the major reservoir for Streptococcus pneumoniae in the community. Although eliminating this reservoir would greatly reduce disease occurrence, no suitable intervention has been available for this purpose. We show here that seconds after contact, a purified pneumococcal bacteriophage lytic enzyme (Pal) is able to kill 15 common serotypes of pneumococci, including highly penicillin-resistant strains. In vivo, previously colonized mice revealed undetectable pneumococcal titers 5 hours after a single enzyme treatment. Pal enzyme had little or no effect on microorganisms normally found in the human oropharynx, and Pal-resistant pneumococci could not be detected after extensive exposure to the enzyme.