Vincent A. Fischetti

Streptococcal M protein: molecular design and biological behavior.

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. Images

A bacteriolytic agent that detects and kills Bacillus anthracis

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 γ 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.

alpha-enolase, a novel strong plasmin(ogen) binding protein on the surface of pathogenic streptococci.

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 α-enolase, a key glycolytic enzyme. Dose-dependent α-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 SEN-bound plasmin and SEN-specific immune responses, provide evidence for an important role of SEN in the disease process and post-streptococcal autoimmune diseases.

Sorting of protein a to the staphylococcal cell wall

The cell wall of gram-positive bacteria can be thought of as representing a unique cell compartment, which contains anchored surface proteins that require specific sorting signals. Some biologically important products are anchored in this way, including protein A and fibronectin binding protein of Staphylococcus aureus and streptococcal M protein. Studies of staphylococcal protein A and Escherichia coli alkaline phosphatase show that the signal both necessary and sufficient for cell wall anchoring consists of an LPXTGX motif, a C-terminal hydrophobic domain, and a charged tail. These sequence elements are conserved in many surface proteins from different gram-positive bacteria. We propose the existence of a hitherto undescribed sorting mechanism that positions proteins on the surface of gram-positive bacteria.

Prevention and elimination of upper respiratory colonization of mice by group A streptococci by using a bacteriophage lytic enzyme

Bacteriophage lytic enzymes quickly destroy the cell wall of the host bacterium to release progeny phage. Because such lytic enzymes specifically kill the species in which they were produced, they may represent an effective way to control pathogenic bacteria without disturbing normal microflora. In this report, we studied a murein hydrolase from the streptococcal bacteriophage C1 termed lysin. This enzyme is specific for groups A, C, and E streptococci, with little or no activity toward several oral streptococci or other commensal organisms tested. Using purified lysin in vitro, we show that 1,000 units (10 ng) of enzyme is sufficient to sterilize a culture of ≈107 group A streptococci within 5 seconds. When a single dose of lysin (250 units) is first added to the oral cavity of mice, followed by 107 live group A streptococci, it provides protection from colonization (28.5% infected, n = 21) compared with controls without lysin (70.5% infected, n = 17) (P < 0.03). Furthermore, when lysin (500 units) was given orally to 9 heavily colonized mice, no detectable streptococci were observed 2 h after lysin treatment. In all, these studies show that lysin represents a unique murein hydrolase that has a rapid lethal effect both in vitro and in vivo on group A streptococci, without affecting other indigenous microorganisms analyzed. This general approach may be used to either eliminate or reduce streptococci from the upper respiratory mucosal epithelium of either carriers or infected individuals, thus reducing associated disease.

Conservation of a hexapeptide sequence in the anchor region of surface proteins from gram-positive cocci.

A highly conserved hexapeptide sequence (both at the protein‐and DNA level) has been identified within the C‐terminal end of all 11 known surface proteins from Gram‐positive cocci. The hexapeptide, with the consensus sequence LPXTGE, is located about 9 amino acids N‐terminal from the C‐terminal hydrophobic domain which is found in all these surface molecules. The conservation of the hexapeptide, despite sequence variation within the regions flanking it, suggests that it is important for the attachment of these proteins within the cell.

Bacteriophage lysins as effective antibacterials

Lysins are highly evolved enzymes produced by bacteriophage (phage for short) to digest the bacterial cell wall for phage progeny release. In Gram-positive bacteria, small quantities of purified recombinant lysin added externally results in immediate lysis causing log-fold death of the target bacterium. Lysins have been used successfully in a variety of animal models to control pathogenic antibiotic resistant bacteria found on mucosal surfaces and infected tissues. The advantages over antibiotics are their specificity for the pathogen without disturbing the normal flora, the low chance of bacterial resistance to lysins, and their ability to kill colonizing pathogens on mucosal surfaces, a capacity previously unavailable. Thus, lysins may be a much needed anti-infective in an age of mounting antibiotic resistance.

Bacteriophage endolysins: A novel anti-infective to control Gram-positive pathogens

Endolysins (or lysins) are highly evolved enzymes produced by bacteriophage (phage for short) to digest the bacterial cell wall for phage progeny release. In Gram-positive bacteria, small quantities of purified recombinant lysin added externally results in immediate lysis causing log-fold death of the target bacterium. Lysins have been used successfully in a variety of animal models to control pathogenic antibiotic-resistant bacteria found on mucosal surfaces and infected tissues. Their specificity for the pathogen without disturbing the normal flora, the low chance of bacterial resistance, and their ability to kill colonizing pathogens on mucosal surfaces, a capacity previously unavailable, make them ideal anti-infectives in an age of mounting resistance. Here we review the current literature showing the effectiveness of these enzymes in controlling a variety of infections.

Exploiting CRISPR-Cas nucleases to produce sequence-specific antimicrobials

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 Cas9 (refs.1,2) delivered by a bacteriophage. We show that Cas9, reprogrammed to target virulence genes, kills virulent, but not avirulent, Staphylococcus aureus. Reprogramming the nuclease to target antibiotic resistance genes destroys staphylococcal plasmids that harbor antibiotic resistance genes and immunizes avirulent staphylococci to prevent the spread of plasmid-borne resistance genes. We also show that CRISPR-Cas9 antimicrobials function in vivo to kill S. aureus in a mouse skin colonization model. This technology creates opportunities to manipulate complex bacterial populations in a sequence-specific manner.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.

Genetic Diversity among Enterococcus faecalis

Enterococcus faecalis, a ubiquitous member of mammalian gastrointestinal flora, is a leading cause of nosocomial infections and a growing public health concern. The enterococci responsible for these infections are often resistant to multiple antibiotics and have become notorious for their ability to acquire and disseminate antibiotic resistances. In the current study, we examined genetic relationships among 106 strains of E. faecalis isolated over the past 100 years, including strains identified for their diversity and used historically for serotyping, strains that have been adapted for laboratory use, and isolates from previously described E. faecalis infection outbreaks. This collection also includes isolates first characterized as having novel plasmids, virulence traits, antibiotic resistances, and pathogenicity island (PAI) components. We evaluated variation in factors contributing to pathogenicity, including toxin production, antibiotic resistance, polymorphism in the capsule (cps) operon, pathogenicity island (PAI) gene content, and other accessory factors. This information was correlated with multi-locus sequence typing (MLST) data, which was used to define genetic lineages. Our findings show that virulence and antibiotic resistance traits can be found within many diverse lineages of E. faecalis. However, lineages have emerged that have caused infection outbreaks globally, in which several new antibiotic resistances have entered the species, and in which virulence traits have converged. Comparing genomic hybridization profiles, using a microarray, of strains identified by MLST as spanning the diversity of the species, allowed us to identify the core E. faecalis genome as consisting of an estimated 2057 unique genes.