Lytic enzyme discovery through multigenomic sequence analysis in Clostridium perfringens.
ABSTRACT: With their ability to lyse Gram-positive bacteria, phage lytic enzymes (or lysins) have received a great deal of attention as novel anti-infective agents. The number of known genes encoding these peptidoglycan hydrolases has increased markedly in recent years, due in large part to advances in DNA sequencing technology. As the genomes of more and more bacterial species/strains are sequenced, lysin-encoding open reading frames (ORFs) can be readily identified in lysogenized prophage regions. In the current study, we sought to assess lysin diversity for the medically relevant pathogen Clostridium perfringens. The sequenced genomes of nine C. perfringens strains were computationally mined for prophage lysins and lysin-like ORFs, revealing several dozen proteins of various enzymatic classes. Of these lysins, a muramidase from strain ATCC 13124 (termed PlyCM) was chosen for recombinant analysis based on its dissimilarity to previously characterized C. perfringens lysins. Following expression and purification, various biochemical properties of PlyCM were determined in vitro, including pH/salt-dependence and temperature stability. The enzyme exhibited activity at low ?g/ml concentrations, a typical value for phage lysins. It was active against 23 of 24 strains of C. perfringens tested, with virtually no activity against other clostridial or non-clostridial species. Overall, PlyCM shows potential for development as an enzybiotic agent, demonstrating how expanding genomic databases can serve as rich pools for biotechnologically relevant proteins.
Project description:Clostridium perfringens is a Gram-positive anaerobic spore-forming bacterium capable of producing four major toxins that are responsible for disease symptoms and pathogenesis in a variety of animals, humans, and poultry. The organism is the third leading cause of human foodborne bacterial disease, and C. perfringens is the presumptive etiologic agent of necrotic enteritis among chickens, which in the acute form can cause increased mortality among broiler flocks. Countries that have complied with the ban on antimicrobial growth promoters (AGP) in feeds have had increased incidences of C. perfringens-associated necrotic enteritis in poultry. To address this issue, new antimicrobial agents, putative lysins from the genomes of bacteriophages, are identified. Two putative phage lysin genes (ply) from the clostridial phages phiCP39O and phiCP26F were cloned and expressed in Escherichia coli , and the resultant proteins were purified to near homogeneity. Gene and protein sequencing revealed that the predicted and chemically determined amino acid sequences of the two recombinant proteins were homologous to N-acetylmuramoyl-l-alanine amidases. The proteins were identical in the C-terminal putative cell-wall binding domain, but only 55% identical to each other in the presumptive N-terminal catalytic domain. Both recombinant lysins were capable of lysing both parental phage host strains of C. perfringens as well as other strains of the bacterium in spot and turbidity reduction assays. The observed reduction in turbidity was correlated with up to a 3 log cfu/mL reduction in viable C. perfringens on brain-heart infusion agar plates. However, other member species of the clostridia were resistant to the lytic activity by both assays.
Project description:In the billion years that bacteriophage (or phage) have existed together with bacteria the phage have evolved systems that may be exploited for our benefit. One of these is the lytic system used by the phage to release their progeny from an infected bacterium. Endolysins (or lysins) are highly evolved enzymes in the lytic system produced to cleave essential bonds in the bacterial cell wall peptidoglycan for progeny release. Small quantities of purified recombinant lysin added externally to gram-positive bacteria results in immediate lysis causing log-fold death of the target bacterium. Lysins have now been used successfully in a variety of animal models to control pathogenic antibiotic resistant bacteria found on mucosal surfaces and in infected tissues. The advantages over antibiotics are 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. Lysins therefore, may be a much-needed anti-infective (or enzybiotic) in an age of mounting antibiotic resistance.
Project description:Bacteriophage lysins are compelling antimicrobial proteins whose biotechnological utility and evolvability would be aided by elevated stability. Lysin catalytic domains, which evolved as modular entities distinct from cell wall binding domains, can be classified into one of several families with highly conserved structure and function, many of which contain thousands of annotated homologous sequences. Motivated by the quality of these evolutionary data, the performance of generative protein models incorporating coevolutionary information was analyzed to predict the stability of variants in a collection of 9,749 multimutants across 10 libraries diversified at different regions of a putative lysin from a prophage region of a Clostridium perfringens genome. Protein stability was assessed via a yeast surface display assay with accompanying high-throughput sequencing. Statistical fitness of mutant sequences, derived from second-order Potts models inferred with different levels of sequence homolog information, was predictive of experimental stability with areas under the curve (AUCs) ranging from 0.78 to 0.85. To extract an experimentally derived model of stability, a logistic model with site-wise score contributions was regressed on the collection of multimutants. This achieved a cross-validated classification performance of 0.95. Using this experimentally derived model, 5 designs incorporating 5 or 6 mutations from multiple libraries were constructed. All designs retained enzymatic activity, with 4 of 5 increasing the melting temperature and with the highest-performing design achieving an improvement of +4°C.IMPORTANCE Bacteriophage lysins exhibit high specificity and activity toward host bacteria with which the phage coevolved. These properties of lysins make them attractive for use as antimicrobials. Although there has been significant effort to develop platforms for rapid lysin engineering, there have been numerous shortcomings when pursuing the ultrahigh throughput necessary for the discovery of rare combinations of mutations to improve performance. In addition to validation of a putative lysin and stabilization thereof, the experimental and computational methods presented here offer a new avenue for improving protein stability and are easily scalable to analysis of tens of millions of mutations in single experiments.
Project description:Beta-hemolytic Streptococcus agalactiae is the leading cause of bacteremia and invasive infections. These diseases are treated with β-lactams or macrolides, but the emergence of less susceptible and even fully resistant strains is a cause for concern. New bacteriophage lysins could be promising alternatives against such organisms. They hydrolyze the bacterial peptidoglycan at the end of the phage cycle, in order to release the phage progeny. By using a bioinformatic approach to screen several beta-hemolytic streptococci, a gene coding for a lysin was identified on a prophage carried by Streptococcus dysgalactiae subsp. equisimilis SK1249. The gene product, named PlySK1249, harbored an original three-domain structure with a central cell wall-binding domain surrounded by an N-terminal amidase and a C-terminal CHAP domain. Purified PlySK1249 was highly lytic and bactericidal for S. dysgalactiae (2-log10 CFU/ml decrease within 15 min). Moreover, it also efficiently killed S. agalactiae (1.5-log10 CFU/ml decrease within 15 min) but not several streptococcal commensal species. We further investigated the activity of PlySK1249 in a mouse model of S. agalactiae bacteremia. Eighty percent of the animals (n = 10) challenged intraperitoneally with 10(6) CFU of S. agalactiae died within 72 h, whereas repeated injections of PlySK1249 (45 mg/kg 3 times within 24 h) significantly protected the mice (P < 0.01). Thus, PlySK1249, which was isolated from S. dysgalactiae, demonstrated high cross-lytic activity against S. agalactiae both in vitro and in vivo. These encouraging results indicated that PlySK1249 might represent a good candidate to be developed as a new enzybiotic for the treatment of systemic S. agalactiae infections.
Project description:Clostridium perfringens is a Gram-positive, anaerobic spore-forming bacterium commonly found in soil, sediments, and the human gastrointestinal tract. C. perfringens is responsible for a wide spectrum of disease, including food poisoning, gas gangrene (clostridial myonecrosis), enteritis necroticans, and non-foodborne gastrointestinal infections. The complete genome sequences of Clostridium perfringens strain ATCC 13124, a gas gangrene isolate and the species type strain, and the enterotoxin-producing food poisoning strain SM101, were determined and compared with the published C. perfringens strain 13 genome. Comparison of the three genomes revealed considerable genomic diversity with >300 unique "genomic islands" identified, with the majority of these islands unusually clustered on one replichore. PCR-based analysis indicated that the large genomic islands are widely variable across a large collection of C. perfringens strains. These islands encode genes that correlate to differences in virulence and phenotypic characteristics of these strains. Significant differences between the strains include numerous novel mobile elements and genes encoding metabolic capabilities, strain-specific extracellular polysaccharide capsule, sporulation factors, toxins, and other secreted enzymes, providing substantial insight into this medically important bacterial pathogen.
Project description:Lysins are phage-encoded, peptidoglycan (cell wall) hydrolases that accumulate in the bacterial cytoplasm during a lytic infection cycle. Late during infection, the lysins undergo holin-mediated translocation across the inner membrane into the peptidoglycan matrix where they cleave cell wall covalent bonds required for wall stability and allow bacterial lysis and progeny phage release. This potent hydrolytic activity is now the foundation of a powerful genetic-based screening process for the identification and analysis of phage lysin proteins. Here, we describe a method for identifying a lysin, PlyG, from a bacteriophage that specifically infects the Gram-positive organism Bacillus anthracis; however, the techniques described can be adapted to clone, express, and analyze lysins from any phage infecting Gram-positive bacteria or possibly even Gram-negative bacteria.
Project description:Most bacteriophages (phages) release their progeny through the action of holins that form lesions in the cytoplasmic membrane and lysins that degrade the bacterial peptidoglycan. Although the function of each protein is well established in phages infecting Streptococcus pneumoniae, the role--if any--of the powerful bacterial autolysin LytA in virion release is currently unknown. In this study, deletions of the bacterial and phage lysins were done in lysogenic S. pneumoniae strains, allowing the evaluation of the contribution of each lytic enzyme to phage release through the monitoring of bacterial-culture lysis and phage plaque assays. In addition, we assessed membrane integrity during phage-mediated lysis using flow cytometry to evaluate the regulatory role of holins over the lytic activities. Our data show that LytA is activated at the end of the lytic cycle and that its triggering results from holin-induced membrane permeabilization. In the absence of phage lysin, LytA is able to mediate bacterial lysis and phage release, although exclusive dependence on the autolysin results in reduced virion egress and altered kinetics that may impair phage fitness. Under normal conditions, activation of bacterial LytA, together with the phage lysin, leads to greater phage progeny release. Our findings demonstrate that S. pneumoniae phages use the ubiquitous host autolysin to accomplish an optimal phage exiting strategy.
Project description: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.
Project description:Methicillin-resistant Staphylococcus aureus (MRSA) can cause a wide range of infections from mild to life-threatening conditions. Its enhanced antibiotic resistance often leads to therapeutic failures and therefore alternative eradication methods must be considered. Potential candidates to control MRSA infections are bacteriophages and their lytic enzymes, lysins. In this study, we isolated a bacteriophage against a nosocomial MRSA strain belonging to the ST45 epidemiologic group. The phage belonging to Caudovirales, Siphoviridae, showed a narrow host range and stable lytic activity without the emergence of resistant MRSA clones. Phylogenetic analysis showed that the newly isolated Staphylococcus phage R4 belongs to the Triavirus genus in Siphoviridae family. Genetic analysis of the 45?kb sequence of R4 revealed 69 ORFs. No remnants of mobile genetic elements and traces of truncated genes were observed. We have localized the lysin (N-acetylmuramoyl-L-alanine amidase) gene of the new phage that was amplified, cloned, expressed, and purified. Its activity was verified by zymogram analysis. Our findings could potentially be used to develop specific anti-MRSA bacteriophage- and phage lysin-based therapeutic strategies against major clonal lineages and serotypes.
Project description:The emergence of multidrug-resistant bacteria has made minor bacterial infections incurable with many existing antibiotics. Lysins are phage-encoded peptidoglycan hydrolases that have demonstrated therapeutic potential as a novel class of antimicrobials. The modular architecture of lysins enables the functional domains - catalytic domain (CD) and cell wall binding domain (CBD) - to be shuffled to create novel lysins. The CD is classically thought to be only involved in peptidoglycan hydrolysis whereas the CBD dictates the lytic spectrum of a lysin. While there are many studies that extended the lytic spectrum of a lysin by domain swapping, few have managed to introduce species specificity in a chimeric lysin. In this work, we constructed two chimeric lysins by swapping the CBDs of two parent lysins with different lytic spectra against enterococci and staphylococci. We showed that these chimeric lysins exhibited customized lytic spectra distinct from the parent lysins. Notably, the chimeric lysin P10N-V12C, which comprises a narrow-spectrum CD fused with a broad-spectrum CBD, displayed species specificity not lysing <i>Enterococcus faecium</i> while targeting <i>Enterococcus faecalis</i> and staphylococci. Such species specificity can be attributed to the narrow-spectrum CD of the chimeric lysin. Using flow cytometry and confocal microscopy, we found that the <i>E. faecium</i> cells that were treated with P10N-V12C are less viable with compromised membranes yet remained morphologically intact. Our results suggest that while the CBD is a major determinant of the lytic spectrum of a lysin, the CD is also responsible in the composition of the final lytic spectrum, especially when it pertains to species-specificity.