Use of a Bacteriophage Lysin,PlyC, as an Enzyme Disinfectant against Streptococcus equi

Streptococcus equi is the causative agent of the purulent infection equine strangles. This disease is transmitted through shedding of live bacteria from nasal secretions and abscess drainage or by contact with surfaces contaminated by the bacteria. Disinfectants are effective against S. equi, but inactivation by environmental factors, damage to equipment, and toxicity are of great concern. Bacteriophage-encoded lysins (cell wall hydrolases) have been investigated as therapeutic agents due to their ability to lyse susceptible gram-positive organisms. Here, we investigate the use of one lysin, PlyC, as a narrow-spectrum disinfectant against S. equi. This enzyme was active against >20 clinical isolates of S. equi, including both S. equi subsp. equi and S. equi subsp. zooepidemicus. Significantly, PlyC was 1,000 times more active on a per weight basis than Virkon-S, a common disinfecting agent, with 1 μg of enzyme able to sterilize a 10⁸ CFU/ml culture of S. equi in 30 min. PlyC was subjected to a standard battery of tests including the Use Dilution Method for Testing Disinfectants and the Germicidal Spray Products Test. Results indicate that aerosolized PlyC can eradicate or significantly reduce the S. equi load on a variety of materials found on common stable and horse-related equipment. Additionally, PlyC was shown to retain full activity under conditions that mimic a horse stable, i.e., in the presence of nonionic detergents, hard water, or organic materials. We propose PlyC as the first protein-based, narrow-spectrum disinfectant against S. equi, which may augment or supplement the use of broad-spectrum disinfectants in barns and stables where equine strangles is prevalent.Equine strangles is a highly contagious lymphadenitis of the head and neck that is uniquely caused by Streptococcus equi, predominantly S. equi subsp. equi, with some disease associated with S. equi subsp. zooepidemicus (24, 29). Progression of the purulent infection leads to acute swelling and subsequent abscess formation of the submaxillary, submandibular, and retropharyngeal lymph nodes causing a “strangling” of the pharynx, with severe cases necessitating a tracheotomy (24). Serious complications occur in approximately 20% of infected horses, and the overall mortality rate has been reported to be as high as 8% on farms where the infection is endemic (25). Even after recovery from infection, long-lasting, immune-mediated complications such as progressive muscle atrophy have been reported, which can adversely affect the career of a racing, show, or work horse (27).The route of S. equi transmission is through nasal secretions and drainage from abscesses. Infected horses can nasally shed bacteria for weeks, contaminating surfaces through which other horses can become infected. S. equi extract and attenuated-live vaccines exist, but they are often associated with abscess formation at the site of injection, short duration of immunity, poor efficacy, and the very real threat of a nascent infection from the vaccine (15, 24, 29). Thus, strangles continues to be a serious and widespread infectious disease of horses despite the presence of multiple commercially available vaccines.Strangles prevention strategies include good disinfection/hygiene practices, isolation of infected animals, and removal of equipment for sanitization where possible (9, 26; also R. E. Holland, D. G. Harris, and A. Monge, presented at the 52nd Annual Convention of the American Association of Equine Practitioners, San Antonio, TX, 2 to 6 December 2006). Current broad-spectrum disinfectants belong to one of several chemical categories including alcohols, aldehydes, biguanides, halogens, oxidizing agents, phenols, or quaternary ammonium compounds (6, 8). To various degrees, these compounds have been shown to be flammable, light sensitive, carcinogenic, corrosive to metals, irritating to mucous membranes, and/or toxic to livestock and humans (8, 10). Additionally, many factors that are often associated with cleaning stalls/barns (e.g., hard water, organic load, or detergents) can reduce or even ablate efficacy of chemical disinfectants (8). Importantly, studies have shown that these commonly used disinfectants can select for mutant bacteria with decreased susceptibility to biocides and antibiotics without compromising virulence (21).Recently, bacteriophage-encoded peptidoglycan hydrolases, collectively termed lysins and often referred to as “enzybiotics,” have been investigated as potential therapeutic agents against pathogens due to their ability to lyse the bacterial cell wall (12). These enzymes not only exert their lethal effects in the absence of bacteriophage (cause “lysis from without”) but also display specificity for a bacterial host, often for a particular genus, species, or even a subspecies depending on the lysin (11). For example, one lysin, PlyC, is known to lyse streptococcal species bearing a polyrhamnose epitope, which include group C streptococcus (i.e., S. equi subsp. equi) among other streptococci (19). As an adjunct to broad-spectrum disinfectants, we investigate here the use of the PlyC enzyme to help control the acquisition and spread of S. equi subsp. equi in horse stalls and barns.     

Origin of a Core Bacterial Gene via Co-option and Detoxification of a Phage Lysin

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Combined Antibacterial Activity of Phage Lytic Proteins Holin and Lysin from Streptococcus suis Bacteriophage SMP

Development of novel antibacterial agents is required to control infection with multidrug-resistant Streptococcus suis. HolSMP and LySMP, the holin and lysin of S. suis serotype 2 bacteriophage, named SMP, are responsible for lysis of host cells and release of progeny phage. HolSMP and LySMP expressed in Escherichia coli BL21(DE3) exerted efficient activity at 37?°C, pH 5.2, with addition of 0.8?% β-mercaptoethanol. Lytic spectra of purified HolSMP, LySMP or HolSMP?+?LySMP mixture were investigated. HolSMP, exhibiting a narrow lytic spectrum, was effective against Staphylococcus aureus and Bacillus subtilis, which were insensitive to LySMP. Moreover, HolSMP was identified as a promising antibacterial agent which was able to extend the spectrum of LySMP. The data suggest that combined use of holin and lysin could be a candidate strategy for resolution of drug resistance.     

Phage Lysin LysK Can Be Truncated to Its CHAP Domain and Retain Lytic Activity against Live Antibiotic-Resistant Staphylococci

A truncated derivative of the phage endolysin LysK containing only the CHAP (cysteine- and histidine-dependent amidohydrolase/peptidase) domain exhibited lytic activity against live clinical staphylococcal isolates, including methicillin-resistant Staphylococcus aureus. This is the first known report of a truncated phage lysin which retains high lytic activity against live staphylococcal cells.     

Application of the C3-Binding Motif of Streptococcal Pyrogenic Exotoxin B to Protect Mice from Invasive Group A Streptococcal Infection

Group A streptococcus (GAS) is an important human pathogen that produces several extracellular exotoxins to facilitate invasion and infection. Streptococcal pyrogenic exotoxin B (SPE B) has been demonstrated to be an important virulence factor of GAS. Our previous studies indicate that SPE B cleaves complement 3 (C3) and inhibits the activation of complement pathways. In this study, we constructed and expressed recombinant fragments of SPE B to examine the C3-binding site of SPE B. Using enzyme-linked immunosorbent assays and pull-down assays, we found that the C-terminal domain, containing amino-acid residues 345–398, of SPE B was the major binding site of human serum C3. We further identified a major, Ala³⁷⁶-Pro³⁹⁸, and a minor C3-binding motif, Gly³⁴⁶-Gly³⁶⁰, that both mediated the binding of C3 complement. Immunization with the C3-binding motifs protected mice against challenge with a lethal dose of non-invasive M49 strain GAS but not invasive M1 strains. To achieve higher efficiency against invasive M1 GAS infection, a combination of synthetic peptides derived from C-terminal epitope of streptolysin S (SLSpp) and from the major C3-binding motif of SPE B (PP6, Ala³⁷⁶-Pro³⁹⁸) was used to elicit specific immune response to those two important streptococcal exotoxins. Death rates and the severity of skin lesions decreased significantly in PP6/SLSpp-immunized mice that were infected with invasive M1 strains of GAS. These results indicate a combination of the C3-binding motif of SPE B and the protective epitope of SLS could be used as a subunit vaccine against invasive M1 strains group A streptococcal infection.     

Intersubunit Hydrophobic Interactions in Pf1 Filamentous Phage

Magic angle spinning solid-state NMR has been used to study the structural changes in the Pf1 filamentous bacteriophage, which occur near 10 °C. Comparisons of NMR spectra recorded above and below 10 °C reveal reversible perturbations in many NMR chemical shifts, most of which are assigned to atoms of hydrophobic side chains of the 46-residue subunit. The changes mainly involve groups located in patches on the interfaces between neighboring capsid subunits. The observations show that the transition adjusts the hydrophobic interfaces between fairly rigid subunits. The low temperature form has been generally more amenable to structure determination; spin diffusion experiments on this form revealed unambiguous contacts between side chains of neighboring subunits. These contacts are important constraints for structure modeling.     

辅助噬菌体在噬菌体展示中的应用及研究进展

噬菌体展示技术是一种将外源肽或蛋白质与特定噬菌体衣壳蛋白相融合,展示于噬菌体表面来构建蛋白质或多肽文库,并从中筛选目的蛋白、多肽或抗体的基因工程高新技术。噬菌粒/辅助噬菌体系统是最常用的噬菌体展示系统,此系统中辅助噬菌体对噬菌粒的复制和组装发挥着至关重要的作用。本文结合当今该领域的最新研究动态,概述了噬菌粒和辅助噬菌体双基因组系统,着重介绍了不同辅助噬菌体的特点及其突变机制,并对其应用前景进行了展望,以期为该技术的进一步完善提供一定的借鉴作用。     

Influence of the Chitosan Oligomer on the Phage Particles and Reproduction of Phage 1-97A in the Culture of Bacillus thuringiensis

The causes of bacteriophage 1-97A inactivation by the chitosan oligomer with a polymerization degree of 15 and the influence of the oligomer on the phage reproduction in the culture of Bacillus thuringiensissubsp. galleriae, strain 1-97, were studied. The study of the inactivation kinetics showed that, in 1 h, virtually all chitosan was bound to the phage particles, causing, as evidenced by electron microscopy, DNA release from the phage head, destruction of the phage particles, and agglutination of the phage particles or of their tails in the region of the basal plate. High-polymeric chitosan caused more pronounced destruction of the phage particles than the oligomer. It was established that chitosan prevented the production of complete phage particles. One of the mechanisms of such an influence may be the production in the presence of chitosan of phage particles devoid of DNA.     

Streptococcal M protein: structural studies of the hypervariable region, free and bound to human C4BP

Streptococcus pyogenes is a Gram-positive bacterium that causes several diseases, including acute tonsillitis and toxic shock syndrome. The surface-localized M protein, which is the most extensively studied virulence factor of S. pyogenes, has an approximately 50-residue N-terminal hypervariable region (HVR) that plays a key role in the escape of the host immunity. Despite the extensive sequence variability in this region, many HVRs specifically bind human C4b-binding protein (C4BP), a plasma protein that inhibits complement activation. Although the more conserved parts of M protein are known to have dimeric coiled-coil structure, it is unclear whether the HVR also is a coiled coil. Here, we use nuclear magnetic resonance (NMR) to study the conformational properties of HVRs from M4 and M22 proteins in isolation and in complex with the M protein binding portion of C4BP. We conclude that the HVRs of M4 and M22 are folded as coiled coils and that the folded nucleus of the M4 HVR has a length of approximately 27 residues. Moreover, we demonstrate that the C4BP binding surface of M4-N is found within a region of four heptad repeats. Using molecular modeling, we propose a model for the structure of the M4 HVR that is consistent with our experimental information from NMR spectroscopy.     

Unstable Lysogeny and Pseudolysogeny in Vibrio harveyi Siphovirus-Like Phage 1

Exposure of Vibrio harveyi (strain VH1114) to V. harveyi siphovirus-like phage 1 (VHS1) resulted in the production of a low percentage of lysogenized clones of variable stability. These were retrieved most easily as small colonies within dot plaques. Analysis revealed that VHS1 prophage was most likely carried by VH1114 as an episome rather than integrated into the host chromosome. In the late exponential growth phase, lysogenized VH1114 continuously produced VHS1 but also gave rise to a large number of cured progeny. The absence of phage DNA in the cured progeny was confirmed by the absence of VHS1 DNA in Southern blot and PCR assays. Curiously, these very stable, cured subclones did not show the parental phenotype of clear plaques with VHS1 but instead showed turbid plaques, both in overlaid lawns and in dot plaque assays. This phenotypic difference from the original parental isolate suggested that transient lysogeny by VHS1 had resulted in a stable genetic change in the cured clones. Such clones may be called pseudolysogens (i.e., false lysogens), since they have undergone transient lysogeny and have retained some resistance to full lytic phage development, despite the loss of viable or detectable prophage.     

Genomic,Proteomic, Morphological,and Phylogenetic Analyses of vB_EcoP_SU10, a Podoviridae Phage with C3 Morphology

A recently isolated phage, vB_EcoP_SU10 (SU10), with the unusual elongated C3 morphotype, can infect a wide range of Escherichia coli strains. We have sequenced the genome of this phage and characterized it further by mass spectrometry based proteomics, transmission electron microscopy (TEM), scanning electron microscopy (SEM), and ultra-thin section electron microscopy. The genome size is 77,327 base pairs and its genes, and genome architecture, show high similarity to the phiEco32 phage genes and genome. The TEM images reveal that SU10 have a quite long tail for being a Podoviridae phage, and that the tail also changes conformation upon infection. The ultra-thin section electron microscopy images of phages at the stage of replication within the host cell show that the phages form a honeycomb-like structure under packaging of genomes and assembly of mature capsids. This implies a tight link between the replication and cutting of the concatemeric genome, genome packaging, and capsid assembly. We have also performed a phylogenetic analysis of the structural genes common between Podoviridae phages of the C1 and C3 morphotypes. The result shows that the structural genes have coevolved, and that they form two distinct groups linked to their morphotypes. The structural genes of C1 and C3 phages appear to have diverged around 280 million years ago applying a molecular clock calibrated according to the presumed split between the Escherichia – Salmonella genera.     

Cloning of Rhizobium meliloti Locus sxf C Involved in Symbiosis and Phage Sensitivity

A symbiotic mutant of Rhizobium meliloti Rmd438 (sxf C:: Tn5) which was phage resistant against RMP64, failed to utilize galactose as carbon source as reported earlier (21). The Bg/ll gene bank of wild type R. meliloti was mobilized into Rmd438 and a clone pSP676 which complemented for phage sensitivity was isolated. In order to characterize this clone, a Bg/ll and EcoRI map was constructed. The insert of 13.2 kb had three Bg/ll fragments of 4.0, 3.6 and 5.6 kb in this order. All three fragments were subcloned on the vector pRK290 and mobilized into Sxf-mutants. The complementation for phage sensitivity, symbiosis and galactose utilization properties are discussed.     

Transduction of Various R Factors by Phage P1 in Escherichia coli and by Phage P22 in Salmonella typhimurium

R factors fi(+) and fi(-), with various combinations of drug-resistance markers and isolated from independent sources, were transduced by phage P1kc in Escherichia coli and by phage P22 in Salmonella typhimurium. Usually the entire R factor was transduced by P1kc in E. coli, as indicated by the absence of segregation of the drug-resistance markers from their conjugal transferability. In contrast, the patterns of segregation of the drug-resistance markers and their conjugal transferability differed considerably among various R factors after transduction by P22 in S. typhimurium. Transduction frequencies varied among R factors in both transduction systems.     

Fluid-phase interaction of C1 inhibitor (C1 Inh) and the subcomponents C1r and C1s of the first component of complement, C1.

Interactions between proenzymic or activated complement subcomponents of C1 and C1 Inh (C1 inhibitor) were analysed by sucrose-density-gradient ultracentrifugation and sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. The interaction of C1 Inh with dimeric C1r in the presence of EDTA resulted into two bimolecular complexes accounting for a disruption of C1r. The interaction of C1 Inh with the Ca2+-dependent C1r2-C1s2 complex (8.8 S) led to an 8.5 S inhibited C1r-C1s-C1 Inh complex (1:1:2), indicating a disruption of C1r2 and of C1s2 on C1 Inh binding. The 8.5 S inhibited complex was stable in the presence of EDTA; it was also formed from a mixture of C1r, C1s and C1 Inh in the presence of EDTA or from bimolecular complexes of C1r-C1 Inh and C1s-C1 Inh. C1r II, a modified C1r molecule, deprived of a Ca2+-binding site after autoproteolysis, did not lead to an inhibited tetrameric complex on incubation with C1s and C1 Inh. These findings suggest that, when C1 Inh binds to C1r2-C1s2 complex, the intermonomer links inside C1r2 or C1s2 are weakened, whereas the non-covalent Ca2+-independent interaction between C1r2 and C1s2 is strengthened. The nature of the proteinase-C1 Inh link was investigated. Hydroxylamine (1M) was able to dissociate the complexes partially (pH 7.5) or totally (pH 9.0) when the incubation was performed in denaturing conditions. An ester link between a serine residue at the active site of C1r or C1s and C1 Inh is postulated.