LysK CHAP endopeptidase domain is required for lysis of live staphylococcal cells

LysK is a staphylococcal bacteriophage endolysin composed of three domains: an N-terminal cysteine, histidine-dependent amidohydrolases/peptidases (CHAP) endopeptidase domain, a midprotein amidase 2 domain, and a C-terminal SH3b_5 (SH3b) cell wall-binding domain. Both catalytic domains are active on purified peptidoglycan by positive-ion electrospray ionization MS. The cut sites are identical to LytA (phi11 endolysin), with cleavage between d -alanine of the stem peptide and glycine of the cross-bridge peptide, and N -acetylmuramoyl- l -alanine amidase activity. Truncations of the LysK containing just the CHAP domain lyse Staphylococcus aureus cells in zymogram analysis, plate lysis, and turbidity reduction assays but have no detectable activity in a minimal inhibitory concentration (MIC) assay. In contrast, truncations harboring just the amidase lytic domain show faint activity in both the zymogram and turbidity reduction assays, but no detectable activity in either plate lysis or MIC assays. A fusion of the CHAP domain to the SH3b domain has near full-length LysK lytic activity, suggesting the need for a C-terminal binding domain. Both LysK and the CHAP-SH3b fusion were shown to lyse untreated S. aureus and the coagulase-negative strains. In the checkerboard assay, the CHAP-SH3b fusion achieves the same level of antimicrobial synergy with lysostaphin as the full-length LysK.     

Characterization of a lysin from deep-sea thermophilic bacteriophage GVE2

Thermostable enzymes from thermophiles have attracted extensive studies. However, little is known about thermophilic lysin of bacteriophage obtained from deep-sea hydrothermal vent. In this study, a lysin from deep-sea thermophilic bacteriophage Geobacillus virus E2 (GVE2) was characterized for the first time. It was found that the GVE2 lysin was highly homologous with N-acetylmuramoyl-L-alanine amidases. After expression in Escherichia coli, the recombinant GVE2 lysin was purified. The recombinant lysin was active over a range of temperature from 40 °C to 80 °C, with an optimum at 60 °C. Its optimal pH was 6.0, and it was stable over a wide range of pH from 4.0 to 10.0. The lysin was highly active when some enzyme inhibitors or detergents (phenylmethylsulfonyl fluoride, Tween 20, Triton X-100, and chaps) were used. However, it was strongly inhibited by sodium dodecyl sulfate and ethylene diamine tetraacetic acid. Its enzymatic activity could be slightly stimulated in the presence of Na⁺ and Li⁺. But the metal ions Mg²⁺, Ba²⁺, Zn²⁺, Fe³⁺, Ca²⁺, and Mn²⁺ at concentrations of 1 or 10 mM showed inhibitions to the lysin activity. Our study demonstrated the first characterization of lysin from deep-sea thermophilic bacteriophage.     

噬菌体裂解酶应用研究进展

近年来,随着抗生素的滥用,导致多重耐药性菌株出现的频率加快。因细菌感染导致死亡的人数逐年增多,人类健康面临巨大挑战,因此研制新型抗菌药物刻不容缓。噬菌体裂解酶因其高效的杀菌能力及高度的宿主专一性而成为新一代抗菌制剂的候选之一。其是一种细胞壁水解酶,在双链DNA噬菌体复制后期被合成,通过水解细胞壁肽聚糖上的化学键,从而裂解细菌细胞壁,释放出子代噬菌体。本文系统地介绍了噬菌体裂解酶的研究进展,为相关裂解酶抗菌药物的研发做出有益探索。     

Properties of the endolytic transglycosylase encoded by gene 144 of Pseudomonas aeruginosa bacteriophage phiKZ

Bacteriophage endolysins degrading bacterial cell walls are prospective enzymes for therapy of bacterial infections. The genome of the giant bacteriophage phiKZ of Pseudomonas aeruginosa encodes two endolysins, gene products (g.p.) 144 and 181, which are homologous to lytic transglycosylases. Gene 144 encoding a 260 amino acid residue protein was cloned into the plasmid expression vector. Recombinant g.p. 144 purified from Escherichia coli effectively degrades chloroform-treated P. aeruginosa cell walls. The protein has predominantly α-helical conformation and exists in solution in stoichiometric monomer: dimer: trimer equilibrium. Antibodies against the protein bind the phage particle. This demonstrates that g.p. 144 is a structural component of the phiKZ particle, presumably, a phage tail. Published in Russian in Biokhimiya, 2006, Vol. 71, No. 3, pp. 379–385.     

Lysis of staphylococcal mastitis pathogens by bacteriophage phi11 endolysin

The Staphylococcus aureus bacteriophage phi11 endolysin has two peptidoglycan hydrolase domains (endopeptidase and amidase) and an SH3b cell wall-binding domain. In turbidity reduction assays, the purified protein can lyse untreated staphylococcal mastitis pathogens, Staphylococcus aureus and coagulase-negative staphylococci (Staphylococcus chronogenes, Staphylococcus epidermidis, Staphylococcus hyicus, Staphylococcus simulans, Staphylococcus warneri and Staphylococcus xylosus), making it a strong candidate protein antimicrobial. This lytic activity is maintained at the pH (6.7), and the "free" calcium concentration (3 mM) of milk. Truncated endolysin-derived proteins containing only the endopeptidase domain also lyse staphylococci in the absence of the SH3b-binding domain.     

The truncated phage lysin CHAP(k) eliminates Staphylococcus aureus in the nares of mice

The endolysin LysK derived from staphylococcal phage K has previously been shown to have two enzymatic domains, one of which is an N-acetylmuramoyl-L-alanine amidase and the other a cysteine/histidine-dependant amidohydrolase/peptidase designated CHAP(k). The latter, when cloned as a single-domain truncated enzyme, is conveniently overexpressed in a highly-soluble form. This enzyme was shown to be highly active in vitro against live cell suspensions of S. aureus. In the current study, the IVIS imaging system was used to demonstrate the effective elimination of a lux labeled S. aureus from the nares of BALB/c mice.     

链球菌噬菌体裂解酶在大肠杆菌中的表达、纯化及活性检测

噬菌体裂解酶是噬菌体产生的细胞壁水解酶,通过水解宿主菌细胞壁使子代噬菌体释放,在体外能高效且特异性地杀死细菌。本研究旨在克隆和表达链球菌噬菌体裂解酶PlyC,并测定其生物学活性。利用PCR方法扩增PlyC的2条肽链PlyCA和PlyCB,构建表达载体pET-32a(+)-PlyCA和pET-32a(+)-PlyCB,分别转化至大肠杆菌BL21(DE3)中,以0.7 mmol/L IPTG在30 oC诱导7 h实现了高效表达,SDS-PAGE分析表明PlyCA和PlyCB表达量均可达菌体总蛋白的30%以上。采用Ni2+-NTA亲和层析法纯化目的蛋白,其纯度大于95%。用透析复性方法得到目的产物重组链球菌噬菌体裂解酶PlyC,以浊度法和平板计数法检测其体外抗菌效果,扫描电子显微镜观察裂解酶作用前后细菌细胞形态变化。结果表明重组PlyC能特异性裂解化脓性链球菌(A组β-溶血性链球菌),以4μg/mL浓度作用于OD600为0.56的菌液60 min后杀菌率达99.6%,扫描电镜观察结果显示该酶作用于菌体后,链球菌细胞裂解,呈碎片状态。本研究为开发一种新型、高效的链球菌感染疾病治疗药物打下了基础。     

PlyC, a novel bacteriophage lysin for compartment-dependent proteomics of group A streptococci

Streptococcus pyogenes (Spy) (group A streptococci) is an important and exclusively human bacterial pathogen, which uses secreted and surface-associated proteins to circumvent the innate host defense mechanisms and to adhere and internalize into host cells. Thus, investigation of the bacterial extracellular compartments, including secreted and cell wall-associated subproteomes, is crucial for understanding adherence, invasion, and internalization mechanisms as major steps of Spy pathogenesis. Here, we compared a bacteriophage encoded cell wall hydrolase, PlyC, a multimeric lysin of the C1 bacteriophage, with the established glycosidase, mutanolysin, from Streptomyces globisporus for their suitability to efficiently digest Spy cell walls and release cell wall-anchored Spy proteins for subsequent proteome research. Our results show that PlyC is superior for cell wall protein extraction compared to mutanolysin due to its higher activity and specificity as an N-acetylmuramoyl-L-alanine amidase. Furthermore, our experimental design allowed us to delineate the actual localization of the proteins despite contamination with intracellular proteins.     

Prevalence, genetic conservation and transmissibility of the Chlamydia pneumoniae bacteriophage (phiCpn1)

The Chlamydia pneumoniae bacteriophage was first identified in isolate AR-39. Its relevance for chlamydial biology and pathogenicity remains unknown. In this study, a collection of 36 C. pneumoniae isolates was screened and the phage was detected in eight. As the positive isolates differed by several polymorphisms, they presumably belonged to different genetic lineages. It was investigated whether different genotypes of the phage also existed and whether they could be assigned to chlamydial genotypes as evidence of coevolution. Sequencing of >3000 bp of the 4524 bp phage genome revealed complete identity to the published sequences. Thus, it was hypothesized that the genetic conservation was related to easy transmissibility of the phage between C. pneumoniae isolates. Cocultivation of phage positive and negative isolates followed by cloning and identification of different C. pneumoniae genotypes demonstrated for the first time transmissibility of the bacteriophage from one isolate to the other. These observations indicate that the phage is capable of infecting C. pneumoniae isolates of different genetic backgrounds and suggest that all C. pneumoniae strains might be susceptible. The successful in vitro infection of C. pneumoniae with the phage provides the basis for studying its pathogenetic relevance in isolates of identical genetic background and provides a potential tool for genetic manipulation of C. pneumoniae.     

Complete genome sequence of a newly isolated lytic bacteriophage,EFAP‐1 of Enterococcus faecalis,and antibacterial activity of its endolysin EFAL‐1

Aims: In this work, we aimed to identify an effective treatment of infections caused by Enterococcus spp. strains resistant to conventional antibiotics. Methods and Results: We report the isolation and characterization of a new lytic bacteriophage, designated bacteriophage EFAP‐1, that is capable of lysing Enterococcus faecalis bacteria that exhibit resistance to multiple antibiotics. EFAP‐1 has low sequence similarity to all known bacteriophages. Transmission electron microscopy confirmed that EFAP‐1 belongs to the Siphoviridae family. A putative lytic protein of EFAP‐1, endolysin EFAL‐1, is encoded in ORF 2 and was expressed in Escherichia coli. Recombinant EFAL‐1 had broad‐spectrum lytic activity against several Gram‐positive pathogens, including Ent. faecalis and Enterococcus faecium. Conclusions: The complete genome sequence of the newly isolated enterococcal lytic phage was analysed, and it was demonstrated that its recombinant endolysin had broad lytic activity against various Gram‐positive pathogens. Significance and Impact of the Study: Bacteriophage EFAP‐1 and its lytic protein, EFAL‐1, can be utilized as potent antimicrobial agents against Enterococcus spp. strains resistant to conventional antibiotics in hospital infections and also as environmental disinfectants to control disease‐causing Enterococcus spp. in dairy farms.     

Inhibition of bacteriophage K proliferation on Staphylococcus aureus in raw bovine milk

AIMS: To assess the ability of staphylococcal bacteriophage K to inhibit Staphylococcus aureus in raw milk. METHODS AND RESULTS: The ability of bacteriophage (phage) to replicate in milk is important in situations where phage might be used as a therapeutic for bovine mastitis. Phage K was able to replicate normally, leading to elimination of the host culture in milk, which had been previously heat-treated. When raw milk was used under identical conditions, the phages were unable to replicate. Phage adsorption assays were performed and these demonstrated that adsorption of phage was significantly reduced in the raw milk while it was restored in the heat-treated sample (86.50% compared with 99.96% adsorption respectively). When confocal microscopy with a Live/Dead Bac light staining system was employed, it was observed that in raw milk S. aureus formed clusters associated with fat globules, while in heat-treated milk, bacterial agglutination had not occurred. CONCLUSIONS: Raw milk inhibits staphylococcal phage K proliferation. Significance and Impact of the Study: This observation has implications for the exploitation of staphylococcal therapeutic phage in milk.     

Characterisation of coliphage K30, a bacteriophage specific for Escherichia coli capsular serotype K30

Abstract Coliphage K30, a bacteriophage specific for strains bearing the Escherichia coli serotype K30 capsular polysaccharide, produced plaques surrounded by extensive haloes, a characteristic of phage which produce capsule depolymerase (glycanase) enzymes. Klebsiella K20, a strain producing a capsular polysaccharide chemically identical to that of E. coli K30, was not lysed by coliphage K30, although the bacteriophage encoded glycanase enzyme did degrade the K20 polysaccharide. Morphologically, coliphage K30 belonged to Bradley group C. The coliphage K30 particle comprised 20 structural polypeptides which varied from 9.5–136 kDa and genomic DNA of 38.7 ± 1.0 kb.     

Identification and characterization of the two‐component cell lysis cassette encoded by temperate bacteriophage ϕPYB5 of Lactobacillus fermentum

Aims: To characterize the two‐component cell lysis cassette comprised of holin (Hyb5) and endolysin (Lyb5) encoded by Lactobacillus fermentum temperate bacteriophage ?PYB5, and illustrate the potential application of Lyb5 as therapeutic agents. Methods and Results: The hyb5–lyb5 cassette was cloned from the genome library of ?PYB5, and the hyb5, lyb5 and hyb5–lyb5 cassette were expressed in E. coli BL21, respectively. The molecular weight of Hyb5 indicated by SDS‐PAGE was 19 kDa, and Lyb5 was 45 kDa. Both Hyb5 and Lyb5 protein could induce cell lysis alone, resulting in the leakage of β‐galactosidase. However, the Hyb5–Lyb5 cassette lysed the host cells more rapidly and extensively. By zymogram analysis, Lyb5 exhibited a broad lytic spectrum. Conclusions: Overexpression of hyb5, lyb5 and hyb5–lyb5 cassette were carried out in E. coli and Lyb5 exhibited a broad lytic spectrum. Significance and Impact of the Study: The Lyb5 produced in E. coli exhibited a broad lytic spectrum against Gram‐positive strains including Staphylococcus aureus as well as Gram‐negative strains such as Salmonella typhi, suggesting that Lyb5 provides a potential alternative of diagnostic tools and therapeutic agents.     

一株产1，3-丙二醇的克雷伯氏菌的噬菌体生物学特性

[目的]克雷伯氏菌发酵生产1,3-丙二醇过程中发生噬菌体感染会严重影响宿主菌的生长和目标产物的生成,因此分离克雷伯氏菌噬菌体并考察其生物学特性对预防和控制噬菌体感染具有重要意义.[方法]采用敏感指示菌法及Adams双层平板法从感染噬菌体的肺炎克雷伯氏杆菌发酵液中分离得到一株噬菌体;纯化后用磷钨酸负染法电镜观察;手工法提取噬菌体核酸,酶切后琼脂糖凝胶电泳分析;同时考察了其最佳感染复数、一步生长曲线及对温度、pH、紫外线、乙醚和氯仿等理化因素的敏感性等生理特性;最后考察了噬菌体对肺炎克雷伯氏杆菌生长和发酵的影响.[结果]分离出一株肺炎克雷伯氏杆菌溶源性噬菌体,其噬菌斑为无晕环透明圆斑,直径约1.5 mm;其头部为直径约60 nm-70 nm的球体,有一长约160 nm的丝状长尾;基因组核酸能被双链DNA内切酶EcoR Ⅰ及HindⅢ切开,大小约42 kb;对高温和紫外线敏感,耐碱性而受强酸抑制,对氯仿不敏感;最佳感染复数为1,潜伏期与裂解期均为50 min,裂解量为343个;感染噬菌体的肺炎克雷伯氏杆菌的细胞生长延滞约8h,代谢流偏向乳酸途径.[结论]该噬菌体属于无包膜长尾噬菌体,能改变克雷伯氏菌发酵生产1,3-丙二醇的代谢规律,为1,3-丙二醇发酵生产过程中噬菌体感染的预防和控制研究奠定了基础.     

Characterization of filamentous bacteriophage PE226 infecting Ralstonia solanacearum strains

Aims: The aim of this study was to isolate and characterize new bacteriophages that infect a wide range of plant pathogenic Ralstonia solanacearum strains. Methods and Results: Fifteen bacteriophages were isolated from pepper, tomato and tobacco plant rhizospheres infected with R. solanacearum. A host specificity analysis of the isolated phages using nine strains of R. solanacearum indicated great phage diversity in a single soil. Two phages, PE226 and TM227, showed clear plaques on all nine bacterial hosts tested and were virtually identical in morphology and genome. PE226, an Inovirus, is a long, flexible, filamentous phage carrying a circular (+) sense single‐strand DNA genome of 5475 nucleotides. DNA sequences of PE226 exhibited nine open reading frames (ORF) that were not highly similar to those of other phages infecting R. solanacearum. The genome organization of PE226 was partially similar to that of p12J of Ralstonia pickettii. One ORF of PE226 showed identity to the zot gene encoding zonula occludens toxin of Vibrio cholera. Orf7 of PE226 was also present in the genome of R. solanacearum strain SL341. However, SL341, a highly virulent strain in tomato, was still sensitive to phage PE226. Conclusions: A new, flexible, filamentous phage PE226 infected wide range of R. solanacearum strains and carried unique circular single‐strand DNA genome with an ORF encoding Zot‐like protein. Significance and Impact of the Study: PE226 may be a new type of temperate phage, based on its lytic nature on a wide range of hosts and the presence of a zot homologue in a host bacterial genome.     

Susceptibility of Staphylococcus epidermidis planktonic cells and biofilms to the lytic action of staphylococcus bacteriophage K

AIMS: To evaluate differences in biofilm or planktonic bacteria susceptibility to be killed by the polyvalent antistaphylococcus bacteriophage K. METHODS AND RESULTS: In this study, the ability of phage K to infect and kill several clinical isolates of Staphylococcus epidermidis was tested. Strains were grown in suspension or as biofilms to compare the susceptibility of both phenotypes to the phage lytic action. Most strains (10/11) were susceptible to phage K, and phage K was also effective in reducing biofilm biomass after 24 h of challenging. Biofilm cells were killed at a lower rate than the log-phase planktonic bacteria but at similar rate as stationary phase planktonic bacteria. CONCLUSIONS: Staphylococcus epidermidis biofilms and stationary growth phase planktonic bacteria are more resistant to phage K lysis than the exponential phase planktonic bacteria. SIGNIFICANCE OF STUDY: This study shows the differences in Staph. epidermidis susceptibility to be killed by bacteriophage K, when grown in biofilm or planktonic phenotypes.     

The P5 protein from bacteriophage phi-6 is a distant homolog of lytic transglycosylases

Peptidases are classical objects of enzymology and structural studies. However, a few protein families with experimentally characterized proteolytic activity, but unknown catalytic mechanism and three-dimensional structures, still exist. Using comparative sequence analysis, we deduce spatial structure for one of such families, namely, U40, which contains just one P5 protein from bacteriophage phi-6. We show that this singleton sequence possesses conserved sequence motifs characteristic of lysozymes and is a distant homolog of lytic transglycosylases that cleave bacterial peptidoglycan. The structure of the P5 protein is therefore predicted to adopt the lysozyme-like fold shared by T4, lambda, C-type, G-type lysozymes, and lytic transglycosylases. Since previous biochemical experiments with P5 of phi-6 have indicated that the purified enzyme possesses endopeptidase activity and not glycosidase activity, our results point to the possibility of a newly evolved molecular function and call for further experimental characterization of this unusual P5 protein.     

Bovine whey proteins inhibit the interaction of Staphylococcus aureus and bacteriophage K

AIMS: To understand the potential use of bacteriophage K to treat bovine Staphylococcus aureus mastitis, we studied the role of whey proteins in the inhibition of the phage-pathogen interaction in vitro. METHODS AND RESULTS: The interaction of bacteriophage K and S. aureus strain Newbould 305 was studied in raw bovine whey and serum. Incubation of S. aureus with phage in whey showed that the bacteria are more resistant to phage lysis when grown in whey and also bovine serum. Whey collected from 23 animals showed a wide variation in the level of phage-binding inhibition. The role of the protein component of milk whey in this inhibition was established; treatment of the whey by heat, proteases and ultrafiltration removed the inhibitory activity. Brief exposure of S. aureus cells to whey, followed by resuspension in broth, also reduced phage binding. Microscopy showed the adhesion of extracellular material to the S. aureus cell surface following exposure to whey. Chromatographic fractionation of the whey demonstrated that the inhibitory proteins were present in the high molecular weight fraction. CONCLUSIONS: The adsorption of whey proteins to the S. aureus cell surface appeared to inhibit phage attachment and thereby hindered lysis. The inhibitory whey proteins are of high molecular weight in their native form and may sterically block phage attachment sites on the cell surface. SIGNIFICANCE AND IMPACT OF THE STUDY: These findings have implications for any future use of phage therapy in the treatment of mastitis, and other diseases, caused by S. aureus. This pathogen is predicted to be much more resistant to phage treatment in vivo than would be expected from in vitro broth culture experiments.     

Characterization of induced Staphylococcus aureus bacteriophage SAP-26 and its anti-biofilm activity with rifampicin

Lytic bacteriophages (phages) have been investigated as treatments for bacterial infectious diseases. An induced phage, SAP-26, was isolated from a clinical isolate of Staphylococcus aureus. It belongs to the family Siphoviridae and its genome consists of double-stranded 41,207 bp DNA coding for 63 open reading frames. The phage SAP-26 showed a wide spectrum of lytic activity against both methicillin-resistant S. aureus and methicillin-susceptible S.aureus. Furthermore, combined treatment with a phage and antimicrobial agents showed a strong biofilm removal effect which induced structural changes in the biofilm matrix and a substantial decrease in the number of bacteria. Such a broad host range in S. aureus and biofilm removal activity of the phage SAP-26 suggests the possibility of its use as a therapeutic phage in combination with appropriate antimicrobial agent(s). Among the three antimicrobial agents combined with phage, the combination of rifampicin showed the best biofilm removal effect. To the authors' knowledge, this study showed for the first time that S. aureus biofilm could be efficiently eradicated with the mixture of phage and an antimicrobial agent, especially rifampicin.     

Endopeptidase and glycosidase activities of the bacteriophage B30 lysin

Synthetic peptides corresponding to portions of group B streptococcal peptidoglycan were used to show that the endopeptidase activity of bacteriophage B30 lysin cleaves between D-Ala in the stem peptide and L-Ala in the cross bridge and that the minimal peptide sequence cleaved is DL-gamma-Glu-Lys-D-Ala-Ala-Ala. The only glycosidase activity present is that of N-acetyl-beta-D-muramidase.