Factors Influencing the Adsorption of Bacteriophage 2 to Cells of Pseudomonas aeruginosa

Phage 2 adsorbed to Pseudomonas aeruginosa strain BI in 5 mM Tris buffer, providing that cations like Na(+), Mg(2+), or Ca(2+) were present. Adsorption was observed over a broad pH range, reaching a maximum level around pH 7.5, which coincided with the pH required for maximal activity of the phage 2-associated slime polysaccharide depolymerase. Mutants of strain BI and other strains of P. aeruginosa possessing slime layers that were devoid of phage 2 depolymerase substrate were incapable of adsorbing phage 2. On the other hand, those strains containing substrate for the phage 2 depolymerase in the slime layer were capable of adsorbing phage 2. The same relationship of phage depolymerase-substrate interaction to phage adsorption was observed with Pseudomonas phage 8, which possesses a depolymerase that differs in its specificity from the phage 2 depolymerase. The receptor-like activity of purified slime containing the specific substrate for the phage-associated depolymerase was demonstrable by its ability to inactivate phage. However, receptor-like activity or phage inactivation was not observed with those slimes that were devoid of the depolymerase substrate.     

Independent functions of viral protein and nucleic acid in growth of bacteriophage

1. Osmotic shock disrupts particles of phage T2 into material containing nearly all the phage sulfur in a form precipitable by antiphage serum, and capable of specific adsorption to bacteria. It releases into solution nearly all the phage DNA in a form not precipitable by antiserum and not adsorbable to bacteria. The sulfur-containing protein of the phage particle evidently makes up a membrane that protects the phage DNA from DNase, comprises the sole or principal antigenic material, and is responsible for attachment of the virus to bacteria. 2. Adsorption of T2 to heat-killed bacteria, and heating or alternate freezing and thawing of infected cells, sensitize the DNA of the adsorbed phage to DNase. These treatments have little or no sensitizing effect on unadsorbed phage. Neither heating nor freezing and thawing releases the phage DNA from infected cells, although other cell constituents can be extracted by these methods. These facts suggest that the phage DNA forms part of an organized intracellular structure throughout the period of phage growth. 3. Adsorption of phage T2 to bacterial debris causes part of the phage DNA to appear in solution, leaving the phage sulfur attached to the debris. Another part of the phage DNA, corresponding roughly to the remaining half of the DNA of the inactivated phage, remains attached to the debris but can be separated from it by DNase. Phage T4 behaves similarly, although the two phages can be shown to attach to different combining sites. The inactivation of phage by bacterial debris is evidently accompanied by the rupture of the viral membrane. 4. Suspensions of infected cells agitated in a Waring blendor release 75 per cent of the phage sulfur and only 15 per cent of the phage phosphorus to the solution as a result of the applied shearing force. The cells remain capable of yielding phage progeny. 5. The facts stated show that most of the phage sulfur remains at the cell surface and most of the phage DNA enters the cell on infection. Whether sulfur-free material other than DNA enters the cell has not been determined. The properties of the sulfur-containing residue identify it as essentially unchanged membranes of the phage particles. All types of evidence show that the passage of phage DNA into the cell occurs in non-nutrient medium under conditions in which other known steps in viral growth do not occur. 6. The phage progeny yielded by bacteria infected with phage labeled with radioactive sulfur contain less than 1 per cent of the parental radioactivity. The progeny of phage particles labeled with radioactive phosphorus contain 30 per cent or more of the parental phosphorus. 7. Phage inactivated by dilute formaldehyde is capable of adsorbing to bacteria, but does not release its DNA to the cell. This shows that the interaction between phage and bacterium resulting in release of the phage DNA from its protective membrane depends on labile components of the phage particle. By contrast, the components of the bacterium essential to this interaction are remarkably stable. The nature of the interaction is otherwise unknown. 8. The sulfur-containing protein of resting phage particles is confined to a protective coat that is responsible for the adsorption to bacteria, and functions as an instrument for the injection of the phage DNA into the cell. This protein probably has no function in the growth of intracellular phage. The DNA has some function. Further chemical inferences should not be drawn from the experiments presented.     

Efficient identification of phosphatidylserine-binding proteins by ORF phage display

To efficiently elucidate the biological roles of phosphatidylserine (PS), we developed open-reading-frame (ORF) phage display to identify PS-binding proteins. The procedure of phage panning was optimized with a phage clone expressing MFG-E8, a well-known PS-binding protein. Three rounds of phage panning with ORF phage display cDNA library resulted in ∼300-fold enrichment in PS-binding activity. A total of 17 PS-binding phage clones were identified. Unlike phage display with conventional cDNA libraries, all 17 PS-binding clones were ORFs encoding 13 real proteins. Sequence analysis revealed that all identified PS-specific phage clones had dimeric basic amino acid residues. GST fusion proteins were expressed for 3 PS-binding proteins and verified for their binding activity to PS liposomes, but not phosphatidylcholine liposomes. These results elucidated previously unknown PS-binding proteins and demonstrated that ORF phage display is a versatile technology capable of efficiently identifying binding proteins for non-protein molecules like PS.     

Isolation and Characterization of a New Generalized Transducing Bacteriophage Different from Pl in Escherichia coli

A new generalized transducing bacteriophage in the Escherichia coli system was isolated and characterized. This phage, designated D108, makes clear plaques on E. coli K-10, K-12, K-12(P1kc), K-12(D6), B/r, C, and 15 T(-), and Shigella dysenteriae. The plaque of phage D108 is larger in size than that of phage P1kc. Electron-microscopic observation revealed that phages D108 and P1kc are morphologically different from each other, suggesting that phage D108 belongs to a phage group different from phage P1. The fact that all of the 10 markers tested were transduced by phage D108 indicates that this phage is a generalized transducing phage in the E. coli system. The transduction frequency by phage D108 of chromosomal markers and of a drug resistance factor (R factor) ranged from 2 x 10(-6) to 3 x 10(-8) and 3 x 10(-9) to 6 x 10(-10) per phage, respectively. The cotransduction frequency of the thr and leu markers was 2.8% for phage P1kc and 1.5% for phage D108. The CM and TC markers (chloramphenicol-resistant and tetracycline-resistant markers, respectively) of the R factor were not cotransduced by phage D108, but the markers were generally cotransduced by phage P1kc. The results suggest that the transducing particle of phage D108 contains a smaller amount of host deoxyribonucleic acid than does phage P1kc.     

Structural and energetic basis of infection by the filamentous bacteriophage IKe

Filamentous phage use the two N‐terminal domains of their gene‐3‐proteins to initiate infection of Escherichia coli. One domain interacts with a pilus, and then the other domain binds to TolA at the cell surface. In phage fd, these two domains are tightly associated with each other, which renders the phage robust but non‐infectious, because the TolA binding site is inaccessible. Activation for infection requires partial unfolding, domain disassembly and prolyl isomerization. Phage IKe infects E. coli less efficiently than phage fd. Unlike in phage fd, the pilus‐ and TolA‐binding domains of phage IKe are independent of each other in stability and folding. The site for TolA binding is thus always accessible, but the affinity is very low. The structures of the two domains, analysed by X‐ray crystallography and by NMR spectroscopy, revealed a unique fold for the N‐pilus‐binding domain and a conserved fold for the TolA‐binding domain. The absence of an activation mechanism as in phage fd and the low affinity for TolA probably explain the low infectivity of phage IKe. They also explain why, in a previous co‐evolution experiment with a mixture of phage fd and phage IKe, all hybrid phage adopted the superior infection mechanism of phage fd.     

Factors affecting survival of bacteriophage on tomato leaf surfaces

The ability of bacteriophage to persist in the phyllosphere for extended periods is limited by many factors, including sunlight irradiation, especially in the UV zone, temperature, desiccation, and exposure to copper bactericides. The effects of these factors on persistence of phage and formulated phage (phage mixed with skim milk) were evaluated. In field studies, copper caused significant phage reduction if applied on the day of phage application but not if applied 4 or 7 days in advance. Sunlight UV was evaluated for detrimental effects on phage survival on tomato foliage in the field. Phage was applied in the early morning, midmorning, early afternoon, and late evening, while UVA plus UVB irradiation and phage populations were monitored. The intensity of UV irradiation positively correlated with phage population decline. The protective formulation reduced the UV effect. In order to demonstrate direct effects of UV, phage suspensions were exposed to UV irradiation and assayed for effectiveness against bacterial spot of tomato. UV significantly reduced phage ability to control bacterial spot. Ambient temperature had a pronounced effect on nonformulated phage but not on formulated phages. The effects of desiccation and fluorescent light illumination on phage were investigated. Desiccation caused a significant but only slight reduction in phage populations after 60 days, whereas fluorescent light eliminated phages within 2 weeks. The protective formulation eliminated the reduction caused by both of these factors. Phage persistence was dramatically affected by UV, while the other factors had less pronounced effects. Formulated phage reduced deleterious effects of the studied environmental factors.     

Factors Affecting Survival of Bacteriophage on Tomato Leaf Surfaces

The ability of bacteriophage to persist in the phyllosphere for extended periods is limited by many factors, including sunlight irradiation, especially in the UV zone, temperature, desiccation, and exposure to copper bactericides. The effects of these factors on persistence of phage and formulated phage (phage mixed with skim milk) were evaluated. In field studies, copper caused significant phage reduction if applied on the day of phage application but not if applied 4 or 7 days in advance. Sunlight UV was evaluated for detrimental effects on phage survival on tomato foliage in the field. Phage was applied in the early morning, midmorning, early afternoon, and late evening, while UVA plus UVB irradiation and phage populations were monitored. The intensity of UV irradiation positively correlated with phage population decline. The protective formulation reduced the UV effect. In order to demonstrate direct effects of UV, phage suspensions were exposed to UV irradiation and assayed for effectiveness against bacterial spot of tomato. UV significantly reduced phage ability to control bacterial spot. Ambient temperature had a pronounced effect on nonformulated phage but not on formulated phages. The effects of desiccation and fluorescent light illumination on phage were investigated. Desiccation caused a significant but only slight reduction in phage populations after 60 days, whereas fluorescent light eliminated phages within 2 weeks. The protective formulation eliminated the reduction caused by both of these factors. Phage persistence was dramatically affected by UV, while the other factors had less pronounced effects. Formulated phage reduced deleterious effects of the studied environmental factors.     

Primary structure of an EcoRI fragment of lambda imm434 DNA containing regions cI-cro of phage 434 and cII-o of phage lambda.

Digestion of phage lambda imm434 DNA with restriction endonuclease EcoRI yields 7 fragments. The shortest among them (1287 bp) contains the right part of the phage 434 immunity region and the phage DNA portion proximal to it. The complete primary structure of this fragment has been determined using the chemical method of DNA sequencing. Hypothetical amino-acid sequences of proteins coded by the cro gene of phage 434 and the cII gene of phage lambda, as well as NH2-terminal amino-acid sequences of the cI protein of phage 434 and the O protein of phage lambda, have been deduced solely on the basis of the DNA sequence. The fragment studied contains also the pR and probably prm promoters and the oR operator of phage 434. The sequence coding for them differs from the respective DNA sequence of phage lambda.     

A guard-killer phage cocktail effectively lyses the host and inhibits the development of phage-resistant strains of Escherichia coli

In recent years, after the emergence of a large number of multidrug-resistant bacteria, phages and phage-associated products for the prevention and control of bacterial disease have revealed prominent advantages as compared with antibiotics. However, bacteria are susceptible to becoming phage-resistant, thus severely limiting the application of phage therapy. In this study, Escherichia coli cells were incubated with lytic bacteriophages to obtain mutants that were resistant to the lytic phages. Then, bacteriophages against the phage-resistant variants were isolated and subsequently mixed with the original lytic phage to prepare a novel phage cocktail for bactericidal use. The data showed that our phage cocktail not only had notable bactericidal effects, including a widened host range and rapid lysis, but also decreased the generation and mutation frequency of phage-resistant strains in vitro. In addition, we tested our cocktail in a murine bacteremia model. The results suggested that compared with the single phage, fewer phage-resistant bacteria appeared during the treatment of phage cocktail, thus prolonging the usable time of the phage cocktail and improving its therapeutic effect in phage applications. Importantly, our preparation method of phage cocktail was proved to be generalizable. Because the bacteriophage against the phage-resistant strain is an ideal guard that promptly attacks potential phage resistance, this guard-killer dual-function phage cocktail provides a novel strategy for phage therapy that allows the natural ecology to be sustained.

     

Direct measurement via phage titre of the dissociation constants in solution of fusion phage-substrate complexes.

Studies of interactions between filamentous fusion phage particles and protein or nucleic acid molecules have gained increasing importance with recent successes of screening techniques based upon random phage display libraries (biopanning). Since a number of different phage are usually obtained by biopanning, it is useful to compare quantitatively the binding affinities of individual phage for the substrate used for selection. A procedure is described for determination of relative dissociation constants (KdRel) between filamentous phage carrying peptide fusions to the coat protein gpIII and substrates in solution. This novel method is based on the measurement of phage titres. Phage selected from a random fusion phage library for binding to a monoclonal antibody or a viral structural protein exhibited KdRel values in the nanomolar and micromolar ranges for their respective substrates, thus validating the method over a wide range of binding affinities.     

Cleavage of DNA from 2 phages of Bacillus thuringiensis by EcoRI and HindIII endonucleases

The DNA of two Bacillus thuringiensis phages was restricted by endonucleases EcoRI and HindIII and the electrophoretic distribution of the fragments in agarose gel was studied. EcoRI was shown to restrict the DNA of phage 1-97A into 8 fragments and the DNA of phage 1-97B into 12 fragments. Restriction with HindIII results in the formation of 22 and 9 fragments for phage 1-97A and phage 1-97B, respectively. The molecular mass of the DNAs determined by summing up EcoRI restricts is 80.87 MDa for phage 1-97A and 32.45 MDa for phage 1-97B.     

The Mechanism of Inactivation of T4 Bacteriophage by Tritium Decay

Coliphage T4 was used as a model system to study the mechanism of biological inactivation produced by tritium decay. Experimentally, tritiated precursors were incorporated into phage DNA (thymidine-³H) or into phage protein (³H-amino acids). The ratio of killing efficiencies for decays originating in phage DNA to those originating in phage protein was 2.6. Inactivation by decays from labeled amino acids was assumed to occur exclusively from β-particle irradiation of phage DNA. If decays originating in DNA are due solely to irradiation of DNA, then the killing efficiencies reflect the energy transfer paths in phage DNA for decays originating in phage DNA and in the protein coat. The energy transfer paths were determined for the two cases with the help of a computer and found to be very nearly equal to the experimentally determined ratio (2.6). The killing efficiencies for decays originating in phage DNA were 0.12 and for decays originating in protein 0.046.     

Inhibition of Host Deoxyribonucleic Acid Synthesis by T4 Bacteriophage in the Absence of Protein Synthesis

The requirement for phage protein synthesis for the inhibition of host deoxyribonucleic acid synthesis has been investigated by using a phage mutant unable to catalyze the production of any phage deoxyribonucleic acid. It has been concluded that the major pathway whereby phage inhibit host syntheses requires protein synthesis. The inhibition of host syntheses by phage ghosts is not affected by inhibitors of protein synthesis.     

Encapsulation of Bacteriophage in Liposome Accentuates Its Entry in to Macrophage and Shields It from Neutralizing Antibodies

Phage therapy has been a centre of attraction for biomedical scientists to treat infections caused by drug resistant strains. However, ability of phage to act only on extracellular bacteria and probability of interference by anti-phage antibodies in vivo is considered as a important limitation of bacteriophage therapy. To overcome these hurdles, liposome were used as delivery vehicle for phage in this study. Anti-phage antibodies were raised in mice and pooled serum was evaluated for its ability to neutralize free and liposome entrapped phage. Further, ability of phage and liposome-entrapped phage to enter mouse peritoneal macrophages and kill intracellular Klebsiella pneumoniae was compared. Also, an attempt to compare the efficacy of free phage and liposome entrapped phage, alone or in conjunction with amikacin in eradicating mature biofilm was made. The entrapment of phage in liposome provided 100% protection to phage from neutralizing antibody. On the contrary un-entrapped phage got neutralized within 3 h of its interaction with antibody. Compared to the inability of free phage to enter macrophages, the liposome were able to deliver entrapped phage inside macrophages and cause 94.6% killing of intracellular K. pneumoniae. Liposome entrapped phage showed synergistic activity along with amikacin to eradicate mature biofilm of K. pneumoniae. Our study reinforces the growing interest in using phage therapy as a means of targeting multidrug resistant bacterial infections as liposome entrapment of phage makes them highly effective in vitro as well as in vivo by overcoming the majority of the hurdles related to clinical use of phage.     

Development of a broad-host-range phage cocktail for biocontrol

The aims of this study were to investigate the incidence of different resistance mechanisms to phage K in a bank of Irish Staph aureus hospital strains; and to develop a broad host-range phage cocktail with enhanced lytic activity against those strains which were previously phage resistant. A bank of 180 Staph aureus strains, which included all the sequence types currently in existence in Ireland, were tested for sensitivity to phage K. Twenty nine strains were identified, which did not permit plaque formation. The phage resistance systems in the 29 strain were investigated and it was found that restriction modification (r-m) was evident in 24, an adsorption inhibition mechanism was evident in three, while two were resistant by an unidentified mechanism. Seventeen modified derivatives of phage K were developed which could circumvent all the r-m systems. Nevertheless, six of the modified phage were considered superior in terms of their individual host ranges. These six were pooled as a cocktail with phage K, which then lysed 24 of the 29 resistant strains (97.2% of the entire staphylococcal bank). In conclusion, phage resistant systems affecting phage K are common in Staph. aureus but it is possible to significantly broaden the host-range of this phage for biocontrol applications.     

噬菌体抗菌治疗安全性评估体系的建立

人类已经进入后抗生素时代,噬菌体治疗近年来重新备受重视,噬菌体制剂不同于传统抗菌药物,已有对传统抗菌药物的安全性评估体系不适合用于对噬菌体治疗制剂的评估,需要建立对噬菌体治疗安全性评估的体系。本文就噬菌体治疗所涉及的安全性问题进行系统分析研究,通过噬菌体本身的选择、噬菌体制剂制备、制剂形式、制剂给予途径、剂量和频次等,以及噬菌体治疗细菌感染性疾病患者选择等所涉及的安全性和噬菌体治疗对周围微环境的影响等进行全面分析。建立噬菌体治疗安全性评估体系,为噬菌体治疗尽早进入临床奠定基础。     

bFGF特异性结合噬菌体的活性研究

研究从噬菌体随机7肽库中筛选的bFGF特异性结合噬菌体的活性。采用ELISA检测3轮淘选后得到的阳性噬菌体克隆对bFGF的结合曲线。通过竞争抑制实验,分析bFGF结合噬菌体与bFGF的竞争结合活性。采用MTT法检测bFGF结合噬菌体对由bFGF诱导的Balb/c3T3细胞增殖的影响。从噬菌体肽库中淘选获得的阳性噬菌体克隆能够特异地与bFGF结合,并呈剂量依赖性,其与固相bFGF的结合能被游离的bFGF竞争抑制,并可抑制由bFGF诱导的Balb/c3T3细胞增殖。bFGF特异性噬菌体可拮抗bFGF的活性,为开发针对bFGF的抗肿瘤短肽类新药提供基础。     

Mapping of the c-region of phage phi80

A set of c-mutants of the phage phi80 is isolated. These mutants fit into three genes. According to plaque morphology and frequency of lysogenization of mutants, the genes were named cI, cII and cIII as it was previously done for phage lambda. Their order, determinated by mutant phage crosses, is cIII-sus326-cI-cII-sus250. Sus326 is a mutation in the gene 15, so it is probably an analogue of the N gene of the phage lambda. Thermoinducible mutants of the phage phi80 cts11 and cts12 correspond to the mutant types cItsB and cItsA of the phage lambda and they complement each other. Thus, it is supposed that phi80 phage repressor molecules consist of few protein subunits.     

Control of the Replication Complex of Bacteriophage P22

A replication complex for the vegetative synthesis of the deoxyribonucleic acid (DNA) of the temperate phage P22 previously has been described. This complex is an association of parental phage DNA, most of the newly synthesized phage DNA made during pulses with (3)H-thymidine, and other cell constituents, and has a sedimentation rate in neutral sucrose gradients of at least 1,000S. The complex is one of the intermediates, intermediate I, in the synthesis and maturation of phage P22 DNA after infection or induction. Evidence supporting the replicative nature of intermediate I is presented. Phage replication is repressed in lysogenic bacteria. On superinfection of P22 lysogens with nonvirulent phage, little association of the input phage DNA with a rapidly sedimenting fraction is demonstrable. However, after induction with ultraviolet light, the superinfecting parental phage DNA quickly acquires the rapid sedimentation rate characteristic of intermediate I; phage DNA synthesis follows; and progeny phages are produced. Infection with a virulent mutant of P22 produces progeny phages in lysogens. Its DNA associates with intermediate I. In mixed infection with the virulent phage, replication of nonvirulent phage P22 is still repressed, even though the virulent replicates normally. The nonvirulent input DNA does not associate with intermediate I. The repressor of the lysogenic cell prevents replication by interfering with the physical association of template material with intermediate I. A phage function is required for association of phage template with the replication machinery.     

细菌群体感应系统与噬菌体的相互作用机制研究进展

细菌在与噬菌体的长期共进化过程中形成多种抵抗噬菌体侵染的机制,其中群体感应参与的细菌抵御噬菌体侵染机制成为近年来的研究热点。群体感应与噬菌体之间的相互作用是复杂和多样的,本文将重点综述群体感应在噬菌体侵染中的作用、调控在噬菌体裂解-溶源转变的作用,以及群体感应与噬菌体的其他相互影响等内容,为噬菌体在细菌性疾病的治疗提供理论依据。