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。     

Engineering dihydropteroate synthase (DHPS) for efficient expression on M13 phage

Phage display is a commonly used selection technique in protein engineering, but not all proteins can be expressed on phage. Here, we describe the expression of a cytoplasmic homodimeric enzyme dihydropteroate synthetase (DHPS) on M13 phage, established by protein engineering of DHPS. The strategy included replacement of cysteine residues and screening for periplasmic expression followed by random mutagenesis and phage display selection with a conformation-specific anti-DHPS antibody. Cysteine replacement alone resulted in a 12-fold improvement in phage display of DHPS, but after random mutagenesis and three rounds of phage display selection, phage display efficiency of the library had improved 280-fold. Most of the selected clones had a common Asp96Asn mutation that was largely responsible for the efficient phage display of DHPS. Asp96Asn affected synergistically with the cysteine replacing mutations that were needed to remove the denaturing effect of potential wrong disulfide bridging in phage display. Asp96Asn alone resulted in a 1.8-fold improvement in phage display efficiency, but in combination with the cysteine replacing mutations, a total of 130-fold improvement in phage display efficiency of DHPS was achieved.     

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.

     

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.     

In vitro selection of a peptide with high selectivity for cardiomyocytes in vivo

One approach to targeted therapies for cardiovascular disease relies on isolating ligands that enhance the tissue-specific uptake of genes or drugs by heart cells. To obtain heart-targeting ligands, phage display biopanning was used to isolate a 20-mer peptide that binds to isolated primary cardiomyocytes. The isolated phage, PCM.1, displays the peptide WLSEAGPVVTVRALRGTGSW, and binds these cells 180 times better than a control phage from the library. Furthermore, phage displaying this peptide preferentially bind to cardiomyocytes when compared with a panel of other cell types. A BLAST search revealed that this peptide contains a 12 amino acid segment with sequence identity to a peptide in tenascin-X, an extracellular matrix protein. Synthetic peptides containing the complete 20-mer or a 12-mer tenascin peptide partially blocked phage binding to the cardiomyocytes. We developed a quantitative real-time PCR assay to assess uptake of this phage by tissues in vivo. Using this assay, preferential localization of the PCM.1 phage in heart was observed compared to the uptake of this phage by other tissues or other phage by heart. Furthermore, PCM.1 phage was associated with cardiomyocytes isolated from mice treated with a phage in vivo. These results demonstrate the utility of biopanning on isolated cells for identifying specific binding peptides that can target a tissue in vivo.     

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.     

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.     

Cloning of genomic DNA of Lactococcus lactis that restores phage sensitivity to an unusual bacteriophage sk1-resistant mutant

An unusual, spontaneous, phage sk1-resistant mutant (RMSK1/1) of Lactococcus lactis C2 apparently blocks phage DNA entry into the host. Although no visible plaques formed on RMSK1/1, this host propagated phage at a reduced efficiency. This was evident from center-of-infection experiments, which showed that 21% of infected RMSK1/1 formed plaques when plated on its phage-sensitive parental strain, C2. Moreover, viable cell counts 0 and 4 h after infection were not significantly different from those of an uninfected culture. Further characterization showed that phage adsorption was normal, but burst size was reduced fivefold and the latent period was increased from 28.5 to 36 min. RMSK1/1 was resistant to other, but not all, similar phages. Phage sensitivity was restored to RMSK1/1 by transformation with a cloned DNA fragment from a genomic library of a phage-sensitive strain. Characterization of the DNA that restored phage sensitivity revealed an open reading frame with similarity to sequences encoding lysozymes (beta-1,4-N-acetylmuramidase) and lysins from various bacteria, a fungus, and phages of Lactobacillus and Streptococcus and also revealed DNA homologous to noncoding sequences of temperate phage of L. lactis, DNA similar to a region of phage sk1, a gene with similarity to tRNA genes, a prophage attachment site, and open reading frames with similarities to sun and to sequences encoding phosphoprotein phosphatases and protein kinases. Mutational analyses of the cloned DNA showed that the region of homology with lactococcal temperate phage was responsible for restoring the phage-sensitive phenotype. The region of homology with DNA of lactococcal temperate phage was similar to DNA from a previously characterized lactococcal phage that suppresses an abortive infection mechanism of phage resistance. The region of homology with lactococcal temperate phage was deleted from a phage-sensitive strain, but the strain was not phage resistant. The results suggest that the cloned DNA with homology to lactococcal temperate phage was not mutated in the phage-resistant strain. The cloned DNA apparently suppressed the mechanism of resistance, and it may do so by mimicking a region of phage DNA that interacts with components of the resistance mechanism.     

Effect of rifampicin on the infectivity of RNA bacteriophage f2.

RNA bacteriophage f2, treated in vitro with rifampicin, loses infectivity dramatically. Rifampicin interacts with phage RNA, binding to a few specific sites. Inhibition of phage RNA infectivity occurs at 10-100 times lower molar excess of rifampicin than inhibition of infectivity of intact phage particles. Thus the phage capsid acts as a barrier, diminishing interaction of the drug with phage RNA.     

INCREASE IN BACTERIOPHAGE AND GELATINASE CONCENTRATION IN CULTURES OF BACILLUS MEGATHERIUM

1. The increase in bacteria, phage concentration, and gelatinase concentration in cultures of B. megatherium has been determined. 2. With lysogenic cultures the phage concentration, gelatinase concentration, and bacteria concentration increase logarithmically at first. The phage and gelatinase concentration then decrease while the bacteria concentration increases to a maximum. 3. The results are the same with sensitive cultures if the ratio of phage to bacteria is small. If the ratio of phage to bacteria is large phage, gelatinase, and bacteria concentration all increase at first and then decrease. The maximum rate of increase coincides approximately with the maximum rate of oxygen consumption of the culture. 4. 60–90 per cent of the phage is free from the cells. 5. The amount of phage produced is determined by the combined phage and not by the total phage. 6. Phage is produced during growth of the cells and not during lysis. 7. In a very narrow range of pH near 5.55 no increase in bacteria occurs but large increases in phage may be obtained.     

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.     

Excision of bacteriophage lambda from a site in the arabinose B gene.

A lambda lysogen with the prophage inserted into the arabinose B gene of Escherichia coli strain K-12 has been prepared. Induction of the phage from this lysogen yields viable phage at a frequency 4 X 10(-6) that found for induction of lysogens with phage inserted at the normal attachment site. Over 30% of the phage particles induced from the insertion in ara are arabinose-transducing phage. The excision end points of 62 independently isolated, nondefective araC-transducing phage containing less than the entire araC gene were genetically determined and were found to be randomly distributed through the araC gene. The amount of arabinose deoxyribonucleic acid contained on four selected transducing phage was determined by electron microscopy of deoxyribonucleic acid heteroduplexes, providing a physical map of the araC gene. The efficiency with which these phage transduce araC and araB point mutations was found to be approximately proportional to the homology length available for recombination.     

Effect of ozone on bacteriophages in the phage--host-cell system

The antiviral action of ozone was studied using Escherichia coli K-12 AB1157 virulent phage T4 and Pseudomonas aeruginosa PAO1 temperate phage SM as models depending on the phage state during the action: a free phage, a phage in the presence of sensitive host cells, or a vegetative phage. Bacteriophages T4 and SM were found to be much more sensitive to ozone as compared to the bacterial strains AB1157 and PAO1. The latter protected phage particles against the activation by ozone at a concentration which effectively inactivated these phages in the absence of the bacteria. Ozone also exerted an inhibiting effect on vegetative phage SM, and the degree of inhibition decreased with the termination of intracellular growth stages.     

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.     

Adapter-Directed Display: A Modular Design for Shuttling Display on Phage Surfaces

A novel adapter-directed phage display system was developed with modular features. In this system, the target protein is expressed as a fusion protein consisting of adapter GR1 from the phagemid vector, while the recombinant phage coat protein is expressed as a fusion protein consisting of adapter GR2 in the helper phage vector. Surface display of the target protein is accomplished through specific heterodimerization of GR1 and GR2 adapters, followed by incorporation of the heterodimers into phage particles. A series of engineered helper phages were constructed to facilitate both display valency and formats, based on various phage coat proteins. As the target protein is independent of a specific phage coat protein, this modular system allows the target protein to be displayed on any given phage coat protein and allows various display formats from the same vector without the need for reengineering. Here, we demonstrate the shuttling display of a single-chain Fv antibody on phage surfaces between multivalent and monovalent formats, as well as the shuttling display of an antigen-binding fragment molecule on phage coat proteins pIII, pVII, and pVIII using the same phagemid vectors combined with different helper phage vectors. This adapter-directed display concept has been applied to eukaryotic yeast surface display and to a novel cross-species display that can shuttle between prokaryotic phage and eukaryotic yeast systems.     

Improved adsorption of an Enterococcus faecalis bacteriophage ΦEF24C with a spontaneous point mutation

Some bacterial strains of the multidrug-resistant Gram-positive bacteria Enterococcus faecalis can significantly reduce the efficacy of conventional antimicrobial chemotherapy. Thus, the introduction of bacteriophage (phage) therapy is expected, where a phage is used as a bioagent to destroy bacteria. E. faecalis phage ΦEF24C is known to be a good candidate for a therapeutic phage against E. faecalis. However, this therapeutic phage still produces nonuniform antimicrobial effects with different bacterial strains of the same species and this might prove detrimental to its therapeutic effects. One solution to this problem is the preparation of mutant phages with higher activity, based on a scientific rationale. This study isolated and analyzed a spontaneous mutant phage, ΦEF24C-P2, which exhibited higher infectivity against various bacterial strains when compared with phage ΦEF24C. First, the improved bactericidal effects of phage ΦEF24C-P2 were attributable to its increased adsorption rate. Moreover, genomic sequence scanning revealed that phage ΦEF24C-P2 had a point mutation in orf31. Proteomic analysis showed that ORF31 (mw, 203 kDa) was present in structural components, and immunological analysis using rabbit-derived antibodies showed that it was a component of a long, flexible fine tail fiber extending from the tail end. Finally, phage ΦEF24C-P2 also showed higher bactericidal activity in human blood compared with phage ΦEF24C using the in vitro assay system. In conclusion, the therapeutic effects of phage ΦEF24C-P2 were improved by a point mutation in gene orf31, which encoded a tail fiber component.     

Diversity in CRISPR‐based immunity protects susceptible genotypes by restricting phage spread and evolution

Diversity in host resistance often associates with reduced pathogen spread. This may result from ecological and evolutionary processes, likely with feedback between them. Theory and experiments on bacteria–phage interactions have shown that genetic diversity of the bacterial adaptive immune system can limit phage evolution to overcome resistance. Using the CRISPR–Cas bacterial immune system and lytic phage, we engineered a host–pathogen system where each bacterial host genotype could be infected by only one phage genotype. With this model system, we explored how CRISPR diversity impacts the spread of phage when they can overcome a resistance allele, how immune diversity affects the evolution of the phage to increase its host range and if there was feedback between these processes. We show that increasing CRISPR diversity benefits susceptible bacteria via a dilution effect, which limits the spread of the phage. We suggest that this ecological effect impacts the evolution of novel phage genotypes, which then feeds back into phage population dynamics.     

Localization and functional role of the pseudomonas bacteriophage 2 depolymerase.

The adsorption apparatus of phage 2 consits of a symmetrical base plate of snowflake appearance, composed of six droplike spikes 7.0 to 7.5 nm in length with a maximum diameter of 4.5 to 5.0 nm. The spikes are attached by their narrow ends to a central ring 7.0 to 7.5 nm in diameter. Phage 2 deopolymerase, a phage 2-induced hydrolytic enzyme, was found to be a structural protein of phage 2 or in close association with the base plate. Pdp1, a phage 2 mutant, possesses a polypeptide that is antigenically similar to the depolymerase, but devoid of hydrolytic activity. This polypeptide was found to be located in the region of the base plate of pdp1. Treatment of intact cells of strain BI with purified phage 2 depolymerase inhibited the adsorption of phage 2. When phage receptor-containing fractions of slime glycolipoprotein and lipopolysaccharide were hydrolyzed by the depolymerase, amino sugars were released, and the phage-inactivating activities of these fractions were lost. The depolymerase was also observed to induce the lysis of strain BI cells in hypotenic medium. The phage 2 depolymerase appears to play a role in adsorption and release of phage.     

Characterization of a bacteriophage related to R-type pyocins.

A bacteriophage with a contractile tail which shows very similar features to R-type pyocins was isolated and characterized. This phage, named PS17,was purified by DEAE-cellulose chromatography and CsCl density gradient centrifugation. It was a DNA-containing phage, and the density of the purified particles in CsCl was found to be 1.468. DNA from this phage had a density of 1.720 in CsCl, indicating its guanine plus cytosine content to be 61.2%. The head was polyhedral, 69 nm in diameter, and the tail was 150 nm in length. This phage was neutralized by antiserum preparations against five R-type pyocins, and the antiserum against this phage was active in neutralizing R-type pyocins. The properties of this phage, PS17, were compared with another similar phage, PS3, which was previously reported.     

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.