Temperate Bacteriophages-The Powerful Indirect Modulators of Eukaryotic Cells and Immune Functions.

Bacteriophages are natural biological entities that limit the growth and amplification of bacteria. They are important stimulators of evolutionary variability in bacteria, and currently are considered a weapon against antibiotic resistance of bacteria. Nevertheless, apart from their antibacterial activity, phages may act as modulators of mammalian immune responses. In this paper, we focus on temperate phages able to execute the lysogenic development, which may shape animal or human immune response by influencing various processes, including phagocytosis of bacterial invaders and immune modulation of mammalian host cells.

Coordination of cohabiting phage elements supports bacteria-phage cooperation.

Bacterial pathogens often carry multiple prophages and other phage-derived elements within their genome, some of which can produce viral particles in response to stress. Listeria monocytogenes 10403S harbors two phage elements in its chromosome, both of which can trigger bacterial lysis under stress: an active prophage (ϕ10403S) that promotes the virulence of its host and can produce infective virions, and a locus encoding phage tail-like bacteriocins. Here, we show that the two phage elements are co-regulated, with the bacteriocin locus controlling the induction of the prophage and thus its activity as a virulence-associated molecular switch. More specifically, a metalloprotease encoded in the bacteriocin locus is upregulated in response to stress and acts as an anti-repressor for CI-like repressors encoded in each phage element. Our results provide molecular insight into the phenomenon of polylysogeny and its intricate adaptation to complex environments.

Conditional tolerance of temperate phages via transcription-dependent CRISPR-Cas targeting.

A fundamental feature of immune systems is the ability to distinguish pathogenic from self and commensal elements, and to attack the former but tolerate the latter. Prokaryotic CRISPR-Cas immune systems defend against phage infection by using Cas nucleases and small RNA guides that specify one or more target sites for cleavage of the viral genome. Temperate phages include viruses that can integrate into the bacterial chromosome, and they can carry genes that provide a fitness advantage to the lysogenic host. However, CRISPR-Cas targeting that relies strictly on DNA sequence recognition provides indiscriminate immunity both to lytic and lysogenic infection by temperate phages-compromising the genetic stability of these potentially beneficial elements altogether. Here we show that the Staphylococcus epidermidis CRISPR-Cas system can prevent lytic infection but tolerate lysogenization by temperate phages. Conditional tolerance is achieved through transcription-dependent DNA targeting, and ensures that targeting is resumed upon induction of the prophage lytic cycle. Our results provide evidence for the functional divergence of CRISPR-Cas systems and highlight the importance of targeting mechanism diversity. In addition, they extend the concept of 'tolerance to non-self' to the prokaryotic branch of adaptive immunity.

RS1 satellite phage promotes diversity of toxigenic Vibrio cholerae by driving CTX prophage loss and elimination of lysogenic immunity.

In El Tor biotype strains of toxigenic Vibrio cholerae, the CTXϕ prophage often resides adjacent to a chromosomally integrated satellite phage genome, RS1, which produces RS1ϕ particles by using CTX prophage-encoded morphogenesis proteins. RS1 encodes RstC, an antirepressor against the CTXϕ repressor RstR, which cooperates with the host-encoded LexA protein to maintain CTXϕ lysogeny. We found that superinfection of toxigenic El Tor strains with RS1ϕ, followed by inoculation of the transductants into the adult rabbit intestine, caused elimination of the resident CTX prophage-producing nontoxigenic derivatives at a high frequency. Further studies using recA deletion mutants and a cloned rstC gene showed that the excision event was recA dependent and that introduction of additional copies of the cloned rstC gene instead of infection with RS1ϕ was sufficient to enhance CTXϕ elimination. Our data suggest that once it is excised from the chromosome, the elimination of CTX prophage from host cells is driven by the inability to reestablish CTXϕ lysogeny while RstC is overexpressed. However, with eventual loss of the additional copies of rstC, the nontoxigenic derivatives can act as precursors of new toxigenic strains by acquiring the CTX prophage either through reinfection with CTXϕ or by chitin-induced transformation. These results provide new insights into the role of RS1ϕ in V. cholerae evolution and the emergence of highly pathogenic clones, such as the variant strains associated with recent devastating epidemics of cholera in Asia, sub-Saharan Africa, and Haiti.

[Functional organization of prophage and lysogeny in Erwinia carotovora with participation of a temperate bacteriophage ZF40].

Functional organization of a prophage of the temperate bacteriophage ZF40 of Erwinia carotovora subsp. carotovora which includes its immunity and inducibility as well as its effect on the host phenotype. It was established that the prophage ZF40 forms several different states in E. carotovora which are distinguished by the indices of spontaneous and lysogenic induction. In contrast to other prophages, including the lambdoid ones, the prophage ZF40 is capable to establish cytoplasmic overimmunity which protects the lysogenic system from superinfection by virulent mutants or other homoimmune bacteriophages. An increase of sensitivity of ZF40-lysogens to killing activity of colicino-like carotovoricin (CCTV) and destabilization of defective lysogeny, or resistant MCTV-prophages are related to the phenomenon of the phage lysogenic conversion of E. carotovora.

The potential role of endogenous bacteriophages in controlling invading pathogens.

Bacteriophages (phages) are omnipresent in our environment, and recent studies highlight their potential impact on the microbial world. Phages can also be present in mammalian organisms, including man (intestines, oral cavity, urine, sputum and serum). Data are available which suggest that those endogenous phages could play an important role in eliminating bacteria and regulating the body ecosystem. Furthermore, our most recent findings suggest that phages can exert immunosuppressive action in the gut, helping control local inflammatory and autoimmune reactions, and demonstrate anticancer activity. We hypothesize that phages could act in concert with the immune system in immunosurveillance against bacteria, viruses and cancer.

Preventing phage lysis of Lactococcus lactis in cheese production using a neutralizing heavy-chain antibody fragment from llama.

Bacteriophage infection is still a persistent problem in large dairy processes despite extensive studies over the last decades. Consequently, new methods are constantly sought to prevent phage infection. In this paper, we show that phage neutralizing heavy-chain antibody fragments, obtained from Camelidae and produced at a large scale in the generally regarded as safe microorganism Saccharomyces cerevisiae, can effectively be used to impede phage induced lysis during a cheese process. The growth inhibition of the cheese starter culture by 10(5) pfu/ml cheese-milk of the small isometric-headed 936-type phage p2 was prevented by the addition of only 0.1 microg/ml (7 nM) of the neutralizing antibody fragment. The use of such antibody fragments in cheese manufacturing are a realistic and interesting option because of the small amount of antibody fragments that are needed. Moreover the antibodies are produced in a food grade microorganism and can easily be isolated from the fermentation liquid in a pure and DNA free form.

Characterization of the lysogenic repressor (c) gene of the Pseudomonas aeruginosa transposable bacteriophage D3112.

Bacteriophage D3112 is a Mu-like temperate transposable phage of Pseudomonas aeruginosa. Genetic mapping and DNA sequence analysis have identified the left end of the phage genome as encoding the transposase enzyme (A) and the lysogenic (c) repressor. The c open reading frame (ORF), located at the leftmost end of the phage genome and transcribed from right to left, has four possible GTG initiation codons. Using site-directed mutagenesis, each of the four GTG codons was modified to GTA, which cannot serve as an initiation codon. Plasmids were constructed expressing either the wild-type repressor ORF or the ORFs containing the mutated GTA codons. When introduced into Pseudomonas aeruginosa, no immunity to superinfection by D3112 was observed when the second GTG had been mutated. Northern blotting analysis demonstrated that the D3112 c repressor is transcribed as a 900-nt mRNA. The promoter region was defined by transcriptional lacZ fusions and primer extension analyses to bp 972-940 from the left end of the phage genome. When the D3112 c repressor was overexpressed and purified as a fusion protein with a C-terminal six-histidine extension (cts15-His6), it showed high affinity for a 261-bp PvuII fragment localized directly upstream of the c repressor ORF. Our results indicate that although D3112 c shows higher amino acid similarity to the lambda family of repressors than it does to those of Mu and D108, it appears that its structure and function more accurately reflect an evolutionary ancestry with those from transposable coliphages Mu and D108.

The primary immunity determinant in modulating the lysogenic immunity of the filamentous bacteriophage cf.

Bacteriophage cf is the first single-stranded DNA phage that has been shown to set up a stable lysogenic state with its genome integrated into the host chromosome. From the isolation and characterization of a virulent mutant, cf-tv2, we report the first investigation into the mechanisms of the immunity established by the filamentous bacteriophage. The mutation in cf-tv2 enables the phage to produce plaques on lawns of cf lysogenic cells. The mutation was defined as a 49-nucleotide deletion located in a 0.59 kb NcoI/KpnI fragment of cf replicative form DNA. Two messages, cM1 and cM2, transcribed from the immunity region of wild-type cf but in opposite directions, were detected. In cf-tv2, the 49-nucleotide deletion abolishes cM2 transcription. The primer extension assay suggests a possible RNA-RNA interaction directed by base-pairing of the cM1 and cM2 RNAs. A frameshift mutation of the open reading frame ORF 165, encoded by cM2, resulted in a 10(5) plating efficiency on the cf lysogen. These observations suggest that both RNA-RNA interaction and repressor protein inhibition are involved in the mechanism of cf immunity. A model is proposed for the regulation of cf immunity.

Gene organization in a central DNA fragment of Oenococcus oeni bacteriophage fOg44 encoding lytic, integrative and non-essential functions.

The nucleotide sequence of a DNA fragment previously shown to contain the attachment site (attP) of Oenococcus oeni phage fOg44 (. Arch. Virol. 143, 523-536) has been determined. Sequence analysis indicated that this 6226bp EcoRI fragment harbours an integrase gene, in the vicinity of a direct repeat rich region defining attP, as well as genes encoding a muramidase-related lysin (Lys) and a holin polypeptide (Hol). Transcriptional studies suggested that lys and hol are mainly co-expressed, late in the lytic cycle, from a promotor located upstream of lys. Between the lytic cassette and the phage integration elements three additional open reading frames were found: orf217 and orf252 of unknown function and orf72, the putative product of which bears 32% identity with acidic excisionases from other Gram positive phages. We have established that the first two orfs, as well as the predicted promotor of orf72, are included in a 2143-bp DNA segment missing from the genome of the deletion mutant fOg44Delta2. Although lysogens of fOg44 and fOg44Delta2 exhibited similar properties, each phage produced two distinguishable types of lysogenic strains, differing in inducibility and immunity to other oenophages.

Producción y análisis de proteínas recombinantes obtenidas de la cepa lisogénica Ro-531

La expresión del cDNA que codifica al antígeno Ro, fue evaluada usando la clona Ro=531 gt11 cuyos productos de traducción fueron inmunorreconocidos por un suero anti-Ro. La producción de proteína Ro recombinante, fue inducida en la cepa lisogénica E. coli Y1089. La antigenicidad de la proteína fue probada por Western blot y por ELISA. En la primera prueba, 15 de los 20 sueros anti-Ro positivos presentaron fuerte reactividad a la proteína de funsión Ro y 4 de los 20 sueros controles, mostraron reactividad débil. Por la técnica de ELISA, se observó una reacción más específica, ya que 15 de los 20 sueros anti-Ro positivos exhibieron afinidad por la proteína Ro recombinante y ninguno de los controles presentó falsos positivos

On the toxinogenicity of Pseudomonas aeruginosa strains.

Investigation of 367 P. aeruginosa strains primarily isolated from clinical and other biological material as well as from the environment yielded results suggesting a substantial toxinogenic potential. 92.6% of the assayed culture filtrates derived from the strains under investigation proved positive in the early skin tests on rabbits. 49.7% of the assayed material induced cytotoxic alterations on Vero cells, the rates for Y1 and CHO cells being 50.3% and 43.5% respectively. 54.3% culture filtrates caused haemolysis of rabbit RBC and 52.7% lysed horse RBC. Gelatinase activity was found in 96.3% of tested material, protease in 89.8%, lecithinase in 62.4% and elastase in 29.6%. 12.6% of tested material induced fluid accumulation in a ligated intestinal loop. None of the culture filtrates elicited a positive reaction in the suckling mice test suggesting the absence of the thermostable enterotoxin.

Lysogenization of Salmonella choleraesuis by phage 14 increases average length of O-antigen chains, serum resistance and intraperitoneal mouse virulence.

Three clones from a strain of Salmonella choleraesuis (serogroup C1) were lysogenized with phage 14 (P14) which converts the O-antigen of serogroup C1 salmonellae from O-6,7 to O-6,7,14. The lysogens were compared with their parental non-lysogenic clones with respect to the following properties: average length of O-antigen polysaccharide chains, sensitivity to normal human serum, and mouse-virulence. SDS-polyacrylamide gel electrophoresis of lipopolysaccharides extracted from these bacteria showed that samples from lysogens consisted mainly of long-chained molecules whereas those from non-lysogens contained mainly short-chained molecules. The O-antigen polysaccharide from a lysogen was estimated by chemical analysis to be six times as long as that from a non-lysogen. Lysogens were serum-resistant whereas non-lysogens were serum-sensitive. About 10 times more colony forming units of a lysogen than of a non-lysogen were recovered from the livers and spleens of mice on day 1 and 3 after intraperitoneal inoculation of equal doses. By comparison with S. choleraesuis, lysogenization of S. typhimurium with phage P22 or phage A4 did not affect the chain-length distribution of O-antigen polysaccharide. Our data suggest that phage 14-coded determinants increase efficiency of O-antigen biosynthesis in S. choleraesuis leading to increase in average length of O-polysaccharide chains. Increased serum resistance and mouse virulence are logical consequences of increase in average length of O-polysaccharide chains and represent phage-conferred selective advantage not previously described in Salmonella.

[The role of the antilysozyme activity of Shigella flexneri as a factor controlling the degree of its resistance to the lytic action of bacteriophages]. / Rol' antilizotsimnoi aktivnosti Shigella flexneri kak faktora, kontroliruiushchego stepen' ikh ustoichivosti k liticheskomu deistviiu bakteriofagov.

The level of the antilysozyme activity of S. flexneri in ensuring the high level of their phage resistance has been studied. The realization of the phage protective effect of antilysozyme activity has been noted to occur due to disturbances in the lysis of infected bacteria by phage-synthetized lysozyme-like enzyme. The direct relationship between the level of the lysozyme production of bacteriophages and their capacity for overcoming the antilysozyme protection of the host bacterium has been shown.

[Bacteriophage conversion as a factor modifying the intensity of phagocytosis of Staphylococcus aureus by human leukocytes]. / Konwersja fagowa jako czynnik modyfikujacy intensywnosc fagocytozy Staphylococcus aureus przez leukocyty ludzkie.

Staphylococcus aureus NCTC 8325-4 and its eight variants lysogenized with phages responsible for the synthesis of staphylococcal staphylokinase were used for the study. Influence of phage conversion of S. aureus on its interaction with human leucocytes and influence of prophage on strain susceptibility to intracellular killing by human granulocytes without opsonins were evaluated. It was found that lysogenization of the strain with the bacteriophages decreased in each case reactivity of human leucocytes for staphylococcal strain what was expressed by lower bioluminescence values and by lower percentage of intracellular killing of bacterial cells carrying prophage.