Uncovering the determinants of model Escherichia coli strain C600 susceptibility and resistance to lytic T4-like and T7-like phage.

As antimicrobial resistance (AMR) continues to increase, the therapeutic use of phages has re-emerged as an attractive alternative. However, knowledge of phage resistance development and bacterium-phage interaction complexity are still not fully interpreted. In this study, two lytic T4-like and T7-like phage infecting model Escherichia coli strain C600 are selected, and host genetic determinants involved in phage susceptibility and resistance are also identified using TraDIS strategy. Isolation and identification of the lytic T7-like show that though it belongs to the phage T7 family, genes encoding replication and transcription protein exhibit high differences. The TraDIS results identify a huge number of previously unidentified genes involved in phage infection, and a subset (six in susceptibility and nine in resistance) are shared under pressure of the two kinds of lytic phage. Susceptible gene wbbL has the highest value and implies the important role in phage susceptibility. Importantly, two susceptible genes QseE (QseE/QseF) and RstB (RstB/RstA), encoding the similar two-component system sensor histidine kinase (HKs), also identified. Conversely and strangely, outer membrane protein gene ompW, unlike the gene ompC encoding receptor protein of T4 phage, was shown to provide phage resistance. Overall, this study exploited a genome-wide fitness assay to uncover susceptibility and resistant genes, even the shared genes, important for the E. coli strain of both most popular high lytic T4-like and T7-like phages. This knowledge of the genetic determinants can be further used to analysis the behind function signatures to screen the potential agents to aid phage killing of MDR pathogens, which will greatly be valuable in improving the phage therapy outcome in fighting with microbial resistance.

[Ambivalent bacteriophages of different species active on Escherichia coli K12 and Salmonella sps. strains].

A study was made of several bacteriophages (including phages U2 and LB related to T-even phages of Escherichia coli) that grow both on E. coli K12 and on some Salmonella strains. Such phages were termed ambivalent. T-even ambivalent phages (U2 and LB) are rare and have a limited number of hosts among Salmonella strains. U2 and LB are similar to canonical E. coli-specific T-even phages in morphological type and size of the phage particle and in reaction with specific anti-T4 serum. Phages U2 and LB have identical sets of structural proteins, some of which are similar in size to structural proteins of phages T2 and T4. DNA restriction patterns of phages U2 and LB differ from each other and from those of T2 and T4. Still, DNAs of all four phages have considerable homology. Unexpectedly, phages U2 and LB grown on Salmonella bungori were unstable during centrifugation in a CsCl gradient. Ambivalent bacteriophages were found in species other than T-even phages and were similar in morphotype to lambdoid and other E. coli phages. One of the ambivalent phages was highly similar to well-known Felix01, which is specific for Salmonella. Ambivalent phages can be used to develop a new set for phage typing in Salmonella. An obvious advantage is that ambivalent phages can be reproduced in the E. coli K12 laboratory strain, which does not produce active temperate phages. Consequently, the resulting typing phage preparation is devoid of an admixture of temperate phages, which are common in Salmonella. The presence of temperate phages in phage-typing preparations may cause false-positive results in identifying specific Salmonella strains isolated from the environment or salmonellosis patients. Ambivalent phages are potentially useful for phage therapy and prevention of salmonellosis in humans and animals.

Evidence of neutralizing activity against T3 coliphage in oyster Crassostrea gigas hemolymph.

To investigate defense reactions of bivalve molluscs against viruses, experimental in vitro assays have been developed using T3 coliphage as a test virus. A native neutralizing factor in oyster Crassostrea gigas serum showed high individual variability and was enhanced significantly by repeated sampling of hemolymph from the same oysters. The responsible factor is apparently thermolabile and sensitive to EDTA treatment. Because of an inhibitory effect by the enzymatic inhibitor, phenylmethylsulphonyl fluoride (PMSF), the T3-neutralizing factor may be related to serine protease.

[Host-dependent modifications of bacteriophage T3 expressing changes in adsorption properties (serological study)]. / Khoziainzavisimye modifikatsii bakteriofaga T3, vyrazhaiushchiesia v izmenenii adsorbtsionnoi sposobnosti (serologicheskie issledovaniia).

The ability of the bacteriophage T3 to adsorb on the host cells of Escherichia coli W1655 changes depending on the host strain in which the phage was propagated before. This phenomenon is termed "non-classical" host-controlled modification in contrast to "classical" DNA modification. We demonstrate here that T3 phages with various non-classical modifications as well as the host range mutant T3hW differ from each other in the antigenic determinants of the phage adsorption protein.

T-even-type bacteriophages use an adhesin for recognition of cellular receptors.

Protein 38 of the Escherichia coli phage T4 is thought to be required catalytically for the assembly of the long tail fibers of this phage. It is shown that this protein of phage T2 and the T-even-type phage K3 and Ox2 act differently. It was found that NH2-terminal fragments of the protein, expressed from cloned fragments of gene 38 of phage K3, bind to gene 38 amber mutants of phage T2. Such phage or T2 gene 38 amber mutants, grown on a non-permissive host, possess a complete set of six tail fibers but are non-infectious. Both types of non-infectious phage could be repaired by incubation with an extract of cells harboring a cloned gene 38 of a host range mutant of phage K3, K3hx. The repaired phages had the host range of K3hx and not of T2. Immuno-electron microscopy showed that protein 38 is located at the free ends of the long tail fibers of phages T2, K3 and Ox2. The protein serves the recognition of the cellular receptor, i.e. it acts as an adhesin.

Production and detection of coliphage T4 endonuclease V polyclonal and monoclonal antibodies using staphylococcal protein-A hybrid proteins.

To facilitate the production of antibodies against endonuclease V, a pyrimidine dimer-specific DNA glycosylase produced in bacteriophage T4-infected Escherichia coli, we constructed plasmids containing protein-A-endonuclease V fusion genes under control of the E. coli tac promoter. Induction with isopropyl-beta-D-thiogalactopyranoside produced large amounts of fusion proteins, which could easily be purified on human IgG agarose columns. The affinity-purified fusion proteins were injected into rabbits and mice to produce polyclonal and monoclonal antibodies, and also used for the screening of the monoclonal antibodies. These antibodies recognized endonuclease V on immunoblots, and also inhibited the DNA-glycosylase activity in vitro. Epitope mapping of monoclonal antibodies showed that they all (6/6) recognized determinants in the C-half of endonuclease V. A convenient way to detect primary antibodies on nitrocellulose was also developed using a crude protein extract containing protein-A-beta-galactosidase fusion protein and subsequent detection with a mixture of dyes.

Defective humoral immunity in pediatric acquired immune deficiency syndrome.

Specific antibody production was assessed in six young children with the acquired immune deficiency syndrome (AIDS). All patients were immunized with bacteriophage phi X 174, a T cell-dependent neoantigen. In addition, antibody responses to pneumococcal vaccine and tetanus toxoid, lymphocyte responses to mitogens, and serum immunoglobulin levels were determined. Polyclonal hypergammaglobulinemia was documented in three patients. Responses to bacteriophage phi X 174 were abnormal in all patients: primary responses were blunted, secondary responses were markedly decreased, and the class switch (IgM-IgG) was absent in five of six patients. Antibody formation to pneumococcal vaccine and tetanus toxoid was also diminished. Lymphocyte mitogenic responses to phytohemagglutinin, concanavalin A, pokeweed mitogen, and staphylococcal Cowan A were generally decreased. These findings confirm that pediatric patients with AIDS have significant abnormalities in humoral immunity. Dysfunction of both T cells and B cells plays a role in the resultant poor specific antibody production.

[Evaluation of the functional activity of peritoneal macrophages using the tetrazolium test]. / Otsenka funktsional'noi aktivnosti peritoneal'nykh makrofagov s pomoshch'iu tetrazolievogo testa.

The redox activity of peritoneal macrophages has been evaluated in the modified NBT test using tetranitro blue tetrazolium. This method permits the calculation of the total amount of peritoneal macrophages and the determination of their glass adherence per cent and their cytochemical activity value. The advantages of this method over the study of the phagocytic activity by means of phage T2 is shown.

Specific labeling of protein domains with antibody fragments.

Monovalent antibody Fab fragments, prepared from antisera raised against two different types of crystalline arrays made of either intact, or a proteolytic fragment of bacteriophage T4 major capsid protein, gp23*, were employed to stoichiometrically label different gp23* protein domains on the outer surface of a tubular variant (i.e., "polyheads") of bacteriophage T4 capsids. Computer filtrations of both negatively stained and freeze-dried/metal-shadowed specimens permitted approximate mapping of the Fab binding sites within the capsomere of the polyheads.

Immunocompetence of chimeric rabbits. I. Participation of donor-derived B lymphocytes in immunoglobulin, but not in antibody, synthesis.

Stable and lasting B lymphocyte chimerism induced in newborn rabbits through the introduction of spleen or lymph node cells from adult donors matched with the recipients for major histocompatibility antigens, is characterized by an apparent immunodeficiency of donor-derived cells. However, priming of the donor with an antigen that is subsequently used to immunize the recipients results in the selective and effective participation of donor cells in the chimera's antibody response to this antigen. These findings are ascribed to limitations in the repertoire of cells from the unprimed donor that colonize the recipients. Polyclonal stimulation secondary to allogeneic effects has been suggested as an explanation for the participation of donor-derived B cells noted in occasional recipients of cells from unprimed donors matched with recipients with respect to major but not minor histocompatibility antigens, and seen more regularly in surviving recipients of unmatched or mismatched donor cells.

[Intracellular neutralization of bacteriophage T4 by antiphagic serum]. / Vnutrikletochnaia neitralizatsiia bakteriofaga T4 antifagovoi syvorotkoi.

Specific antiserum, introduced into the spheroplasts of Escherichia coli B infected with bacteriophage T4, has been shown to neutralize phage particles formed within the cells.

[Intercellular information transfer in the process of immunogenesis. IX. The induction of the synthesis of antiphage antibodies in lymphosarcoma cells of rats as affected by different fractions of immune nuclear RNA]. / O mezhkletochnoi peredache informatsii v protsesse immunogeneza. IX. Induktsiia sinteza antifagovykh antitel v kletkakh limfosarkomy krys pod vliianiem razlichnykh fraktsii immunoi iadernoi RNK.

Various fractions of the immune nuclear RNA were isolated from spleens of phage T2 immunized rats. The fractions were compared for their ability to induce anti-phage T2 antibody synthesis in transplantable lymphosarcoma cells. The most active proved to be the nuclear sap RNA and its subfraction with sedimentation constant of 10 S. The 4S and 26S subfractions RNA were less stable and in some experiments failed to induce antibody synthesis.

Gene 20 product of bacteriophage T4. II. Its structural organization in prehead and bacteriophage.

The location of gene 20 product of bacteriophage T4 in phage and phage percursors has been determined by immunochemical analysis of polyacrylamide gels, immunoturbidimetry and immunoelectron microscopy. The protein is present at the membrane attachment site of the prehead, a head precursor, and is accessible to the antibodies in the solution. It is present at the tail attachment site of the capsid, partially buried in the structure. In complete phage particles it is totally buried in the structure. It is in contact with the major shell proteins, gp23 and gp23*, respectively, in preheads and capsids, as revealed by partial crosslinking experiments. It forms the upper collar of the neck in necked tails. The lower collar is constructed from other gene products. On the basis of these data a structural model of the neck region of the phage has been derived. This model is consistent with a number of events in phage assembly, such as the role of gp20 in head assembly and DNA packaging, prehead detachment from the bacterial membrane and head-tail attachment. The symmetry mismatch known to occur between head and tail has been localized at the gp20-gp23* contact area.

Specific antibodies and immunoglobulins in the oviductal fluid of the rhesus monkey.

Levels of specific antibodies against model antigens, immunoglobulins G and A and also albumin, in oviductal fluid were studied in the rhesus monkey during the periovulatory period. Animals were systemically or intravaginally immunized against T4 coliphages. Attempts to induce ovulation were made with human menopausal gonadotropin (hMG) and human chorionic gonadotropin (hCG) monitored by radioimmunoassay of serum estrogen and progesterone. Collection of tubal fluid over 6 to 13 days was accomplished by surgical cannulation using a refrigerated extracorporeal collection device for each side. The results indicated the following: (1) The levels of specific antibodies against T4 coliphage and immunoglobulins (IgG, IgA) in the oviduct fluid averaged approximately one tenth of the serum values and showed a characteristic decrease and subsequent increase by a factor of 4 to 5 during and following treatment with hMG/hCG. The nadir was observed on the first or second day after hCG injection. (2) This pattern was similar in both ovulatory or nonovulatory cycles; therefore, these changes seem to be associated with the changes in serum estrogen levels. (3) There was a striking difference in serum and tubal fluid antibody levels after systemic versus after vaginal immunization by a factor of 10(3) and 10(4); however, the patterns in tubal fluid under treatment with hMG/hCG were very similar. (4) Specific antibodies in oviductal fluid and serum were mainly of the IgG class. (5) A concomitant change of total protein and albumin in oviduct fluid was also observed. The presence of sperm agglutination antibody in oviductal fluid was demonstrated in two monkeys after systemic immunization with homologous spermatozoa. The sperm antibody titers showed a similar pattern of change after hMG/hCG treatment.

Degrees of relatedness of T-even type E. coli phages using different or the same receptors and topology of serologically cross-reacting sites.

The relatedness of a series of T-even like phages which use the Escherichia coli outer membrane protein OmpA as a receptor, and the classical phages T2, T4 and T6 has been investigated. Immunoelectron microscopy and the pattern of phage resistance in bacterial mutants revealed that: (i) phages of this morphology do not necessarily cross-react serologically; (ii) phages using different receptors may bind heterologous IgG everywhere except to the tip (comprising approximately 10% of one fiber polypeptide) of the long tail fibers; (iii) cross-reacting OmpA-specific phages may bind heterologous IgG only to the tip of these fibers: (iv) OmpA-specific phages not cross-reacting at the tip of the tail fibers use different receptor sites on the protein. Absence of cross-reactivity appears to reflect high degrees of dissimilarity. A DNA probe consisting of genes encoding the two most distal tail fiber proteins of T4 detected homologies only in DNA from phages serologically cross-reacting at this fiber. Even under conditions of low stringency, allowing the formation of stable hybrids with almost 30% base mismatch, no such homologies could be found in serologically unrelated phages. Thus, in the collection of phages examined, there are sets of very similar and very dissimilar tail fiber genes and even of such gene segments.

Specificity for tail fibers of a humoral crab factor neutralizing T2 bacteriophage.

It is now becoming clear that invertebrates, animals lacking the ability to make humoral antibodies, are capable of recognizing self from non-self. In several instances this recognition is manifest in normal ("unimmunized") animals by a rapid clearance of certain foreign materials from the circulation One example of such a rapid innate reaction involves the clearance of T2 bacteriophage from the blue crab (1). Other studies have indicated that blue crab plasma contained a factor capable of neutralizing in vitro T2 bacteriophage and hence, by inference, was responsible for the in vivo clearance reaction with this virus (2). The purpose of the work undertaken here was to establish the nature of the specificity of this in vitro neutralizing factor. The strategy involved utilizing purified T2 bacteriophage tail fibers to inhibitthe neutralization of T2 bacteriophage by crab plasma.

[Serologic affinity and specificity of action of pseudotuberculosis and coli-dysentery phages]. / Serologicheskoe rodstvo i spetsifichnost' deistviia psevdotuberkuleznykh i koli-dizenteriinykh fagov.

The study of serological properties, specificity and the range of action has revealed affinity between Y. pseudotuberculosis phages (PST, 3M, Kotlyarova, 2344, 2391), some coliphages (T2, T3, T4) and Sh. dysenteriae phage (dd IV). The existence of serovar III of Y. pseudotuberculosis phages has been established; to this serovar phage PST belongs. Newly isolated 2344 and 2391 belong to serovar I. The problem of the existence of Y. pseudotuberculosis phages as an independent group is discussed.