Min Of Infection Research Articles

Evaluation of a lytic bacteriophage, Φ st1, for biocontrol of Salmonella enterica serovar Typhimurium in chickens

In this study, a Salmonella Typhimurium lytic bacteriophage, Φ st1, which was isolated from chicken faecal material, was evaluated as a candidate for biocontrol of Salmonella in chickens. The morphology of Φ st1 showed strong resemblance to members of the Siphoviridae family. Φ st1 was observed to be a DNA phage with an estimated genome size of 121kbp. It was found to be able to infect S. Typhimurium and S. Hadar, with a stronger lytic activity against the former. Subsequent characterisation of Φ st1 against S. Typhimurium showed that Φ st1 has a latent period of 40min with an average burst size of 22 particles per infective centre. Approximately 86.1% of the phage adsorbed to the host cells within the initial 5min of infection. At the optimum multiplicity of infection (MOI) (0.1), the highest reduction rate of S. Typhimurium (6.6log10CFU/ml) and increment in phage titre (3.8log10PFU/ml) was observed. Φ st1 produced adsorption rates of 88.4–92.2% at pH7–9 and demonstrated the highest bacteria reduction (6.6log10CFU/ml) at pH9. Φ st1 also showed an insignificant different (P>0.05) reduction rate of host cells at 37°C (6.4log10CFU/ml) and 42°C (6.0log10CFU/ml). The in vivo study using Φ st1 showed that intracloacal inoculation of ~1012PFU/ml of the phage in the chickens challenged with ~1010CFU/ml of S. Typhimurium was able to reduce (P<0.05) the S. Typhimurium more rapidly than the untreated group. The Salmonella count reduced to 2.9log10CFU/ml within 6h of post-challenge and S. Typhimurium was not detected at and after 24h of post-challenge. Reduction of Salmonella count in visceral organs was also observed at 6h post-challenge. Approximately 1.6log10PFU/ml Φ st1 was found to persist in the caecal wall of the chicks at 72h of post-challenge. The present study indicated that Φ st1 may serve as a potential biocontrol agent to reduce the Salmonella count in caecal content of chickens.

An Agrobacterium-mediated transformation system of pyrethrum (Tanacetum cinerariifolium) based on leaf explants

Pyrethrum (Tanacetum cinerariifolium) is well-known for the production of natural pyrethrin insecticides, which are isolated from the dried flower heads. To allow genetic analysis and improvement of pyrethrum in terms of yield and quality by molecular breeding a transformation protocol would be desirable. To achieve this, first an efficient direct shoot regeneration system was set up. Leaf explants formed shoots within 4 weeks on MS medium supplemented with 2.0mgl−1 6-BA and 0.2mgl−1 IBA. The best regeneration frequency obtained was 87% with an average number of 2 shoots per explant. Subsequently, a genetic transformation protocol was developed. Kanamycin added to the medium significantly reduced explant regeneration, and no green shoots were formed after 4 weeks incubation at 10mgl−1 kanamycin. Severe phytotoxic effects of cefotaxime were observed at concentrations >300mgl−1 to control Agrobacterium. Thus, 10mgl−1 kanamycin and 200mgl−1 cefotaxime were selected as optimal for genetic transformation. Agrobacterium-mediated genetic transformation was carried out with a GUS gene under the control of the chrysanthemum RbcS promoter. Two days of pre-culture and 5min of infection were found to result in the most efficient transformation rates. Ninety-nine percent of leaf explants infected with Agrobacterium showed GUS expression 3–5 days after transfection and 12% of the regenerated shoots had a stable GUS-positive phenotype. The other plants had a transient kanamycin-resistant phenotype and eventually lost the NPT-II and GUS gene expression.

Involvement of Ca 2+ signaling in intracellular invasion of non-phagocytic host cells by Orientia tsutsugamushi

Calcium signaling has been implicated in various steps in bacterial pathogenesis. Here, we investigated the role of Ca 2+ signaling in intracellular invasion of non-phagocytic host cells infected with Orientia tsutsugamushi, the causative agent of scrub typhus. The bacteria induced a transient Ca 2+ increase in HeLa cells immediately after infection and the source of the mobilized Ca 2+ appears to be intracellular stores, not the extracellular milieu, as determined using extracellular (EGTA) or intracellular (BAPTA-AM) Ca 2+ chelators. O. tsutsugamushi rapidly induced activation of PLC-γ1, as indicated by tyrosine phosphorylation. PLC-γ1 activity increased within 1 min of infection and returned to the basal level by 5 min. Pretreatment of host cells with inhibitors of PLC-γ1 ( U73122) or IP3R channel activity (2-APB) significantly blocked bacterial entry without affecting bacterial attachment. In addition, these chemical inhibitors were effective in suppressing not only the activation of ERK MAP kinase but also the expression of the chemokine MCP-1. Taken together, Ca 2+ signaling induced by O. tsutsugamushi may play a crucial role in bacterial pathogenesis including inflammatory reactions as well as intracellular invasion into non-phagocytic host cells.

Transcript map of the temperate Lactobacillus gasseri bacteriophage phiadh.

Temporal transcription of phage phiadh was analysed during lytic reproduction. Based on Northern hybridizations the phage genome was divided into regions of early, middle and late transcription. Eight groups of overlapping transcripts, probably originating from common precursors, were distinguished. Early transcription of a 10.9 kb region adjacent to the lytic/lysogenic switch started within the first 10 min of infection and produced three groups of mRNAs mostly related to DNA replication. Four middle transcripts were observed 30 min after infection, corresponding to an 8.5 kb genomic region, which started at the replication origin (ori) and encompassed a DNA packaging function and the cos site. Three groups of late transcripts were first observed 50 min after infection, corresponding to a 21.1 kb region between the middle region and the attachment site (attP), encoding functions for capsid morphogenesis and host cell lysis. A fourth group of late-appearing mRNAs was divergently transcribed from the 3.2 kb section between attP and the lytic/lysogenic switch, including the repressor and integrase genes. Except for one set of early mRNAs, all the transcripts persisted until the end of the reproduction cycle. Two confirmed and two predicted promoters were assigned to transcript 5' ends in the early region.

Effects of herpes simplex virus on structure and function of nectin-1/HveC.

Herpes simplex virus (HSV) entry requires the interaction between the envelope glycoprotein D (gD) and a cellular receptor such as nectin-1 (also named herpesvirus entry mediator C [HveC]) or HveA/HVEM. Nectin-1 is a cell adhesion molecule found at adherens junctions associated with the cytoplasmic actin-binding protein afadin. Nectin-1 can act as its own ligand in a homotypic interaction to bridge cells together. We used a cell aggregation assay to map an adhesive functional site on nectin-1 and identify the effects of gD binding and HSV early infection on nectin-1 function. Soluble forms of nectin-1 and anti-nectin-1 monoclonal antibodies were used to map a functional adhesive site within the first immunoglobulin-like domain (V domain) of nectin-1. This domain also contains the gD-binding site, which appeared to overlap the adhesive site. Thus, soluble forms of gD were able to prevent nectin-1-mediated cell aggregation and to disrupt cell clumps in an affinity-dependent manner. HSV also prevented nectin-1-mediated cell aggregation by occupying the receptor. Early in infection, nectin-1 was not downregulated from the cell surface. Rather, detection of nectin-1 changed gradually over a 30-min period of infection, as reflected by a decrease in the CK41 epitope and an increase in the CK35 epitope. The level of detection of virion gD on the cell surface increased within 5 min of infection in a receptor-dependent manner. These observations suggest that cell surface nectin-1 and gD may undergo conformational changes during HSV entry as part of an evolving interaction between the viral envelope and the cell plasma membrane.

YopH dephosphorylates Cas and Fyn-binding protein in macrophages

The tyrosine phosphatase YopH is an essential virulence effector of pathogenic Yersinia spp. YopH, which is translocated from extracellularly located bacteria into interacting target cells, blocks phagocytosis by professional phagocytes. We show here that immunoprecipitation of YopH from lysates of J774 cells infected with Y. pseudotuberculosis expressing an inactive form of YopH resulted in co-precipitation of certain phosphotyrosine proteins. The association between the inactive YopH and phosphotyrosine proteins in the 120 kDa range was rapid and could be detected after 2 min of infection. The proteins were identified as the docking proteins Cas and Fyn-binding protein (FYB). Upon infection of J774 cells with Y. pseudotuberculosis lacking YopH expression both of these proteins became tyrosine phosphorylated. Moreover, this infection caused recruitment of Cas to peripheral focal complexes, and FYB was relocalized to areas surrounding these structures. Both Cas and FYB became dephosphorylated upon infection with Y. pseudotuberculosis expressing active YopH, and this was associated with disruption of focal complexes. With regard to the previous identification of Cas and focal complexes as targets of YopH in HeLa cells, the present study supports an important role for these targets in a general mechanism of bacterial uptake.

Transcription analysis of the prolate-headed lactococcal bacteriophage c2.

A detailed transcription map of the prolate-headed lactococcal phage c2 has been constructed. Transcription of about one-third of the genome, encoding 22 open reading frames, began within the first 2 min of infection and produced at least 12 overlapping transcripts that persisted until lysis occurred at 30 min after initiation of infection. The remaining two-thirds of the genome, encoding 17 open reading frames, was divergently transcribed, beginning between 4 and 6 min after initiation of infection, and resulted in at least 18 overlapping transcripts that persisted until lysis. Five very strong, simultaneously active, and probably unregulated early promoters and a single positively regulated late promoter were identified. The late promoter had an extended -10 sequence, had a significant basal level of activity in the uninduced state, and was induced to high activity by a phage gene product. The complex overlapping pattern of transcripts resulted from the action of the multiple early promoters, inefficient termination of transcription, and (possibly) processing of a late precursor transcript(s). Phage proteins were not required for these processes, and the host RNA polymerase was probably used for both early and late transcription.

Maturation of bacteriophage 9NA DNA is influenced by the fatty acid composition of the host cell membrane

The fatty acid composition of the membrane of the conditional auxotroph fabB2 can be altered by allowing the cells to grow at non-permissive temperature (37°C) in the presence of a cis-unsaturated fatty acid. The phage 9NA, a virulent phage of Salmonella typhimurium, can not multiply in fabB2. Synthesis and maturation of the phage DNA are differentially affected by variation in the fatty acid composition of the cell membrane. The replicating DNA associates with the membrane complex, the site of DNA synthesis. The association is comparatively weak in oleic, claidic, palmitoleic, palmitelaidic and linolelaidic acid enriched cells. When the cells are grown in the presence of palmitoleic acid, a large pool of concatemeric phage DNA accumulates in the cytoplasm within 10 min of infection. The conversion of concatemeric DNA to monomeric one i.e., mature phage length DNA, is inhibited in such cells. The presence of concatemeric DNA can be visualized by electron microscope. Such a situation is not observed when the cells are grown in media supplemented with other types of unsaturated fatty acids. The mechanism by which the host cell membrane lipid controls phage development is yet to be worked out.

Phage DNA synthesis and host DNA degradation in the life cycle of Lactococcus lactis bacteriophage c6A.

Bacteriophage c6A is a lytic phage that infects strains of Lactococcus lactis. Infection of L. lactis strain C6 resulted in inhibition of culture growth within 10 min, mature intracellular phage particles appeared after 17.5 min, and cell lysis occurred after 25 min. A culture of strain C6 carrying 3H-labelled DNA was infected with c6A, and the fate of the radiolabel was monitored. The results showed that degradation of host cell DNA began within 6 min of infection and that the breakdown products were incorporated into progeny c6A DNA. Quantitative DNA hybridizations indicated that synthesis of phage DNA began within 6 min of infection and continued at an approximately constant rate throughout the latent period.

Molecular cloning of .PHI.80 adsorption-inhibiting cor gene.

It was reported that φ80 lysogens did not adsorb superinfecting phage φ80 and a gene responsible to the phenomenon was named cor (Kozyrev et al., 1982). We found that E. coli containing φ80 genome in lytic cycle also began to show resistance to superinfecting φ80 after 5min of infection. We cloned the cor gene and determined its nucleotide sequence. A putative cor gene product should be 92 amino acids long and have three hydrophobic cores and one hydrophilic region, indicating a possibility of membrane binding protein. The product may interact with φ80 receptor on inner- or outer-membrane of E. coli cells and inhibit the adsorption of superinfecting φ80. The cor gene is supposed to be transcribed from a unique promoter located close to the gene.

Requirement for protein synthesis for survival of unmodified bacteriophage T1 in a restricting host.

At high multiplication of infection, a substantial fraction of restricting cells (P1 lysogens) could be productively infected by unmodified coliphage T1 (T1.0) provided that protein synthesis was uninhibited during the first 5 min of infection. Successful infection under restricting conditions was accompanied by more genetic recombination than was seen under nonrestricting host, the recombination frequency declined for markers on T1.0 genomes; no effect was seen on recombination between markers on modified (T1.P) genomes. This suggested that recombination between unmodified genomes may be essential for their survival under conditions of host restriction. In a restricting host, genetic markers on T1.0 could recombine with T1.P even when the rescuing phage was added 6 min after T1.0 infection. However, even marker rescue recombination was diminished when protein synthesis was inhibited during early infection. Since DNA restriction is an early event, protein synthesis may be required soon after infection of a restricting host by T1.0 in order to preserve restriction-damaged DNA in a form that can participate in recombination. Experiments are also described that rule out some possibilities for the role of such a protein(s).

Alteration of [formula omitted] RNase D by infection with bacteriophage T4

Infection of a variety of E. coli strains with bacteriophage T4 leads to about a 25,000 dalton increase in the apparent molecular weight of RNase D based on gel filtration on Ultrogel AcA44. No alteration occurs when infection is carried out in the presence of chloramphenicol. The change in RNase D is substantially completed by 7.5 min of infection. Chromatography of the altered RNase D on the adsorbant, Affi-gel Blue, restores the enzyme to its original molecular weight of 40,000, indicating that the modification is reversible. Mixing an extract from infected cells with one from uninfected cells converts a portion of the uninfected cell enzyme to the higher molecular weight form. No conversion takes place if the infected cell extract is first treated with phenol to inactivate proteins. Preliminary analysis indicates that the factor in infected cell extracts responsible for the conversion is a heat-labile, relatively low-molecular weight protein, and that RNase D is modified by association with this phage-specific component. The potential role of RNase D in the 3′ processing of bacteriophage T4 tRNA precursors, and the involvement of a phage gene product in this process, are discussed.

Host transcription in bacteriophage P22-infected Salmonella typhimurium

The kinetics of host RNA synthesis, as measured by pulse-label kinetics and RNA-DNA hybridization, is inhibited 10-fold shortly after infection with bacteriophage P22. This early inhibition lasts through the first 6 min of infection and affects not only RNA synthesis but several other energy-requiring cellular functions. In lysogenic infections, the rate of bacterial transcription rapidly recovers to the value of uninfected controls. In lytic infections, the rate of host transcription increases only to about 20 to 25% of the original level, indicating a second mechanism for the inhibition of RNA synthesis in the lytic response. The early inhibition is multiplicity dependent, bhloramphenicol insensitive, and independent of phage gene 24. The lytic inhibition is dependent upon the expression of gene 24 but independent of gene 23.

The influence of differing ionic environments on the cercarial, post-cercarial and adult stages of the ectoparasitic digenean Transversotrema patialense

Survival of cercariae of Transversotrema patialense was enhanced in saline with an ionic concentration equivalent to 25 % sea water whilst infectivity was unchanged up to the maximum salinity tolerance of the fish experimental host ( Brachydanio rerio). Adult survival in vitro increased little at ionic concentrations above 12.5% of that of sea water but fell sharply at lower concentrations. Survival in vitro was not enhanced by sterile conditions and only increased slightly with increasing age of worms. Adult parasites became water sensitive within 5 min of infection. However mechanically decaudated cercariae showed no water sensitivity. To survive on freshwater hosts the adult parasite must experience a degree of physiological isolation from its environment despite its apparent ectoparasitic microhabitat.

Etude de la relation parasitaire initiale entre un virus du groupe L 1 de Gourlay et une population cellulaire d'Acholeplasma laidlawii

The study of the relationship between MVL 51 virus, of the Gourlay group L 1, and the Mycoplasma cells of A. laidlawii during the first 35 min of infection leads to the conclusion that a "carrier state" has been established, free virus as well as infected and noninfected cells being present at the same time in the cultures. The evidence of a virus growth after 20 min of infection and the high level of remaining viable cells (1.15 x 10(8) CFU) at the end of the experiment also indicate that the carrier state should persist, at least for some time, being probably an important factor of the virus-cell relationship in the viral group L 1.

Fast sedimenting bacteriophage T7 DNA from T7-infected Escherichia coli

The sedimentation rate of bacteriophage T7 DNA from T7-infected Escherichia coli has been determined after lysis with the nonionic detergent Brij 58. After 8.5 min of infection at 37°, most of newly replicated T7 DNA sedimented more rapidly than 100 S(100 S + DNA). Some of the T7 100 S + DNA sedimented in a broad peak between 100 Sand 600 S; the remainder sedimented at speeds greater than 600 S. In the electron microscope 100 S + T7 DNA appeared to be a massive DNA complex containing up to 30 phage equivalents of DNA in a single structure. In these complexes densely packed cores of DNA were surrounded by more loosely packed DNA. Kinetic labeling experiments strongly suggest that 100 S + DNA is a precursor for DNA in progeny phage. It was also shown that: (i) Non-DNA substances, possibly membrane fragments and/or proteins, are bound to 100 S + T7 DNA, but are probably not the primary cause of the rapid sedimentation of this DNA. (ii) Single-chain interruptions exist in the predominantly duplex structure of T7 100 S + DNA. (iii) When cells are lysed with the ionic detergent, Sarkosyl, the 100 S + DNA is obtained, though in reduced yield. Phenol extraction eliminates virtually all 100 S + DNA. (iv) Some of the 100 S + T7 DNA is more shear-resistant than linear, duplex DNA.

Localization of membrane protein synthesized after infection with bacteriophage T4.

The synthesis of membrane protein after infection with bacteriophage T4 was examined. Protein constituents of both the cytoplasmic and outer membrane are made during the infective cycle. In addition, newly synthesized membrane protein is found in material which has a buoyant density greater than that of either of the two host membrane fractions. Polyacrylamide gel analyses and solubilization studies using the detergent Sarkosyl indicate that synthesis of most of the membrane proteins made during the first 5 min of infection is directed by bacterial genes. New membrane proteins synthesized at times greater than 6 min after infection appear to be distinct from those of the host, and new proteins of the outer membrane are different from those of the inner. Proteins in the new dense membrane fraction are similar to those of the outer membrane.

Effect of Calcium Ions on Synthesis of T5-Specific Ribonucleic Acid

Abstract The arrest in the development of bacteriophage T5 and in the synthesis of phage-specific proteins in Escherichia coli F cells infected at a low calcium ion concentration (10-4 m) has been traced to a deficiency in the system for synthesis of a specific class of viral RNA. Previous studies have recognized that during an infection under standard conditions at 10-3 m Ca++ three classes of RNA are formed in sequence during the infective cycle. They are Class I RNA, which appears between 0 and 4 min after infection, and Class II and Class III RNA, commencing at 4 and 9 min, respectively. The predominant RNA species formed during the first 4 min of infection at 10-4 m Ca++ appears to be identical with that synthesized immediately after infection of cells at 10-3 m Ca++ and is therefore Class I RNA. By this time, however, the rate of RNA synthesis has fallen rapidly and thereafter there is a marked change in the composition of the RNA species. At 20 min after infection 75% of the product is Class II RNA. Upon elevation of the Ca++ concentration to 10-3 m the rate of RNA synthesis increases markedly. Class II RNA at greatly elevated levels is the immediate product, and 5 to 6 min later Class III is formed. The arrest in RNA synthesis at 10-4 m Ca++ does not result from the failure of the T5 DNA to inject completely into the host cell nor from a loss of the metabolic capacity of cells to generate nucleoside triphosphates and maintain them within the cell. The interruption in viral development at low calcium ion concentrations probably represents a malfunction of the machinery for DNA transcription beginning with the synthesis of Class II RNA. The cessation of Class I RNA synthesis at either high or low Ca++ concentration probably results from the action of a protein formed early in the infective cycle.

Intracellular inactivation of bacteriophage T4 early deoxyribonucleic acid.

As previously shown, a small amount of polynucleotide material is added to parental T4 deoxyribonucleic acid (DNA) molecules within the first 5 min of infection. I have asked whether this process is essential for phage replication. Two approaches-one involving decay of (32)P incorporated into this "early DNA" and the other involving photoinactivation of bromodeoxyuridine-containing early DNA-indicate that it is.

Transcription of the bacteriophage T4 template: strand selection by E. coli RNA polymerase in vitro.

THE prereplicative viral messenger RNA that is made in bacteriophage T4 infected E. coli during the first 5 min of infection (at 30° C) hybridizes almost exclusively with the l strand of its DNA1,2. This strand selection results solely from the interaction of the bacterial RNA synthesizing machinery and the infecting phage DNA; at least it does not require any protein programmed by the virus, for it takes place even when the infection is carried out in the presence of chloramphenicol. At later times of the normal viral development, several new RNA species, some of which are transcribed off the viral r strand, appear (refs. 1, 2 and results of A. G. W. Salser, Bolle, W. Epstein, Pulitzer, E. P. G. and W. S., to be published). In this communication we report that the in vitro transcription of the mature T4 viral DNA template by purified E. coli RNA polymerase is restricted almost absolutely to the l strand of the DNA.