Charles C. Brinton

Efficacy trial of a parenteral gonococcal pilus vaccine in men

A randomized, placebo-controlled, double-blind efficacy trial of a purified gonococcal pilus vaccine composed of a single pilus type was tested in 3123 men and 127 women volunteers. Either 100 micrograms of vaccine or a placebo was given intradermally on day 1 and day 14. Each group was evenly matched with respect to age, sex, prior history of a sexually transmitted disease, sexual exposure during the study and attrition from the study. None of the women volunteers acquired gonorrhoea during the trial. In the male volunteers, 108 vaccine and 102 placebo recipients acquired gonorrhoea 15 days or later after the initial immunization. Vaccines developed a sustained ELISA antibody response to homologous and heterologous pili, but the latter titres were approximately 40% as high as the homologous pilus antibody rises. There were, however, no increases in inhibition of attachment antibody (IEA) titres. Local antibodies (semen) against homologous and heterologous strains were also elicited (ELISA). The vaccine was safe and did not alter the clinical expression of disease. This gonococcal pilus vaccine composed of a single pilus type failed to protect men against gonococcal urethritis.

Electrophoresis and phage susceptibility studies on a filament-producing variant of the E. coli B bacterium.

Abstract A variant of E. coli B bacterium, which we have called the F variant, has been found to exist in two distinct forms, fast and slow, which differ by a factor of almost two in electrophoretic mobility. The lower mobility of the slow form has been shown to be due to the presence of filaments, visible in electron micrographs, which exert a viscous drag. The filaments can be stripped off by mechanical agitation. De-filamented slow bacteria have the same mobility as non-filamented fast bacteria, indicating that the surface charge density is the same for the two forms. The bacteria are not killed by the stripping process and their progeny are filamented. The F variant appears to be similar to a phage semi-resistant variant, reported by Wahl 10 , which can exist in two states, refractory and receptive. The filamented bacteria are less susceptible to infection by a number of the T phages than de-filamented bacteria, and a preparation of filaments inactivated these phages. Bacteria selected for resistance to T 1 were found to be non-filamented and remained so on subculturing. The transition between the filamented and the non-filamented forms is rapid as is the transition between the refractory and receptive states. The probability of the transition is of the order of IO −3 per bacterium per division and is the same in both directions. It was noted that the filamented bacteria have a greater tendency to agglutinate spontaneously than the non-filamented. The same difference exists between smooth and rough forms. However, no detailed study was made of the colony morphology of the filamented variants.

Identification of the structural gene for F-pilin

Mutants in 12 cistrons of the sex factor F are deficient in transfer (Tra−) and nine of these fail to produce the hair-like appendages, F-pili, which are obligatory for both conjugation and male-specific phage infection. As a first step in providing a detailed characterization of the molecular basis of conjugation we have examined the number of radioactively labeled [125I]tyrosine peptides obtainable from purified pili produced by serine and tyrosine suppressed tra amber mutants. This has permitted the identification of traA as the structural gene for F-pilin, the protein subunit comprising F-pili.

A STUDY OF PARTICLE SIZES, SHAPES AND TOXICITIES PRESENT IN A BOIVIN‐TYPE ENDOTOXIC PREPARATION

When Escherichia coli Boivin endotoxic preparations

The infection of Pseudomonas aeruginosa by RNA pilus phage PP7: The adsorption organelle and the relationship between phage sensitivity and the division cycle☆

were analyzed by sedimentation in sucrose density gradients,’ 40 to 50 per cent of the material consisted of a monodisperse nontoxic and nonimmunogenic, but serologically reactive component (“hapten”) .’ The remainder, containing the toxic components, sedimented more rapidly and was very inhomogeneous. Highest toxicity was found associated with material of intermediate sedimentation constants in parallel with the maximum radioactivity of externally ”Cr-labelled endotoxin. These findings stimulated us to attempt the separation and physical characterization of the macromolecules contained in Boivin endotoxic preparations and to determine their toxicities and relationships to each other and to other components of the endotoxic preparations. In a first phase of the study, the endotoxic preparations were fractionated into several components by means of sucrose-density-gradient centrifugation. Again, large amounts of a nontoxic “hapten” were recovered. The toxic part of the parent material could be separated into two fractions of different particle size and toxicity, One of these contained all the very large heterogeneous aggregates. The other consisted mainly of smaller spherical and rod-shaped particles. With this technique, the toxic components were not obtainable in a homogeneous state. In a second phase of the investigation, it was found that dialysis against weak alkali dissociated the large polydisperse aggregates into smaller particles according to a characteristic pattern. This dissociation by alkali was accompanied by a marked increase in toxicity.

Removal of the coat protein of bacteriophages M13 or fd from the exterior of the host after infection

Abstract Evidence was obtained in favor of the proposal that FP pili are the adsorption organelle for the RNA containing Pseudomonas aeruginosa phage PP7. A pili-less mutant derived from the phages piliated host was found to be phage resistant and unable to adsorb phage. Spheroplasts prepared from either the mutant or the sensitive strain could be infected by naked PP7 RNA with equal efficiency, demonstrating that the phage resistance of the mutant was due to a defect in the adsorption-penetration mechanism and not due to an inability to replicate the phage. Two types of experiments with synchronously dividing cells demonstrated that the phages host was susceptible to infection only during a brief phase of the cellular growth cycle, beginning shortly before division. In the first, exposure of an asynchronously growing culture to phage infection for most of one generation time selected for cells in approximately the same growth phase as evidenced by the observation that the survivors grew synchronously for three generations after the phage exposure was terminated. In the second type of experiment, a culture which had been induced to divide synchronously by a period of starvation showed cyclic fluctuations in phage susceptibility with the maxima occurring just before cell division. Because of the short duration of the phage-susceptible phase, the susceptible cells extant in an exponentially growing culture constituted only a small minority of the cell population. Quantitative models were constructed in which phage susceptibility was defined as a particular phase of the cellular growth cycle. These models correctly predicted the rate at which phage-resistant cells became phage susceptible, were infected and accumulated as infected cells after phage had been added to an exponentially growing culture.

Infection of Pseudomonas aeruginosa spheroplasts by RNA from a pilus phage

Abstract The protein coat of the filamentous DNA-containing bacteriophages M13 and fd could be removed from the exterior of the host cell after successful infection had been initiated. M13 and fd thus behaved similarily to other known Escherichia coli bacteriophages. If the disassembled coat protein subunits were not removed from the surface of the infected cell, they were eventually ingested and utilized by the host and appeared partially in progeny phage.

Assay of influenza virus infectivity with chicken embryonic membranes.

Abstract Lysozyme-EDTA treatment rendered Pseudomonas aeruginosa cells susceptible to infection by RNA which had been phenol extracted from the P. aeruginosa pilus phage PP7. The efficiency of lysozyme-EDTA conversion was one competent spheroplast from every 10 5 bacteria. Competence rapidly decayed after 20 min at either 25 ° or 4 °. When spheroplasts were challenged with different concentrations of RNA, the infected spheroplast yield was directly proportional to the RNA concentration over a 10-fold range. The efficiency with which infectious RNA was extracted and assayed was one infected spheroplast from every 10 6 plaque-forming phage particles. This low efficiency was partially due to the presence of a contaminating RNase in the assay system. Three sequential steps in the infection of spheroplasts by phage RNA were differentiated. In the first step, RNA was rapidly bound to competent spheroplasts to form RNA-spheroplast complexes which were sensitive to pancreatic RNase. The first order rate constant describing the inactivation of these complexes by RNase was 12.5 log 10 units sec −1 (μg RNase per ml) −1 . In the second step, which occurred when the complexes were subjected to a positive osmotic shock, the RNA of the complexes became resistant to pancreatic RNase. The time required for this step was calculated to be 0.56 sec. In the third step, which required 1 min of incubation after the second step had occurred, the complexes became resistant to an unidentified inactivating agent in bovine serum albumin.

THE STRUCTURE, FUNCTION, SYNTHESIS AND GENETIC CONTROL OF BACTERIAL PILI AND A MOLECULAR MODEL FOR DNA AND RNA TRANSPORT IN GRAM NEGATIVE BACTERIA*

Summary An assay technic has been developed for titrating PR8 influenza A virus infectivity on preparations of chorioallantoic membranes removed from chicken embryos. This technic is simpler and less laborious than the standard assay method involving the use of chicken embryos. It is comparable in accuracy to the embryo technic although appreciably less sensitive. Evidence is submitted to show that both technics measure the same infectious entity.