Thomas C. Montie

Flagella, Motility and Invasive Virulence of Pseudomonas aeruginosa

The role of motility as a virulence factor in Pseudomonas aeruginosa burn wound sepsis was examined using mutants deficient in the Fla or Mot phenotype. Physiological profiles of parental strains and Fla- and Mot- mutants were similar with respect to antibiograms, O antigen types, growth rates, and proteolytic, exotoxin A and phospholipase activities, providing evidence for isogenicity. Lethality studies using a subcutaneous mouse burn model showed that three Fla- mutants and one Mot- mutant were much less virulent (10(2) to 10(5) times) than the parent wild-type. Topical challenges in the flame burn model showed that a Fla- mutant of strain M-2 was approximately tenfold less virulent. A reduction in virulence, although somewhat less than tenfold, was also observed in the scald burn model for M-2 Fla-, and Mot- strains. Tissue colonization experiments revealed a characteristic, rapidly systemic infection in burned mice challenged with wild-type organisms. Nonmotile mutants similarly proliferated in the burn wound, but the characteristic bacteraemia and systemic invasion were markedly absent. The infection remained localized in the skin wound and the mice survived. The pattern of infection by nonmotile mutants in the colonization studies was very similar to that obtained with Fla+ cells in burned animals passively treated with antiflagellar antibody. These results add substantial support to the concept of motility as a P. aeruginosa virulence factor in invasive infections.

Isolation of toxic subunits from two murine-toxic proteins from Pasteurella Pestis

Abstract Two mouse-toxic proteins, Toxin A (240,000 molecular weight) and Toxin B (120,000 molecular weight), were dissociated in sodium dodecyl sulfate to small molecular weight subunits. Subunit size from 10,000 to 12,000 molecular weight is proposed based on gel electrophoresis, ultracentrifugation and dialysis experiments. The subunits retained approximately 60% of the toxic activity of the large molecular weight toxins.

A comparison of the characteristics of two murine-toxic proteins from Pasteurella pestis.

Abstract Two murine toxic proteins of relatively equivalent specific toxicity, previously isolated from an avirulent strain of Pasteurella pestis , have been further characterized. Molecular weights determined on Sephadex G-200 columns were 240 000 for Toxin A and 120 000 for Toxin B. Whereas this information, together with the sedimentation values, amino acid composition and ultraviolet spectra suggests a polymeric relationship between the two toxins, the 30% higher tryptophan content in Toxin A is not in accord with this notion. The selective inhibition of Toxin A formation in vivo on the addition of tryptophan analogues may be related to this difference in tryptophan content. Other differences include the increased lability of Toxin A and the high absorption of Toxin B in the 200 to 230 mμ ultraviolet range. These latter differences, along with the previously reported responses to -SH and other denaturing reagents, may be reflecting primarily differences in tertiary structure and amino acid sequences. Toxin B has been found to be identical with the earlier isolated plague murine toxin.

Action of Sulfhydryl and other Denaturing Agents on the Protein Toxins of Pasteurella pestis.

Abstract The interaction of various denaturing agents with proteins from Pasteurella pestis was studied with use of biological, immunological, and electrophoretic techniques. Only the larger molecular weight toxin, Toxin A, was sensitive to deoxycholate. Both the smaller molecular weight Toxin B, and Toxin A, were inactivated by urea and sulfhydryl reagents such as p -chloromercuribenzoate (CMB), N -ethylmaleimide, and silver ions. Toxin B was particularly sensitive to low concentrations of reagents for sulfhydryl groups. Increasing the concentration of these reagents resulted in the complete loss of Toxin Bs electrophoretic and immunologic identity. Loss in toxicity of both toxins was directly related to the concentration of these inhibitors. Increased silver ion concentration resulted in an increased amount of toxin precipitation. The resolubilized silver precipitate was biologically inactive, but activity was retained in the supernatant fluid. The resolubilized precipitate could be reactivated with glutathione, but not by dialysis. At low concentrations of CMB, one to several SH groups reacted per milligram of toxin as assayed by mercaptide formation. More of these groups reacted per milligram of Toxin B than A. The addition of 0.5% of lithium bromide increased by 5- to 8-fold the number of available SH groups to react with CMB. Under the latter conditions all activity of the toxin is lost. Therefore a certain number of SH groups apparently must be available to maintain toxicity of both Toxin A and B.

Early Cell Envelope Alterations by Tobramycin Associated with its Lethal Action on Pseudomonas aeruginosa

The immediate activities of the aminoglycoside antibiotic tobramycin were investigated in Pseudomonas aeruginosa PAO1. The lethal action of a low concentration of tobramycin (8 micrograms ml-1) occurred rapidly (1-3 min) and was associated with leakage of certain cellular components into the supernatant. The presence of magnesium at the time of initial exposure protected cells by preventing uptake of tobramycin; however, magnesium addition following a brief exposure did not restore viability. Analyses of supernatant material revealed a rapid 2-fold increase in protein released following tobramycin treatment. A prominent 29 kDa protein, observed by SDS-PAGE in the released material was identified as the periplasmic beta-lactamase. Brief exposure to tobramycin did not result in major morphological damage or cell lysis as observed by transmission electron microscopy, and release of LPS was not a primary event. Although activity at the ribosomal level was observed by 2-3 min, leakage was detected after only 1 min. These data indicate that leakage of cellular components, particularly beta-lactamase, occurs simultaneously, if not prior to inhibition of protein synthesis by tobramycin.

THE MOLECULAR BIOLOGY OF PLAGUE TOXIN

This chapter discusses the molecular biology of plague toxin. Studies on the chemical nature and biological action of plague toxin require adequately pure material. The chapter lists some pertinent properties of the best samples that have been subjected to various electrophoretic procedures. The most purified material contained approximately 95% protein and was found to be free of nucleic acids, carbohydrates, lipids and capsular material. It had an intravenous LD 50 for 16 gram to 18 gram Swiss albino mice of 0.1 μg of protein and exhibited one band in the sensitive Oudin and Ouchterlony gel diffusion precipitation reactions. To determine whether plague toxin possessed any unusual components that could account for its high toxicity, detailed elemental and amino acid analyses were performed on the most highly purified samples of toxin available. In the study, 18 amino acids and a number of elements were identified. On a dry weight basis, over 98% of the toxin molecule was accounted for by organic analysis including ammonia and ash content. There was nothing unusual about the toxin molecule aside from the high proportion of acidic amino acids, which verified the previously observed isoelectric point of the toxin of 4.7.