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Studies on the isolation and characterization of Proteus mirabilis siderophores provided no evidence that these bacteria synthesize catechol- or hydroxamate-type siderophores. However, gas chromatograph analysis in conjunction with mass spectroscopy revealed the presence of α-hydroxyisovaleric acid, a previously unknown metabolite. Additional substantiating evidence for the presence of α-hydroxyisovaleric acid in these bacteria was obtained from experiments involving the use of thin-layer chromatography and an ultraviolet absorption spectrum. This compound was found to be capable of removing iron from the synthetic chelator, ethylene-diamine-di-orthohydroxyphenyl acetic acid, and supplying that iron to the bacteria both in a solid agar medium and in a liquid medium. Proteus mirabilis was found to possess an enzyme capable of catalyzing the reaction by which α-hydroxyisovaleric acid is converted to α-ketoisovaleric acid, an intermediate in the valine biosynthetic pathway.

The therapeutic efficacies of procaine penicillin G and acetohydroxamic acid, when administered alone or in combination, were evaluated in rats with experimentally induced pyelonephritis at 24 h and 4 days after intrabladder inoculation with Corynebacterium renale. Penicillin alone prevented the development of pyelonephritis when administered in a once daily dose of 50 000 units for 5 days beginning within 24 h of infection challenge. However, if therapy was not initiated until the 4th day after induction of infection, pyelonephritis ensued which could not be differentiated from untreated rat pyelonephritis. Acetohydroxamic acid, when administered by itself in a once daily dose of 100 mg for 5 days, was completely ineffective whether therapy was started at 24 h or 4 days after initiation of infection. On the other hand, the combination of penicillin and acetohydroxamic acid prevented progression of pyelonephritic infection and facilitated healing when rat kidneys were examined at 14 and 30 days after initiation of infection. The kidneys of those rats sacrificed at 30 days after cessation of treatment revealed the presence of increased interstitial fibrous connective tissue in the renal medulla suggesting prior damage followed by recovery and repair. In vitro studies on the action of penicillin and acetohydroxamic acid against C. renale revealed no signs of synergism or antagonism.

The role of Corynebacterium renale urease in the establishment of pyelonephritis was studied by the oral administration of acetohydroxamic acid (AHA), a urease inhibitor, to experimentally infected rats. The bacteria were introduced by surgical insertion of a zinc disc containing 1 × 106 colony-forming units of C. renale into the urinary bladder whereas sterile discs were implanted in the bladders of the control animals. Daily administration of AHA via the drinking water did not halt the development of pyelonephritis. Larger doses, given by gavage, did accomplish this goal; that is, the pH of the urine was lowered, the number of colony-forming units of C. renale in the kidney was reduced drastically, and pyelonephritic lesions were observed in the kidney by light-microscopic examination. All experimental rats developed cystitis in varying degrees of severity. About 70% of the intact AHA given by gavage was excreted in the urine 24 h after administration of this compound. Rats implanted with a urease-negative mutant of C. renale displayed no signs of pyelonephritis but did develop cystitis.

A chemically defined medium (CDM) has been developed which supports both growth and hemolysin production by Haemophilus pleuropneumoniae. Although the growth rate in stationary cultures was substantially slower in CDM than in trypticase soy broth plus 0.6% yeast extract (TSBYE) and slightly slower than in heart infusion broth (HIB), extracellular hemolysin activity in CDM was slightly higher than in HIB and 16-fold greater than in TSBYE. Maximum hemolytic activity was produced in CDM in early to mid log phase of growth. Hemolytic activity in sterile, cell-free culture supernatant fluids persisted for over 10 days at 4 degrees C and 3-5 days at 37 degrees C, but was completely destroyed at 56 degrees C after 30 min. Total hemolysin inactivation was also achieved in the presence of trypsin or pronase (10 units/mL), but no decrease in hemolytic activity was noted in the presence of DNase or RNase. Iron had little effect on the hemolytic activity in the early stages of growth. However, in the later stages of growth, iron had a pronounced effect with hemolytic activity decreasing as the iron concentration increased from 1 to 500 microM. None of these iron concentrations had any effect on the hemolytic activity when added directly to prepared cell-free culture supernatant fluids. The extracellular hemolysin produced by H. pleuropneumoniae in CDM appears to be a heat-labile protein the activity of which is influenced by iron at certain phases of growth.

The solubility of the homogeneous, nonenzymatic structural protein isolated and purified from bovine heart mitochondria when incubated with the murine toxin of Pasteurella pestis at pH 11.0 increases as the concentration of the toxin is increased. This indicates that a protein-protein interaction occurs between the monomeric units of the structural protein and the toxin. This interaction results in the decline of the toxicity of the toxin. The extent to which the toxicity is diminished depends upon the ratio of structural protein to toxin in the incubation mixture. Double diffusion gel precipitation reactions with plague murine toxin incubated with structural protein revealed that the decrease in the toxicity of the toxin is correlated with the disappearance of the bands exhibited by both toxin A and toxin B, the two protein species comprising plague murine toxin. No interaction is observed between the toxin and polymeric structural protein. There is, however, an indication of a slight decrease in the toxicity of the toxin. Under the latter conditions, the band for toxin A is not visible in double diffusion gel precipitation reactions whereas the band for toxin B remains intact. Bovine serum albumin does not interact with plague murine toxin nor does it affect its toxicity. These results suggest that the interaction between plague murine toxin and monomeric structural protein involves binding of the two proteins. The relationship that this protein-protein interaction may have to the known inhibitory effect of the toxin on the electron transport system is discussed.

1. 1. The inhibition of rat-liver mitochondrial respiration in the presence of a-ketoglutarate, glutamate, succinate and β-hydroxybutyrate as a substrates by the murine toxin of Pasteurella pestis is not relieved by 2,4-dinitrophenol indicating that the toxin exerts its inhibitory effect somewhere in the electron-transport chain. 2. 2. Absolute and difference spectra of the cytochrome components of toxin-treated rat-heart and -liver mitochondria reveal that toxin addition causes on oxidation of cytochromes a, b and c suggesting that the toxin acts between reduced nicotinamide-adenine dinucleotide or succinate and cytochrome b. 3. 3. The oxidation of ascorbate by rat and liver mitochondria in the presence of tetramethylphenylenediamine and cytochrome c as carriers is not affected by the toxin. 4. 4. The reduction of cytochrome c in the presence of reduced nicotinamide-adenine dinucleotide by rat-heart mitochondria and electron-transport particles is inhibited by the toxin.

Plague murine toxin can inhibit to a significant extent the uptake of Ca ++ and inorganic phosphate (P i ) by rat heart mitochondria in the presence of succinate, α-ketoglutarate, malate, or β-hydroxybutyrate as the respiratory substrate. When these mitochondrial suspensions are incubated with ATP, but without a respiratory substrate, the uptake of these ions is also inhibited by the toxin. Although the toxin does not alter rat heart mitochondrial respiration supported by the oxidation of reduced cytochrome c, it does inhibit the uptake of Ca ++ and P i supported by this segment of the electron transport chain. When toxin is inactivated by treatment with formalin, it has little or no effect on the uptake of Ca ++ or P i by rat heart mitochondria in any of the systems investigated. The uptake of these ions by heart mitochondria isolated from the toxin-resistant rabbit is either not inhibited at all by the toxin or is inhibited to a much lesser extent as compared with rat heart mitochondria. Ethylenediaminetetraacetic acid, at a concentration which does not inhibit Ca ++ or P i uptake but does prevent swelling, prevents to a large extent the inhibitory effect of the toxin on the accumulation of these ions by rat heart mitochondria.

Studies were conducted on the effect of four different hydroxamic acids (HA), hydroxyurea, acetohydroxamic acid, p-fluorobenzoylhydroxamic acid and sorbylhydroxamic acid, on the growth and urease activity of Corynebacterium renale. The addition of each of these HA, at concentrations ranging from 10−3 to 10−5 M, to medium containing urea as the sole nitrogen source resulted in a lengthened lag period of growth the extent of which depended upon the concentration of each HA tested as well as the structure of the compound; that is, the size and (or) complexity of the side chain attached to the common terminal group of the molecule. However, the maximal growth levels achieved following conclusion of the exponential phase were not affected by the HA. Investigations on the effect of these HA on the urease activity of intact cells as well as cell-free extracts revealed that in each case the enzymatic activity was inhibited by each of the HA tested. The extent of inhibition with the intact cells was about one-half of that observed with cell-free extracts. Direct incubation of cell-free extracts as well as intact cells with each of the HA tested was required for maximal inhibition.

Moderately iron-deficient rats were significantly less susceptible to experimentally induced Proteus mirabilis pyelonephritis than iron-sufficient or severely iron-deficient littermates, as indicated by significantly lower prevalence of infection, mean pyelonephritic lesion scores, and numbers of P. mirabilis cells recovered from the kidneys.

Kadis, Solomon(Albert Einstein Medical Center, Philadelphia, Pa.),Samuel J. Ajl, and James H. Rust, Jr.Action of plague murine toxin on mitochondria from resistant and susceptible animals. J. Bacteriol.86:757–765. 1963.—Purified murinePasteurella pestistoxin inhibited the respiration of liver mitochondria isolated from both the rat and the rabbit. Toxin had little or no effect on the respiration of rabbit sarcosomes, but when they were disrupted with either sodium deoxycholate or sonic vibration their respiration was inhibited. The respiration of heart mitochondria from rats immunized with toxoid-adjuvant mixtures was inhibited only slightly by the toxin, whereas the respiration of liver mitochondria from immunized rats was inhibited to the same degree as that from nonimmunized animals. Toxin caused the heart mitochondria of the rat to swell, but had little or no effect on the heart mitochondria of the rabbit. Also, brain mitochondria were not swollen by the 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.

The murine toxin of Pasteurella pestis inhibited the respiration of heart mitochondria from the rat and the mouse but had little or no effect on the respiration of mitochondria from the rabbit, chimpanzee, dog, and monkey. Alterations occurred in tile S-T segments of the electrocardiogramus recorded corded from rats injected with (1/4) to 10 LD(50) of toxin, but not in those from rats dying of hemorrhagic shock, hypoxia, intoxication with glucose, or Escherichia coli endotoxin. No abnormalities were observed in electrocardiograms from rabbits injected with large amounts of toxin.