Richard A. Finkelstein

The critical role of iron in host-bacterial interactions.

The ability of potential pathogens to acquire iron in a host is an important determinant of both their virulence and the nature of the infection produced. Virulent gram-negative bacteria are capable of acquiring sufficient iron from the host because their virulence (for chick embryos) is unaffected by exogenous iron. Avirulent mutants which are apparently limited in their ability to acquire iron could be isolated from the virulent strains. The lethality of these mutants was significantly enhanced by exogenous iron. Reduction of the relatively high serum iron saturation of chick embryos (to levels more closely approximating those in man) by pretreatment with iron-binding proteins or endotoxin inhibits the lethality of some virulent bacteria. Those bacteria whose virulence was reduced include the Shigella, Vibrio cholerae and strains of Neisseria gonorrhoeae, all of which are nondisseminating pathogens in the normal human host. Pathogens which produce septicemic and disseminating infections such as Neisseria meningitidis, Haemophilus influenzae type B, Escherichia coli possessing K-1 antigen, Pseudomonas aeruginosa and Salmonella typhimurium and disseminating strains of N. gonorrhoeae were, in general, unaffected by reduced serum iron saturation. These disseminating bacteria appeared to produce greater quantities of compounds (siderophores) which stimulated microbial growth in low-iron media than did the nondisseminating pathogens. Thus, the gram-negative bacteria tested can be divided into four major classes according to their responses to modifications in iron levels in the chick embryo model and these results correlate with the nature of the infections which they typically produce in man.

Chemical and physical properties of cholera exo-enterotoxin (choleragen) and its spontaneously formed toxoid (choleragenoid)

Abstract The chemical properties of two proteins, cholera enterotoxin (choleragen) and a spontaneously formed toxoid (choleragenoid), have been studied. The toxin has a molecular weight of 84 000 and most likely consists of six subunits with molecular weights of approx. 15 000. The toxin molecule appears to consist of more than one type of subunit. Choleragenoid consists of a mixture of at least three proteins with molecular weights of 58 000 and 4 subunits of approximately equal size. Neither protein contains significant quantities of lipid or hexose.

Studies on toxinogenesis in Vibrio cholerae. III. Characterization of nontoxinogenic mutants in vitro and in experimental animals.

Spontaneous and chemically induced mutants with reduced ability to produce cholera enterotoxin (choleragen) as an extracellular protein were isolated from Vibrio cholerae strains 569B Inaba, a classical cholera vibrio, and 3083-2 Ogawa, an El Tor vibrio. By qualitative and quantitative immunological assay in vitro such mutants could be separated into different classes characterized either by production of no detectable choleragen (tox minus), or of small quantities of extracellular choleragen, or of large quantities of cell-associated choleragen but little extracellular choleragen. Analysis of proteins in concentrated culture supernates by electrophoresis in polyacrylamide gels showed that cultures from tox minus strains lacked proteins with electrophoretic mobilities corresponding with choleragen or the spontaneously formed toxoid (choleragenoid). Infant rabbits infected with the tox minus strains remained asymptomatic or developed milder symptoms than rabbits infected with the tox+ parental strains. When symptoms of cholera developed after inoculation with tox minus mutants, detectable numbers of tox+ revertants could be isolated from the intestines of the infected animals. Two tox minus strains, designated M13 and M27, caused no sumptoms and showed no evidence of reversion to tox+ during single passage in infant rabbits, and mutant M13 also remained avirulent and stably tox minus during six cycles of serial passage in infant rabbits. Strains M13 and M27 were also noncholeragenic in acult rabbit ileal loops. Quantitative cultures of the intestines from infected infant rabbits demonstrated that the avirulent mutant M13 can multiply in vivo and can persist in the intestinal tract for at least 48 h.

Crystalline cholera toxin and toxoid.

The exo-enterotoxin of Vibrio cholerae has been obtained in crystalline form. A solution of the crystalline protein was equal in potency to the parent pure toxin in both choleragenicity and skin reactivity. Crystals of the natural toxoid, choleragenoid, resemble those of the toxin in appearance. A solution of crystalline choleragenoid was equivalent to the parent preparation in the flocculation test.

Ion transport during cholera-induced ileal secretion in the dog

To assess the ion transport mechanism by which cholera causes the small bowel to secrete, ion transport rates and electrical potential difference (PD) were determined simultaneously in the normal and choleragen-treated dog ileum in vivo. The results indicate that, during cholera, HCO(3) is actively secreted (i.e., against both an electrical and a concentration gradient); Cl is also actively secreted, against a modest electrochemical gradient. Electrogenic pumping of one or both of these anions is probably responsible for an observed PD change of approximately 13 mv (lumen negative). Na secretion can be accounted for entirely by passive ion movement. K secretion can be partly explained by passive diffusion secondary to the negative intraluminal PD; however, its concentration in the secreted fluid is two to three times higher than expected on the basis of passive forces, suggesting a component of active K secretion. The PD response of the choleragen-treated ileum is normal in response to glucose, but there was no PD response to saline-free mannitol perfusion. This suggests that the normal differential permeability of the ileum to anions and cations may be altered by choleragen, although other explanations of this finding are also possible.

Pathogenesis of experimental cholera: choleragen-induced rat foot edema; a method of screening anticholera drugs.

Summary This preliminary study suggests that the rat foot edema test may provide a useful means of screening pharmacological agents potentially capable of preventing or reversing the specific metabolic lesions induced by cholera enterotoxin (choleragen). The first inhibitor selected for study, cyclo-heximide, prevented or delayed the onset of choleragen-induced edema in the rat foot test and also prevented the development of choleraic diarrhea in the infant rabbit assay. However, it did not alter the course of the established lesion in the rat foot. These observations suggest that a choleragen-induced, host-produced protein mediator may be involved. Additional studies, involving this model, directed toward understanding the pathogenic mechanism of cholera toxin and to develop potentially more effective methods of treatment of cholera patients, are in progress. We wish to thank Miss Mary Knoohuizen for her assistance in helping to set up these assays. Support was provided by a grant, 1 R22 AI08877-01, from the National Institute of Allergy and Infectious Diseases.

Subunit structure and N-terminal amino acid sequence of the three chains of cholera enterotoxin☆

Abstract The cholera enterotoxin (choleragen) was separated into its A, or biologically active, region, and its B, or binding, region by gel filtration in 5 M guanidine. The A region was further separated into its component peptides, A 1 and A 2 , after complete reduction and 14 C-alkylation. The amino acid composition and the N-terminal amino acid sequences of the component peptides, A 1 , A 2 and B, were determined. The A 1 (7300 daltons) and A 2 (21,300 daltons) peptides are joined by a disulfide bond involving residue 5 of A 2 . The B chains (10,000 daltons) each have an intra-chain disulfide loop involving position 9. Slightly over 5 B chains are associated non-covalently with one A region in the average molecule of intact toxin. The B region is entirely equivalent to choleragenoid, the spontaneously formed enterotoxoid. The amino acid sequences reported herein differ significantly from those reported previously by others.