Conformational studies of Actinobacillus actinomycetemcomitans leukotoxin: partial denaturation enhances toxicity.
A 114 kDa, water-soluble, cytotoxin secreted by the Gram-negative bacterium Actinobacillus actinomycetemcomitans (Aa) is similar in sequence to Escherichia coli alpha-hemolysin, but is non-hemolytic, killing leukocytes of select species, including humans. In this work, we investigated aspects of the water-soluble conformation of Aa toxin which relate to its biological, pore-forming activity. The toxin has five native tryptophans and fluorescence spectra were monitored in aqueous solutions in the presence of varying denaturants. Significant changes in the fluorescence spectra, without significant wavelength shifts, were induced by small additions of denaturants and changes in the temperature or pH. The fluorescence changes suggested that small perturbations in the aqueous environment resulted in structural changes in the toxin related not to a large unfolding but to more subtle conformational changes. Analytical ultracentrifugation showed the toxin to be a globular monomer in dilute aqueous solution. Circular dichroism spectroscopy showed about 25% alpha-helical structure which is largely maintained up to a temperature (65 degrees C) known to deactivate toxin activity. Changes in the cytotoxic properties of the toxin were monitored with flow cytometric analysis following preincubation of the toxin under mild conditions similar to those used in the fluorescence studies. These experiments showed that the pretreated toxin exhibited enhanced cell-killing potency on toxin-sensitive cells. The correlation of cytotoxicity with the changes in Trp fluorescence is consistent with the idea that partial unfolding of Aa toxin is an early, obligate step in toxin-induced cell kill.
Lear
J D
JD
The Johnson Foundation, Department of Biochemistry and Biophysics, University of Pennsylvania School of Medicine, Philadelphia, PA 19104-6059, USA. lear@mail.med.upenn.edu
Karakelian
D
D
Furblur
U
U
Lally
E T
ET
Tanaka
J C
JC
D016976:Aggregatibacter actinomycetemcomitans / Q000378:metabolism*; D001427:Bacterial Toxins / Q000737:chemistry / Q000378:metabolism; D004926:Escherichia coli; D005098:Exotoxins / Q000737:chemistry* / Q000378:metabolism; D005453:Fluorescence; D011487:Protein Conformation; D011489:Protein Denaturation
D001427:Bacterial Toxins; D005098:Exotoxins; C018579:leukotoxin