Autoinducer 2 is required for biofilm growth of Aggregatibacter (Actinobacillus) actinomycetemcomitans

Abstract

Autoinducer 2 (AI-2) is required for the growth of Aggregatibacter (Actinobacillus) actinomycetemcomitans in culture under conditions of iron limitation. However, in vivo this organism thrives in a complex multispecies biofilm that forms in the human oral cavity. In this report, we show that adherent growth of A. actinomycetemcomitans on a saliva-coated surface, but not planktonic growth under iron-replete conditions, is defective in a LuxS-deficient background. Biofilm growth of the luxS mutant exhibited lower total biomass and lower biofilm depth than those for the wild-type strain. Normal biofilm growth of the luxS mutant was restored genetically by introduction of a functional copy of luxS and biochemically by addition of partially purified AI-2. Furthermore, introduction of S-adenosylhomocysteine hydrolase, which restores the metabolism of S-adenosylmethionine in the absence of LuxS, into A. actinomycetemcomitans did not complement the luxS mutation unless AI-2 was added in trans. This suggests that AI-2 itself is required for biofilm growth by A. actinomycetemcomitans. A biofilm growth deficiency similar to that of the LuxS-deficient strain was also observed when a gene encoding the AI-2-interacting protein RbsB or LsrB was inactivated. Biofilm formation by A. actinomycetemcomitans was virtually eliminated upon inactivation of both rbsB and lsrB. In addition, biofilm growth by wild-type A. actinomycetemcomitans was reduced in the presence of ribose, which competes with AI-2 for binding to RbsB. These results suggest that RbsB and LsrB function as AI-2 receptors in A. actinomycetemcomitans and that the development of A. actinomycetemcomitans biofilms requires AI-2.

FIG. 1.

AI-2 influences the biofilm growth of A. actinomycetemcomitans. (A) (Top) Representative confocal images of biofilms formed by wild-type (strain JP2) (left) and LuxS-deficient (luxS mutant) (right) A. actinomycetemcomitans. (Bottom) Biofilm growth of the luxS mutant complemented with a plasmid-borne copy of luxS (luxS-pluxS) (left) or in the presence of growth medium supplemented with partially purified A. actinomycetemcomitans AI-2 (luxS mutant + AI-2) (right). The upper images are in the x/y plane, and the lower images show the corresponding x/z reconstruction from a Z-stack from 0 to 100 μm in 1-μm increments above the saliva-coated coverglass. Scale bar, 100 μm. (B) Graphic representation of biofilm biomass and depth expressed as integrated fluorescence of wild-type (•) or LuxS-deficient A. actinomycetemcomitans grown either in medium alone (▪) or in medium supplemented with partially purified AI-2 (▴). Total fluorescence in each Z-stack image was measured using the Fluoview software provided with the Olympus Fluoview 500 laser scanning confocal microscope and was plotted (as relative fluorescence units) against distance above the saliva-coated coverglass (z axis depth).

FIG. 2.

Inactivation of lsrB and rbsB results in reduced biofilm growth. (A) Representative confocal images of 60-h biofilms formed by wild-type A. actinomycetemcomitans (JP2), the lsrB mutant, and the lsrB rbsB double mutant. Biofilm growth of the lsrB mutant was also complemented with a plasmid-borne copy of lsrB. The top panels represent images in the x/y plane, and the lower panels show the corresponding x/z reconstruction from a Z-stack from 0 to 100 μm in 1-μm increments above the saliva-coated coverglass. Scale bar, 100 μm.(B) The biomass of each biofilm was determined using COMSTAT image-processing software as described in Materials and Methods.

FIG. 3.

Ribose inhibits the development of A. actinomycetemcomitans biofilms. A. actinomycetemcomitans JP2 biofilms were grown in the presence of 50 mM or 100 mM ribose and were analyzed by laser scanning confocal microscopy. The top panels show representative images in the x/y plane; the lower panels show the corresponding x/z reconstruction from a Z-stack from 0 to 100 μm in 1-μm increments above the saliva-coated coverglass. Scale bar, 100 μm.