The Actinobacillus actinomycetemcomitans Ribose Binding Protein RbsB Interacts with Cognate and Heterologous Autoinducer 2 Signals

Abstract

Autoinducer 2 (AI-2) produced by the oral pathogen Actinobacillus actinomycetemcomitans influences growth of the organism under iron limitation and regulates the expression of iron uptake genes. However, the cellular components that mediate the response of A. actinomycetemcomitans to AI-2 have not been fully characterized. Analysis of the complete genome sequence of A. actinomycetemcomitans (www.oralgen.lanl.gov) indicated that the RbsB protein was related to LuxP, the AI-2 receptor of Vibrio harveyi. To determine if RbsB interacts with AI-2, the bioluminescence of the reporter strain V. harveyi BB170 (sensor 1؊, sensor 2؉) was determined after stimulation with partially purified AI-2 from A. actinomycetemcomitans or conditioned medium from V. harveyi cultures in the presence and absence of purified six-His-tagged RbsB. RbsB efficiently inhibited V. harveyi bioluminescence induced by both A. actinomycetemcomitans AI-2 and V. harveyi AI-2 in a dose-dependent manner, suggesting that RbsB competes with LuxP for AI-2. Fifty percent inhibition occurred with approximately 0.3 nM RbsB for A. actinomycetemcomitans AI-2 and 15 nM RbsB for V. harveyi AI-2. RbsB-mediated inhibition of V. harveyi bioluminescence was reversed by the addition of 50 mM ribose, suggesting that A. actinomycetemcomitans AI-2 and ribose bind at the same site of RbsB. The RbsB/AI-2 complex was thermostable since A. actinomycetemcomitans AI-2 could not be recovered by heating. This was not due to heat inactivation of A. actinomycetemcomitans AI-2 since signal activity was unaffected by heating in the absence of RbsB. Furthermore, an isogenic A. actinomycetemcomitans mutant that was unable to express rbsB was deficient in depleting A. actinomycetemcomitans AI-2 from solution relative to the wild-type organism. Inactivation of rbsB also influenced the ability of the organism to grow under iron-limiting conditions. The mutant strain attained a cell density of approximately 30% that of the wild-type organism under iron limitation. In addition, real-time PCR showed that the expression of afuABC, encoding a major ferric ion transporter, was reduced by approximately eightfold in the rbsB mutant. This phenotype was similar to that of a LuxS-deficient mutant of A. actinomycetemcomitans that is unable to produce AI-2. Together, our results suggest that RbsB may play a role in the response of A. actinomycetemcomitans to AI-2.

Autoinducer 2 Is Required for Biofilm Growth ofAggregatibacter(Actinobacillus)actinomycetemcomitans

“…Introduction of S-adenosylhomocysteine hydrolase to A. actinomycetemcomitans, which restores the metabolism of SAM in the absence of LuxS, did not complement the luxS mutation unless AI-2 was added in trans. The biofilm growth defect was also observed in RbsB-and LsrB-deficient backgrounds, as well as with wild-type A. actinomycetemcomitans in the presence of ribose, which competes with AI-2 for binding to RbsB (21). Together, these results suggest that AI-2 itself is required for A. actinomycetemcomitans biofilm growth and that RbsB and LsrB function as AI-2 receptors in A. actinomycetemcomitans.…”

“…2B) and also significantly reduced the rate of AI-2 depletion from solution by intact cells (40). We also previously showed that ribose and AI-2 compete for the same binding site of RbsB (21), suggesting that ribose may block AI-2 interaction with RbsB and LsrB and interfere with AI-2-dependent phenotypes. To determine if ribose inhibits A. actinomycetemcomitans biofilm growth, biofilms of strain JP2 were generated by feeding adherent cells with BHI containing 50 mM or 100 mM ribose.…”

Autoinducer-2 influences interactions amongst pioneer colonizing streptococci in oral biofilms

“…We hypothesize that the reduction in AI-2 activity is due to an active uptake system such as that which exists in Salmonella typhimurium and Aggregatibacter actinomycetemcomitans. These species can remove AI-2 from solution through the expression of ABC-type transporters (James et al, 2006;Shao et al, 2007;Taga et al, 2001Taga et al, , 2003. Assuming that the reduction of AI-2 activity by S. gordonii DL1 and S. oralis 34 is also due to the removal by transporters, the difference in rates of removal along with different rates of production of AI-2 will contribute to the amount of AI-2 that is bioavailable for cell-cell communication.…”

Quorum Sensing of Periodontal Pathogens

“…Spojevi iz češnjaka, ekstrakta vanilije, papra i halogenirani furanoni koje proizvodi morska alga Delisea pulchra pokazali su quorum sensing inhibitorna svojstva (37 -44). Istraživanja su također pokazala da dodatak spojeva sličnih ribozi u kulturu Aggregatibacter actinomycetemcomitans negativno utječu na formaciju biofilma, s obzirom na to da receptori autoinduktor-2 iskazuju određenu homolognost receptorima koji vežu ribozu, zbog čega riboza djeluje kao receptorni antagonist (45). Dokazano je i da halogenirani furanoni, konkretno furanoni s bromom, negativno djeluju na formaciju biofilma Porphyromonas gingivalis bez utjecaja na rast bakterijske populacije (46).…”