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. 2007 Aug;189(15):5559-65.
doi: 10.1128/JB.00387-07. Epub 2007 May 25.

Differential interaction of Aggregatibacter (Actinobacillus) actinomycetemcomitans LsrB and RbsB proteins with autoinducer 2

Hanjuan Shao  1 , Deanna James, Richard J Lamont, Donald R Demuth
Affiliations

Differential interaction of Aggregatibacter (Actinobacillus) actinomycetemcomitans LsrB and RbsB proteins with autoinducer 2

Hanjuan Shao et al. J Bacteriol. 2007 Aug.

Abstract

Our previous studies showed that the Aggregatibacter actinomycetemcomitans RbsB protein interacts with cognate and heterologous autoinducer 2 (AI-2) signals and suggested that the rbsDABCK operon encodes a transporter that may internalize AI-2 (D. James et al. , Infect. Immun. 74:4021-4029, 2006. ). However, A. actinomycetemcomitans also possesses genes related to the lsr operon of Salmonella enterica serovar Typhimurium which function to import AI-2. Here, we show that A. actinomycetemcomitans LsrB protein competitively inhibits the interaction of the Vibrio harveyi AI-2 receptor (LuxP) with AI-2 from either A. actinomycetemcomitans or V. harveyi. Interestingly, LsrB was a more potent inhibitor of LuxP interaction with AI-2 from V. harveyi whereas RbsB competed more effectively with LuxP for A. actinomycetemcomitans AI-2. Inactivation of lsrB in wild-type A. actinomycetemcomitans or in an isogenic RbsB-deficient strain reduced the rate by which intact bacteria depleted A. actinomycetemcomitans AI-2 from solution. Consistent with the results from the LuxP competition experiments, the LsrB-deficient strain depleted AI-2 to a lesser extent than the RbsB-deficient organism VSports手机版. Inactivation of both lsrB and rbsB virtually eliminated the ability of the organism to remove AI-2 from the extracellular environment. These results suggest that A. actinomycetemcomitans possesses two proteins that differentially interact with AI-2 and may function to inactivate or facilitate internalization of AI-2. .

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Figures

FIG. 1.
FIG. 1.
The lsrACDBFG and lsrRK operons of A. actinomycetemcomitans. (A) Comparison of the lsr operons of S. enterica serovar Typhimurium (top) and A. actinomycetemcomitans (bottom). The numbers represent percent amino acid sequence identity between the corresponding S. enterica and A. actinomycetemcomitans genes. The A. actinomycetemcomitans lsr operon lacks the lsrE gene present in S. enterica. (B) Expanded view of the A. actinomycetemcomitans lsrDB region showing the locations of oligonucleotide primers (see Materials and Methods) used to clone lsrB gene fragments for expression in E. coli and gene inactivation and verification in A. actinomycetemcomitans.
FIG. 2.
FIG. 2.
A. actinomycetemcomitans LsrB competes with V. harveyi LuxP for AI-2 derived from A. actinomycetemcomitans JP2 (A and B) or from V. harveyi BB170 (C). In panels A and C, LsrB (0 to 200 ng) was incubated for 30 min at 30°C with AI-2 that was partially purified from a mid-exponential culture of A. actinomycetemcomitans JP2. Purification of A. actinomycetemcomitans AI-2 was carried out as described by James et al. (13). Samples were then added to V. harveyi BB170 cells that were diluted 1:25,000 from an overnight culture in a microtiter plate and incubated at 30°C for up to 7 h. Bioluminescence was determined at hourly intervals by luminometry using a Perkins-Elmer Wallac Victor multilabel counter. The data shown represent the 6-h time point and are expressed as a percentage of the positive control. The positive control reaction was comprised of V. harveyi BB170 cells that were stimulated with A. actinomycetemcomitans AI-2 in the absence of LsrB. Light production in the positive control reaction at the time point shown was >400-fold higher than that measured from V. harveyi BB170 cells in the absence of the A. actinomycetemcomitans AI-2. Panel B shows a direct competition of LsrB and LuxP for A. actinomycetemcomitans AI-2. V. harveyi BB170 cells were stimulated with A. actinomycetemcomitans AI-2 and incubated for 3 h at 30°C; then 1 μg (▾), 2 μg (▪), or 5 μg (⧫) of LsrB was added to the reaction mixtures, and incubation was continued for an additional 4 h. Samples were assayed for induction of bioluminescence at hourly intervals as described in Materials and Methods. The positive control reaction that did not receive LsrB protein (•) exhibited a 420-fold stimulation of bioluminescence relative to V. harveyi BB170 cells that were incubated in the absence of AI-2.
FIG. 3.
FIG. 3.
Comparison of LsrB- (•) and RbsB-mediated (▾) inhibition of V. harveyi BB170 bioluminescence stimulated by A. actinomycetemcomitans AI-2 (A) and V. harveyi AI-2 (B). V. harveyi bioluminescence was determined as described in Materials and Methods. Percent inhibition was calculated by relating the bioluminescence of experimental samples with the bioluminescence of the positive control, which did not receive inhibitor, using the following formula: [1 − (experimental bioluminescence/control bioluminescence)] × 100. Bioluminescence of the positive control was >400-fold higher than that observed in the absence of AI-2.
FIG. 4.
FIG. 4.
LsrB- and RbsB-mediated depletion of A. actinomycetemcomitans AI-2. Intact cells of A. actinomycetemcomitans JP2 (•), isogenic LsrB-deficient (⧫), RbsB-deficient (▪), or LsrB- and RbsB-deficient strains (▴) and the lsrB mutant complemented with a plasmid borne copy of lsrB (○) were incubated with partially purified AI-2 from a mid-exponential culture of A. actinomycetemcomitans for 0 to 15 min. Bacterial cells were then removed by centrifugation, and the supernatant was filtered through a 0.22-μm-pore-size filter. The AI-2 bioactivity at each time point was determined by incubating an aliquot of each filtered sample with V. harveyi BB170 for 6 h as described in Materials and Methods and measuring total bioluminescence. Data are expressed as a percentage of the light production induced by AI-2 in the positive control, in which V. harveyi BB170 was stimulated with A. actinomycetemcomitans AI-2 that was not preincubated with A. actinomycetemcomitans cells. In these reactions, the total bioluminescence in the positive control reactions was approximately 400-fold greater than background levels observed with V. harveyi BB170 in the absence of AI-2. All reactions were carried out in triplicate.
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References

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