AI-2 of Aggregatibacter actinomycetemcomitans inhibits Candida albicans biofilm formation

Abstract

Aggregatibacter actinomycetemcomitans, a Gram-negative bacterium, and Candida albicans, a polymorphic fungus, are both commensals of the oral cavity but both are opportunistic pathogens that can cause oral diseases. A. actinomycetemcomitans produces a quorum-sensing molecule called autoinducer-2 (AI-2), synthesized by LuxS, that plays an important role in expression of virulence factors, in intra- but also in interspecies communication. The aim of this study was to investigate the role of AI-2 based signaling in the interactions between C. albicans and A. actinomycetemcomitans. A. actinomycetemcomitans adhered to C. albicans and inhibited biofilm formation by means of a molecule that was secreted during growth. C. albicans biofilm formation increased significantly when co-cultured with A. actinomycetemcomitans luxS, lacking AI-2 production. Addition of wild-type-derived spent medium or synthetic AI-2 to spent medium of the luxS strain, restored inhibition of C. albicans biofilm formation to wild-type levels. Addition of synthetic AI-2 significantly inhibited hypha formation of C. albicans possibly explaining the inhibition of biofilm formation. AI-2 of A. actinomycetemcomitans is synthesized by LuxS, accumulates during growth and inhibits C. albicans hypha- and biofilm formation. Identifying the molecular mechanisms underlying the interaction between bacteria and fungi may provide important insight into the balance within complex oral microbial communities.

Keywords: interspecies interaction; oral microbiology; quorum sensing.

Figure 1

Real-time microscopic analysis of adhesion of A. actinomycetemcomitans to C. albicans SC5314. Hyphae of C. albicans SC5314 were allowed to form on the bottom of a microfluidics plate. Bacteria were stained with Syto9 and PI and allowed to adhere to C. albicans SC5314 while flowing at 0.5 dyne cm⁻². Images were captured every 30 s for a total of 10 min (montage shows images every 210 s). Images were edited for brightness and contrast.

Figure 2

AI-2 plays a role in biofilm formation of A. actinomyc-etemcomitans. Biofilm formation of A. actinomycetemcomitans luxS is restored to near wild-type levels by the addition of sterile spent medium (left panel) as well as by the addition of 100 nM synthetic DPD (right panel). ^*Indicates significantly different from control.

Figure 3

The effect of co-culture with A. actinomycetemcomitans on C. albicans ATCC 10231 biofilm formation. (A) C. albicans biofilms were grown without any bacteria or with A. actinomycetemcomitans wild-type or luxS mutant. Biofilm formation, quantified using the MTT assay, was measured after 24 h of growth. Data represent the mean and standard deviations of six biofilms grown on two separate occasions. (B) The effect of spent medium on co-cultures between C. albicans and A. actinomycetemcomitans luxS. Spent medium of A. actinomycetemcomitans wild-type cultures grown for different times was added to the mixed species culture of C. albicans and A. actinomycetemcomitans luxS. Biofilm formation was quantified after 24 h of growth using the MTT assay. The relative biofilm formation compared to C. albicans + A. actinomycetemcomitans luxS without any spent medium was calculated. Data represent the mean and standard deviations of six biofilms grown on two separate occasions. ^*Indicates significantly different from control.

Figure 4

Effect of synthetic DPD on C. albicans SC5134. Hypha-formation was induced by switching fresh cultures to 37°C for 3–4 h. Representative microscopic images show a decreased hypha formation (A = control, B = 0.1 μM DPD, C = 1.0 μM DPD; bar represent 40 μm for all images). Hyphae and yeast morphologies were counted and plotted as % of all cells (D). The results represent the mean of two independent experiments, each consisting of at least 100 cells per sample. Synthetic DPD added to spent medium of A. actinomycetemcomitans luxS inhibits C. albicans SC5314 biofilm formation (E). ^*Indicates significantly different from control.

Figure 5

Effect of spent medium of A. actinomycetemcomitans wt and luxS on preformed C. albicans ATCC 10231 biofilms. Biofilms of C. albicans were grown for 24 h after which they were exposed to spent medium of A. actinomycetemcomitans cultures of increasing age for an additional 24 h. Total amount of biofilm was determined using the MTT assay and results depict the averages of a total of 4 wells in 2 independent experiments. ^*Indicates significantly different from control as determined using a One-Way ANOVA followed by a TUKEY HSD test.