A novel exclusion mechanism for carbon resource partitioning in Aggregatibacter actinomycetemcomitans

Abstract

The bacterium Aggregatibacter actinomycetemcomitans is a common commensal of the human oral cavity and the putative causative agent of the disease localized aggressive periodontitis. A. actinomycetemcomitans is a slow-growing bacterium that possesses limited metabolic machinery for carbon utilization. This likely impacts its ability to colonize the oral cavity, where growth and community composition is mediated by carbon availability. We present evidence that in the presence of the in vivo relevant carbon substrates glucose, fructose, and lactate A. actinomycetemcomitans preferentially metabolizes lactate. This preference for lactate exists despite the fact that A. actinomycetemcomitans grows faster and obtains higher cell yields during growth with carbohydrates. The preference for lactate is mediated by a novel exclusion mechanism in which metabolism of lactate inhibits carbohydrate uptake. Coculture studies reveal that A. actinomycetemcomitans utilizes lactate produced by the oral bacterium Streptococcus gordonii, suggesting the potential for cross-feeding in the oral cavity.

FIG. 1.

Growth of A. actinomycetemcomitans in CDM containing glucose, fructose, l-lactate, and no energy source (no addition). Bacteria were grown shaking (165 rpm) at 37°C with a 10% CO₂ atmosphere with 20 mM carbon source. Growth curves were determined at least three times, and representative data are provided.

FIG. 2.

Glucose, fructose, and l-lactate consumption by A. actinomycetemcomitans. A. actinomycetemcomitans was grown in the presence of equimolar (2 mM) glucose, fructose, and l-lactate, and the disappearance of these carbon substrates was evaluated over time. Experiments were performed in duplicate. Error bars represent the standard deviation and in some cases are too small to be seen.

FIG. 3.

Addition of lactate inhibits glucose and fructose consumption by A. actinomycetemcomitans. A. actinomycetemcomitans was inoculated into CDM containing glucose (A) or fructose (B) at time zero. After growth for 1 h, l-lactate was added (where designated by the arrow), and the consumption of each carbon source was monitored for 3 h. Experiments were performed in duplicate. Error bars represent standard deviation and in some cases are too small to be seen.

FIG. 4.

Lactate inhibits uptake of glucose in A. actinomycetemcomitans strains VT1169 (A) and Y4 (B). A. actinomycetemcomitans was suspended in CDM containing fructose, l-lactate, or no energy source (control). Radiolabeled glucose ([U-¹⁴C]glucose) was then added, and uptake was examined as outlined in Materials and Methods. Uptake of radiolabeled glucose is expressed as cpm/μg of A. actinomycetemcomitans protein. Experiments were performed in triplicate. Error bars represent the standard deviation and in some cases are too small to be seen. The background radioactivity using heat-killed cells was 187 ± 27 cpm/μg of protein.

FIG. 5.

Pyruvate inhibits uptake of glucose in A. actinomycetemcomitans. A. actinomycetemcomitans was suspended in CDM and incubated with no energy source (control) or pyruvate (A) or with no energy source (control), acetate, or propionate (B). Radiolabeled glucose ([U-¹⁴C]glucose) was then added, and uptake was examined as outlined in Materials and Methods. The uptake of radiolabeled glucose is expressed as cpm/μg of A. actinomycetemcomitans protein. Formate and succinate also had no effect on glucose transport (data not shown). Experiments were performed in duplicate. Error bars represent the standard deviation and in some cases are too small to be seen.

FIG. 6.

Lactate dehydrogenase is required for the inhibition of glucose uptake. The A. actinomycetemcomitans l-lactate dehydrogenase (ldhA) mutant was suspended in CDM and incubated with no energy source (control) or l-lactate. Radiolabeled glucose was then added, and uptake was examined as outlined in Materials and Methods. Uptake of radiolabeled glucose is expressed as cpm/μg of A. actinomycetemcomitans protein. Experiments were performed in duplicate. Error bars represent the standard deviation and in some cases are too small to be seen.

FIG. 7.

Lactate-grown A. actinomycetemcomitans contain higher levels of pyruvate. Intracellular levels of pyruvate were measured for logarithmic glucose-, fructose-, and l-lactate-grown A. actinomycetemcomitans as previously described (1) and are presented as fold increases during growth with l-lactate. Error bars represent the standard deviation, and experiments were performed in triplicate. Intracellular volume was calculated based on normal size measurements of A. actinomycetemcomitans (1.6 μm by 0.5 μm by 0.5 μm).

FIG. 8.

A. actinomycetemcomitans utilizes lactate produced by S. gordonii. S. gordonii was grown in CDM containing sucrose, and sucrose and/or lactate levels were measured throughout the growth period. After 8 h, A. actinomycetemcomitans was added (as designated by the arrow), and lactate levels were measured. Experiments were performed in duplicate. Standard deviations are <5% of the mean for sucrose and <9% of the mean for lactate and were not included for the sake of clarity.

FIG. 9.

Model for lactate-mediated inhibition of glucose transport in A. actinomycetemcomitans during growth with streptococci. Lactate produced by Streptococcus spp. is transported into A. actinomycetemcomitans by lactate permease (LctP). Once inside the cell, intracellular pyruvate levels increase due to the conversion of lactate to pyruvate by lactate dehydrogenase (LdhA). Protein E1 normally autophosphorylates using PEP as the phospho-donor; however, elevated intracellular pyruvate levels inhibit this autophosphorylation. Since E1 is the first step in transport of PTS sugars, inhibition of E1 autophosphorylation inhibits the uptake of all PTS carbohydrates.