Frontiers in Microbiology | www.frontiersin.org 1 March 2020 | Volume 11 | Article 401 Ahn et al. Environmental Triggers of lrgAB Expression 600 ) was measured at 37 • C at 30 min-intervals using a Bioscreen C growth curve analysis system. At least three independent experiments, each in quadruplicate, wereFrontiers in Microbiology | www.frontiersin.org absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
Lack of LrgAB renders cariogenic Streptococcus mutans more sensitive to oxidative stress, as well as limits the capacity of this organism to re-uptake pyruvate upon starvation. This study was aimed at investigating the ecological and metabolic contribution of LrgAB to competitive fitness, using S. mutans strains, that either lack or overexpress lrgAB. These experiments revealed that impaired aerobic growth of the lrgAB mutant can be effectively restored by supplementation of pyruvate, and that perturbated expression of lrgAB significantly affects pyruvate flux and the conversion of pyruvate to acetyl-CoA by the Pdh pathway, verifying that LrgAB is closely linked to pyruvate catabolism. In vitro competition assays revealed that LrgAB plays an important role in S. mutans competition with H 2 O 2-producing S. gordonii, an interaction which can also be modulated by external pyruvate. However, no obvious competitive disadvantage was observed against S. gordonii by either the S. mutans lrgAB mutant or lrgAB overexpression strain in vivo using a mouse caries model. Organic acid analysis of mouse dental biofilms revealed that metabolites produced by the host and/or dental plaque microbiota could complement the deficiency of a lrgAB mutant, and favored S. mutans establishment compared to S. gordonii. Collectively, these results reinforce the importance of the oral microbiota and the metabolic environment in the oral cavity battleground, and highlight that pyruvate uptake through LrgAB may be crucial for interspecies competition that drives niche occupancy.
The ability of Streptococcus mutans to persist in a variety of adverse environments and to emerge as a numerically dominant member of stable oral biofilm communities are essential elements for its cariogenicity. The S. mutans Cid/Lrg system has been studied as a key player in the integration of complex environmental signals into regulatory networks that modulate virulence and cell homeostasis. Cid/Lrg has also been shown to be closely associated with metabolic pathways of this organism, due to distinct patterns of cid and lrg expression in response to growth phase and glucose/oxygen levels. In this study, a comparison of cid and lrg promoter regions with conserved CodY (a regulator which responds to starvation stress)‐binding motifs revealed the presence of a potential CodY‐binding site, which is arranged similarly in both cid and lrg promoters. Electrophoretic mobility shift assays (EMSAs) and promoter reporter assays demonstrated that expression of the cid and lrg operons is directly mediated by the global transcriptional regulator CodY. DNase I footprinting analyses confirmed the predicted binding sequences for CodY in both the cid and the lrg promoter regions. Overexpression of CodY had no obvious effect on lrgAB expression, but deficiency of CodY still affected lrgAB expression in a lytST ‐overexpressing strain, suggesting that CodY is required for the full regulation of lrgAB by LytST. We also demonstrated that both CodY and CcpA are involved in regulating pyruvate flux and utilization. Collectively, these data show that CodY directly regulates cid and lrg expression, and together with CcpA (previously shown to directly regulate cid and lrg promoters) contributes to coordinating pyruvate uptake and utilization in response to both the external environment and the cellular metabolic status.
Pyruvate forms the central node of carbon metabolism and promotes growth as an alternative carbon source during starvation. We recently revealed that LrgAB functions as a stationary phase pyruvate uptake system in Streptococcus mutans, the primary causative agent of human dental caries, but its underlying regulatory mechanisms are still not clearly understood. This study was aimed at further characterizing the regulation of LrgAB from a metabolomic perspective. We utilized a series of GFP quantification, growth kinetics, and biochemical assays. We disclosed that LrgAB is critical for pyruvate uptake especially during growth under low-glucose stress. Inactivation of the Pta-Ack pathway, responsible for the conversion of acetyl-CoA to acetate, completely inhibits stationary phase lrgAB induction and pyruvate uptake, and renders cells insensitive to external pyruvate as a signal. Inactivation of Pfl, responsible for the conversion of pyruvate to acetyl-CoA under anaerobic conditions, also affected stationary phase pyruvate uptake. This study explores the metabolic components of pyruvate uptake regulation through LrgAB, and highlights its potential as a metabolic stimulator, contributing to the resuscitation and survival of S. mutans cells during nutritional stress.
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