Characterization of LrgAB as a stationary phase-specific pyruvate uptake system in Streptococcus mutans

Abstract

Background Our recent ‘-omics’ comparisons of Streptococcus mutans wild-type and lrgAB-mutant revealed that this organism undergoes dynamic cellular changes in the face of multiple exogenous stresses, consequently affecting its comprehensive virulence traits. In this current study, we further demonstrate that LrgAB functions as a S. mutans pyruvate uptake system. Results S. mutans excretes pyruvate during growth as an overflow metabolite, and appears to uptake this excreted pyruvate via LrgAB once the primary carbon source is exhausted. This utilization of excreted pyruvate was tightly regulated by glucose levels and stationary growth phase lrgAB induction. The degree of lrgAB induction was reduced by high extracellular levels of pyruvate, suggesting that lrgAB induction is subject to negative feedback regulation, likely through the LytST TCS, which is required for expression of lrgAB. Stationary phase lrgAB induction was efficiently inhibited by low concentrations of 3FP, a toxic pyruvate analogue, without affecting cell growth, suggesting that accumulated pyruvate is sensed either directly or indirectly by LytS, subsequently triggering lrgAB expression. S. mutans growth was inhibited by high concentrations of 3FP, implying that pyruvate uptake is necessary for S. mutans exponential phase growth and occurs in a Lrg-independent manner. Finally, we found that stationary phase lrgAB induction is modulated by hydrogen peroxide (H2O2) and by co-cultivation with H2O2-producing S. gordonii. Conclusions Pyruvate may provide S. mutans with an alternative carbon source under limited growth conditions, as well as serving as a buffer against exogenous oxidative stress. Given the hypothesized role of LrgAB in cell death and lysis, these data also provide an important basis for how these processes are functionally and mechanically connected to key metabolic pathways such as pyruvate metabolism.

Acetate and Potassium Modulate the Stationary-Phase Activation of lrgAB in Streptococcus mutans

“…When bacterial cells experience nutrient limitation (during the transition to stationary phase), they rapidly initiate re-uptake of previously excreted pyruvate through LrgAB (Paczia et al, 2012;Ahn et al, 2019). Consistent with the observation of stationary phase uptake of pyruvate, we recently found that although supplemented pyruvate had no impact on the growth rate of S. mutans cells, it did prolong the exponential phase of growth (Ahn et al, 2019), presumably enabling cells to take up pyruvate according to their needs and ensuring longterm survival. Along with other α-keto acids (i.e., α-ketoglutarate, oxaloacetate), pyruvate is also known to effectively scavenge ROS (Reactive oxygen species), including hydrogen peroxide (H 2 O 2 ), through a non-enzymatic oxidative decarboxylation mechanism (Constantopoulos and Barranger, 1984;O'Donnell-Tormey et al, 1987;Desagher et al, 1997;Mizunoe et al, 1999).…”

“…LrgAB homologs were recently reported to function as a pyruvate transporter in Bacillus subtilis (Charbonnier et al, 2017;van den Esker et al, 2017), and most recently in Streptococcus mutans (Ahn et al, 2019), a primary causative agent of human dental caries. Although pyruvate has a large potential to modulate various virulence traits via cell homeostasis for improved survival and persistence of various bacteria (Busuioc et al, 2010;Chaudhari et al, 2016;Hawver et al, 2016;Vilhena et al, 2018Vilhena et al, , 2019, to date little is known about the role and regulation of pyruvate in these organisms.…”

“…Pyruvate is excreted as an overflow metabolite (Paczia et al, 2012), thus a common nutrient in microbiome environments such as the oral cavity (Takahashi et al, 2010;Gawron et al, 2019). When bacterial cells experience nutrient limitation (during the transition to stationary phase), they rapidly initiate re-uptake of previously excreted pyruvate through LrgAB (Paczia et al, 2012;Ahn et al, 2019). Consistent with the observation of stationary phase uptake of pyruvate, we recently found that although supplemented pyruvate had no impact on the growth rate of S. mutans cells, it did prolong the exponential phase of growth (Ahn et al, 2019), presumably enabling cells to take up pyruvate according to their needs and ensuring longterm survival.…”

“…Along with other α-keto acids (i.e., α-ketoglutarate, oxaloacetate), pyruvate is also known to effectively scavenge ROS (Reactive oxygen species), including hydrogen peroxide (H 2 O 2 ), through a non-enzymatic oxidative decarboxylation mechanism (Constantopoulos and Barranger, 1984;O'Donnell-Tormey et al, 1987;Desagher et al, 1997;Mizunoe et al, 1999). We recently demonstrated that stationary phase lrgAB induction is modulated by H 2 O 2 and by co-cultivation with the H 2 O 2 -producing oral commensal, Streptococcus gordonii (Ahn et al, 2019), buffering external sources of oxidative stress. Also, of note, the reaction of pyruvate with H 2 O 2 produces water, CO 2 , and acetate (CH 3 -CO-COOH + H 2 O 2 → CH 3 -COOH + H 2 O + CO 2) (Giandomenico et al, 1997), and acetate has also reported to be taken up into bacterial cells in parallel with pyruvate under nutrient limited growth conditions (Jolkver et al, 2009;Paczia et al, 2012), as well as to cause cell death in S. aureus (Sadykov et al, 2013;Thomas et al, 2014;Chaudhari et al, 2016).…”

“…Due to its central role in metabolism, Pdh is known to be regulated at both the biochemical and genetic levels in Escherichia coli (Hansen and Henning, 1966;Schwartz and Reed, 1970;Shen and Atkinson, 1970;Datta, 1991). In S. mutans, the genes encoding the Pdh complex are also dramatically upregulated in response to carbohydrate depletion, an expression pattern that is shared with lrgAB, encoding a pyruvate uptake system (Busuioc et al, 2010;Ahn et al, 2019).…”