Inactivation of the Pta-AckA pathway causes cell death in Staphylococcus aureus

Abstract

During growth under conditions of glucose and oxygen excess, Staphylococcus aureus predominantly accumulates acetate in the culture medium, suggesting that the phosphotransacetylase-acetate kinase (Pta-AckA) pathway plays a crucial role in bacterial fitness. Previous studies demonstrated that these conditions also induce the S. aureus CidR regulon involved in the control of cell death. Interestingly, the CidR regulon is comprised of only two operons, both encoding pyruvate catabolic enzymes, suggesting an intimate relationship between pyruvate metabolism and cell death. To examine this relationship, we introduced ackA and pta mutations in S. aureus and tested their effects on bacterial growth, carbon and energy metabolism, cid expression, and cell death. Inactivation of the Pta-AckA pathway showed a drastic inhibitory effect on growth and caused accumulation of dead cells in both pta and ackA mutants. Surprisingly, inactivation of the Pta-AckA pathway did not lead to a decrease in the energy status of bacteria, as the intracellular concentrations of ATP, NAD(+), and NADH were higher in the mutants. However, inactivation of this pathway increased the rate of glucose consumption, led to a metabolic block at the pyruvate node, and enhanced carbon flux through both glycolysis and the tricarboxylic acid (TCA) cycle. Intriguingly, disruption of the Pta-AckA pathway also induced the CidR regulon, suggesting that activation of alternative pyruvate catabolic pathways could be an important survival strategy for the mutants. Collectively, the results of this study demonstrate the indispensable role of the Pta-AckA pathway in S. aureus for maintaining energy and metabolic homeostasis during overflow metabolism.

Fig 1

Inactivation of the Pta-AckA pathway has drastic effects on the growth characteristics of S. aureus. (A) Growth curves of the wild-type (wt) strain UAMS-1 and mutant strains UAMS-1-ackA and UAMS-1-pta grown aerobically in TSB containing 0.25% glucose. The OD₆₀₀ and the pH of the culture medium were determined at the indicated times. (B) Temporal accumulation and depletion of acetic acid in the culture media of strains UAMS-1, UAMS-1-ackA, and UAMS-1-pta. (C) Temporal depletion of glucose from the culture media of strains UAMS-1, UAMS-1-ackA, and UAMS-1-pta. (D) Concentrations of glucose in the culture media of UAMS-1, UAMS-1-ackA, and UAMS-1-pta plotted as a function of growth. The results are presented as the means ± standard errors of the mean for at least three independent experiments.

Fig 2

Inactivation of the Pta-AckA pathway causes cell death. (A) Number of viable cells per OD₆₀₀ unit determined for strains UAMS-1, UAMS-1-ackA, and UAMS-1-pta after 3 h of growth. (B) Quantitation of dead cells in the strains UAMS-1, UAMS-1-ackA, and UAMS-1-pta after 3 h of growth by flow cytometry using the LIVE/DEAD BacLight viability kit. The results are presented as the means plus standard errors of the mean of duplicate determinations for at least three independent experiments.

Fig 3

Impact of Pta-AckA pathway inactivation on the energy status of bacteria. (A) Intracellular ATP concentrations determined for strains UAMS-1, UAMS-1-ackA, and UAMS-1-pta after 3 h of growth. (B) Relative transcript levels of pfkA and citZ genes in strains UAMS-1, UAMS-1-ackA, and UAMS-1-pta determined by quantitative RT-PCR after 3 h of growth. (C) Intracellular NAD⁺ and NADH concentrations determined for strains UAMS-1, UAMS-1-ackA, and UAMS-1-pta after 3 h of growth. (D) Relative oxygen consumption rates in strains UAMS-1, UAMS-1-ackA, and UAMS-1-pta were determined using an oxygen-sensitive probe after 3 h of growth. The results are presented as the means plus standard errors of the mean of duplicate determinations for at least two independent experiments. Statistical significance between the wild-type strain and pta and ackA mutants was determined by Student's t test (*, P ≤ 0.001) and by ANOVA with heterogeneous variances (**, P < 0.005) (see Materials and Methods).

Fig 4

Pta-AckA pathway inactivation alters the metabolic status of bacteria. (A) Heat map generated from the normalized mean peak intensities for each metabolite identified from the triplicate set of 2D ¹H-¹³C HSQC NMR experiments. The normalized mean intensities are plotted on a color scale from −1 (red) to 1 (green). The red and blue asterisks denote statistical significance at the 90% confidence level (P < 0.10). (B) Metabolic pathway depicting the metabolites identified in the UAMS-1, UAMS-1-ackA, and UAMS-1-pta cellular metabolome by the 2D ¹H-¹³C HSQC NMR experiments. The up arrows correspond to a relative increase in the metabolite concentration, and the down arrows correspond to a relative decrease in the metabolite concentration. Statistical significance at the 90% confidence level (P < 0.10) is denoted by asterisks above the arrows. Acetyl-P, acetyl-phosphate; (1,3)BP-G, 1,3-bisphosphoglycerate; F6-P, fructose 6-phosphate; FB-P, fructose 1,6-bisphosphate; DHAP, dihydroxyacetone phosphate; G6-P, glucose 6-phosphate; G1-P, glucose 1-phosphate; GlcN-6-P, glucosamine-6-phosphate; GlcN-1-P, glucosamine-1-phosphate; GA3-P, glyceraldehyde 3-phosphate; GlcNAc-1-P, N-acetyl-glucosamine-1-phosphate; 3P-G, 3-phosphoglycerate; 2P-G, 2-phosphoglycerate; PEP, phosphoenolpyruvate; Ru5P, ribulose 5-phosphate; S7-P, sedoheptulose 7-phosphate; UDP-GlcNAc, UDP N-acetylglucosamine; X5-P, xylulose 5-phosphate.

Fig 5

Disruption of the Pta-AckA pathway alters carbon flow at the pyruvate node and activates the CidR regulon. (A) Intracellular Ac-CoA concentrations determined for strains UAMS-1, UAMS-1-ackA, and UAMS-1-pta after 3 h of growth. (B) Intracellular pyruvate concentrations determined for strains UAMS-1, UAMS-1-ackA, and UAMS-1-pta after 3 h of growth. (C) Concentrations of pyruvate in the culture medium determined for strains UAMS-1, UAMS-1-cidC, UAMS-1-pta, and UAMS-pta-cidC after 3 h of growth. (D) Relative transcript levels of the alsSD and cidABC operons in strains UAMS-1, UAMS-1-ackA, and UAMS-1-pta determined by quantitative RT-PCR after 3 h of growth. (E) Concentrations of acetoin in the culture medium determined for strains UAMS-1, UAMS-1-ackA, and UAMS-1-pta after 3 h of growth. (F) Concentrations of acetate in the culture medium determined for strains UAMS-1, UAMS-1-cidC, UAMS-1-pta, and UAMS-pta-cidC after 5 h of growth. All results are presented as the means plus standard errors of the mean of duplicate determinations for at least three independent experiments.