Comparison of the regulation, metabolic functions, and roles in virulence of the glyceraldehyde-3-phosphate dehydrogenase homologues gapA and gapB in Staphylococcus aureus

Abstract

The Gram-positive bacterium Staphylococcus aureus contains two glyceraldehyde-3-phosphate dehydrogenase (GAPDH) homologues known as GapA and GapB. GapA has been characterized as a functional GAPDH protein, but currently there is no biological evidence for the role of GapB in metabolism in S. aureus. In this study we show through a number of complementary methods that S. aureus GapA is essential for glycolysis while GapB is essential in gluconeogenesis. These proteins are reciprocally regulated in response to glucose concentrations, and both are influenced by the glycolysis regulator protein GapR, which is the first demonstration of the role of this regulator in S. aureus and the first indication that GapR homologues control genes other than those within the glycolytic operon. Furthermore, we show that both GapA and GapB are important in the pathogenesis of S. aureus in a Galleria mellonella model of infection, showing for the first time in any bacteria that both glycolysis and gluconeogenesis have important roles in virulence.

FIG. 1.

Enzyme activity of S. aureus GAPDH proteins in the presence of either NAD⁺ or NADP⁺ as a cofactor. (A) GAPDH activity of N-terminal His-tagged proteins His₆-GapA, His₆-GapB, and His₆-GapR. Whole-cell NAD⁺-dependent GAPDH activity (B) and whole-cell NADP⁺-dependent GAPDH activity (C) were determined for the 8325-4, 8325-4 ΔgapA, 8325-4 ΔgapB, and 8325-4 ΔgapA ΔgapB strains. Cells were grown overnight in TSB medium and equalized for growth. The data show the average result of three repeats, and the error bars indicate the standard error. Significant differences relative to the wild-type (WT) strain are indicated as follows: *, P < 0.01; **, P < 0.001.

FIG. 2.

Growth of the 8325-4 (black line; •), 8325-4 ΔgapA (gray line; ▪), 8325-4 ΔgapB (dashed line; ▴), and 8325-4 ΔgapA ΔgapB (dotted line; X) strains in TSB without the addition of any additional carbon source (A) or with the addition of 0.056% pyruvate (B) or 0.75% glucose (C), added at time zero. Growth of the same strains, indicated as above, was measured in TM medium without the addition of an additional carbon source (D) and with the addition of 0.056% pyruvate (E) or 1% glucose (F), added at time zero. Cultures were grown at 37°C with aeration, and growth was measured as the optical density of the culture at 600 nm. Experiments were repeated a minimum of three times on different days, and the data presented are the average of three repeats, with error bars indicating the standard deviations.

FIG. 3.

(A) Flow chart indicating where the various carbon sources can enter glycolysis and gluconeogenesis during carbon metabolism. (B) Graph showing the level of growth of the 8325-4, 8325-4 ΔgapA, 8325-4 ΔgapB, and 8325-4 ΔgapA ΔgapB strains after a 5-h incubation in TM medium supplemented with an additional carbon source. Cultures were grown at 37°C with aeration, and growth was measured as the optical density of the culture at 600 nm. Each experiment was repeated three times on different days, and the data presented are averages, with error bars indicating the standard deviations. CoA, coenzyme A.

FIG. 4.

(A) Schematic representation of the glycolytic operon with a ΔgapR mutation, indicating the position of the gapR and gapA probes used for Northern blot analysis. Genes are putative operon regulator (gapR), glyceraldehyde-3-phosphate dehydrogenase (gapA), phosphoglycerate kinase (pgk), triphosphate isomerase (tpi), phosphoglycerate mutase (pgm), and enolase (eno). Northern blot analysis is shown of gapA transcript (B) and gapB transcript (C) expression in response to glucose induction in strain 8325-4 and gapR transcript (D) and gapB transcript (E) expression in response to glucose induction in both wild-type (WT) 8325-4 and 8325-4 ΔgapR strains. Total RNA was extracted from cells grown for 5 h in TM broth with 1% glucose added at time zero (G), 1% glucose added 1 h before cells were harvested (GP), and without glucose (−). Gels presented are representative of experiments that were repeated two times using RNA extracts from cultures grown on different days, with similar results observed each time. Blots were stripped and rehybridized with a control probe (16S) to ensure equal loading of RNA in each case.

FIG. 5.

(A) Growth of 8325-4 and isogenic ΔgapA, ΔgapB, and ΔgapA ΔgapB mutants after 5 h of incubation in TM medium with varied concentrations of glucose. Cultures were grown at 37°C with aeration, and growth was measured as the optical density of the culture at 600 nm. Each experiment was repeated three times on different days, and results are presented as averages with error bars indicating the standard deviations. (B) Effect of the GAPDH mutations on the pathogenesis of S. aureus infection in G. mellonella. The graph indicates the percent viability of infected G. mellonella larvae at 24, 48, and 72 h postinfection with S. aureus 8325-4 wild-type strain and the isogenic ΔgapA, ΔgapB, and ΔgapA ΔgapB mutants. With the PBS negative control, larvae showed 100% survival at all time points. The results from three independent experiments were combined and used to produce survival curves using the Kaplan-Meier method.