Metabolic sensor governing bacterial virulence in Staphylococcus aureus

Abstract

An effective metabolism is essential to all living organisms, including the important human pathogen Staphylococcus aureus. To establish successful infection, S. aureus must scavenge nutrients and coordinate its metabolism for proliferation. Meanwhile, it also must produce an array of virulence factors to interfere with host defenses. However, the ways in which S. aureus ties its metabolic state to its virulence regulation remain largely unknown. Here we show that citrate, the first intermediate of the tricarboxylic acid (TCA) cycle, binds to and activates the catabolite control protein E (CcpE) of S. aureus. Using structural and site-directed mutagenesis studies, we demonstrate that two arginine residues (Arg145 and Arg256) within the putative inducer-binding cavity of CcpE are important for its allosteric activation by citrate. Microarray analysis reveals that CcpE tunes the expression of 126 genes that comprise about 4.7% of the S. aureus genome. Intriguingly, although CcpE is a major positive regulator of the TCA-cycle activity, its regulon consists predominantly of genes involved in the pathogenesis of S. aureus. Moreover, inactivation of CcpE results in increased staphyloxanthin production, improved ability to acquire iron, increased resistance to whole-blood-mediated killing, and enhanced bacterial virulence in a mouse model of systemic infection. This study reveals CcpE as an important metabolic sensor that allows S. aureus to sense and adjust its metabolic state and subsequently to coordinate the expression of virulence factors and bacterial virulence.

Keywords: Staphylococcus aureus; bacterial virulence; iron acquisition; metabolism; virulence gene expression.

Fig. 1.

CcpE negatively modulates pigment production of S. aureus by activating the expression of citB. (A) Pigmentation of S. aureus Newman strain and its derivatives grown on TSA without glucose supplement, at 37 °C for 24 h. The wild-type Newman, ΔccpE, and ΔcitB strains harbor plasmid pYJ335. (B) Electropherograms show the protection pattern of the citB promoter after digestion with DNaseI following incubation in the absence and the presence of 2 µM 6His-CcpE. There are two CcpE-protected regions (I and II) in the promoter of the citB. CcpE-protected region I harbors a box I-like sequence (shown in bold letters) [ATAA-N₇-TTAT, where N is any nucleotide; the potential LysR-type transcriptional regulator (LTTR) box is underlined]. Protected region II contains a box II-like sequence (AATA, shown in bold letters), which can be found in the CcpC-binding sites of citB in B. subtilis. (C) Assessment of mutations or deletion of the protected region I on CcpE binding. EMSA assays were performed using a wild-type fragment of the citB promoter DNA (citB-p, from nucleotides −151 to +88 of the start codon of citB) or the same fragment with ATAAGTTTTGCTTAT mutated to CGCCACTTTGCTTAT (citB-M-p). Test DNA was citB-p or citB-M-p, as indicted. citB-U, a DNA fragment of the citB promoter DNA (from nucleotides −128 to +88 of the start codon of citB) containing no CcpE-protected region. (D) Effects of mutations to the protected region I on the promoter activity of citB. ATAAGTTTTGCTTAT of the citB promoter (nucleotides −767 to +78 of the start codon of citB) was mutated to CGCCACTTTGCTTAT. Bacteria were grown in TSB at 37 °C with shaking, 250 rpm of aeration, and sampled at 6 h. Values are relative to the wild-type Newman strain (set to 1). The expression level of transcriptional fusion citB-lacZ is high enough that the introduction of ccpE-null could have a detectable reducing effect. Results represent means ± SD, and data are representative of three independent experiments.

Fig. 2.

CcpE is a citrate-sensing regulator. (A) EMSA showing that sodium citrate increases the DNA-binding ability of 6His-CcpE (0.2 µM) in a dose-dependent manner. citB-L-p, a DNA fragment of the citB promoter DNA (from nucleotides −194 to +88 of the start codon of citB) containing CcpE-protected region I. citB-U, a DNA fragment of citB promoter DNA (from nucleotides −128 to +88 of the start codon of citB) containing no CcpE-protected region. (B) Gel filtration analysis of 6His-CcpE oligomerization in the absence (continuous line) and presence (dashed line) of 10 mM sodium citrate as described in SI Appendix, Experimental Procedures. HMW, high molecular weight. (C) Thermal denaturation curves for 6His-CcpE alone or in the presence of either sodium citrate or sodium isocitrate, as indicated. (D) Intracellular citrate concentrations of the S. aureus wild-type Newman, ΔcitB, and ΔccpEΔcitB strains. The intracellular citrate concentration was estimated according to the assumptions that S. aureus cell volume is 5 × 10⁻¹³ mL (57) and that 1 A₆₀₀ corresponds to 2 × 10⁸ cells/mL. (E) The expression of citB-lacZ in S. aureus wild-type Newman, ΔcitB, and ΔccpEΔcitB strains. In D and E, bacteria were grown in TSB without glucose at 37 °C with shaking, 250 rpm of aeration, and were sampled at 6 h. In E, values are relative to the wild-type Newman strain (set to 1). Results represent means ± SEM, and data are representative of three independent experiments. ***P < 0.001. The statistical difference was determined by unpaired two-tailed Student t test.

Fig. 3.

Arg145 and Arg256, located within the putative inducer-binding cavity of CcpE, are critical for the activation of CcpE by citrate. (A, Left) Overall protein folding of the inducer-binding fragment of CcpE is presented in a cartoon. One monomer is colored in magenta and the other in cyan. Each monomer possesses two domains separated by a hinge formed from the central regions of β4 and β9. (Right) A local view of the putative inducer-binding cavity of CcpE. Protein is shown in gray, amino acids in magenta sticks, water as red spheres, chloride ions as green spheres, and hydrogen bonds as black dashed lines. (B and C) Gel filtration analysis of 6His-CcpE_R145A (B) and 6His-CcpE_R256A (C) oligomerization in the absence (continuous line) and presence (dashed line) of 10 mM sodium citrate. (D and E) Thermal denaturation curves for 6His-CcpE_R145A (D) and 6His-CcpE_R256A (E) in the absence and presence of 20 mM sodium citrate. (F) Effect of the amino acid substitutions in CcpE on its ability to promote the expression of citB-lacZ in the ΔccpE mutant. ccpE_R145A, arginine 145 of ccpE mutated to alanine; ccpE_R256A, arginine 256 of ccpE mutated to alanine. The wild-type Newman and ΔccpE strains harbor the control plasmid pYJ335-Tc. S. aureus was grown in TSB at 37 °C with shaking, 250 rpm of aeration, and was sampled at 6 h. Values are relative to the wild-type Newman strain (set to 1). Results represent means ± SEM, and data are representative of three independent experiments.

Fig. 4.

CcpE is a global regulator of virulence gene expression in S. aureus. (A) Grouping CcpE-regulated genes according to their annotated function shows that the CcpE regulon consists predominantly of genes involved in the pathogenesis of S. aureus. Numbers of genes whose expressions are down-regulated or up-regulated in the ΔccpE strain compared with the wild-type Newman strain are shown. (B) EMSA showing that 6His-CcpE binds to the promoter DNA (cap5A-p) of cap5A (NWMN_0095) but not to a DNA fragment (ccpE-O) amplified from the ccpE (NWMN_0641) gene in the absence of citrate. (C) EMSA showing that 6His-CcpE binds to the promoter DNA (SAV1168-p) of NWMN_1077 but not to a DNA fragment (citB-U) of the citB promoter (from nucleotides −128 to +88 of the start codon of citB) containing no CcpE-protected region in the absence of citrate. (D–G) The expression of transcriptional cap5A-lacZ and SAV1168-lacZ fusions in the wild-type Newman strain and its derivatives, as indicated. In F and G the wild-type Newman and ΔccpE strains harbor the control plasmid pYJ335-Tc. S. aureus was grown in TSB at 37 °C with shaking, 250 rpm of aeration, and sampled at 6 h. Values are relative to the wild-type Newman strain (set to 1). Results represent means ± SEM, and data are representative of three independent experiments. The statistical difference was determined by unpaired two-tailed Student t test (**P < 0.01, ***P < 0.001). NWMN_1077 gene of the S. aureus Newman strain is corresponding to the SAV1168 locus of S. aureus Mu50.

Fig. 5.

Deletion of ccpE results in improved ability of S. aureus to acquire iron. In all panels, the wild-type Newman and ΔccpE strains harbor the plasmid pYJ335. (A) Assessment of the siderophore production using a chrome azurol S agar diffusion assay as described in SI Appendix, Experimental Procedures. The orange halos formed around the wells correspond to the iron-chelating activity of the siderophores. (B) Siderophore levels in spent culture supernatants of the Newman strain and its derivatives, as indicated. Siderophore units were calculated as described in SI Appendix, Experimental Procedures. (C and D) Representative growth curves for S. aureus grown in iron-limited (C) and in iron-sufficient (D) medium. (E) Determination of intracellular iron content of the wild-type Newman strain and its derivatives. Bacteria were grown in RPMI medium for 24 h with shaking, 250 rpm of aeration. Then the cells were collected, prepared for, and run on atomic absorption spectroscopy. Results show iron content as a percentage of the dry weight. Values represent means ± SEM. (F) Iron release from transferrin mediated by various spent media from the wild-type Newman strain and its derivatives. A decrease in optical density signifies a release of iron from transferrin. Data are representative of three independent experiments.

Fig. 6.

Deletion of ccpE causes increased resistance to whole-blood–mediated killing and enhanced bacterial virulence. (A) Whole-blood survival in the wild-type Newman (harboring pYJ335) and ΔccpE strains complemented with vector (pYJ335) alone or with p-ccpE. Values represent means ± SEM, and data are representative of three independent experiments. (B) Virulence of the S. aureus wild-type Newman strain (harboring pCL-lacZ) and the ΔccpE strain complemented with vector (pCL-lacZ) alone or with pCL::ccpE (ΔccpE-C) (SI Appendix, Table S1). BALB/c mice were infected by retroorbital injection of staphylococcal suspensions. Inocula of 3 × 10⁶ cfu staphylococci per mouse were used. S. aureus colonization in murine kidney or liver was measured by tissue homogenization, dilution, and colony formation on TSA plates after 5 d of infection. Each circle, triangle, and square represents data from one experimental animal. Horizontal bars indicate observation means, and dashed lines mark limits of detection. *P < 0.05; **P < 0.01; n.s., not significant; statistical difference was determined by unpaired two-tailed Student t test.

Fig. 7.

Proposed role of CcpE in metabolite sensing and the information it transfers. Through the use of citrate as a key inducer, S. aureus CcpE is involved in the regulation of TCA-cycle activity, the production of staphyloxanthin, iron acquisition, virulence gene expression, and bacterial virulence, as described in the main text. CitZ, citrate synthase; CitB, aconitase; CitC, isocitrate dehydrogenase. CitB is the second enzyme of the TCA cycle and is responsible for the interconversion of citrate to isocitrate. Acetyl-CoA is the precursor for staphyloxanthin production via the mevalonate pathway (58). Lactate facilitates the release of iron from the host iron-sequestering protein transferrin (51).