Staphylococcus aureus uses a GGDEF protein to recruit diacylglycerol kinase to the membrane for lipid recycling

Abstract

Staphylococcus aureus is a Gram-positive pathogen responsible for numerous antibiotic-resistant infections. Identifying vulnerabilities in S. aureus is crucial for developing new antibiotics to treat these infections. With this in mind, we probed the function of GdpS, a conserved Staphylococcal membrane protein containing a cytoplasmic GGDEF domain. These domains are canonically involved in cyclic-di-GMP signaling processes, but S. aureus is not known to make cyclic-di-GMP. Using a transposon screen, we found that loss of GdpS is lethal when combined with disruption in synthesis of the glycolipid anchor of a cell surface polymer called lipoteichoic acid (LTA) or with deletion of genes important in cell division. Taking advantage of a small molecule that inhibits LTA glycolipid anchor synthesis, we selected for suppressors of ΔgdpS lethality. The most prevalent suppressors were hypermorphic alleles of dgkB, which encodes a soluble diacylglycerol (DAG) kinase required to recycle DAG to phosphatidylglycerol. By following up on these suppressors, we found that the GGDEF domain of GdpS interacts directly with DgkB, orienting its active site at the membrane to promote DAG recycling. DAG kinase hypermorphs also suppressed the lethality caused by combined loss of gdpS and cell division factors, highlighting the importance of lipid homeostasis for cell division. GdpS' positive regulation of DAG kinase function was dependent on the GGDEF domain but not its catalytic residues. As the sole conserved GGDEF-domain protein in Staphylococci, GdpS promotes an enzymatic process independent of cyclic-di-GMP signaling, revealing a new function for the ubiquitously conserved GGDEF domain.

Keywords: Gram-positive membrane lipid homeostasis; Staphylococcus aureus GGDEF; diacylglycerol kinase; diguanylate cyclase; phospholipid recycling.

Fig. 1.

The GGDEF domain of GdpS, but not its catalytic residues, is required when lipoteichoic acid (LTA) synthesis is disrupted. (A) Plot of transposon insertions within individual genes (gray and colored circles) from a ΔgdpS transposon library compared to a WT control library. Genes that are >4-fold depleted and meet statistical cutoffs (q-val < 0.05) are colored by pathway (See Datasets 1 for more information). (B) Schematic summarizing the role of UgtP and LtaS in the LTA biosynthetic pathway. UgtP, which forms a synthetic lethal pair with GdpS, makes the glycolipid anchor for LTA (Glc₂DAG) on the inner leaflet of the membrane. Following translocation of Glc₂DAG to the cell surface, the LTA polymerase LtaS uses the headgroup of phosphatidylglycerol to synthesize LTA, releasing DAG at each elongation step. The inhibitor GLI-2 blocks Glc₂DAG synthesis by interacting with UgtP, forcing the assembly of defective LTA on an alternative membrane anchor. See SI Appendix, Fig. S15 for detailed overview of LTA pathway. (C) Bacterial spot assay confirming synthetic lethality between ugtP and gdpS. Spotted strains include HG003 (WT) and derivatives lacking gdpS (AM0207), ugtP (AM0206), or both ugtP and gdpS (AM0209). The ugtP mutants lack endogenous ugtP but include an aTc-inducible copy of ugtP at the geh locus. Strains were grown to saturation in TSB containing 0.4 μM aTc before spotting on TSA plates without inducer and incubating overnight. (D) Derivatives lacking ugtP and gdpS can be rescued by expressing either WT GdpS (AM0211) or variants lacking key active site residues (GGEEF → GDSIF = strain AM0215; GGEEF → GGAAL = strain AM0247), but not empty vector (AM0244). Strains were spotted and incubated on TSA with erm and 1,000 μM IPTG (E) Proteins with a GGDEF domain typically function as enzymes that synthesize a signaling molecule, c-di-GMP, but GdpS has unknown noncatalytic functions. (F) Cells lacking gdpS can be complemented by the GGDEF domain, provided it contains a transmembrane helix that localizes it to the membrane. Strains spotted include WT HG003 and ΔgdpS containing either an empty vector (AM0243) or vectors expressing full-length GdpS (AM0097), the transmembrane domain (TMD; AM0150), the GGDEF domain (AM0149), or the GGDEF domain fused to the 1st TM helix of GdpS (AM0151). Strains were spotted and incubated on TSA with erm and 1,000 μM IPTG.

Fig. 2.

The conditional essentiality of GdpS can be bypassed by a DAG kinase (DgkB) variant that has increased affinity for membranes. (A) A selection plate showing suppressors that rescue growth of ΔgdpS plated on the UgtP inhibitor GLI-2 (see arrows; frequency of resistance, FOR: ~1:500,000). Whole-genome sequencing revealed that ~half the suppressors from the initial selection had mutations at the same position in dgkB (encoding DgkB^T237I). (B) DgkB is a membrane-associated enzyme that converts DAG to phosphatidic acid (PA) to recycle phosphatidylglycerol (PG). (C) Spot assay showing that hydrophobic residues in place of T237 in DgkB suppress ΔugtP ΔgdpS synthetic lethality. Strains spotted are HG003 (WT) and derivatives lacking gdpS (AM0274) or with dgkB mutations: T237I (AM0432), T237W (AM0430), or T237L (AM0431). Strains were spotted and incubated on TSA with 2 μg/mL GLI-2. (D) A previously solved crystal structure (2QV7) shows that DgkB T237 is a surface-exposed residue on the same face of the enzyme as the active site. (E) A vesicle cosedimentation assay shows that DgkB T237W has increased membrane affinity compared with WT DgkB. Purified Staphylococcus aureus DgkB or DgkB T237W were mixed with or without vesicles made from S. aureus lipids and subjected to ultracentrifugation (I = input; S = soluble fraction; P = pelleted vesicle fraction). (F) Bar graph summarizing DAG abundance compared to WT for ΔgdpS and ΔgdpS with a dgkB* allele encoding DgkB T237W (n = three biological replicates; error bars = mean ± SD). DAG was quantified by LC-MS (see also SI Appendix, Fig. S6 and S7). (G) Spot assay showing that overexpression of WT DgkB suppresses synthetic lethality due to loss of UgtP and GdpS. Strains spotted include HG003 (WT, AM0241) and derivatives of ΔugtP ΔgdpS containing an empty vector (AM0244) or vectors expressing either dgkB (AM0279) or a dgkB* allele encoding dgkB^T237W (AM0280). Strains were grown overnight in TSB containing 0.4 μM aTc overnight to permit ugtP expression, and then spotted on TSA plates containing IPTG to induce DgkB or DgkB* expression.

Fig. 3.

The GGDEF domain of GdpS directly interacts with DgkB via an interface conserved across Staphylococcaceae. (A) AlphaFold2 predicts with high confidence (i.e., low position alignment error; see plot) that GdpS uses its GGDEF domain to form a complex with DgkB. The predicted interaction would orient DgkB’s active site, which is on the same face of the protein as T237, toward the cytoplasmic membrane. Selected polar contacts that mediate the predicted interaction are shown (boxed region). (B) Sequence logos comparing sequences for GdpS homologs in Staphylococcaceae (Bottom) and in other Bacillales (Top). Residues of the GGDEF domain that are predicted to interact with DgkB are conserved only in Staphylococcaceae. Red arrows indicate invariant residues at the interaction interface, and black arrows indicate other conserved interacting residues. (C) Top panel: changing the invariant residues GdpS N255 and GdpS H300 disrupts GdpS complementation. Strains spotted are HG003 (WT; AM0241) or ΔugtP ΔgdpS containing an empty vector (AM0244) or a vector expressing either WT GdpS (AM0211) or GdpS with H300E (AM0485) or N255K (AM0486) mutations. Strains were spotted on TSA with 1 mM IPTG. Bottom panel: altering DgkB residues predicted to interact with GdpS results in synthetic lethality with ΔugtP even though the DgkB variants are enzymatically active (SI Appendix, Fig. S11). Strains spotted are HG003 (WT, AM0241), ΔugtP (AM0206), ΔdgkB harboring a vector expressing DgkB (AM0560), or ΔugtP ΔdgkB containing a vector expressing either WT DgkB (AM0591), DgkB S261R (AM0592), or DgkB N281K (AM0593). Strains were spotted on TSA with 10 µM IPTG. (D) GdpS-Strep and FLAG-DgkB variants containing cysteine substitutions at positions predicted to be in close proximity in the GdpS–DgkB complex (GdpS^Q251C and DgkB^K277C) form crosslinks in cells. Anti-Strep and anti-FLAG blots show the presence of a high molecular weight band following immunoprecipitation of FLAG-DgkB from cells treated with the oxidant bis(1,10-phenanthroline)copper(2+). Crosslinking of the GdpS–DgkB complex was not observed when the binding interface was disrupted through mutation of GdpS residue H300.

Fig. 4.

Staphylococci employ a unique strategy of membrane recruitment of DAG kinase by the GGDEF domain of GdpS. (A) The membrane-interacting α7 helix of S. aureus (RCSB: 2QV7), L. ivanovii (AlphaFoldDB: G2ZBD7), B. subtilis (AlphaFoldDB: O31502), and P. polymyxa (AlphaFoldDB: A0A0F0G959). Residues that when changed to hydrophobic amino acids can suppress gdpS synthetic lethality with GLI-2 are labeled. The orthologs are numbered as the equivalent from S. aureus. (B) Logo showing sequence conservation for the predicted membrane-interacting helix of DgkB. Arrows point to residues on the surface-exposed face of helix α7, with red arrows indicating positions substituted with at least one hydrophobic residue in other Bacillales. (C) Changes to hydrophobic residues at any one of the four positions on the surface-exposed face of DgkB’s α7 helix bypass the need for GdpS in S. aureus. Spot assay shows HG003 (WT; AM0275) and strains lacking gdpS (AM0276), or lacking gdpS but containing additional mutations in dgkB mediated by ssDNA recombineering (A230W, AM0472; G233L, AM0510; S240W, AM0470; and S240F, AM0469). Strains were spotted and incubated on TSA with 2 μg/mL GLI-2 (D) A working model for GdpS function in Staphylococcaceae and other Bacillales. GdpS recruits DgkB to the membrane in Staphylococcaceae. Other families of bacteria in the order Bacillales contain hydrophobic residues on the membrane-docking α7 helix that bypass the need for a recruitment mechanism.