Role of the twin-arginine translocation pathway in Staphylococcus

Abstract

In Staphylococcus, the twin-arginine translocation (Tat) pathway is present only in some species and is composed of TatA and TatC. The tatAC operon is associated with the fepABC operon, which encodes homologs to an iron-binding lipoprotein, an iron-dependent peroxidase (FepB), and a high-affinity iron permease. The FepB protein has a typical twin-arginine (RR) signal peptide. The tat and fep operons constitute an entity that is not present in all staphylococcal species. Our analysis was focused on Staphylococcus aureus and S. carnosus strains. Tat deletion mutants (DeltatatAC) were unable to export active FepB, indicating that this enzyme is a Tat substrate. When the RR signal sequence from FepB was fused to prolipase and protein A, their export became Tat dependent. Since no other protein with a Tat signal could be detected, the fepABC-tatAC genes comprise not only a genetic but also a functional unit. We demonstrated that FepABC drives iron import, and in a mouse kidney abscess model, the bacterial loads of DeltatatAC and Deltatat-fep mutants were decreased. For the first time, we show that the Tat pathway in S. aureus is functional and serves to translocate the iron-dependent peroxidase FepB.

FIG. 1.

(A) Gene organization of fepABC and tat operons of Staphylococcus. The fepABC operon encodes a lipoprotein, an iron-dependent peroxidase, and a high-affinity iron transporter. Arrows indicate the orientation of the genes, and the circle represents a putative Fur box. (B) Allelic replacement of tatAC operon by an erythromycin resistance cassette in S. aureus and S. carnosus. (C) Allelic replacement of fepABC and tatAC operons by a kanamycin resistance cassette in S. aureus. (D) Relevant part of plasmid pTX-tat, carrying tatAC genes under the control of a xylose-inducible promoter.

FIG. 2.

The iron-dependent peroxidase FepB is a Tat substrate. (A) Multiple amino acid sequence alignment of the N terminus of FepB of S. aureus, S. haemolyticus, and S. carnosus, using Clustal W. Asterisks indicate identical amino acid residues. The twin-arginine motif in the sequence is boxed. The arrow indicates the processing site. (B) Culture supernatants of S. carnosus WT, the S. carnosus Δtat mutant, and its complemented mutant, S. carnosus Δtat(pTX-tat), were tested for the presence of peroxidase by use of an Amplex red peroxidase assay kit.

FIG. 3.

Expression of lipase fused to RR-SP. (A) Illustration of the primary structures of lipase with the Tat SP and propeptide, as expressed with plasmid pCXRR-lip, and the lipase fused to the Sec SP and propeptide, as expressed with plasmid pCX19. (B) p-Nitrophenyl-caprylate-based lipase assay with culture supernatants of S. carnosus(pCX19) (⧫), S. carnosus ΔtatAC(pCX19) (⋄), S. carnosus(pCXRR-lip) (•), and S. carnosus ΔtatAC(pCXRR-lip) (○). (C) Lipase activity staining (zymogram) of culture supernatants and whole-cell extracts. Lane 1, S. carnosus(pCX19); lane 2, S. carnosus(pCXRR-lip); lane 3, S. carnosus ΔtatAC(pCX19); lane 4, S. carnosus ΔtatAC(pCXRR-lip). Plasmid pCX19 encodes lipase with a Sec SP, while pCXRR-lip encodes lipase with the Tat SP of FepB. The expression of lipase was induced by the addition of 0.5% xylose.

FIG. 4.

Expression of pCXRR-spa. (A) Relevant part of plasmid pCXRR-spa encoding protein A under the control of a xylose-inducible promoter. Amino acids at the fusion site are indicated. (B) Western blot analysis of protein A expression in the culture supernatant, cell wall (CW)-anchored, and cytoplasmic fractions of S. carnosus(pCXRR-spa) and S. carnosus ΔtatAC(pCXRR-spa). S. aureus SA113 was used as a control. (C) Immunofluorescence labeling of protein A anchored to the cell wall, using Alexa Fluor 594-conjugated antibody. SC, S. carnosus WT. (1) Phase-contrast image. (2) Protein A visualized by immunofluorescence following antibody labeling.

FIG. 5.

(A) Iron transport. ⁵⁵Fe uptake was measured using cells grown under iron-sufficient (closed symbols) and iron-deficient (open symbols) conditions. The strains employed were S. aureus WT (▵), S. aureus ΔtatAC (○), and S. aureus Δtat-fep (□). Values presented are representative of three independent experiments. (B) Role of S. aureus RN1HG TatAC and FepABC in kidney infection. Bacterial loads of parental S. aureus and the tatAC and tat-fep mutant strains in the kidneys of BALB/c mice (n = 7) were determined 5 days after intravenous infection with 7 × 10⁷ CFU. Each dot represents the bacterial load for one animal per gram of kidney.