Staphylococcus aureus FadB is a dehydrogenase that mediates cholate resistance and survival under human colonic conditions

Abstract

Staphylococcus aureus is a common colonizer of the human gut and in doing so it must be able to resist the actions of the host's innate defences. Bile salts are a class of molecules that possess potent antibacterial activity that control growth. Bacteria that colonize and survive in that niche must be able to resist the action of bile salts, but the mechanisms by which S. aureus does so are poorly understood. Here we show that FadB is a bile-induced oxidoreductase which mediates bile salt resistance and when heterologously expressed in Escherichia coli renders them resistant. Deletion of fadB attenuated survival of S. aureus in a model of the human distal colon.

Keywords: FadB; Staphylococcus aureus; bile acids; cholate; colon; dehydrogenase.

Fig. 1.

Cell envelope proteins in bile-treated S. aureus . Coomassie-stained SDS-PAGE (12 %, w/v) gel of cell envelope extracts grown in the absence (lane 1) or presence (lane 2) of bovine bile (8 %, w/v).

Fig. 2.

Transcription of fadB is upregulated in the presence of bile acids. qRT-PCR was performed to quantify amounts of transcript in S. aureus SH1000. Data represent means±sd from three independent experiments. White circles indicate data points. P<0.01, Student’s t-test.

Fig. 3.

FadB protects S. aureus against the bactericidal activity of bile salts. Viability of S. aureus SH1000 (■) and ΔfadB (•) treated with (a) 2 mM cholic acid, (b) 0.25 mM deoxycholic acid and (c) 25 mM glycocholic acid. Data represent means±sd from three independent experiments. *P<0.05; all other time points P>0.05, Student’s t-test.

Fig. 4.

Heterologous expression of FadB in E. coli protects against the bacteriostatic effects of bile salts. Data show the viability of E. coli JW3822 (fadB) and E. coli JW3822 (pfadB) cells in LB medium containing cholic acid (CA, 10 mM), deoxycholic acid (DCA, 2 mM), glycocholic acid (GCA, 50 mM) and taurocholic acid (TCA, 50 mM) and then grown for 16 h at 37 °C. Cell counts were determined by viable plate counting. Data represent means±sd from three independent experiments. *P<0.005, Student’s t-test of arabinose treated versus no arabinose.

Fig. 5.

Hydroxyacyl-CoA dehydrogenase enzyme assay. (a) Conversion of acetoacetyl-CoA to hyrdoxybutyryl-CoA in the presence of β-NADH. (b) The catalytic activity of the enzyme by converting acetoacetyl-CoA to hydroxybutyryl-CoA in the presence of NADH as a cofactor was determined spectrometrically (A ₃₄₀). The serial dilution of the substrate (acetoacetyl-CoA) was used to measure the activity rate of the enzyme. Data represent means±sd from three independent experiments. No activity was observed in the absence of enzyme.

Fig. 6.

FadB is required for S. aureus survival in a human distal colon model. Survival of S. aureus SH1000 (■) and ΔfadB (×) cells in a human colonic model. Samples were taken at 4, 8, 24 and 48 h post-infection. Data represent means±sd from three independent experiments. *P<0.01, Student’s t-test.