Fur is required for the activation of virulence gene expression through the induction of the sae regulatory system in Staphylococcus aureus

Abstract

Our previous studies showed that both Sae and Fur are required for the induction of eap and emp expression in low iron. In this study, we show that expression of sae is also iron-regulated, as sae expression is activated by Fur in low iron. We also demonstrate that both Fur and Sae are required for full induction of the oxidative stress response and expression of non-covalently bound surface proteins in low-iron growth conditions. In addition, Sae is required for the induced expression of the important virulence factors isdA and isdB in low iron. Our studies also indicate that Fur is required for the induced expression of the global regulators Agr and Rot in low iron and a number of extracellular virulence factors such as the haemolysins which are also Sae- and Agr-regulated. Hence, we show that Fur is central to a complex regulatory network that is required for the induced expression of a number of important S. aureus virulence determinants in low iron.

Fig. 1

(A) Structure of the sae operon, showing positions of promoters (P₁ and P₃) and sae operon transcripts (A-D). (B and C) Northern blot analysis of total RNA prepared from S. aureus Newman and its isogenic fur and agr mutants growing exponentially in CRPMI (−) or CRPMI with 50 μM Fe₂(SO₄)₃ (+). Ten μg of total RNA were resolved by agarose gel electrophoresis and hybridised with both the saeR and saeP DNA probes. The blot was then stripped and rehybridised with the 16S rRNA control probe.

Fig. 2

Northern blot analysis of total RNA prepared from S. aureus Newman and its isogenic sae, agr, and fur mutants growing exponentially in CRPMI (−) or CRPMI with 50 μM Fe₂(SO₄)₃ (+). Ten μg of total RNA were resolved by agarose gel electrophoresis and hybridised with (B) fur and (C) xerD DNA probes. The blots were then stripped and rehybridised with the 16s rRNA control probe. (A) Genetic organisation of fur xerD operon.

Fig. 3

(A) Cell wall. (B) SDS surface extract obtained from S. aureus Newman and its isogenic mutants sae, fur, and sae/fur grown for 16 h in iron-restricted CRPMI (−) or CRPMI with 50 μM Fe₂ (SO₄)₃ (+) added to give iron-replete growth conditions. All lanes are equivalently loaded. Gels presented are representative of experiments repeated at least 3 times using protein extracted from post-exponential phase cultures grown on different days with similar results observed each time. (C) Northern blot analysis of total RNA prepared from S. aureus Newman and its isogenic sae mutant growing exponentially in CRPMI (−). Ten μg of total RNA were resolved by agarose gel electrophoresis and hybridised with (C) isdA or isdB DNA probes. The blots were then stripped and rehybridised with the 16S rRNA control probe.

Fig. 4

Model of a regulatory network involving Fur, Sae, and Agr showing that Fur is a central regulator of virulence gene expression. Positive and negative regulatory pathways are indicated with either a + or −, respectively.