Activation of the latent PlcR regulon in Bacillus anthracis

Abstract

Many genes in Bacillus cereus and Bacillus thuringiensis are under the control of the transcriptional regulator PlcR and its regulatory peptide, PapR. In Bacillus anthracis, the causative agent of anthrax, PlcR is inactivated by truncation, and consequently genes having PlcR binding sites are expressed at very low levels when compared with B. cereus. We found that activation of the PlcR regulon in B. anthracis by expression of a PlcR-PapR fusion protein does not alter sporulation in strains containing the virulence plasmid pXO1 and thereby the global regulator AtxA. Using comparative 2D gel electrophoresis, we showed that activation of the PlcR regulon in B. anthracis leads to upregulation of many proteins found in the secretome of B. cereus, including phospholipases and proteases, such as the putative protease BA1995. Transcriptional analysis demonstrated expression of BA1995 to be dependent on PlcR-PapR, even though the putative PlcR recognition site of the BA1995 gene does not exactly match the PlcR consensus sequence, explaining why this protein had escaped recognition as belonging to the PlcR regulon. Additionally, while transcription of major PlcR-dependent haemolysins, sphingomyelinase and anthrolysin O is enhanced in response to PlcR activation in B. anthracis, only anthrolysin O contributes significantly to lysis of human erythrocytes. In contrast, the toxicity of bacterial culture supernatants from a PlcR-positive strain towards murine macrophages occurred independently of anthrolysin O expression in vitro and in vivo.

Fig. 1.

Characterization of PlcR–PapR-expressing strains. (a) Haemolysis on sheep blood agar by Sterne strain and Sterne expressing PlcR–PapR. (b) Microscopic analysis of spores formed by Sterne and the isogenic PlcR–PapR-expressing strain. (c) Efficacy of sporulation. Shown are mean±sd from one representative experiment out of three. (d) Western blot analysis of AtxA activity by determining PA and LF expression in response to bicarbonate/CO₂. Control (CTR) bands represent 1 μg recombinant protein.

Fig. 2.

2D SDS-PAGE separation of proteins secreted by Sterne and Sterne pFP12 grown in BHI broth to late exponential phase. Boxed spots indicate proteins identified by MS. Proteins are numbered according to B. anthracis Ames (BA) gene ID. See Table 2 for gene descriptions. Molecular mass (in kDa) is given on the left and the pH range is indicated at the top.

Fig. 3.

Identification and analysis of the PlcR-dependent protease BA1995. (a) Putative protease BA1995 identified by 2D SDS-PAGE in supernatants of Sterne pFP12. Matched peptides are shown in bold type. (b) Semiquantitative RT-PCR of BA1995 transcription in response to PlcR–PapR expression (lower band). The gene for gyrase A (gyrA) served as internal control. Fold change indicates gyrA-normalized differences in BA1995 transcript between Sterne and Sterne pFP12. (c) Sequence comparisons of the PlcR box present in the promoter region of BA1995 of B. anthracis strain Ames and the homologue BC1991 in B. cereus. The PlcR consensus sequence is indicated. The nucleotide differing from the consensus is shown in bold type, nucleotides differing between B. cereus and B. anthracis are shaded, and putative −10, −35 regions and the start codon (ATG) are indicated.

Fig. 4.

Transcription and expression of haemolysins in PlcR–PapR-expressing B. anthracis. (a) Semiquantitative RT-PCR of B. anthracis putative haemolysins anthrolysin (alo) and sphingomyelinase (sph) in response to PlcR–PapR expression in Sterne and Sterne pFP12. Transcript levels of bacteria harvested at different growth phases were compared with those for gyrase A (gyrA). exp., exponential; stat., stationary. (b) Haemolytic activity (as a percentage) towards human erythrocytes of bacterial supernatants derived from Sterne or anthrolysin knockout mutant (Alo) in the presence or absence of PlcR–PapR. Supernatants were harvested at different OD₆₀₀. Shown are mean±sd from one representative experiment out of three. •, Sterne; ▪, Sterne pFP12; ▴, Alo; ▾, Alo pFP12. (c) Haemolytic activity (%) of bacterial supernatants in the presence or absence of 25 μM cholesterol. Shown are mean±sd from one representative experiment out of three.

Fig. 5.

In vitro toxicity towards macrophages of proteins secreted by Bacillus. (a) MTT stain of BMDMs treated for 4 h with cell-free bacterial culture supernatants derived from late exponential phase B. cereus BC569 (a2), B. anthracis (a3), B. anthracis pFP12 (a4), B. anthracis anthrolysin knockout (a5), B. anthracis anthrolysin knockout harbouring pFP12 (a6), and broth-treated control (N.T.) (a1). (b) Quantification of surviving macrophages treated with bacterial culture supernatants before (N.T.; grey bars) and after (H.I.; black bars) heat treatment at 95 °C for 10 min. Shown are mean±sd from one representative experiment out of three.

Fig. 6.

In vivo macrophage toxicity of proteins secreted by Bacillus. (a) Intraperitoneal (IP) washes of mice (n=2) treated with 500 μl filter-sterilized bacterial culture supernatants derived from B. anthracis or B. anthracis anthrolysin knockout strain in the presence or absence of PlcR–PapR. Control animals were treated with BHI broth alone. (b) Flow cytometry analysis showing forward and side scatter of IP washes derived from mice treated with cell-free bacterial culture supernatants. Arrows indicate macrophage (F4/80+) populations lacking in washes treated with supernatants derived from PlcR–PapR-expressing bacteria. (c) Chart of macrophage (F4/80+) populations present in IP washes of mice treated with bacterial supernatants or with BHI alone. (d) Quantification of macrophages (%) present in the IP washes of mice treated with bacterial culture supernatants. Percentages represent the number of F4/80+ cells relative to the entire cell population present in the IP wash. Shown are mean±sd from one representative experiment out of three.