plcR papR-independent expression of anthrolysin O by Bacillus anthracis

Abstract

Cholesterol-dependent cytolysins (CDCs) are secreted, pore-forming toxins that are associated with pathogenesis in a variety of gram-positive bacteria. Bacillus anthracis produces anthrolysin O (ALO), a CDC that is largely responsible for the hemolytic activity of culture supernates when the bacterium is cultured in appropriate conditions. B. cereus and B. thuringiensis, species closely related to B. anthracis, produce CDCs with significant amino acid sequence homology to ALO. Transcription of the B. cereus and B. thuringiensis CDC genes is controlled by PlcR, a transcription regulator that requires a pentapeptide derived from the papR gene product for binding to a consensus sequence (PlcR box) and transcriptional activation of downstream genes. A PlcR box precedes the B. anthracis alo gene, and the B. anthracis genome contains three plcR-like genes, one of which harbors a nonsense mutation that is predicted to result in a truncated, nonfunctional protein. We detected mRNA of alo, papR, and the three plcR-like genes in spleens of B. anthracis-infected mice, indicating gene expression in vivo. Analysis of alo transcription in batch culture revealed a potential transcription start located between the PlcR box and the translational start. Nevertheless, steady-state levels of alo transcripts and ALO protein were unaffected by deletion of papR or disruption of the PlcR box. Our data indicate that despite the presence of the transcriptionally active plcR and papR genes in B. anthracis and a PlcR box in the promoter region of the alo gene, alo expression is independent of this control system.

FIG. 1.

alo expression in a B. anthracis-infected mouse. RNA was extracted from the spleen of a mouse 48 h after intratracheal inoculation with UT500 spores. Transcript was detected using RT-PCR. Lane 1, RNA template control; lane 2, cDNA template; lane 3, molecular mass marker; lane 4, DNA template PCR control. Sizes of markers are indicated.

FIG. 2.

alo transcript analysis. (A) 5′-end mapping of alo mRNA transcripts. Primer CR16 was employed. RNA was obtained from a culture of B. anthracis 7702 during exponential (E) and stationary (S) phases of growth. (B) Schematic representation of the alo locus. Locations of the PlcR box and the apparent transcriptional start site (arrows) are shown.

FIG. 3.

Comparison of the PlcR sequences from B. anthracis, B. cereus, and B. thuringiensis. Sequences were aligned using CLUSTAL multiple alignment software (31). Identical and similar residues were shaded using BOXSHADE (K. Hofmann and M. D. Baron, BOXSHADE 3.21, pretty printing and shading of multiple-alignment files, 1996; http://www.ch.embnet.org/software/BOX_form.html). The line above 53 amino acids near the amino termini designates a predicted DNA-binding region.

FIG. 4.

papR, plcR1, plcR2, and plcR3 expression in a B. anthracis-infected mouse. Transcript was detected using RT-PCR. Lane 1, RNA template control; lane 2, cDNA template; lane 3, molecular mass marker; lane 4, DNA template PCR control. RNA was extracted from the spleen of a mouse 48 h after intratracheal inoculation with UT500 spores.

FIG. 5.

Comparison of alo expression in parent and papR strains. (A) Semiquantitative RT-PCR results comparing alo transcript levels during growth in batch culture. The PCR product sizes are as indicated in Table 2. (B) Detection of ALO in culture supernates using anti-ALO serum. Sizes are indicated in kilodaltons. Strains and growth phases were as shown.

FIG. 6.

alo expression in the absence of a PlcR box. (A) Parent and mutant sequences corresponding to the putative PlcR box upstream from alo. (B) Western hybridization. Culture supernates from UT231 (alo) containing the plasmids shown were probed with anti-ALO serum. pUTE674, alo locus with an unaltered PlcR box; pUTE673, alo locus with a mutated PlcR box. pHT304 is the empty vector control. Sizes are indicated in kilodaltons.