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Journal of Bacteriology, November 2006, p. 7823-7829, Vol. 188, No. 22
0021-9193/06/$08.00+0     doi:10.1128/JB.00525-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

plcR papR-Independent Expression of Anthrolysin O by Bacillus anthracis{triangledown}

Caná L. Ross and Theresa M. Koehler*

Department of Microbiology and Molecular Genetics, The University of Texas-Houston Health Science Center Medical School, Houston, Texas 77030

Received 12 April 2006/ Accepted 29 August 2006


   ABSTRACT
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
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.


   INTRODUCTION
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Cholesterol-dependent cytolysins (CDCs) are pore-forming toxins that are secreted by numerous species of gram-positive bacteria. CDC monomers bind to cholesterol present in eukaryotic cell membranes. Upon binding, the monomers oligomerize and create large pores in the cell membrane. CDCs have been reported to contribute to the virulence of a number of pathogens (21). Bacillus anthracis, the causative agent of anthrax, produces anthrolysin O (ALO), a CDC that is secreted during growth in specific conditions during batch culture (28).

A recent report by Park et al. (22) demonstrated that ALO is a Toll-like receptor 4 (TLR4) agonist. Apoptosis of bone marrow-derived macrophages (BMDMs) infected with an alo mutant is significantly reduced compared to apoptosis of BMDMs infected with the parent strain. Additionally, BMDMs treated with lethal toxin require the addition of recombinant ALO or B. anthracis culture supernate for an apoptotic response. These investigators also demonstrated that CDC family members known to play roles in virulence of other species induce TLR4 gene expression in BMDMs. It has been established that macrophage apoptosis requires activation through TLR4 (32). Furthermore, TLR4-mediated macrophage apoptosis could allow pathogenic bacteria to avoid the host's innate immune system (15, 32). Thus, the results implicate ALO as a potential virulence factor for B. anthracis.

Despite growing interest in ALO, little is known regarding alo gene expression by B. anthracis either during infection or during growth in batch culture. Shannon et al. demonstrated that the hemolytic activity of B. anthracis supernate required growth in rich media and was dependent on growth phase (28). The CDC genes of B. cereus and B. thuringiensis, species closely related to B. anthracis, are members of plcR papR regulons (13, 14, 26). In the current model for function of the transcriptional activator, PlcR, a pentapetide derived from cleavage of the papR gene product, is required for PlcR binding to a consensus sequence, the PlcR box, generally located 41 to 58 bp upstream from the transcriptional start site of the target gene (1, 30). The alo gene of B. anthracis is preceded by sequences bearing similarity to the PlcR box, but some published reports have indicated that the plcR papR system may not function in B. anthracis (1, 20). Agaisse et al. reported that the B. anthracis plcR gene harbors a nonsense mutation predicted to result in a truncated protein (1). They showed that the B. thuringiensis and B. cereus plcR genes activated a B. thuringiensis reporter gene when cloned in B. subtilis but that the B. anthracis plcR gene did not affect expression of the reporter. Mignot et al. demonstrated that the relatively low steady-state level of alo transcript increased significantly when the B. thuringiensis plcR and papR genes were cloned in multiple copies in B. anthracis (20). Nevertheless, experiments directly assessing native plcR papR function in B. anthracis have not been reported. The potential role of alo in virulence prompted us to test for alo expression in vivo and to further examine regulation of the alo gene. In addition to the previously reported plcR, the B. anthracis genome harbors two plcR-like genes. We hypothesized that despite the sequence differences between the B. anthracis genes and those of the related species, one or more of the B. anthracis genes may contribute to expression of the alo gene in the native species background. Our data indicate that although all of these genes are expressed by B. anthracis grown in vivo and in vitro, the B. anthracis plcR papR system does not affect alo expression.


   MATERIALS AND METHODS
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Media and growth conditions. B. anthracis was cultured in LB medium (Difco, Detroit, MI) containing 0.5% glycerol and appropriate antibiotics at 30°C with shaking (200 rpm). Following overnight incubation, cultures were transferred to brain heart infusion medium (Difco, Detroit, MI) containing 0.5% glycerol such that the starting optical density at 600 nm (OD600) was 0.06 to 0.08. Cultures were incubated at 37°C with shaking, and samples were obtained at the exponential (OD600 = 2.0 to 2.8) and stationary (OD600 = 4.8 to 6.5) growth phases. When appropriate, kanamycin and spectinomycin (Fisher) were used at a final concentration of 100 µg/ml.

Strain and plasmid constructions. Strains are listed in Table 1. Plasmids were introduced into B. anthracis by electroporation as described previously (16).


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  		TABLE 1. Plasmids and strains used in this study

 
Plasmid pUTE674 contains the alo locus from 513 nucleotides (nt) upstream of the predicted translational start site to 41 nt downstream of the apparent translational stop codon. Sequences corresponding to the alo locus were amplified using oligonucleotide primers CR24 and CR25 (Table 2) and high-fidelity Easy-A cloning enzyme (Strategene, La Jolla, CA). The PCR product was ligated into pGEMT-Easy (Promega, Madison, WI) and cloned into Escherichia coli TG1. A SalI-SphI fragment containing the alo locus was subcloned into pHT304 (2).


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  		TABLE 2. Oligonucleotide primers used for PCRa

 
Strain UT253, in which papR sequences are replaced with the {Omega}sp cassette, was created using a protocol described previously (27). The gene replacement vector was created as follows. DNA sequences flanking papR from –1,592 to 61 nt relative to the translational start were amplified using ES47 and ES45 (Table 2). Primers ES46 and ES48 (Table 2) were used to amplify DNA sequences from 80 nt to 1,384 nt downstream from the translational start of papR. pUTE29, the vector containing the {Omega}sp cassette flanked by the papR upstream and downstream regions, was electroporated into B. anthracis 7702.

RNA purification. RNA was extracted from midexponential and stationary phase cultures grown in BHI broth at 37°C with shaking at 200 rpm using a method described previously (9). RNA was extracted from the spleen of a mouse harvested 48 h after intratracheal inoculation with UT500 spores by methods described previously (12).

RT-PCR. alo transcript levels were compared using a semiquantitative reverse transcriptase PCR (RT-PCR) protocol described previously (28). Reverse transcriptase reactions were performed using SuperScript III reverse transcriptase (Invitrogen, Carlsbad, CA) and 240 ng random primers (Invitrogen) as described in the protocols of the manufacturers. Oligonucleotide primers were as shown in Table 2.

Primer extension reactions. 5'-end mapping of the alo message was carried out using primer extension analysis as described previously (3). Briefly, primer CR16 (5'-AATATTCAGAAAAATCACCCC-3'), corresponding to sequences 17 bases downstream from the translational start site of alo, was labeled using [{gamma}-32P]ATP and T4 polynucleotide kinase (Invitrogen, Carlsbad, CA). In separate experiments, 10 µg of RNA was hybridized to an oligonucleotide primer and extended at 50°C using SuperScript III reverse transcriptase (Invitrogen). Sequencing of the 5' ends of the alo gene by use of the same oligonucleotide, CR16, was carried out using an fmol DNA cycle sequencing system (Promega, Madison, WI). Products obtained from the primer extension and sequencing reaction were electrophoresed on 0.5% Tris-borate-EDTA-6% polyacrylamide-urea gels.

Western hybridization. Samples of culture supernates were concentrated using Microcon centrifugal filter devices from Millipore (Bedford, MA) prior to electrophoresis on sodium dodecyl sulfide-polyacrylamide gels. Proteins were transferred to nitrocellulose membranes and probed with rabbit anti-ALO serum kindly provided by R. Rest of Drexel University. Goat anti-rabbit immunoglobulin G-horseradish peroxidase from Bio-Rad (Hercules, CA) was used in conjunction with an ECL Plus Western blotting detection system (Amersham, Piscataway, NJ) to detect and visualize cross-reacting material.

Site-directed mutagenesis. A QuickChange site-directed mutagenesis kit (Stratagene La Jolla, CA) was used to create plasmid pUTE673. Primers CR89 and CR90 (Table 2) anneal to opposite strands of vector pUTE674 and contain the desired point mutations. The primers are extended in a 12-cycle PCR program using PfuTurbo (Stratagene, La Jolla, CA), the high-fidelity DNA polymerase. The PCR product containing the newly generated plasmid pUTE673 was treated with DpnI (New England Biolabs, Beverly, MA), a restriction enzyme specific for methylated DNA. E. coli TG1 was transformed with 2 µl of the DpnI-treated PCR product. Plasmids from erythromycin-resistant transformants were extracted using a Promega Miniprep kit (Madison, WI) and sequenced using primer CR16.


   RESULTS
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
alo expression in vivo. Compared to the results seen with CDCs of other species, the level of synthesis of ALO by B. anthracis is relatively low when the bacterium is cultured in laboratory media (13, 20, 28). In vivo expression has not been assessed. Considering that CDCs of numerous bacterial pathogens have been reported to play roles in pathogenesis (21), we tested for alo expression during infection in a murine model for inhalation anthrax. Previous studies employing this intratracheal infection model indicated that spleens of infected mice contain vegetative B. anthracis cells as early as 24 h postinfection (12). We isolated RNA from the spleen of a mouse 48 h after infection with spores of the virulent B. anthracis strain UT500. The RNA was used as a template in RT-PCRs with primers specific for the alo gene. As shown in Fig. 1, alo transcript was present in the mRNA extracted from the mouse spleen, indicating that alo is expressed during infection.


Figure 1
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  		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.

 
alo transcript analysis. CDC gene expression in some pathogens is tightly controlled by trans-acting regulatory proteins that bind specific sequences upstream of the CDC coding sequence (6, 11). It was noted previously that a 14-bp palindromic sequence that matches the consensus sequence, TATGNAN4TNCATA, for binding of the transcriptional activator PlcR was located upstream of the predicted translational start site of alo (20, 26, 28). Moreover, there is no apparent recognition sequence for the housekeeping sigma factor {sigma}A or any other sigma factor in the –35 region upstream from the alo coding region (26, 28).

We used primer extension analysis to map the 5' ends of the alo transcripts in cells grown in batch culture. RNA was extracted from cells grown in BHI broth at 37°C, optimal conditions for alo expression during exponential and stationary growth phases (28). As shown in Fig. 2A, a major band representing RNA with a 5' end mapping 178 nt upstream of the alo translational start was apparent. Interestingly, this site is 52 nt downstream from the putative PlcR box in the alo promoter region (Fig. 2B). In addition, the intensity of the 5' end is much greater in stationary phase samples than in samples taken from exponential phase, indicating a greater abundance of this transcript during stationary phase growth.


Figure 2
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  		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.

 
plcR papR-independent alo expression. Considering the location of the major apparent start site for alo transcription, we wanted to determine whether this transcript was affected by plcR papR in B. anthracis. The B. anthracis genome harbors 58 putative PlcR-binding motifs (26). It has been reported that the low-level expression of these genes is due to the lack of a functional plcR gene product in B. anthracis (1, 20). The 858-nt B. anthracis plcR gene bears a nonsense mutation at nt 640 that is predicted to result in a truncated protein (1). However, the function of the B. anthracis plcR papR system has not been tested directly in B. anthracis.

Analysis of the B. anthracis genome revealed that in addition to the plcR gene first reported by Lereclus and coworkers (18) and referred to here as plcR1, two other genes predicted to encode proteins with similarity to PlcR, plcR2 (YP_027352) (26) and plcR3 (YP_027872), are located on the B. anthracis chromosome. As shown in Fig. 3, the predicted amino acid sequences of the B. anthracis proteins share significant amino acid sequence identity with those of the PlcR proteins of B. cereus and B. thuringiensis. Although the B. anthracis PlcR1 sequence is very similar to the PlcR sequences of the B. cereus and B. thuringiensis proteins (70% identity-4% similarity and 66% identity-8% similarity, respectively), the B. anthracis protein is predicted to lack the 72 carboxy-terminal amino acids of the B. cereus and B. thuringiensis proteins (1). Although the overall identity of the B. anthracis PlcR2 sequence to the sequences of the B. cereus and B. thuringiensis proteins (28% identity and 41% similarity) is not as high as that of the B. anthracis PlcR1 sequence, the predicted size of the PlcR2 protein (294 amino acids) closely matches those of the B. cereus and B. thuringiensis PlcR proteins. The 403-amino acid sequence of PlcR3 is much larger than the other PlcR sequences and contains five regions ranging from 16 to 26 amino acids in length that do not align with the other PlcR proteins. Nevertheless, the remaining PlcR3 sequences share significant similarity with the B. cereus and B. thuringiensis proteins.


Figure 3
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  		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.

 
In B. cereus and B. thuringiensis, PlcR function is dependent upon a peptide derived from the product of papR (30). The B. anthracis genome contains only one apparent papR gene starting 306 nt downstream of the plcR1 translational stop. The B. cereus and B. thuringiensis papR genes are also located directly downstream from the plcR genes (AY195601, X93361, and AF132086). As shown in Fig. 4, RNA extracted from the spleen of a mouse infected intratracheally with B. anthracis spores contained transcripts corresponding to plcR1, plcR2, plcR3, and papR. Using the RT-PCR and the same primers, we also detected transcript from each of these genes in RNA obtained from B. anthracis grown in batch culture in conditions optimal for alo expression (data not shown).


Figure 4
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  		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.

 
We reasoned that one or more of the B. anthracis plcR genes may encode a protein that functions with the PapR peptide to activate expression of B. anthracis genes preceded by a PlcR box. To determine whether a B. anthracis plcR papR system analogous to those of B. cereus and B. thuringiensis functions to control the alo gene, we created a B. anthracis papR-null mutant by replacing the papR coding sequences with an {Omega} cassette and compared the steady-state levels of alo mRNA and ALO protein produced by the parent and mutant strains. We found no differences in the steady-state levels of alo mRNA (Fig. 5A) or ALO protein (Fig. 5B) in cultures of the parent and papR mutant strains during exponential or stationary growth phases.


Figure 5
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  		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.

 
We also assessed the effect of the putative PlcR box on alo expression. We cloned the alo locus into a low-copy-number plasmid and used site-directed mutagenesis to alter nucleotides within the PlcR box, as shown in Fig. 6A. Within the consensus sequence, five nucleotides were changed from adenine or thymine to guanine or cytosine. The positions of the mutations disrupted the palindromic sequence of the PlcR box. The plasmid containing the altered PlcR box was introduced into B. anthracis strain UT231, from which the alo coding region is deleted. UT231 was also electroporated with the plasmid harboring the unaltered PlcR box sequence. Comparison of steady-state levels of ALO protein in the supernates of the two cultures revealed that disruption of the PlcR box did not affect ALO synthesis (Fig. 6B). Taken together, our data indicate that alo expression in B. anthracis is dependent neither upon the presence of papR nor upon the presence of a PlcR box.


Figure 6
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  		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.

 

   DISCUSSION
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Anthrolysin O is a cholesterol-dependent cytolysin of B. anthracis that is of interest in light of recent studies indicating that this protein may have a role in apoptosis of infected macrophages (22). Previous reports have indicated relatively low expression of the alo gene when B. anthracis is cultured in laboratory media, and investigators have noted the apparent absence of PlcR, a functional activator of alo transcription (1, 20). In studies reported here we determined that the alo gene is expressed in the spleen of mice with inhalation anthrax and, for the first time, assessed function of the putative plcR papR system of B. anthracis. We found that despite the presence of a major alo transcript initiating downstream from a well-conserved plcR box, alo expression is not regulated by the plcR papR system when B. anthracis is grown in batch culture.

We tested for function of the native plcR papR system in B. anthracis, because other investigations providing insight into this regulatory system have focused on plcR papR function in heterologous hosts. By use of a transcriptional fusion of plcA, the B. thuringiensis phosphatidylinositol-specific phospholipase C gene, as a reporter, Agaisse and coworkers (1, 18) assessed function of the plcR papR genes of the B. cereus group species in a B. subtilis strain harboring a chromosomal plcA-lacZ fusion. The B. thuringiensis and B. cereus plcR papR genes positively regulated plcA-lacZ expression when cloned in trans in B. subtilis. However, the B. anthracis plcR and papR genes did not activate expression of the reporter gene. Based on this result and the prediction that expression of the B. anthracis plcR gene would result in a truncated protein, Agaisse et al. postulated that the B. anthracis PlcR was nonfunctional (1).

Mignot et al. (20) further characterized expression of the plcR papR regulon in B. anthracis by introducing the plcR and papR genes from B. thuringiensis into B. anthracis on a multicopy vector. A significant increase in the expression of the alo gene was observed. Moreover, the presence of the B. thuringiensis plcR and papR genes in B. anthracis restored phenotypes commonly associated with B. thuringiensis and B. cerues, including protease, lecithinase, and hemolytic activities. In the same report, these investigators noted that expression of B. thuringiensis plcR and papR genes from a high-copy-number plasmid in a pXO1-containing B. anthracis strain drastically reduced sporulation. This observation led to the hypothesis that incompatibility between the plcR papR and atxA regulons lead to the nonsense mutation in plcR that resulted in the apparent loss of function in the B. anthracis PlcR protein.

Most recently Pomerantsev et al. cloned the B. cereus plcR and papR genes into B. anthracis and monitored expression of B. anthracis genes associated with the plcR papR regulon (24). In contrast to the results of Mignot et al. obtained using the B. thuringiensis-derived genes (20), the presence of the B. cereus plcR and papR genes in trans did not enhance the protease, lecithinase, or hemolytic activities of B. anthracis (24). Curiously, Pomerantsev and coworkers later demonstrated that a PlcR-PapR fusion protein from B. cereus origin induced hemolytic activity, most likely that of ALO, when cloned into B. anthracis (25). These results emphasize the importance of species-specific factors for plcR papR function and indicate important genetic differences between B. cereus and B. anthracis.

Although the plcR papR system appears to be limited to members of the B. cereus group species, expression of CDC homologues in other gram-positive pathogens is also subject to control by trans-acting regulators. Listeriolysin O encoded by the hly gene, as well as several other L. monocytogenes virulence factors, is regulated at the transcriptional level by the PrfA protein (8). PrfA-mediated gene activation requires binding of PrfA to a 14-bp palindromic sequence located in the promoter of target genes (6). Clostridium perfringens regulates expression of perfringolysin O (encoded by pfoA), as well as many other secreted toxins and enzymes, through a two-component signal transduction system comprised of the response regulator VirR and the histidine kinase VirS (4, 29). Activated VirR recognizes and binds to imperfect direct repeats known as VirR box 1 and VirR box 2 located in the pfoA promoter region (11). Expression of pfoA requires both VirR boxes and a functional VirS-VirR two-component system (10).

Overall, we found no evidence for regulation of alo in B. anthracis by its native plcR papR system when the bacterium is grown in optimal conditions for alo expression in batch culture. We note that the function of the B. anthracis plcR papR system has not been assessed during growth of B. anthracis in an animal model. If control of the anthrolysin O synthesis is important for B. anthracis virulence, it is possible that the plcR papR system functions in vivo. Alternatively, alo expression may be regulated by a plcR papR-independent mechanism. Experiments addressing temporal and spatial expression alo in B. anthracis-infected animals are under way and may reveal alternative mechanisms for alo regulation.


   ACKNOWLEDGMENTS
 
This work was supported by National Institutes of Health grant AI033537.

We thank Elke Saile for construction of the papR-null mutant, Melissa Drysdale and C. Rick Lyons for RNA from B. anthracis-infected mice, Rodney Tweten for recombinant ALO, and Elise Bifano and Rick Rest for antisera raised against rALO and helpful discussions.


   FOOTNOTES
 
* Corresponding author. Mailing address: Department of Microbiology and Molecular Genetics, University of Texas—Houston Medical School, 6431 Fannin St., JFB 1.765, Houston, TX 77030. Phone: (713) 500-5450. Fax: (713) 500-5499. E-mail: Theresa.M.Koehler{at}uth.tmc.edu. Back

{triangledown} Published ahead of print on 15 September 2006. Back


   REFERENCES
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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Journal of Bacteriology, November 2006, p. 7823-7829, Vol. 188, No. 22
0021-9193/06/$08.00+0     doi:10.1128/JB.00525-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.



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