Faculty Opinions recommendation of 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.

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Bacillus anthracis anthrolysin O and three phospholipases C are functionally redundant in a murine model of inhalation anthrax

FEMS Microbiology Letters ◽

10.1111/j.1574-6968.2007.00713.x ◽

2007 ◽

Vol 271 (1) ◽

pp. 98-105 ◽

Cited By ~ 31

Author(s):

Brian J. Heffernan ◽

Brendan Thomason ◽

Amy Herring-Palmer ◽

Philip Hanna

Keyword(s):

Bacillus Anthracis ◽

Murine Model ◽

Inhalation Anthrax ◽

Anthrolysin O

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Passive administration of monoclonal antibodies to Anthrolysin O prolong survival in mice lethally infected with Bacillus anthracis

BMC Microbiology ◽

10.1186/1471-2180-8-159 ◽

2008 ◽

Vol 8 (1) ◽

pp. 159 ◽

Cited By ~ 19

Author(s):

Antonio Nakouzi ◽

Johanna Rivera ◽

Richard F Rest ◽

Arturo Casadevall

Keyword(s):

Monoclonal Antibodies ◽

Bacillus Anthracis ◽

Anthrolysin O

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Immunisation with anthrolysin O or a genetic toxoid protects against challenge with the toxin but not against Bacillus anthracis

Vaccine ◽

10.1016/j.vaccine.2007.07.040 ◽

2007 ◽

Vol 25 (41) ◽

pp. 7197-7205 ◽

Cited By ~ 11

Author(s):

Graeme J.M. Cowan ◽

Helen S. Atkins ◽

Linda K. Johnson ◽

Richard W. Titball ◽

Tim J. Mitchell

Keyword(s):

Bacillus Anthracis ◽

Anthrolysin O

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Activation of the latent PlcR regulon in Bacillus anthracis

Microbiology ◽

10.1099/mic.0.041418-0 ◽

2010 ◽

Vol 156 (10) ◽

pp. 2982-2993 ◽

Cited By ~ 14

Author(s):

Inka Sastalla ◽

Lauren M. Maltese ◽

Olga M. Pomerantseva ◽

Andrei P. Pomerantsev ◽

Andrea Keane-Myers ◽

...

Keyword(s):

Bacillus Anthracis ◽

Consensus Sequence ◽

Transcriptional Analysis ◽

Virulence Plasmid ◽

Putative Protease ◽

2D Gel ◽

Culture Supernatants ◽

Anthrolysin O

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.

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Characterization of Listeria monocytogenes Expressing Anthrolysin O and Phosphatidylinositol-Specific Phospholipase C from Bacillus anthracis

Infection and Immunity ◽

10.1128/iai.73.10.6639-6646.2005 ◽

2005 ◽

Vol 73 (10) ◽

pp. 6639-6646 ◽

Cited By ~ 25

Author(s):

Zhengyu Wei ◽

Pamela Schnupf ◽

Mathilde A. Poussin ◽

Lauren A. Zenewicz ◽

Hao Shen ◽

...

Keyword(s):

Listeria Monocytogenes ◽

Bacillus Anthracis ◽

Host Cell ◽

Phospholipase C ◽

Host Cells ◽

Listeriolysin O ◽

Intracellular Growth ◽

Cell Cytosol ◽

Anthrolysin O ◽

Cell Spread

ABSTRACT Two virulence factors of Listeria monocytogenes, listeriolysin O (LLO) and phosphatidylinositol-specific phospholipase C (PI-PLC), mediate escape of this pathogen from the phagocytic vacuole of macrophages, thereby allowing the bacterium access to the host cell cytosol for growth and spread to neighboring cells. We characterized their orthologs from Bacillus anthracis by expressing them in L. monocytogenes and characterizing their contribution to bacterial intracellular growth and cell-to-cell spread. We generated a series of L. monocytogenes strains expressing B. anthracis anthrolysin O (ALO) and PI-PLC in place of LLO and L. monocytogenes PI-PLC, respectively. We found that ALO was active at both acidic and neutral pH and could functionally replace LLO in mediating escape from a primary vacuole; however, ALO exerted a toxic effect on the host cell by damaging the plasma membrane. B. anthracis PI-PLC, unlike the L. monocytogenes ortholog, had high activity on glycosylphosphatidylinositol-anchored proteins. L. monocytogenes expressing B. anthracis PI-PLC showed significantly decreased efficiencies of escape from a phagosome and in cell-to-cell spread. We further compared the level of cytotoxicity to host cells by using mutant strains expressing ALO in combination either with L. monocytogenes PI-PLC or with B. anthracis PI-PLC. The results demonstrated that the mutant strain expressing the combination of ALO and B. anthracis PI-PLC caused less damage to host cells than the strain expressing ALO and L. monocytogenes PI-PLC. The present study indicates that LLO and L. monocytogenes PI-PLC has adapted for L. monocytogenes intracellular growth and virulence and suggests that ALO and B. anthracis PI-PLC may have a role in B. anthracis pathogenesis.

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Characterization of Anthrolysin O, the Bacillus anthracis Cholesterol-Dependent Cytolysin

Infection and Immunity ◽

10.1128/iai.71.6.3183-3189.2003 ◽

2003 ◽

Vol 71 (6) ◽

pp. 3183-3189 ◽

Cited By ~ 79

Author(s):

Jeffrey G. Shannon ◽

Cana L. Ross ◽

Theresa M. Koehler ◽

Richard F. Rest

Keyword(s):

Bacillus Anthracis ◽

Hemolytic Activity ◽

Brain Heart Infusion ◽

Luria Bertani ◽

Dependent Manner ◽

Listeriolysin O ◽

Kanamycin Cassette ◽

Putative Toxin ◽

Anthrolysin O

ABSTRACT We characterized the expression of a putative toxin of Bacillus anthracis, a member of the cholesterol-dependent cytolysin (CDC) family, which includes listeriolysin O, perfringolysin O, and streptolysin O. We named this cytotoxin anthrolysin O (ALO). Although B. anthracis expresses minimal hemolytic activity in clinical settings, we show that Sterne strain 7702 expresses hemolytic activity when grown in brain heart infusion broth or in other rich bacteriologic media, but it secretes barely detectable amounts of hemolysin when grown in Luria-Bertani (LB) broth. Glucose supplementation of LB broth increases the amount of secreted hemolytic activity. Expression of hemolytic activity is maximal during mid- to late-log phase and decreases in the stationary phase. These observations are supported, in part, by semiquantitative reverse transcriptase PCR of alo mRNA. Hemolytic activity in growth supernatants was increased in the presence of reducing agent and almost totally inhibited in a dose-dependent manner by cholesterol; both of these activities are characteristic of a CDC toxin. A mutant of Sterne strain 7702, strain UT231, in which the alo gene was deleted and replaced by a kanamycin cassette, secreted barely detectable hemolytic activity into the growth medium. When strain UT231 was complemented in trans with native alo on a low-copy-number plasmid [strain UT231(pUTE554)], it regained the ability to secrete hemolytic activity, indicating that ALO is the major hemolysin secreted by this strain of B. anthracis in rich media in vitro. To further support the alo gene product being a hemolysin, recombinant B. anthracis ALO (rALO) purified from Escherichia coli was extremely active against washed human erythrocytes, with complete hemolysis detected at ∼30 molecules of rALO per erythrocyte. Considering the virulence roles of CDCs for other gram-positive bacteria, we speculate that ALO may have a role in anthrax virulence.

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