Dr. DeBell is a well-known world authority within this complex area and ASA is very pleased to present his CBMTS V paper for the ASA family of professionals.

Progress in Research with the plcR Gene in Bacillus anthracis

Robert M. DeBell, Ph.D.
Battelle Memorial Institute

Introduction
         Ongoing research with Bacillus anthracis is attempting to expand our understanding of the spectrum of genomic capabilities to include the expression of new virulence factors. Although these studies are academic and valuable from a microbiology perspective, there are concerns in that they demonstrate genetic alterations that could change the pathogenicity of B. anthracis. Knowledge of this work is most important to ensure that anthrax therapies are adequate to treat anthrax or that competent therapies be developed to avoid circumvention by a genetically altered form of B. anthracis.
         Of the more than 50 species within the genus Bacillus, those closely related to B. anthracis include B. cereus, B. thuringiensis, and B. mycoides.(10) All three species produce toxins as a basis for disease. B. thuringiensis with its array of crystal proteins (Cry toxins) is pathogenic to insect larvae.(18) B. cereus produces a wide array of toxins, but the emetic toxin is most noteworthy and is responsible for gastroenteritis caused by this opportunistic pathogen. (2) Two toxins, lethal factor (LF), and edema factor (EF), are produced by B. anthracis along with a third protein, protective antigen (PA). PA facilitates the entry of the toxins into the cells, and if this process is prevented, anthrax does not occur.
         In addition to the immunological, metabolic, and genetic similarities that exist within this group, results from molecular studies have shown the remarkably high degree of relatedness among the species.(20) Analyses using multilocus enzyme electrophoresis suggested these organisms should be considered the same species, and functional differences among these organisms depend largely on the expression of plasmid genes.(7) DNA base composition determinations and DNA-DNA hybridization experiments have shown that three species – anthracis, cereus, and thuringiensis – have a high degree of relatedness.(8,9) Of more than 2900 nucleotides comprising the base sequences of the 23S rRNAs of both B. anthracis and B. cereus, only two small differences, a nucleotide change at one position and a nucleotide insertion in the B. cereus RNA, were found.(3) On the basis that these species are such close “molecular” relatives, researchers have used these relationships to investigate new ways to manipulate B. anthracis to express new factors, and in some cases new virulence factors, which could potentially affect vaccine efficacy. (13,15,16) In other words, expression of these factors may increase the ability of B. anthracis to cause and sustain an infection even in the presence of recognized therapies.

Genetically Modified B. anthracis
          Seminal work for genetically engineered B. anthracis appeared in 1997 when Russian researchers described the main virulence factors for B. anthracis, the capsular polysaccharide that coats the exterior of vegetative forms of the organism and the tripartite toxin secreted by the organism during infection, and then remarked that other factors may play a role in the organism’s pathogenesis.(16) In particular, they focused on the ability of B. anthracis to lyse red blood cells, a characteristic used commonly to differentiate B. anthracis from both B. cereus and B. thuringiensis which are almost always hemolytic. Few strains of B. anthracis have demonstrated hemolytic activity, and then, only under appropriate, but limited, conditions.(5,14)
         The hemolytic property of B. cereus is attributed to the expression of two genes, the cereolysin AB genes which are under the control of a global regulator PlcR. These genes are located in tandem on the B. cereus chromosome, and the expression of both genes is important for effective hemolytic activity.(6) More specifically, the cereolysin AB genes are the plc and sph genes producing phospholipase C and sphingomyelinase, respectively.(6,15) Although these same genes exist in B. anthracis along with other genes known to be under the control of PlcR in B. cereus, these genes are not expressed because the plcR gene in B. anthracis contains a nonsense mutation (13) leading to expression of a non-functional truncated PlcR (1).
         In their 1997 report(16), however, the Russians described the insertion of the cereolysin AB genes from B. cereus into several strains of B. anthracis consisting of the following: a competent pathogen strain (H-7) capable of causing typical anthrax, a live vaccine strain (STI-1), and a control strain designated 221. Each of these strains differs with respect to their genetic make-up responsible for expression of the major virulence factors. H-7 has both the pXO1 and pXO2 plasmids responsible for the toxin complement and capsule, respectively. STI-1, lacking the pXO2 plasmid, is incapable of making a capsular polysaccharide but has the toxin complement. Strain 221 lacks both plasmids and, therefore, lacks the ability to produce the major virulence factors.
         Recombinant and parent H-7 strains with similar mouse LD50 values were used to determine protection provided by both the recombinant and parent STI-1 vaccine strains. Each of the original and each of the recombinant vaccine and control strains were injected into Golden hamsters to determine the level of protection when the hamsters were challenged with either the original or recombinant H-7 pathogen strains. The results were predictable in all cases except one where the parent vaccine strain, STI-1, protected against the typical pathogen, H-7, as would be expected, but did not protect against the genetically engineered H-7. This unanticipated finding is important because it demonstrates that a traditional vaccine could not protect against a modified pathogen. Although these results remain unexplained, the most significant rationale offered was based on work performed in the U.S. that describes the insertion of the hemolysin gene from Listeria monocytogenes into a non-spore forming Bacillus subtilis permitting the organism to grow within mammalian cells. (4) Similarly, the recombinant H-7 B. anthracis might have had a change in its pathogenic character in that its vegetative form could multiply in mammalian cells.

The PlcR Regulon
          Since the 1997 publication by the Russians, much has been done to determine what genes are controlled by the transcriptional activator PlcR, the mechanism responsible for control of plcR, and what happens when a functionally complete PlcR is present in B. anthracis. PlcR is the first pleiotropic regulator in pathogenic Bacillus species found to control the expression of several virulence factors on genes widely dispersed on the chromosome. The product of the plcR gene in both B. cereus and B. thuringiensis is functionally equivalent unlike the non-functional PlcR encoded in B. anthracis.
         In 1996, a study by Lereclus, et. al., showed that transcription of the plcA gene responsible for producing a phospholipase C (PI-PLC) to cleave phosphatidylinositol in B. thuringiensis required a trans activator encoded by the plcR gene in B. thuringiensis.(12) PlcR was found to regulate its own expression and the expression of PI-PLC. A conserved 17 base pair DNA sequence upstream of the plcR and plcA transcriptional start sites was suspected of being the specific recognition sites for PlcR activation. Because the promoter regions of the plcA genes in both B. cereus and B. thuringiensis had previously been shown to be identical, it was concluded that PlcR likely controlled expression of plcA in B. cereus, as well.
         When the plcR gene was disrupted to produce mutant strains of B. cereus and B thuringiensis Cry- which are both normally pathogenic for insect larvae and mice, the mortality of either the insect larvae or mice was reduced by at least 90% when infected with organisms containing the mutant plcR gene.(17) The reduction in mortality occurred with infections caused by the introduction of spores or vegetative cells. Also, the cytolytic activities of either of the mutant strains also greatly decreased when tested against sheep, human, and horse erythrocytes. This work suggested that the pathogenicity of either of the tested organisms was controlled by PlcR.(17)
         This work eventually led to the determination that PlcR controls a large regulon of least 14 genes that encode degradative enzymes, cell surface proteins, and both hemolytic and non-hemolytic enterotoxins.(1) The promoter region of PlcR-regulated genes revealed the presence of a highly conserved palindromic region (TATGNAN4TNCATA) thought to be the PlcR recognition site for gene activation.
         Although PlcR is active in both B. cereus and B. thuringiensis, B. anthracis contains a nonsense mutation in plcR which yields an inactive regulator consisting of approximately 212 amino acids, and this truncated PlcR is likely responsible for the lack of hemolytic activity typical of B. anthracis.(1,13) Although a PlcR regulon exists in B. anthracis, it is silenced by the nonsense mutation of the plcR gene. Repair of the gene results in the expression of a functional PlcR in B. anthracis, but the expression of the complete regulon is comparably weak.(13)
         Regulation of the plcR gene has been shown to be more complex. The expression of plcR is controlled by SpoA~P, a regulator protein for sporulation that likely prevents the PlcR activator from binding to sites necessary for plcR expression.(11,19) In addition, Slamti and Lereclus, 2002, determined PlcR activation of genes in the regulon is under control of a small 48-amino acid peptide, PapR, and disruption of the papR gene abolished expression of the regulon. They found that PapR was secreted from the bacteria, taken back into the bacterial cell by means of the activity of oligopeptide permease (Opp), and processed to a pentapeptide that activated the PlcR regulon by activating PlcR to bind to the appropriate DNA sites. The mechanism was found to be strain specific and did not function, as expected, in B. anthracis because of the lack of a functional plcR gene.
         A comparison of the nucleotide sequences of the plcR and papR genes from Bacillus cereus, thuringiensis, and anthracis showed no differences within the PlcR operon except for the mutated gene in B. anthracis.(15) PlcR from the other species consisted of 285 amino acids, and the papR gene from all species encoded a polypeptide of 48 amino acids.
         Recently, Pomerantsev, et al., 2003, performed gene replacements to determine the effects on expression within the PlcR regulon. When the B. cereus plcR gene was exchanged with the B. anthracis plcR gene that can only produce a PlcR activator truncated at the C-terminus, the activities of phosphatidylcholine-specific phospholipase C (PC-PLC) and sphingomyelinase (SPH) were eliminated. This result was consistent with the previous observations that inactivation of PlcR greatly reduced PC-PLC and SPH expression in B. cereus. If, however, plcR from B. cereus was transferred to B. anthracis either on a multicopy plasmid under control of the B. anthracis protective antigen gene promoter or containing the entire B. cereus plc-sph operon, B. anthracis was found to produce hemolytic activity.
          Coupled with the work published in 1997 by Pomerantsev, et al., the work being performed with the PlcR regulon presently is suggestive that it may be possible to eventually produce modified forms of B. anthracis that will have altered virulence attributed to the expression of genes presently silent in this organism. Ultimately, just as with the STI-1 vaccine in the golden hamsters(16), these studies may lead to an altered pathogenesis in B. anthracis which may alter the efficacy or circumvent vaccines now in use. New approaches to vaccine development may be necessitated if hemolytic activity or other potential virulence factors not usually associated with B. anthracis are now produced through genetic engineering methods.

References

1. Agaisse, H., M. Gominet, O.A. Økstad, A. Kolstø, and D. Lereclus. 1999. PlcR is a pleiotropic regulator of extracellular virulence factor gene expression in Bacillus thruingiensis. Molec. Microbiol. 32:1043-1053.
2. Agata, N., M. Ohta, M. Mori, and M. Isobe. 1995. A novel dodecadepsipeptide, cereulide, is an emetic toxin of Bacillus cereus. FEMS Microbiol. Lett. 129:17-20.
3. Ash, C. and M.D. Collins. 1993. Comparative analysis of 23S ribosomal RNA gene sequences of Bacillus anthracis and emetic Bacillus cereus determined by PCR-direct sequencing. FEMS Microbiol. Lett. 94:75-80.
4. Bielecki, J., P. Youngman, P. Connelly, and D.A. Portnoy. 1990. Bacillus subtilis expressing a haemolysin gene from Listeria monocytogenes can grow in mammalian cells. Nature 345:175-176.
5. Fellows, P.F. 1996. A survey of worldwide strains of Bacillus anthracis. Salisbury Medical Bulletin No. 87 (Special Supplement):31-33.
6. Gilmore, M.S., A.L. Cruz-Rodz, M. Leimeister-Wächter, J. Kreft, and W. Goebel. 1989. A Bacillus cereus cytolytic determinant, cereolysin AB, which comprises the phospholipase C and sphingomyelinase genes: nucleotide sequence and genetic linkage. J. Bacteriol. 171:744-753.
7. Helgason, E., O.A. Økstad, D.A. Caugant, H.A. Johansen, A. Fouet, M. Mock, I. Hegna, and A. Kolstø. Bacillus anthracis, Bacillus cereus, and Bacillus thuringiensis – one species on the basis of genetic evidence. Appl. Environ. Microbiol. 66:2627-2630.
8. Hill, L.R. 1966. An index to deoxyribonucleic acid base compositions of bacterial species. J. Gen. Microbiol. 44:419-437.
9. Kaneko, T., R. Nozaki, and K. Aizawa. 1978. Deoxyribonucleic acid relatedness between Bacillus anthracis, Bacillus cereus, and Bacillus thuringiensis. Microbiol. Immunol. 22:639-641.
10. Keim, P., A. Kalif, J. Schupp, K. Hill, S.E. Travis, K. Richmond, D.M. Adair, M. Hugh-Jones, C.R. Kuske, and P. Jackson. 1997. Molecular evolution and diversity in Bacillus anthracis as detected by amplified fragment length polymorphism markers. J. Bacteriol. 179:818-824.
11. Lereclus, D., H. Agaisse, C. Grandvalet, S. Salamitou, and M. Gominet. 2000. Regulation of toxin and virulence gene transcription in Bacillus thuringiensis. Int. J. Med. Microbiol. 290:295-299.
12. Lereclus, D., H. Agaisse, M. Gominet, S. Salamitou, and V. Sanchis. 1996. Identification of a Bacillus thuringiensis gene that positively regulates transcription of the phosphatidylinositol-specific phospholipase C gene at the onset of the stationary phase. J. Bacteriol. 178:2749-2756.
13. Mignot, T., M. Mock, D. Robichon, A. Landier, D. Lereclus, and A. Fouet. 2001. The incompatibility between the PlcR- and AtxA-controlled regulons may have selected a nonsense mutation in Bacillus anthracis. Molec. Microbiol. 42:1189-1198.
14. Neves, R.D., R. Corseuil, C. Pianta, and T.H. Santos. 1996. Comparison between protection by anthrax vaccines and infection by Bacillus anthracis field strain in Brazil. Salisbury Medical Bulletin No. 87 (Special Supplement):137.
15. Pomerantsev, A.P., K.V. Kalnin, M. Osorio, and S.H. Leppla. 2003. Phosphatidylcholine-specific phospholipase C and sphingomyelinase activities in bacteria of the Bacillus cereus group. Infect. Immun. 71:6591-6606.
16. Pomerantsev, A.P., N.A. Staritsin, Y.V. Mockov, and L.I. Marinin. 1997. Expression of cereolysine AB genes in Bacillus anthracis vaccine strain ensures protection against experimental hemolytic anthrax infection. Vaccine 15:1846-1850.
17. Salamitou, S., F. Ramisse, M. Brehélin, D. Bourguet, N. Gilois, M. Gominet, E. Hernandez, and D. Lereclus. 2000. The plcR regulon is involved in the opportunistic properties of Bacillus thuringiensis and Bacillus cereus in mice and insects. Microbiol. 146:2852-2832.
18. Schnepf, E., N. Crickmore, J. Van Rie, D. Lereclus, J. Baum, J. Feitelson, D.R. Zeigler, and D.H. Dean. 1998. Bacillus thuringiensis and its pesticidal crystal proteins. Microbiol. Mol. Biol. Rev. 62:775-806.
19. Slamti, L. and D. Lereclus. 2002. A cell-cell signaling peptide activates the PlcR virulence regulon in bacteria of the Bacillus cereus group. EMBO J. 21:4550-4559.
20. Turnbull, P.C.B. 1986. Thoroughly modern anthrax. Abstracts on Hygiene and Tropical Diseases. 61:R1-R13.

Key Words
Bacillus anthracis, PlcR, hemolytic activity, virulence

The CBMTS and future meetings:
#11: SISPAT IV/CBMTS: Singapore, 4-10 December 2004
#12: CBMTS-Industry IV: Dubrovnik, Croatia, September 2005
#13: CBMTS VI, The sixth Spiez Laboratory International Plenary on Chemical, Biological and Radiological Science nd Medicine

Planning Meetings:
a. SISPAT 4/CBMTS: NPS and preliminary review at CDC-Atlanta, Aug ‘04
b. CBMTS-Industry IV: Croatia, Sept.’ 04
c. SISPAT 4/CBMTS: In Singapore for general review - early October ‘04