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Highlights
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The main virulence factors for B. anthracis are the anthrax toxin protein and the capsule. |
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The atxA gene is the “elusive master regulator” of toxin and capsule gene expression. |
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Genetically complete strains, containing both toxin and capsule, should be used to study virulence gene expression and function. |
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The B. anthracis capsule is a potential target for detection, treatment, or prevention of anthrax disease. |
A virulent disease
While it has been estimated that 25% of patients with untreated cutaneous anthrax will die, more than 85% of those who go untreated for the inhalational form of the disease will meet this fate. (Click here to see report.) Why is inhalational anthrax so lethal? Can researchers pinpoint the causes and pave the way to new treatment targets?
On January 10, 2005, Theresa M. Koehler, associate professor in the department of microbiology and molecular genetics at the University of Texas-Houston Health Science Center, addressed these and other questions related to anthrax pathogenesis in her thought-provoking presentation on virulence gene expression in anthrax.
Koehler's talk focused on three main elements of current research: the anthrax toxin protein and B. anthracis capsule, which are the main virulence factors for the disease; the atxA gene, dubbed the “elusive master regulator” of both toxin and capsule gene expression; and the new models that are being used in vitro and in vivo to study the basic mechanisms involved in anthrax virulence and to identify potential targets for treatment and prevention. She also described experiments conducted by her group and others as they fill in the knowledge gaps in these complex processes.
Koehler noted that the anthrax toxin is actually comprised of three different proteins: protective antigen (PA), edema factor (EF), and lethal factor (LF). “B. anthracis secretes these three proteins, and entry of the toxin into a susceptible host cell begins when PA binds a receptor on the host cell. Following cleavage of PA by a cell-associated protease, processed PA forms a heptameric ring with high-affinity binding sites for EF and LF,” she explained. The toxin complex is then taken up by endocytosis. Acidification of the endocytic compartment results in translocation of EF and LF to the cytoplasm, ultimately resulting in cell death.
The second virulence factor, the capsule, is unique in that, unlike other bacterial capsules, it is made entirely of protein—specifically, poly-D-glutamic acid. But although its composition is unique, it behaves like other bacterial capsules in that it prevents the host's phagocytes from destroying the vegetative bacterium.
The anthrax toxin protein and the B. anthracis capsule are the main virulence factors for disease.
Discussing the structure of the bacterium further, Koehler explained that B. anthracis harbors two virulent plasmids, pXO1 and pXO2. The structural genes for the anthrax proteins PA, EF, and LF are located on pXO1. An operon of four genes, called capBCAD, is located on pXO2. The operon encodes proteins that are involved in the biosynthesis of the capsule.
 New models
Most studies of virulence gene expression in anthrax use established, attenuated strains of B. anthracis: the Pasteur strain, which is missing the pXO1 plasmid, and thus the anthrax toxin; and the Sterne strain, which is missing the pXO2 plasmid, and thereby, the capsule.
By contrast, Koehler is working with a genetically complete strain of B. anthracis that harbors both virulence plasmids, thereby providing a more complete window into virulence gene expression. For in vivo studies of the function of virulence genes and their regulators, she is collaborating with Rick Lyons at the University of New Mexico Health Science Center, who has developed a new mouse model of inhalation anthrax.
A genetically complete strain of B. anthracis harbors both virulence plasmids.
“The animal studies are also unique,” she said. “Rather than using an injection model for mice, which is commonly seen in the literature, we are using the inhalational model to address how the capsule genes affect pathogenesis, and whether the function of regulators in vitro match what we're seeing in vivo.” Koehler's work thus has the potential to expose the mechanisms behind the most virulent form of the disease.
“Elusive” master regulator
Previous studies suggest that expression of the three toxin genes is controlled by the atxA gene, which also plays a role, along with acpA, in regulating the capsule biosynthetic operon, capBCAD. But just how atxA accomplishes this “master regulator” function is not known. “We've been trying to associate some molecular function to the AtxA protein for a long time, and simply have not been able to do so,” Koehler said.
Nonetheless, her group and others have pinpointed certain characteristics of the gene, which she summarized as follows:
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atxA encodes a 56-kD soluble basic protein. |
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AtxA has amino acid sequence similarity to AcpA. |
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AtxA has no demonstrated, specific nucleic acid-binding activity and atxA-regulated promoters have no obvious similarities. |
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atxA-regulated genes respond to CO2/bicarbonate. |
These findings were a starting point for additional studies. The first question Koehler's group asked was whether AtxA had other targets in addition to the toxin proteins. A series of experiments using the genetically complete anthrax strain revealed that atxA not only controlled the EF, PA, and LF genes, but also a number of other genes on both plasmids—including acpA
Koehler's next question was to inquire why acpA exists. “If atxA can do acpA's job,” she asked, “isn't it redundant to have this gene in B. anthracis?” It turned out that although acpA does not seem to affect expression of genes other than the capsule biosynthetic operon and one other pXO2-encoded gene, the group was excited to find synergy between atxA and acpA.
Equally exciting was the discovery of a gene (formerly called pXO2-63; now called acpB) on pXO2 that is downstream of the capsule biosynthetic operon. “We're interested in the product of this gene because its predicted amino acid sequence is 62% similar to that of AcpA,” she explained, “which suggests that it, too, may affect capsule synthesis.”
Subsequent experiments with various mutant strains revealed that, indeed, acpB does have an effect on the capsule biosynthetic operon; that atxA's effect on capsule gene expression is mediated by acpA or acpB; and that there is some functional similarity between these two regulators, although acpA seems to have a greater effect on capsule gene expression than does acpB.
Although knowledge of the complex regulation of virulence gene expression is increasing, the atxA gene remains in charge, controlling all known virulence factors and potentially some new factors that are yet to be determined. In terms of bacterial targets for specifically shutting down virulence, atxA appears to represent a viable candidate.
Virulence in vivo
The second part of Koehler's presentation summarized studies using the mouse inhalational anthrax model. These showed that atxA controls virulence in mice, just as it does in vitro. Surprisingly, though, acpB has a greater role in virulence in the animal model than predicted from the in vitro studies. In fact, knocking out AcpB from mice increased mean time to death 100-fold compared with the parent (non-mutant) strain, whereas knocking out AcpA had no effect on time to death. Additional studies showed that the capsule is required for dissemination of B. anthracis from the lung to the spleen.
“We're actively engaged now in determining the function of these virulence genes,” Koehler noted, “and in exploring another pathway, sigH, that seems to influence the transcription and expression of the three toxin genes. And we're trying hard to find some link between this pathway and atxA.”
Near the end of her talk, Koehler emphasized two take-home messages for researchers: First, it's important to use genetically complete strains for gene expression studies because of the synergy between two genes (atxA and acpA) encoded on two different plasmids and the presence of other plasmid-encoded regulators. Second, it's also crucial to remember that the Sterne strain, sometimes referred to as a “capsule” mutant, is missing more than the capsule, whereas the Pasteur strain, sometimes considered a “toxin” mutant, is missing more than the toxin. Both are missing whole plasmids “and a lot of genetic information that may be important.”
Finally, Koehler explained that the new inhalational mouse model shows that the capsule is essential for dissemination. Looking ahead towards future research agendas, she concluded, “This really increases our interest in the capsule as a potential target for detection, treatment, or prevention of anthrax disease.” |
