In a study appearing online in advance of print publication of the September 1 issue of the Journal of Clinical Investigation, Stefan Kaufmann and colleagues from the Max Planck Institute devise a strategy to boost the immunogenicity of BCG and describe a novel vaccine strain with high efficacy against tuberculosis. The researchers engineer a BCG strain that secretes the listeriolysin protein, which punches holes in the membranes of phagosomes where M. tuberculosis is located, allowing better T cell-mediated immunity. Because listeriolysin works optimally at a pH of 5.8, the researchers also deleted the urease C gene of BCG, which normally plays a role in pH neutralization of the phagosome. The lack of urease C allows phagosomal acidification and provides an ideal pH environment for listeriolysin.
The new BCG vaccine strain protects mice against tuberculosis significantly better than the parental BCG. Superior protection is not only induced against the laboratory strain of M. tuberculosis but also against a clinical isolate of the Beijing/W family, a straing of tuberculosis that is spreading all over the world, is drug-resistant, and is responsible for the most threatening disease outbreaks.
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The researchers first applied a test that could tell them whether the offending gene was "upstream" or "downstream" from activated FANCD2 -- that is, did action of the mutant gene fall in the molecular pathway before FANCD2 was activated, or after, respectively? The answer was that the problem was located downstream from a normally functioning FANCD2.
The researchers then mapped SNPs in the genome of those patients and families, looking for changes in which a single chemical building block in the DNA differs from the usual building block at that position. Because FA is a recessive genetic disease, an affected child needs to inherit two copies of an errant gene, each from a parent that carried a single mutation.
They were startled to find only one suspect location in the entire genome, on chromosome 17, that was present in all four families. Further research uncovered two candidate genes within that region, and none of the patients had an abnormality in one of them. But they all had mutations in the second gene, BRIP1.
"What was very surprisingly to us is that while all five patients were homozygous for a mutation in the gene, as expected, all had the same mutation in this gene," Auerbach says. In other words, the five patients each inherited two copies of the same mutation, one from each parent.
When the researchers looked at the other families in their registry with no known mutations in any of the genes associated with the disease, they found six more patients with this same BRIP1 mutation, three of whom were homozygous.
Now the story began to make sense to the researchers, since the protein, BACH1, produced by BRIP1, was known to be a DNA helicase, a class of enzymes which unwind the two strands of the DNA double helix so that DNA synthesis can take place. And they knew from the scientific literature that BACH1 interacts with BRCA1 protein.
"This is the first gene associated with Fanconi anemia that we have a defined function for," says Auerbach. "It interacts directly with BRCA1, and is known to play a role in the repair of DNA double-strand breaks."
BACH1 could be the link between FANCD2 and BRCA1, the researchers say.
"It may be that DNA can't be repaired without a normally functioning BACH1," says Auerbach. "So perhaps FANCD2 activation isn't the endpoint, as had been thought, but that it has to do something downstream that can't be accomplished if BACH1 is not present."
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