As early as two weeks after infection, researchers found significantly more bacteria from the organs of mice infected with the mutated tuberculosis (TB) bacteria than for mice infected with the unmodified, or "wild-type," strain. By 27 weeks, the mutant-infected mice started to die, while their counterparts infected with the wild-type strain survived until the end of the experiment at 41 weeks.
"These findings came as a complete surprise to us," said Dr. Lee Riley, professor of epidemiology and infectious diseases at UC Berkeley's School of Public Health and principal investigator of the study. "We thought we had made a mistake, so we repeated the test several times, and we always got the same result."
The researchers say the study, to be published Dec. 8 in Proceedings of the National Academy of Sciences, sheds light on the mechanisms used by a pathogen that now infects one-third of the world's population and kills 2 million people per year. According to the World Health Organization, which in 1993 declared TB a global emergency, an estimated 36 million people could die of TB by 2020 if the disease is not controlled.
The results were unexpected because prior studies pointed to the mce1 operon, the collection of genes that researchers disabled in the TB bacteria, as an important virulence factor that helped the organism invade cells. Researchers expected that mutating the mce1 genes would impair the pathogen's ability to infect the mice. Instead, the bacteria became more deadly.
"This is one of the very few hypervirulent organisms ever created," said Lisa Morici, a lead author of the study who received her Ph.D. in infectious diseases from UC Berkeley in May. "This breaks a long-standing assumption among scientists that disabling a potential virulence gene weakens a pathogen."
Morici and Nobuyuki Shimono, assistant professor of medicine at Kyushu University's Graduate School of Medical Sciences in Japan, are co-lead authors of the paper.
The researchers point out that even though the virulent strain of TB bacteria can be grown in a lab, it is not a likely candidate for use as a biological weapon. "Mycobacterium tuberculosis grows extremely slowly, is hard to aerosolize and, if it is not in a dormant stage, can be treated with antibiotics," said Morici, who is now a post-doctoral fellow at Tulane University's School of Medicine. "There are several other virulent organisms out there that are easier to manipulate than TB."
The researchers compared the spleens, livers and lungs of mice at various time points throughout the experiment, from 24 hours to 41 weeks after infection. They found that the progression of the unmodified TB strain hit a plateau about 17 weeks after infection, while the mce1 mutated TB strain didn't stop spreading until it killed its host.
The researchers also compared the reactions to normal and mutated forms of bacillus Calmette-Guérin (BCG), a weakened version of the TB bacteria that triggers an immune response but does not lead to disease.
They found that in the unmodified strains of both the TB bacteria and the BCG groups, there were well-defined granulomas, clusters of immune cells that surround TB bacteria to keep it in check. The researchers noticed that granulomas had not formed properly in the mutated strains of both the TB bacteria and the BCG groups.
The differences suggest that the mce1 gene mutations led to changes in the TB bacteria that impacted the host's own immune response. "It appears that the host immune system does not recognize the mutated TB organisms, so the bacteria are left to grow unchecked," said Morici.
While the body typically summons granulomas to keep the TB bacteria from spreading and developing into an active infection, it does not completely eliminate the bug. When encased by granulomas, the bacteria move into a dormant, asymptomatic stage. It is when they sense a change in the host's immune system, caused by such factors as the onset of diseases including AIDS, diabetes or cancer, that they begin to multiply again and cause the active TB disease.
"What we have learned is that the granuloma shield not only protects the host, it also protects the TB bacteria from the host's other immune cells and antibiotic drugs that may otherwise kill the bacteria," said Shimono.
"The hallmark of the TB bacterium is its ability to stay dormant in a person's body for years, making it one of the most successful bacteria around," said Riley. "Even if we could treat all the people now with active infection, we'd never be able to wipe out TB entirely because 60 percent of the people exposed to TB develop latent infections. TB is very difficult to treat not because it kills people rapidly, but because it stays dormant. By understanding the mechanism behind latency, we may also be able to develop new diagnostic tests to predict who will develop the active disease."
Other co-authors of the paper are Nicola Casali, Sally Cantrell and Ben Sidders at UC Berkeley's School of Public Health; and Sabine Ehrt at Cornell University's Weill Medical College.
The National Institute of Allergy and Infectious Diseases, part of the National Institutes of Health, supported this study.
Journal
Proceedings of the National Academy of Sciences