Apr. 15, 2003 CHAPEL HILL -- New research led by a University of North Carolina at Chapel Hill scientist shows for the first time how Mycobacterium tuberculosis, the germ responsible for TB, uses a system for releasing proteins to help it survive the lungs' immune defenses to spread and cause disease.
The study, published online in the April issue of Molecular Microbiology, also adds crucial new knowledge to the molecular factors that underlie the virulence of M. tuberculosis and may aid development of new, targeted treatments for the disease.
"I think this study moves us along in our understanding of TB pathogenesis," said study lead author Dr. Miriam Braunstein, assistant professor of microbiology and immunology at UNC's School of Medicine.
In 2003, 10 years after the World Health Organization declared tuberculosis a global emergency, tuberculosis remains a severe worldwide health threat. More people die from the disease than from any other curable infectious disease. TB kills approximately 2 million people every year, 98 percent in developing countries. One-third of the world's population is infected with the TB bacillus.
In the report, Braunstein and co-authors Drs. William R. Jacobs Jr. and John Chan from New York's Albert Einstein College of Medicine, and Drs. Benjamin Espinosa and John T. Belisle from Colorado State University said numerous disease-causing bacteria "possess specialized protein secretion systems that are dedicated to the export of virulence factors."
The TB bacillus possesses in its genome the genes secA1 and secA2, the researchers said. In a previous report, Braunstein and others had shown that the protein SecA1 is essential for the bacillus, whereas SecA2 is not. Both of these proteins are similar to SecA, a protein that functions in the secretion process of all other bacteria.
However, the presence of multiple SecA proteins in a single bacterium is highly unusual and only shared with a few other pathogenic bacteria.
"In this study, we wanted to see if the two SecAs do the same thing or have different functions. We had a hunch that SecA2 was involved in the bacteria's virulence, that it might be dedicated to secreting a specific subset of proteins involved in virulence," Braunstein said. To test that hypothesis, she and her colleagues genetically engineered a mutant strain of M. tuberculosis that did not have secA2. This gene deletion meant it could not produce the protein SecA2.
"We tested the strain for virulence by infecting mice with it and observed how long the mice survived over time," Braunstein said. "And we found that the mice infected with mutant TB survived longer than 'wild-type' mice infected with TB having a functioning secA2 gene. This told us the mutant strain was not as virulent."
To further examine virulence, the researchers also examined the extent of bacterial growth in the lung, liver and spleen. "Those experiments showed the same thing," she said. "When TB is missing SecA2, it is less virulent. There is less bacterial growth, particularly in the lung."
Having demonstrated that SecA2 is required for virulence, the next step was to identify the virulence factor secreted by the protein. Two of the proteins dependent on SecA2 that were identified were antioxidant molecules: superoxide dismutase-A and catalase-peroxidase.
When M. tuberculosis is inhaled and enters the lungs via the small air sacs called alveoli, the bacteria becomes aggressively attacked and engulfed by macrophages, immune system scavenger cells. But where other bacteria succumb to the attack, TB survives in macrophage, having evolved over the millennia a mechanism to overcome the "oxidative burst" leveled at it.
"These SecA2-dependent secretions, superoxide dismutase and catalase-peroxidase are enzymes that actually scavenge the oxygen radicals that are shot at the bacteria," Braunstein said.
"All mycobacteria strains, including TB, appear to have two secA genes. So I think a long time ago the gene duplicated benignly, but one of those secAs evolved to provide a protective advantage for the pathogen. That's why it's still there and important to pathogen survival in macrophages."
Thus, according to the researcher, the two enzymes secreted by secA2 act as virulence factors contributing to TB's defense against destruction in the macrophage. Moreover, this newly described virulence mechanism may apply to other types of disease-causing bacteria.
"In the last year or so, a number of Gram-positive bacterial pathogens have been identified that have two SecA proteins," Braunstein said. Important Gram-positive pathogens with two SecAs include Listeria monocytogenes, Staphylococcus aureus and Streptococcus pneumoniae.
"Gram-positive" refers to the grouping of bacteria relating to its outer structure.
"In some it has already been shown that the extra secA2 gene is required for virulence. So it might be common to certain bacterial pathogens."
Someday, drugs against TB infection could be developed aimed at blocking this secretion system, Braunstein said. "For now, the results of this study offer some new and important insights into the pathogenesis of this serious health threat."
The research was funded by the National Institute of Allergy and Infectious Diseases and the Howard Hughes Medical Institute.
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