Preventing Bacterial Biofilms Could Help Fight TB

PITTSBURGH and NEW YORK-Howard Hughes Medical Institute (HHMI) researchers at the University of Pittsburgh and the Albert Einstein College of Medicine have identified a gene that enables mycobacteria-the cause of tuberculosis (TB) and leprosy-to form biofilms. Bacterial biofilms help mycobacteria resist treatment. But the researchers found that when mycobacteria closely related to the TB and leprosy pathogens lack one key protein, mature biofilms fail to form. Interrupting the gene that produces this protein, known as GroEL1, could help treat or prevent these dread diseases.

To decipher the protein's role in biofilm construction, Graham F. Hatfull, an HHMI professor at the University of Pittsburgh, collaborated with HHMI investigator William R. Jacobs Jr., at Albert Einstein College of Medicine. They discovered that GroEL1 oversees the production of a particular set of fatty acids called mycolic acids, which are necessary for biofilm growth.

Hatfull is one of 20 scientists nationwide who received $1 million each from HHMI to help bring the excitement of research into the science classroom. He works with undergraduates and Pittsburgh area high school students to identify bacteriophages, common viruses that infect bacteria. A bacteriophage infecting Mycobacterium smegmatis, a nonpathogenic cousin of Mycobacterium tuberculosis, helped launch the study that Hatfull and Jacobs report in the Dec. 2 issue of the journal Cell.

"We've defined one of the first genes and mechanisms through which mycobacteria form biofilms," said Hatfull, who also is Eberly Family Professor of Biotechnology and chair of the Department of Biological Sciences at Pitt. "Understanding biofilms is important because bacteria in biofilms are tolerant to most antibiotics, and this tolerance is a major problem in controlling TB infections."

TB infects one in three people worldwide and kills thousands each day in economically underdeveloped countries. Infections also can persist undetected for a lifetime. Biofilms could play an important role in how TB itself can hunker down and protect itself from drugs and immune effector killing mechanisms. Perhaps TB hangs out in a biofilm somewhere in the body," suggested Jacobs, who also is professor of microbiology and immunology and of molecular genetics at Einstein. "If so, an understanding of biofilm formation will provide novel ways to develop more effective drugs to fight TB and other mycobacterial infections."

Biofilms are associated with antibiotic resistance in some bacterial infections, including Streptococcus and Pseudomonas respiratory infections. For bacteria, biofilms are an important survival tool-consisting of communal layers of bacterial cells attached to a liquid or solid surface.

They stubbornly persist, hindering treatment with antibiotics. Physically, a biofilm forms a stronger, less accessible structure than a loosely clumped colony of bacteria. And metabolically, biofilm cells are believed to function in an energy-saving mode.

Jacobs and Hatfull's current study began with the unexpected observation by Hatfull's postdoctoral fellow, Anil Ohja, that a virus-infected strain of Mycobacterium smegmatis could not form proper biofilms. The virus, the mycobacteriophage Bxb1-named the Bronx Bomber by Jacobs after he isolated it from dirt in his own backyard in the Bronx, N.Y.-integrates its DNA into the middle of the mycobacterium's GroEL1 gene. This integration disrupts production of the GroEL1 protein, which belongs to a class of proteins known as chaperones that help shape and guide other proteins within the cell.

Another chaperone protein, GroEL2, is a general "housekeeping" protein that helps the cell's proteins unfold properly. But GroEL1 has a more specialized role. Without it, the mycobacteria could not construct mature, textured biofilms.

To find out how a chaperone protein might influence different growth phases, Ohja compared proteins made by mycobacteria strains with and without GroEL1. They showed that without the chaperone, the cells were lacking a key part of their fatty acid synthesis machinery. Then the group compared the fatty acids profiles of the two strains. The bacteria without GroEL1 made less fatty acid in general and none of the particular mycolic acids required to produce a biofilm.

"These studies emphasize that fatty acid synthesis is a highly regulated process that depends on the physiological growth state of the cells," said Hatfull. Researchers must do further study to find out how the chaperone causes the change in mycolic acid production, he said, but it is likely that it throws a molecular switch in the synthesis machinery.

Mycobacterium tuberculosis also has two GroEL genes, and its GroEL1 protein is 90 percent identical to the M. smegmatis GroEL1. Even though there is no direct evidence yet that

M. tuberculosis forms biofilms, Hatfull and Jacobs say it is highly likely that the two GroEL1 proteins act in similar ways to change mycolic acid synthesis-a hypothesis they plan to test next. The same mechanism also might be at work in M. ulcerans and M. leprae, which both cause painful, disfiguring diseases.

Additional researchers contributing to the study were Mridula Anand, University of Pittsburgh; Apoorva Bhatt, Albert Einstein College of Medicine; and Laurent Kremer, Universite de Montpellier II in France.

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