Dartmouth Research Offers Clues To New Anti-Microbial Treatments

Dr. Ronald Taylor, professor of microbiology, and Christian LaPointe, a graduate student, report a way to inhibit the enzyme that many types of bacteria need to infect and damage a variety of hosts, from plants to humans. Their work, reported in the January 14 issue of the Journal of Biological Chemistry, could provide a foundation for developing new agents to combat bacterial infections.

"In this age of antibiotics, people have come to expect a ready cure for the majority of common ailments caused by infectious microbes, from acne to ear aches. However, the microbes have been fighting back, and increasing numbers are becoming resistant to all available antibiotics at an alarming rate," says Taylor.

"These recent findings may advance screening for additional compounds that can be developed into novel therapeutic or prophylactic antimicrobial agents, at a time when many of the mainstay antibiotics are no longer useful due to the development of resistant bacteria."

Taylor's laboratory has delineated mechanisms for a common bacterial enzyme or protease that bacteria need to secrete their toxins or other virulent factors that cause damage. Treating bacteria with compounds to prevent protease function could augment therapies against a number of infectious diseases. For example protease inhibitors have been used with success to inhibit replication of the Human Immunodeficiency Virus (HIV-1) in AIDS.

Their work, says Taylor, might be a useful adjunct for cystic fibrosis treatment by inhibiting the growth of Pseudomonas that colonizes patients' lungs and is notable for resistance to antibiotics. It might lead to particularly useful approaches against infections such as meningitis by providing a way to clear bacteria without the potential complication of toxic shock that is associated with conventional treatments.

The researchers have identified the active site and biochemical pathway for type four prepilin peptidase (TFPP), a protease that cleaves the precursor form of pilin and related proteins prior to their secretion by bacteria. Pilins are protein building block subunits of hair-like fibers called pili that protrude from the bacterial surface and allow pathogenic bacteria to colonize on or in their hosts. Related proteins, termed pilin-like proteins, form channels across the bacterial membrane to facilitate the movement of toxins or other virulent factors the bacteria produce. If the TFPP function is absent, neither the pili nor the secretion apparatus can form and the pathogenic bacteria cannot spread or cause disease.

The Dartmouth researchers developed an assay to monitor TFPP activity in the laboratory and used the assay to identify a compound that inhibited the TFPP activity. They found that the compound worked the way their genetic analysis had predicted and demonstrated that the TFPPs represent a novel family, unlike other proteases.

Taylor and his colleagues tested the activity of the TFPP in the Vibrio cholerae bacterium, which the laboratory studies. The organism causes cholera, a severe life-threatening diarrheal disease spread by ingestion of contaminated water or food. Cholera is not common in the United States due to efficient sewage treatment, but it is a large problem in many areas of the world. The recent findings could also lead to therapies for use in conjunction with the primary form of cholera treatment that relies heavily on rehydrating the patient.

V. cholerae bacteria secrete two major virulent factors that are both needed to cause disease. One is cholera toxin (CT), which enters intestinal cells of an infected individual, causing them to lose copious amounts of fluid and electrolytes that leads to rapid dehydration serious enough to be fatal. The second factor is the toxin coregulated pilus (TCP) that allows the bacterium to colonize the human intestine. Without colonization, toxin production and delivery to the host cannot occur.

Each factor utilizes a different member of the TFPPs during transport outside the bacterium and both of these corresponding TFPPs, termed VcpD for toxin secretion and TcpJ for pilin secretion, were first discovered in the Taylor laboratory. These two TFPPs were the model molecules used to work out the mechanisms of action and inhibition for the TFPPs that have been identified in at least 50 bacterial species.

The research was funded by grants from the National Institutes of Health.