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November 20, 2015
THE SEE-THROUGH PHASE
Zebrafish Offer View of TB Pathogenesis

Dr. Lalita Ramakrishnan

Dr. Lalita Ramakrishnan

In addition to killing more than 1.5 million people a year globally—according to 2013 data from the World Health Organization— tuberculosis is a disease of irony and paradox.

Irony because, although effective antibiotic treatments were developed in the 1960s—and are still being used today—TB remains widespread. “It is killing more people today than it has ever killed before,” said Dr. Lalita Ramakrishnan, who presented “The Zebrafish Guide to Tuberculosis” at the Wednesday Afternoon Lecture on Oct. 7. “Multidrug resistant tuberculosis is quite widespread. TB is now the lethal disease that it was in the pre-antibiotic years.”

Paradox because you could drop a bucket of TB-infected material on a person and not transmit the disease. It is the tiny, aerosolized particles of perhaps 1 to 3 bacteria-laden droplets that get into the alveolar spaces of the deep lung that transmit TB. The bacteria enter macrophages, where the bug has learned how to survive, causing pneumonia in the lymphoid tissues of the lungs. From there, it can proceed to other organs in the body.

The worst outcome is when TB migrates to the base of the brain, causing TB meningitis—death is the result in between 20-40 percent of cases.

Now at Cambridge University in the U.K., Ramakrishnan, who is a past winner of an NIH Pioneer Award, almost accidentally discovered zebrafish as a model for studying TB pathogenesis.

She was poolside with a clinical mentor during an infectious diseases fellowship at the University of California, San Francisco, when he suggested that zebrafish, which are susceptible to Mycobacterium marinum—the closest genetic relative of the human TB pathogen—might be a good model organism for studying the TB lifecycle and how to interrupt it.

M. marinum had been identified in 1926 when fish in the now-defunct Philadelphia Aquarium began dying of a TB-like disease. It can infect humans, too, but only causes skin lesions and soft-tissue infections.

“But in fish, it looks very much like the human disease,” said Ramakrishnan.

Ramakrishnan says, “If we didn’t have poverty, I’d be out of business.”
Ramakrishnan says, “If we didn’t have poverty, I’d be out of business.”

PHOTOS: ERNIE BRANSON

During the first 2-3 weeks of life, zebrafish go through a transparent phase. Ramakrishnan recognized this as a perfect opportunity to conduct real-time analysis of the pathogenesis process in live animals. “We can study infection in great depth, from the first phase onward.”

There were technical hurdles to surmount. Pull out a penny and observe the size of Lincoln’s cravat. That is the size of a zebrafish. Ramakrishnan’s colleagues learned to microinject M. marinum into the hindbrain ventricle of the fish, then observe, under high-magnification, the bug’s penchant for invading specific macrophages.

Ramakrishnan has learned much about TB’s evolutionary strategy, which has been successful enough that an estimated one-third of the global population carries latent TB infection. “It’s a good example of pathogen-host co-evolution,” she said. Her work has been like precision medicine in action—there is a high-inflammatory (increased production of tumor necrosis factor) genotype, for which the appropriate therapy is steroids, and a low-inflammatory genotype, for which the leukotriene-inhibitor zilueton is effective, at least in zebrafish.

In an effort to understand why it takes 6-9 months to treat TB, Ramakrishnan and her team learned that bacteria have efflux pumps that get activated when they are inside macrophages and that pump out administered antibiotics.

During a brief Q&A session, Ramakrish-nan divulged that her mother was infected with TB, but was eventually treated successfully with rifampicin.

She also noted, “The TB control program in India is a disaster.”

Social problems confound not just TB programs, but many interventions in global disease. Concluded Ramakrishnan, “If we didn’t have poverty, I’d be out of business.”

The full lecture is available at http://videocast.nih.gov/summary.asp?Live=17256&bhcp=1.

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