By Marla Vacek Broadfoot
An unconventional path to a potential cure
Lalita Ramakrishnan considers herself a jack of all trades and a master of none, which is a funny comment for a scientist who has garnered a number of accolades -- including a Burroughs Wellcome Fund Investigator in the Pathogenesis of Infectious Disease Award. But for a free spirit who has followed her gut instinct to switch career paths, disciplines, even disease areas, that description isn’t too far off the mark. Yet her unconventional approach has landed numerous publications in prestigious scientific journals such as Science and Cell, the most recent of which is turning the field of tuberculosis research on its head.
Ramakrishnan went to medical college when she was just seventeen, which was not atypical in India, where specialized training begins shortly after high school. It didn’t take long for her to see that medical school didn’t suit her. In class, Ramakrishnan felt suffocated by what she described as a rote, repetitive atmosphere and apathetic professors fond of insults, with only a few notable exceptions. In the clinic, she was saddened to witness so much suffering and poverty mixed with social problems that were beyond her reach.
“There was just too little that I could do, and that is when I started to realize that I wanted to understand things more so I could change them,” explained Ramakrishnan.
While in the throes of residency at a city hospital, she applied to a number of Ph.D. programs in the States. Ramakrishnan still considers the day she received her first acceptance letter for graduate school one of the happiest days of her life, because she suddenly saw a way out. But with laughter in her voice, she admits that her first days at Stony Brook, where she chose to matriculate, were “another set of misery.”
Ramakrishnan struggled to keep up with her coursework in the molecular biology graduate program, quickly realizing that her training from medical school was little preparation for advanced courses like physical chemistry and biochemistry. Luckily, she found support among the fellow residents of her dorm, which was full of foreigners like her because as she joked “no self-respecting American graduate student would have lived there.” One of those fellow graduate students, having just escaped the Mao suppression in China, was also her TA. He kindly taught Ramakrishnan bits and pieces of calculus at night. She eventually earned an A in physical chemistry and began to get comfortable. Then an elective course in advanced immunology caused Ramakrishnan to shift gears yet again.
“I could see obvious connections to disease and how to relate all of that information to medical problems that I had seen,” said Ramakrishnan.
So she looked around for immunology programs and was accepted in transfer to graduate school at Tufts, before going on to be the first foreign graduate at the medical residency program at Tufts/New England Medical Center. Deciding to specialize in infectious diseases, she moved to the University of California at San Francisco to combine further clinical training with a postdoc studying hepatitis B, which she considered an “intellectual bridge” from her thesis on immunoglobulin genes and retroviruses. But once she arrived at UCSF, her instinct veered toward tuberculosis, which she realized was in dire need of medical breakthroughs. She was intrigued by several reviews written by Stanley Falkow on how intracellular bacteria (of which the tuberculosis –causing TB bacillus is one) affect the host and how in turn the host affects them. Ramakrishnan convinced him to let her come to his lab, and earned her own keep working ER shifts until she was awarded a Howard Hughes Medical Institute postdoctoral fellowship to study TB.
More than two billion people, one third of the world’s population, are infected with the TB bacillus. The illness is endemic to poverty-stricken regions, where overcrowding and poor ventilation foster its spread. Treating TB takes at least six months and multiple antibiotics, presenting a challenge to patients who can feel cured much earlier. This long, drawn-out, and sometimes inadequate therapy is the perfect breeding ground for multidrug tolerance, giving microbes ample time to evolve into impervious forms.
“The general feeling is if that barrier could be overcome, if the treatment could be substantially shortened, that it would be huge boost for global control of TB,” said Ramakrishnan.
For decades, researchers have thought that TB was tough to treat because the bacteria go to sleep after infecting a host, becoming less sensitive to antibiotics that target actively replicating bugs. But Ramakrishnan’s recent research suggests that explanation gives only part of the picture. While “playing around” with her zebrafish model of the disease, she noticed that the small pockets of bacteria left after antibiotic treatment were not in fact dormant. Rather, they escaped death by growing within the body’s garbage trucks, cells called macrophages that clean up waste and can kill many bacteria.
When one of Ramakrishnan’s graduate students looked at the TB bug in cultured human macrophages, she found that growing populations were more likely to be tolerant than dormant ones, and that this tolerance was enriched within macrophages. Interestingly, being inside a macrophage made bacteria tolerant to multiple antibiotic classes, whether they targeted cell wall agents, gyrase inhibitors, or ribosome blockers.
There were two possible explanations for these findings. In the first, the bacteria would change inside the macrophage, perhaps toughening up their cell wall, such that no antibiotic could get in. In the second, the antibiotics would get in, but then something within the macrophage-dwelling bugs would pump them back out again. The researchers tested the latter by using drugs known to counter such pumps – called “efflux pumps,” – a kind of septic system that pumps out toxic substances from the cell. Lo and behold, the drugs made the TB bug more sensitive to antibiotics. And they found that genetic mutations that disrupt the pump affected both drug susceptibility and growth.
“Once we knew that the efflux pump was also mediating growth within macrophages, suddenly everything made sense,” said Ramakrishnan. “It was one of those moments where all of those pieces of evidence that we had gathered over the years fit together, like a hand in a glove. The pump was being turned on to push out some noxious substance produced by the macrophage that keeps it from growing, and just happens to also pump out the drug. It is because of this result that we understand why it is the growing bacteria that are the tolerant ones.”
The result could lend insight into not just TB but also other intracellular pathogens, which may have similar mechanisms of survival. Though Ramakrishnan laughs that if she could just cure TB, it would be good enough for her. Talk of a cure may seem odd for a self-described jack of all trades, particularly as Ramakrishnan is not so focused on one aspect of scientific inquiry as her older brother, Venkatraman, who won the Nobel Prize in chemistry in 2009.
“If you go after a single biological mechanism, say you want to understand protein secretion, then you may have a little more of a chance of becoming a master,” said Ramakrishnan. “But if you go after TB, then you have to follow whatever the bug is telling you. My three papers in the journal Cell just go to show that I am not master of anything, because they uncover three distinct biological pathways. Yet I am finding my way.”