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TB Wars

The Persister

Williams R. Jacobs, Jr., Ph.D., is obsessed with 0.1 percenters. Not the earners at the top of the income pyramid, but the tiny minority of tuberculosis bacteria that seemingly survive any and all attackers.

Williams R. Jacobs, Jr., Ph.D.
Williams R. Jacobs, Jr., Ph.D.
“If you treat M. tuberculosis with a powerful antibiotic like isoniazid, you kill 99.9 percent of the cells in the first four or five days,” says Dr. Jacobs, professor of microbiology & immunology and of genetics at Einstein and a Howard Hughes Medical Institute investigator. “But you just can’t kill that last 0.1 percent. Whether confronted by drugs or immune cells, these ‘persisters’ are able to activate a genetic program involving hundreds of genes that allows the bacteria to survive.”

If TB bacteria are persistent, they’ve met their match in Dr. Jacobs. For the better part of three decades—all of them spent at Einstein—he has studied these stealthy microbes, finding new and creative ways to exploit their vulnerabilities.

Dr. Jacobs is a pioneering TB researcher who first made his mark on the field in 1987, when he figured out how to manipulate the TB microbe’s genome. He did so using mycobacteriophages (or “phages,” for short), viruses that specifically infect mycobacteria. The phages he engineered were able to penetrate M. tuberculosis’ tough, waxy envelope and insert new mycobacterial DNA.

The word “revolutionary” is overused, but this discovery transformed TB research. Dr. Jacobs’ phages helped reveal how isoniazid (a first-line TB medication) disables TB bacteria. Later, he determined the genetic reason that the BCG strain of TB (used in the first and only TB vaccine) triggers an immune response but does not cause full-blown infection.

More recently, Dr. Jacobs created a rapid fluorophage test that both diagnoses the presence of TB in a sputum sample and determines whether that particular strain is susceptible to antibiotics. To do so, he took phages that infect TB bacteria and engineered them to carry a fluorescent gene taken from fireflies.

Each mycobacterial cell infected with the virus expresses the fluorescent-protein gene, causing it to reveal its presence by glowing green under the microscope (signaling that the patient has an active infection). Upon exposure to antibiotics, the bacterial strain will remain glowing if it’s drug resistant, but the green signal will fade away (along with the bacteria themselves) in the case of an antibiotic-sensitive strain. In 36 hours, the simple and inexpensive fluorophage test can pinpoint MDR-TB and XDR-TB strains. Dr. Jacobs described his fluorophage test in the Journal of Clinical Microbiology in 2012.

If the test proves successful in clinical trials, it could save countless lives in places such as sub-Saharan Africa and Central Asia, where drug-resistant TB strains are a growing concern—and where tests to diagnose active TB infections and determine their drug sensitivity are too costly and technologically complex to be used.

Phages are also playing a role in Dr. Jacobs’ promising new TB vaccine, dubbed IKEPLUS. It’s based on an ingenious transfer of genes from M. tuberculosis to M. smegmatis, a closely related mycobacterial species that is lethal to mice at high doses but does not harm people. To construct the vaccine, Dr. Jacobs and his colleagues first created a version of M. smegmatis lacking a set of genes, known as ESX-3, that enable the bacteria to evade host immunity. (This strain was designated IKE, for “immune killing evasion.”)

When high doses of the ESX-3-deficient M. smegmatis bacteria were infused into mice, the bacteria quickly succumbed to their hosts’ immune systems via a robust T-cell response—the same response a successful TB vaccine would elicit.

In July, Einstein’s William R. Jacobs, Jr., Ph.D.; Michelle Larsen, Ph.D.; Paras Jain, Ph.D.; and Oren Mayer taught the 6th Annual Mycobacterial Genetics Course at the KwaZulu-Natal Research Institute for Tuberculosis and HIV (K-RITH) in Durban, South Africa. “This is how we’re educating the next generation of African scientists,” says Dr. Jacobs.
Unfortunately, removing the same set of genes from M. tuberculosis killed the bacterium—which meant that M. tuberculosis could not be manipulated in this way to make a live vaccine. But the Jacobs team found a workaround. They took the M. smegmatis bacteria lacking ESX-3 and inserted into them the analogous set of M. tuberculosis ESX-3 genes. These viable M. smegmatis bacteria, called IKEPLUS, were then infused into mice, which fought off the infection as before. Eight weeks later, the mice were challenged with high doses of M. tuberculosis, which kills mice as well as people. These “vaccinated” mice lived two and a half times longer than control mice, as Dr. Jacobs reported in 2011 in Nature Medicine.

Equally impressive, says Dr. Jacobs, was the markedly reduced level of TB bacteria found in the animals’ tissues. “Most notably,” he said, “those vaccinated animals that survived for more than 200 days had livers that were completely clear of TB bacteria, and nobody has ever seen that before.” Just one in five mice showed this robust response in the initial experiments, indicating that IKEPLUS must be improved before it can be considered for clinical trials.

In yet another phage-related experiment, Dr. Jacobs and his colleagues are systematically deleting all 4,500 genes in the TB bacterium, one gene at a time. In this way, the researchers hope to learn more about the function of each gene by observing how TB bacteria fare without it.

“We hope our systematic evaluation of the entire TB genome will reveal vulnerabilities that we can target with new and more effective treatments and vaccines,” says Dr. Jacobs, whose work is funded in part by the National Institutes of Health and the Bill and Melinda Gates Foundation.

Quite fittingly, Dr. Jacobs’ first phages were discovered in the Bronx—in a soil sample from his own backyard. He has also found other useful phages at the Bronx Zoo, many with the help of local high school students whom he sponsors in a summer science program called “No Phage Left Behind.”

Dr. Jacobs’ enthusiasm for basic research is, well, infectious. Quite a few of his summer students have gone on to careers in science. One recent “student” was Bill Gates, who invited Dr. Jacobs to his Manhattan office in 2012 so he could learn more about research into TB. Dr. Jacobs ended up spending two hours educating the software pioneer turned global health philanthropist. “He loved it,” says the researcher.

A drug-resistant TB primer:

Multidrug-resistant TB (MDRTB):
TB that does not respond to isoniazid and rifampicin, the two most potent anti-TB drugs. Increasing numbers of TB cases are being diagnosed as MDR-TB. Compared with first-line treatment, second-line drugs for treating MDR-TB require a longer course and are more toxic, more costly and not readily available in resource-limited settings.

Extensively drug-resistant TB (XDR-TB): TB that is resistant to rifampicin and isoniazid, as well as to any member of the quinolone family of antibiotics and at least one of four second-line injectable anti-TB drugs.

  • The World Health Organization calls MDR-TB “a major public health problem” and estimates that there are 650,000 cases worldwide. About 9 percent of these patients have XDR-TB. Only a small proportion of drug-resistant cases are detected and treated appropriately.
  • Resistance to anti-TB drugs can occur when these antibiotics are misused or mismanaged— for example, when patients fail to complete their full course of treatment; when healthcare providers prescribe the wrong treatment, dose or length of time for taking the drugs; when the supply of drugs is not always available; or when the drugs are of poor quality
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