Tuberculosis

It is responsible for the deaths of more people than any other single infectious disease. According to the World Health Organization, globally more than 10 million people become sick with tuberculosis each year and one in six will die from the infection. In addition to those with active disease, more than a quarter of the global population is estimated to be infected with the bacterium but maintain it in a dormant state. This latent infection can convert to active disease if immune function declines due to advanced age or HIV infection.

From 1884, when he established the first laboratory in the United States dedicated to research on tuberculosis, Dr. Edward Livingston Trudeau and his protégés pioneered treatment of the dreaded disease, culminating in antibiotic treatments that led to a cure by the 1950s. This cure, however, has remained lengthy and complex, requiring a minimum of six months of treatment on four different potentially toxic drugs. In much of the world, this treatment is challenging to deliver properly, and incomplete treatment can lead to the emergence of drug-resistant strains. Infections with these drug-resistant strains, now comprising over five percent of new cases, can require over two years to treat with treatment failing in nearly half of cases. In order to turn the tide of the tuberculosis pandemic we must develop new treatments that can effectively eliminate multi-drug resistant bacteria and reduce the treatment time and complexity.

Trudeau scientists are making use of recent developments in our understanding of how tuberculosis bacteria are able to grow and survive in the lungs in order to identify novel treatments to more quickly cure the disease. They are employing new techniques involving genetic manipulation of the bacterium, imaging of the infection within the lungs, computational modeling of protein-drug interactions, whole genome sequencing, and measuring the metabolic state of the bacterium to reveal vulnerabilities that can be exploited in the search for improved treatments.

Persister Cells

Through transcriptional profiling, we were able to identify a gene expression signature of Mycobacterium tuberculosis persister cells that survive four days of treatment with isoniazid, a lytic first-line antitubercular drug. This signature was then used to develop reporter constructs in which the regulatory regions of genes that are induced in persister cells are fused to the coding sequence of fluorescent protein genes. Initially, these constructs were delivered by mycobacteriophage, which allowed us to detect cells that are likely to be persisters in sputum from tuberculosis patients. Time-lapse imaging of M. tuberculosis cells infected with the reporter-bearing mycobacteriophage indicated that cells expressing high levels of the persister-induced reporter construct had enhanced survival during isoniazid treatment. We have employed phage-delivered reporter constructs to investigate several aspects of persister cell biology in vitro and will now extend our studies to the behavior of these cells during experimental infection.

Currently, we know that persister cells are present during infection based on the biphasic response of bacterial burden to antimicrobial drug treatment, but we are in the dark about the niches occupied by persister cells over the course of infection and treatment, particularly in relation to the well-organized structure of the characteristic lesion of human tuberculosis, the necrotic granuloma. To address these unresolved questions, we are using a recently reported iNOS knockout mouse model of tuberculosis that has been shown to develop human-like pathology and to more accurately reproduce the response to drug treatment that has been observed in human patients.

We are infecting these mice with strains bearing persister reporter constructs, and characterizing the occurrence of persister cells during the stages of infection and antitubercular drug treatment by advanced imaging techniques, with particular interest in where persister cells are found in relation to the structural and cellular features of the granuloma. The knowledge gained from these studies will be invaluable in the development of improved therapies that can clear persister cells from the niches where they survive, thus shortening treatment.