Attacking the attacker
Understanding the structure of bacterial cell walls to defeat tuberculosis
June 26, 2017
Story and photo by David Edwards
June's featured paper is titled, "The Mycobacterium tuberculosis MmpL11 cell wall lipid transporter is important for biofilm formation, intracellular growth and non-replicating persistence," published in Infection and Immunity. The project was led by Georgiana Purdy, Ph.D., associate professor of molecular microbiology and immunology, OHSU School of Medicine. Its authors, in descending order, are Catherine Wright, a research associate in the Purdy lab; Fong Fu Hsu, research professor of medicine at the Washington University in St. Louis School of Medicine; Eusondia Arnett, Ph.D., a postdoctoral fellow at The Ohio State University; Jennifer L. Dunaj and Patrick M. Davidson, both research assistants in the Purdy lab; Sophia A. Pacheco, a researcher in the Purdy lab; Melanie J. Harriff, Ph.D., assistant professor of medicine, Division Pulmonary/Critical Care Medicine, OHSU School of Medicine; David Lewinsohn, M.D., Ph.D., assistant professor of medicine, Division Pulmonary/Critical Care Medicine, OHSU School of Medicine; Larry S. Schlesinger, M.D., chair of the Department of Microbial Infection and Immunity, Ohio State University, and Dr. Georgiana Purdy.
Under the cover of tuberculosis
The human body is constantly under assault. Although modern medicine has greatly improved our ability to survive these attacks, there are significant challenges in our constant fight against the attackers, including insufficient access to medical care and the development of drug resistance.
One example of this health care struggle is tuberculosis, a respiratory infection caused by the bacterium Mycobacterium tuberculosis (abbreviated MTB), which affects millions of people around the world. The World Health Organization estimates that one-third of the world's population is infected by MTB. However, drug resistance is a significant problem in treating this disease. To overcome the development of multiple drug-resistant tuberculosis, it is critical to gain a better understanding of basic MTB biology.
One avenue of research is into the outer structure of MTB, called the cell wall, which provides structural integrity to the bacteria. The mycobacterial cell wall is crucial to the interaction between the host and the invading pathogen since it provides a barrier against antibiotics and against the natural immune response from the host itself. In addition, the fat molecules contained in the cell wall, called lipids, are mycobacterial virulence factors, or things that help the bacteria to colonize the host organism and evade its defenses.
The lipids contained in the cell wall are transported by a family of proteins called MmpL (mycobacterial membrane rotein large). MmpLs are not only critical for basic MTB function, but they also contribute to the ability of MTB to spread and sustain tuberculosis.
Recently, one of the MmpL family members, MmpL11, was previously shown to be pathogenic in mice, especially in the later stages of chronic, long-term infection. However, these studies did not identify how genetic mutations in MmpL would impact its function or identify the particular lipid(s) transported by MmpL11.
Behind the lipid transporter
Previous research by Dr. Georgiana Purdy has addressed some outstanding questions of MmpL11 function. For example, her group has shown that MmpL11 is important for a process called "biofilm formation," whereby bacteria form organized communities of cells attached to a surface, one of the hallmarks of drug tolerance.
This prior research was done in a non-pathogenic model organism commonly used to study tuberculosis. (Instead of experimenting on Mycobacterium tuberculosis directly, they use Mycobacterium smegmatis, a similar organism that allows for increased experimental flexibility.) Now, they have characterized the function of this protein in Mycobacterium tuberculosis itself.
To do this, they created a mutated form of MmpL11 in MTB, one which didn't function normally, and compared the effects of the mutant version of MTB to its non-mutated counterpart. Similar to their prior research, they observed that MTB MmpL11 mutants have reduced biofilm formation. In addition, they conducted a biochemical analysis of the lipids on the surface of mutant and non-mutant MTB and found that a group of lipid family members – specifically, long-chain triacylglycerol (TAG) and mycolate wax ester (MWE) molecules – were differentially transported, suggesting that MmpL11 controls their transport.
To investigate the role of MmpL11 in the virulence of tuberculosis, the Purdy group took advantage of another model system, an in vitro granuloma model, created isolating peripheral blood mononuclear cells (PBMCs) from humans with a latent infection of tuberculosis. Using this model, they showed that the MmpL11 mutant cells had a lower survival rate compared to non-mutant cells after exposure to a simulated host immune response, suggesting that MmpL11 is important to MTB in defending itself against an immune system attack.
In addition, they exposed both mutant and non-mutant cells to starvation conditions (lacking both nutrients and oxygen), which is meant to simulate the local environment the cells would experience inside the host. They found that the mutant cells had impaired survival and resuscitation compared to non-mutant cells, again implicating MmpL11 as a critical contributor to MTB survival against a host attack.
Beyond MmpL11 and next steps
The majority of tuberculosis infections result in "latent" tuberculosis, where the bacteria have an altered metabolism and already show signs of drug tolerance. Finding proteins that are required for survival and resuscitation, including MmpL11, are critically important in overcoming that tolerance and represent potential targets for therapy.
"The majority of individuals infected with M. tuberculosis have latent disease where the bacterium is in a 'persistent' state and is contained in a granuloma," wrote Dr. Purdy, senior author of the paper. "Combined these data suggest that MmpL11 and its substrates are important for intracellular growth, non-replicating persistence and latency."
The results of this research, wrote Mary Heinricher, Ph.D., associate dean for basic research, OHSU School of Medicine, are intriguing because they demonstrate "That this single protein has such a pivotal role in these very different but important aspects of mycobacterial function."
In the future, the Purdy group plans on studying other MmpL family members, especially those associated with virulence. Not only will their research to help improve our understanding of how the mycobacterial cell wall changes during different phases of infection, but also to potentially develop new drugs to inhibit similarly critical MTB proteins.
The Mycobacterium tuberculosis MmpL11 cell wall lipid transporter is important for biofilm formation, intracellular growth and non-replicating persistence. Infect Immun, 2017 May. Catherine C. Wright, Fong Fu Hsu, Eusondia Arnett, Jennifer L. Dunaj, Patrick M. Davidson, Sophia A. Pacheco, Melanie J. Harriff, David M. Lewinsohn, Larry S. Schlesinger, and Georgiana E. Purdy.
More Published Papers
Pictured above: Members of the Purdy lab.
About the OHSU School of Medicine Paper of the Month
The OHSU School of Medicine spotlights a recently published faculty research paper each month. The goals are to describe to the public the exceptional research happening at OHSU as well as inform our faculty of the innovative work underway across the school’s departments, institutes and disciplines. The monthly paper is selected by Associate Dean for Basic Research Mary Heinricher, Ph.D. Learn more