Microbiologists Yasu Morita and Sloan Siegrist recently were awarded a two-year, $422,500 Exploratory/Developmental Research Grant from the NIH’s National Institute of Allergy and Infectious Diseases to investigate a new strategy for interfering with a well-proven antibiotic target, cell wall synthesis. If successful, their work may suggest alternative ways to kill or control the pathogen that causes tuberculosis.
They will work in a model mycobacterial system that is a “cousin” to the one that causes tuberculosis, combining the complementary expertise of the two labs and using techniques that have not been previously combined, Morita explains. Mycobacteria cause not only TB but leprosy. Doctoral students Alam García-Heredia and Ian Sparks, with postdoctoral fellow Takehiro Kado, are also part of the team.
Moritais an expert in the lipids and carbohydrate polymers that are made in the plasma membrane, a boundary that surrounds mycobacterial cells. Once these molecules are made in the plasma membrane, they become integral parts of the multi-layered cell envelope that is crucial for mycobacteria in their response against antibiotics and the human immune system. In these experiments, he and Siegrist will focus on peptidoglycan (PG), an essential component of bacterial cell walls. Siegrist is an expert in PG assembly.
As she explains, synthesis of PG precursor building blocks begins inside of the cell, in the cytoplasm, and continues in the plasma membrane. Precursors are then “flipped” across the membrane and inserted into the existing meshwork outside of the cell. The researchers point out that the optimal conditions for these steps are at odds: synthetic reactions are helped by freely moving molecules, but cell wall integrity and bacterial viability require precise precursor insertion.Together, Siegrist and Morita seek to understand the precise coordination of the membrane-bound steps of this pathway.
TB-causing mycobacteria are rod-shaped, and growth only takes place at each end, or pole, Morita says. He explains, “Cell wall synthesis is like constructing a building. Sloan can identify the building materials, which are lipid-based membrane components, and how they are put together. I can identify the workers needed, which are proteins. We’re combining our techniques to get a better picture of the whole process. Knowing the workers and what they are making, and where they are, gives us a more convincing idea of what is happening in the cell.”
He adds, “It turns out that to build a wall specifically at the poles you need teams of different kinds of workers at different locations. Some of the workers make wall-building blocks very fast, then deliver it to the guys who carefully put the block into the wall to make polar growth happen.” Further, Siegrist says, “The overall process is optimized by teamwork, and this wasn’t known before. It turns out that building the cell wall is much more complicated than anyone expected.”
She explains, “For the rest of the field, except for Yasu, it was unexpected to find that there is higher-level organization to cell wall synthesis within the membrane. Yasu laid the groundwork, by discovering that several mycobacterial cell processes take place across different regions of the plasma membrane. Now we know that these membrane domains also organize PG assembly, and we are generalizing this concept to more species.”
Many drugs, including a new class of antimicrobial peptides, have recently been shown to be able to use “focal targeting” to attack disease-causing bacteria, Morita says. “The antimicrobial peptides get into different regions of the membrane, and one of the things they mess up is cell wall synthesis. Understanding the exact mechanism might allow us to design new antimicrobial peptides that can interfere with mycobacterial membrane pathways.”
Siegrist says, “We’re excited about this. We want to know what organizes the domains themselves, and if there is a master regulator.” The two also will study mechanisms of how mycobacteria respond to the stress of being attacked by antimicrobial peptides and other membrane-perturbing chemicals.
Morita notes, “They aren’t going to just die by being exposed to these antimicrobials, they will try to tolerate and fight back because that’s in their nature over their millions of years of experience. We present a new way to kill them, and at the same time, we have to be prepared to understand exactly how they will fight back.”