NDSU professor receives NIH R15 Award to further research in cell surface signaling

Christopher Colbert, North Dakota State University professor of chemistry and biochemistry, has received an Academic Research Enhancement Award (R15) from the National Institutes of Health for his project, “Sigma-regulator recognition and activation of regulated intramembrane proteolysis by a site-1 protease during iron import cell surface signaling in Gram-negative bacteria.”

The award will continue the work of Colbert’s research team on understanding the mechanism of iron import cell-surface signaling in Gram-negative bacteria. Cell surface signaling is the cell's way of reacting quickly to external environmental signals.

Iron is a requirement for all of life’s processes, but it is a limiting nutrient in the environment. Colbert’s research group has been studying how certain bacteria, specifically Gram-negative bacteria, bring iron into their cells and how this process also sends signals inside the cell. Bacteria expend a significant amount of energy in maintaining their iron levels.

“This award is essential for continued progress in understanding iron import by Gram-negative bacteria,” Colbert said. “Gram-negative bacteria play an important role in human health and include both species that are beneficial to humans and others that are among the most dangerous human pathogens, many of which have evolved to produce multidrug-resistant strains.”

Iron is scarce in most environments, so bacteria must work diligently to gather enough of the element to survive. This is even more challenging for Gram-negative bacteria, as they have two protective membranes, an outer membrane and an inner membrane, with a gap between them called the periplasm. To acquire iron from the outside world, they use special barrel-shaped proteins in their outer membrane called TonB-dependent transporters (TBDTs).

“Chris’s work in this area is critical to human health because new understandings of iron uptake by Gram-negative bacteria such as E. coli and Salmonella can lead to new and more effective antibiotics and treatments for these life-threatening bacterial infections,” said NDSU interim vice president for research and creative activity Heidi Grunwald.

Some TBDTS can also signal inside the cell to produce more copies of themselves when there's not enough iron, a process known as cell surface signaling. The signaling system relies on three proteins – a TBDT in the outer membrane, a sigma regulator inserted into the inner membrane, and a sigma factor that must be released into the cytoplasm to activate genes to create more iron-importing machinery.

Colbert’s lab was the first to structurally characterize the intracellular domain of this class of sigma regulators and demonstrate how this domain is functionally distinct from equivalent domains in other bacterial systems.

“We also determined the first atomic-resolution structure of the sigma regulator's periplasmic domain, which we named the C-terminal Cell Surface Signaling Domain (CCSSD), in complex with the amino-terminal signaling domain (NTSD) of the TBDT,” he said.

That structural discovery produced some unexpected findings, Colbert said, that “fundamentally altered the paradigm of iron import cell surface signaling.”

“First, we showed that the CCSSD comprises two subdomains: the carboxy-terminal juxta membrane (CJM) subdomain and a Secretin and TonB short N-terminal (STN) subdomain,” Colbert said. “The STN designation reflects that these domains typically occupy the N-terminus (beginning) of proteins. However, in the sigma regulator, the STN subdomain is paradoxically located at the C-terminus (the very end of the protein), contrary to its canonical position. Second, we discovered that the CJM subdomain adopts an entirely novel fold previously undescribed in any protein. Third, contrary to prevailing expectations, we found that the TBDT NTSD binds the CCSSD tightly and stabilizes it.”

The NIH R15 award will further support the next steps of this project, specifically by studying how the site-1 protease recognizes and interacts with the CCSSD. This interaction is crucial because the site-1 protease prepares the CCSSD for its next step, where the site-2 protease processes it further.

“This work promises to further advance our mechanistic understanding of cell surface signaling,” Colbert said. “Ultimately, these insights may facilitate the targeted antibiotic delivery strategies, such as the ‘Trojan horse’ antibiotic design that hijacks the iron uptake pathways to deliver drugs directly to specific bacterial cells, or phage therapy approaches using special viruses called bacteriophages that fight diseases by attaching to and killing harmful bacteria.”

Colbert also mentioned his team plans to study how E. coli, an important bacterium that can cause disease, utilizes iron import CSS to see if the processes they discovered are the same in this family of bacteria.

“The proposed experiments represent the key next steps in a research program that will undoubtedly generate many additional hypotheses and avenues for investigation,” Colbert said. He added that the research will provide essential knowledge to understand how iron import can be exploited for the development of novel therapeutic strategies targeting pathogenic Gram-negative bacteria. Additionally, this research has another benefit for North Dakota: developing and training personnel for the state’s workforce.

“Training in biochemistry opens numerous career pathways beyond academic research. In addition to pharmaceutical and biotech industries, it provides a strong foundation for fields such as biomedical patent law and science communication,” Colbert said. “The funding will enable me to provide critical research opportunities for undergraduate and graduate students in a STEM field that has experienced continuous growth over the past two decades. This grant supports my mission to train the next generation of scientists, giving them immediate opportunities to contribute to peer-reviewed publications. This credential will prove invaluable to their careers.”

Colbert’s interest in metalloproteins began during his graduate studies at Purdue and further developed during his postdoctoral work at the University of Texas Southwestern Medical Center. When he arrived at NDSU in 2010, this interest led him to establish the study of import cell surface signaling in Gram-negative bacteria as a primary research theme for his laboratory.

Colbert’s research is supported by NIH Grant R15GM160942, a continuation of his recently completed NIH Grant R01GM126207.