Researchers at the University of Notre Dame and collaborators have discovered a key process for how the outer membrane of gram-negative bacteria attaches to the cell wall, advancing the understanding of how these bacteria frequently manifest resistance to antibiotics.
The research , published in the Journal of the American Chemical Society, was carried out in the laboratory of Shahriar Mobashery, Navari Professor of Life Sciences in the Department of Chemistry and Biochemistry, with structural aspects of the study performed by Juan A. Hermoso of the Institute of Physical Chemistry "Blas Cabrera" in Madrid, Spain. They discovered that the protein PA2854 performs the reaction that keeps the outside layers, or envelope, of gram-negative bacteria connected to each other.
Mobashery and collaborators studied the process in Pseudomonas aeruginosa (P. aeruginosa), a ubiquitous antibiotic-resistant bacterium commonly affecting people with cystic fibrosis. P. aeruginosa, like other gram-negative bacteria including E. coli, Klebsiella pneumoniae and Salmonella, are shielded by a three-layer biological envelope that prevent many antibiotics from penetrating and damaging the bacteria. Gram-positive bacteria do not have an outer membrane and are generally more susceptible to antibiotics.
"PA2854 functions as a glue, a way that keeps the outer membrane attached to the cell wall, a process that is necessary for the health of the organism," said Mobashery, who is affiliated with the Eck Institute for Global Health and the Warren Center for Drug Discovery . "When you abolish the ability of the bacterium to make that cross-link, the cell envelope is no longer in good shape."
The research evolved after Luis F. Avila-Cobian, one of the authors and a previous student in Mobashery's lab, identified 71 proteins in P. aeruginosa that interacted with a key, cell wall modifying protein. Avila-Cobian determined that PA2854 (annotated for the position that its gene occupies in the pseudomonal genome), one of the 71, might be a transpeptidase.
Transpeptidases are known as bacterial enzymes that assist with building a rigid, protective cell wall. This discovery led them to investigate its role in the bacterium further.
To keep the cell envelope intact, PA2854 works in tandem with lipoprotein OprI. OprI is a part of the outer membrane, and PA2854 performs a chemical reaction that links Oprl to the cell wall. Amr M. El-Araby, a doctoral student in Mobashery's lab, performed key experiments in both live bacteria and in vitro for the reconstituted system that used purified proteins and cell wall components to demonstrate the process in greater detail.
In the light of the shared structure and function for the envelope, Mobashery asserts that the discovery will apply to more than just P. aeruginosa.
"Experiments such as the one we have described are critical in understanding one by one what each protein in the bacterium does. In other words, we're demystifying functions of the genes of bacteria in hopes of understanding the collective set of biochemical processes that define the organism," said Mobashery, who is also affiliated with the Berthiaume Institute for Precision Health at Notre Dame.
The work in the United States was funded by the National Institutes of Health, the Ruth L. Kirschstein National Research Service Fellowship and the American Heart Association Predoctoral Fellowship. The work in Spain was funded by the Ministry of Science, Innovation and Universities by the State Research Agency.
