In what they labeled a “surprising” finding, Johns Hopkins Medicine researchers studying bacteria from freshwater lakes and soil say they have determined a protein’s essential role in maintaining the microorganism’s shape. And because the integrity of a bacterial cell’s envelope is key to its survival, the finding could advance the search for new and better antibiotics.
The team’s research suggests that loss of a protein called OpgH in a widely studied Gram negative bacterium, Caulobacter crescentus, creates a cascade of activity that disrupts the cell envelope protecting the microorganism, resulting in cell death. OpgH is an enzyme that creates glucose-containing molecules known as osmoregulated periplasmic glucans, OPGs, which are an important constituent of the gelatinous in between space of the protective cell envelope.
“In our experiments, when we get rid of the protein OpgH in Caulobacter bacteria, which halts production of OPG sugar molecules, the bacteria can’t survive,” says Erin Goley, PhD, professor of biochemistry at the Johns Hopkins University School of Medicine.
Caulobacter crescentus bacteria are not generally thought to cause diseases, but OPGs found abundantly in Gram-negative bacteria that have this shell-like cell envelope play a role in antibiotic resistance and disease outcomes. As a result, efforts to better understand the role of the sugar molecules in gram-negative bacteria such as Caulobacter could ultimately help scientists develop new drugs that target disease-causing bacteria that have OPGs, including Brucella, Pseudomonas, Salmonella and E.coli.
If it’s found that the proteins that make or modify these OPG sugar molecules are essential to bacterial survival, Goley notes, they could be good drug targets for antibiotics themselves. Or, in organisms where OPGs are not essential, a drug that targets some part of the OPG pathway might sensitize the cells to existing antibiotics.
Goley is senior author of the team’s published paper in mBio, titled “OpgH is an essential regulator of Caulobacter morphology.” In their report the authors concluded, “Because cell envelope integrity is critical for bacterial survival, understanding how OpgH activity contributes to morphogenesis and maintenance of envelope integrity could aid in the development of antibiotic therapies.”
Bacteria inhabit an impressive range of environments and can adapt to sudden changes in environmental conditions, the authors wrote. “One parameter that can vary significantly in different niches is osmolarity, ranging from dilute freshwater habitats to highly concentrated soil.” Caulobacter crescentus is well-established as a model for physiological adaptation in the face of changing environments, and has distinct cellular structures and processes to aid its survival.
“Notably, the bacterial cell envelope serves as the physical barrier between the cell and its environment,” the team stated. The cell envelope comprises the inner and outer cell membranes and the periplasm between them. Within the periplasm is a peptidoglycan (PG) cell wall that gives the cell structure and shape. “In addition to the cell wall, some bacteria also produce glucose polymers in the periplasm, called osmoregulated periplasmic glucans (OPGs, also referred to as membrane-derived oligosaccharides),” the authors further explained.
For their reported studies the investigators used a molecular tool called an inducible promoter, which allowed them to dial down the presence of the OpgH protein in Caulobacter and observe the effects on the shape of the cell.
The team in addition discovered that loss of OpgH affects a signaling pathway known as CenKR, which identifies and repairs deficits in the cell envelope. They then manipulated the Caulobacter cells to make too much of the protein CenR, effectively hyperactivating the CenKR pathway responsible for regulating the shape of the cell envelope.
After dialing up or down the OpgH or CenR proteins, the scientists placed the bacterial cells on a pad of gel that prevented them from moving. They used a specialized microscope to observe cell shape and activity. “Loss of OpgH causes asymmetric cell bulging and lysis via misregulation of the localization and activity of morphogenetic complexes,” they notes. “Overactivation of the CenKR two-component system involved in envelope homeostasis phenocopies OpgH depletion, suggesting that depletion of OpgH activates CenKR.”
Goley explained, “We found that they became misshapen as we dialed down the OpgH protein and halted production of sugar OPG molecules, or hyperactivated the CenKR signaling pathway that maintains the cell envelope. Then we also looked at where some of the molecular players that helped to grow the cell and keep the shape of the cell were located. The molecular players were not in the correct locations, suggesting that OpgH and CenR are integral to maintaining the cell’s shape.” The authors added, “Ultimately, we determined that the bulging phenotype is due to depletion of OpgH by cenR.”
And once the cell envelope loses its shape, all of the bacteria eventually burst open and die. “We established a model for how either depleting OPGs or activating the signaling pathway affects cell shape and growth,” Goley stated.
While characterizing the sugar molecule’s role in Caulobacter’s cell structure is an important first step, Goley cautioned that “it will take some time to develop a complete picture about how they function across Gram-negative species of bacteria.
In Caulobacter, the sugar molecules resemble closed rings, and in E. coli, they look like trees, with branches sticking off chained structures. Goley acknowledged that understanding the shape and all of the decorations that associate with the molecules can help researchers characterize the cell envelope.
“In the next phase of research, we hope to investigate all of the enzymes that make, decorate and break down these molecules—so we can get a full picture of their metabolism and how they maintain the cell envelope,” Goley noted “Once we uncover how these enzymes function, that’s great, because those are things drugs can target.”
The authors concluded “We have established that OpgH, and likely OPG production, plays a crucial role in Caulobacter morphogenesis and works in concert with CenKR to maintain the integrity of the cell envelope…Our findings have established a fundamental homeostatic role for an essential OpgH homolog and have uncovered a novel connection between the OPG pathway cellular morphology, and CenKR that is ripe for future investigation.”
