The researchers are the first to reproduce a specific component of this natural process in a test tube – an essential step to fully understanding how these structures grow.
With the new method described, these and other researchers now can delve even deeper into the various interactions that must occur for these structures – called lipopolysaccharides – to form, potentially discovering new antibiotic targets along the way.Lipopolysaccharides are composed primarily of polysaccharides – strings of sugars that are attached to bacterial cell surfaces. They help bacteria hide from the immune system and also serve as identifiers of a given type of bacteria, making them attractive targets for drugs. But before a drug can be designed to inhibit their growth, scientists must first understand how polysaccharides are developed in the first place.
The study is published in the April 25 online edition of the journal Nature Chemical Biology.
The researchers used a harmless strain of Escherichia coli as a model for this work, which would apply to other E. coli strains and similar Gram-negative bacteria, a reference to how their cell walls are structured.
The surface of these bacteria house the lipopolysaccharide, which is a three-part molecular structure embedded into the cell membrane. Two sections of this structure are well understood, but the third, called the O-polysaccharide, has to date been impossible to reproduce.
Two significant challenges have hindered research efforts in this area: The five sugars strung together to compose this section of the molecule are difficult to chemically prepare in the lab, and one of the key enzymes that initiates the structure’s growth process doesn’t easily function in a water-based solution in a test tube.
Ohio State synthetic chemists and biochemists put their heads together to solve these two problems, Woodward said.
To produce the five-sugar chain, the researchers started with a chemically prepared building block containing a single sugar and introduced enzymes that generated a five-sugar unit from that single carbohydrate.
“The first part was done chemically, and in the second part, we used the exact same enzymes that are normally present in a bacterial cell to transform the single sugar into a five-sugar string,” Woodward said.
Once these sugars join to make a five-sugar chain, a specific number of these chains are joined together to fully form the O-polysaccharide. A protein is required to connect those chains – the protein that doesn’t respond well to the test-tube environment.
Early attempts to produce this protein in the lab resulted in clumping structures that did not function. So Woodward and colleagues produced this protein in the presence of what are known as “chaperone” proteins.
“And basically what the chaperones do is help the protein fold into its correct state. We were able to produce the desired enzyme and also were able to verify that it was functional,” Woodward said.
This protein is called Wzy. It is a sugar polymerase, or an enzyme that interacts with the five-sugar chain to begin the process of linking several five-sugar units together.
Getting this far into the process was important, but the researchers also completed one additional step to define yet another protein’s role.
Wzy connected the five-sugar chains, but it did so with no defined limit to the number of five-sugar units involved, a feature that does not match the natural process. On an actual bacterial cell wall, the length of the polysaccharide falls within a relatively narrow range of the number of chains connected.
So the scientists introduced another protein, called Wzz, to the mixture. This protein is known as a “chain length regulator.” With this protein in the mix, the lengths of the resulting polysaccharides were confined to a much more narrow range.
“We were able to replicate the exact polysaccharide biosynthetic pathway in vitro, getting the correct lengths,” Woodward said. “This is important because now you can begin to look at a whole host of other properties in the system.”
The group already started trying to answer one compelling question: whether the two proteins, Wzy and Wzz, have to interact to fully achieve formation of the polysaccharide.
“We’ve shown in some preliminary results that they do interact, but we haven’t determined whether that interaction has any functional relevance,” Woodward said.
With this knowledge in hand, researchers now have access to information about how all three parts of the lipopolysaccharide, the large biomolecule on Gram-negative bacteria cell surfaces, is formed. One thing they already knew is that the entire process takes place on an inner membrane and is then exported to the outer membrane on the cell surface.
Now that scientists can reproduce formation of the lipopolysaccharide, they can more directly characterize the export process – a step in the pathway that serves as another potential antibiotic target, Woodward noted.
This work was supported by the National Institutes of Health, including its Predoctoral Trainee Program, the China Scholarship Council, the National Cancer Institute, the National Science Foundation and the Bill & Melinda Gates Foundation.
Co-authors on the study are Wen Yi, Lei Li, Guohui Zhao, Hironobu Eguchi, Perali Ramu Sridhar, Hongjie Guo, Jing Katherine Song, Edwin Motari, Li Cai, Patrick Kelleher, Xianwei Liu, Weiqing Han, Wenpeng Zhang and Mei Li, all former or current Ohio State graduate students or postdoctoral researchers in biochemistry and chemistry; Yan Ding of Shandong University in China; and Peng George Wang, Ohio Eminent Scholar and professor of biochemistry and chemistry at Ohio State.Contact: Robert Woodward, (614) 292-8704; email@example.com
Robert Woodward | EurekAlert!
Nerves control the body’s bacterial community
26.09.2017 | Christian-Albrechts-Universität zu Kiel
Ageless ears? Elderly barn owls do not become hard of hearing
26.09.2017 | Carl von Ossietzky-Universität Oldenburg
Controlling electronic current is essential to modern electronics, as data and signals are transferred by streams of electrons which are controlled at high speed. Demands on transmission speeds are also increasing as technology develops. Scientists from the Chair of Laser Physics and the Chair of Applied Physics at Friedrich-Alexander-Universität Erlangen-Nürnberg (FAU) have succeeded in switching on a current with a desired direction in graphene using a single laser pulse within a femtosecond ¬¬ – a femtosecond corresponds to the millionth part of a billionth of a second. This is more than a thousand times faster compared to the most efficient transistors today.
Graphene is up to the job
At the productronica trade fair in Munich this November, the Fraunhofer Institute for Laser Technology ILT will be presenting Laser-Based Tape-Automated Bonding, LaserTAB for short. The experts from Aachen will be demonstrating how new battery cells and power electronics can be micro-welded more efficiently and precisely than ever before thanks to new optics and robot support.
Fraunhofer ILT from Aachen relies on a clever combination of robotics and a laser scanner with new optics as well as process monitoring, which it has developed...
Plants and algae use the enzyme Rubisco to fix carbon dioxide, removing it from the atmosphere and converting it into biomass. Algae have figured out a way to increase the efficiency of carbon fixation. They gather most of their Rubisco into a ball-shaped microcompartment called the pyrenoid, which they flood with a high local concentration of carbon dioxide. A team of scientists at Princeton University, the Carnegie Institution for Science, Stanford University and the Max Plank Institute of Biochemistry have unravelled the mysteries of how the pyrenoid is assembled. These insights can help to engineer crops that remove more carbon dioxide from the atmosphere while producing more food.
A warming planet
Our brains house extremely complex neuronal circuits, whose detailed structures are still largely unknown. This is especially true for the so-called cerebral cortex of mammals, where among other things vision, thoughts or spatial orientation are being computed. Here the rules by which nerve cells are connected to each other are only partly understood. A team of scientists around Moritz Helmstaedter at the Frankfiurt Max Planck Institute for Brain Research and Helene Schmidt (Humboldt University in Berlin) have now discovered a surprisingly precise nerve cell connectivity pattern in the part of the cerebral cortex that is responsible for orienting the individual animal or human in space.
The researchers report online in Nature (Schmidt et al., 2017. Axonal synapse sorting in medial entorhinal cortex, DOI: 10.1038/nature24005) that synapses in...
Whispering gallery mode (WGM) resonators are used to make tiny micro-lasers, sensors, switches, routers and other devices. These tiny structures rely on a...
19.09.2017 | Event News
12.09.2017 | Event News
06.09.2017 | Event News
26.09.2017 | Life Sciences
26.09.2017 | Physics and Astronomy
26.09.2017 | Information Technology