“Molecular scissors” for plastic waste

A research team from the University of Greifswald and Helmholtz-Zentrum-Berlin (HZB) has solved the molecular structure of the important enzyme MHETase at BESSY II.

MHETase was discovered in bacteria and together with a second enzyme – PETase – is able to break down the widely used plastic PET into its basic building blocks. This 3D structure already allowed the researchers to produce a MHETase variant with optimized activity in order to use it, together with PETase, for a sustainable recycling of PET. The results have been published in the research journal Nature Communications.

Plastics are excellent materials: extremely versatile and almost eternally durable. But this is also exactly the problem, because after only about 100 years of producing plastics, plastic particles are now found everywhere – in groundwater, in the oceans, in the air, and in the food chain. Around 50 million tonnes of the industrially important polymer PET are produced every year. Just a tiny fraction of plastics is currently recycled at all by expensive and energy-consuming processes which yield either downgraded products or depend in turn on adding ‘fresh’ crude oil.

>Read more on the BESSY II at HZB website

Image: At the MX-Beamlines at BESSY II, Gottfried Palm, Gert Weber and Manfred Weiss could solve the 3D structure of MHETase.
Credit: F. K./HZB

Structural insights into tiny bacterial harpoons

Bacteria produce complex nano-harpoons on their cell surface. One of their functions is to harpoon and inject toxins into cells that are close by. Producing such a complex weapon requires lots of different moving components that scientists are still trying to understand. Researchers from the University of Sheffield have been using some of Diamond’s crystallography beamlines to understand a particularly enigmatic piece of this tiny puzzle. The team led by David Rice and Mark Thomas worked on a protein component of the harpoon called TssA which they already knew was an integral piece of the machinery. However, unlike the other components of the harpoon, there are distinct variants of the TssA protein that contain radically different amino acid sequences at one end of the protein. The team showed that the structures of the variable region of two different TssA subunits were completely unrelated and they could assemble into distinctly different multisubunit complexes in terms of their size and geometry. This begged the question as to how different bacteria could use this protein with different structures to produce a harpoon with the same function across all species. They found that despite these differences, there was a very specific conserved region at the other end of the protein. They hypothesise that the conserved region is the part that does the work and helps the harpoon to function whereas the variable region acts as a scaffold. They used I02, I03 and I24 in their study and plan to do follow up work using X-ray crystallography and Cryo-EM such as those at the eBIC centre at Diamond. The research was published in Nature Communications.

>Read more on the Diamond Light Source website

Image: Macromolecular Crystallography (MX) at Diamond reveals the shape and arrangement of biological molecules at atomic resolution, knowledge of which provides a highly accurate insight into function. 

Enzyme structure of bacteria that causes tuberculosis

Results on its interaction with antibiotics may lead to the development of new forms of treatment for this disease.

Tuberculosis is a chronic infection usually caused by a bacterium called Mycobacterium tuberculosis. This bacterium infects cells of the immune system called alveolar macrophages, which are responsible for removing pollutants and microorganisms from the surface of the alveoli, where the exchange of gases occurs during respiration.
It is estimated that approximately two billion people worldwide are infected with M. tuberculosis without symptoms. However, the clinical manifestations of the disease may appear at any time in life, especially when the immune system is weakened, such as due to malnutrition or diseases such as cancer and AIDS.
Tuberculosis is considered a curable disease when the patient is diagnosed and treated promptly with antibiotics. Nevertheless, the chronicity of this infection makes it difficult to eradicate bacteria altogether. Generally, patients must take the medication for several months, making it harder for them to persist in the treatment and favoring the emergence of antibiotic-resistant bacteria. In recent years, the emergence of new bacteria, resistant to routine treatments, has been a worldwide concern and it is imperative to seek new therapeutic strategies against this disease.

>Read more on the Brazilian Synchrotron Light Laboratory (LNLS) 

Image: (extract, full image here) Elements of the secondary structure of L,D-transpeptidase-3 from Mycobacterium tuberculosis acylated by an acetyl fragment derived from faropenem. Beta sheets in red, α-helices in yellow and the loops are shown in green. The figure shows, at the amino terminus (N-ter), the bacterial domain similar to immunoglobulin (BIg) and in the carboxy terminus the catalytic domain (CD). B-loop is a unique structure of this enzyme when compared to the other M. tuberculosis L,D-transpeptidases. In blue is shown an acetyl fragment covalently attached to cysteine 246 at the active site of the enzyme. Figure taken with Pymol.

A two-pronged defense against bacterial self-intoxication

Researchers solved the structure of a bacterial toxin bound to a neutralizing protein, revealing two distinct mechanisms for how the toxin-producing bacteria avoid poisoning themselves.

Microbial communities are of fundamental importance to virtually all natural ecosystems, from the ocean floor to the gastrointestinal tract. Although the term “communities” implies cooperation, scientists now realize that bacterial colonies compete with each other for life-sustaining resources, availing themselves of a variety of strategies to reduce overcrowding. In some cases, they secrete toxins in their fight for survival. Here, researchers studied one such toxin from the bacterium Serratia proteamaculans, various strains of which live inside tree roots or inhabit the digestive tracts of insects and other animals.

Toxin targets cell division

The researchers showed that the toxin, Tre1, targets a bacterial protein, FtsZ, which is analogous to tubulin in human cells. Tubulin molecules are the building blocks of microtubules—long polymers that provide structure and shape to our cells and play an important role in cell division. In bacteria, FtsZ loses the ability to polymerize when attacked by the Tre1 toxin. Instead of dividing, the intoxicated cells grow longer and longer until they eventually split open and die (cellular elongation and lysis).

>Read more on the Advanced Light Source website

Image: Healthy bacteria (left) and bacteria (right) whose cell-division machinery has been disrupted by a toxin newly discovered in some bacterial arsenals.
Credit: Mougous Lab

Know your ennemy

Light source identifies a key protein interaction during E. coli infection

Escherichia coli is a common source for contaminated water and food products, causing the condition known as gastroenteritis with symptoms that include diarrhea, vomiting, fever, loss of energy, and dehydration. In fact, for children or individuals with weakened immune systems, this bacterial infection in the gut can be life-threatening.

One of the microbes responsible for gastroenteritis, known formally as enteropathogenic E. coli (EPEC), causes infections by directing a pointed, needle-like projection into the human intestinal tract, releasing toxins that make people sick.

“Enteropathogenic E. coli can fire toxic proteins from inside the bacterium right into the cells of your gut lining,” says Dustin Little, a post-doctoral researcher in the Brian Coombes lab at McMaster University’s Department of Biochemistry and Biomedical Sciences.

>Read more on the Canadian Light Source website

Image: Dustin Little and Brian Coombes in the lab.
Credit: Dustin Little. 

Scientists produce 3-D chemical maps of single bacteria

Researchers at NSLS-II used ultrabright x-rays to generate 3-D nanoscale maps of a single bacteria’s chemical composition with unparalleled spatial resolution.

Scientists at the National Synchrotron Light Source II (NSLS-II)—a U.S. Department of Energy (DOE) Office of Science User Facility at DOE’s Brookhaven National Laboratory—have used ultrabright x-rays to image single bacteria with higher spatial resolution than ever before. Their work, published in Scientific Reports, demonstrates an x-ray imaging technique, called x-ray fluorescence microscopy (XRF), as an effective approach to produce 3-D images of small biological samples.

“For the very first time, we used nanoscale XRF to image bacteria down to the resolution of a cell membrane,” said Lisa Miller, a scientist at NSLS-II and a co-author of the paper. “Imaging cells at the level of the membrane is critical for understanding the cell’s role in various diseases and developing advanced medical treatments.”
The record-breaking resolution of the x-ray images was made possible by the advanced capabilities of the Hard X-ray Nanoprobe (HXN) beamline, an experimental station at NSLS-II with novel nanofocusing optics and exceptional stability.
“HXN is the first XRF beamline to generate a 3-D image with this kind of resolution,” Miller said.

>Read more on the NSLS-II at Brookhaven National Laboratory website

Image: NSLS-II scientist Tiffany Victor is shown at the Hard X-ray Nanoprobe, where her team produced 3-D chemical maps of single bacteria with nanoscale resolution.

Mycoplasma genitalium’s cell adhesion mechanism revealed

Mycoplasma genitalium is a sexually transmitted bacterium responsible for several genitourinary disorders.

An estimated 1% of the adult population is infected with this bacterium. Using XALOC beamline at the ALBA Synchrotron it has been defined the structure of the protein involved in the pathogen’s adhesion process. The discovery opens the door to defining new therapeutic strategies to fight this pathogen which is becoming more and more resistant to antibiotics.

Researchers from the Molecular Biology Institute of Barcelona (IBMB-CSIC) and the Institute of Biotechnology and Biomedicine (IBB-UAB) have discovered the mechanism by which the bacterium Mycoplasma genitalium (Mgen) adheres to human cells. This adhesion is essential for the onset of bacterial infection and subsequent disease development.
Mgen is an emerging pathogen responsible for several infectious genitourinary disorders. In men, it is the most common cause of urethritis (15-20%) while in women, it has been associated with cervicitis, pelvic inflammatory disease, premature birth and spontaneous abortions. So far, it was known that adherence to the genitourinary tract was possible thanks to proteins known as adhesins, which recognise specific cell surface receptors.
In this study, IBMB-CSIC researchers determined the three-dimensional structure of the Mgen’s P110 adhesins interacting with these cell receptors using X-rays diffraction and protein crystallography at the XALOC beamline. “We made a protein crystal of the P110 adhesin bound to these receptors and diffracted with the synchrotron’s X-rays to determine the exact position of the atoms within the protein, and we were able to decipher the three-dimensional structure”, explains IBMB researcher David Aparicio.

>Read more on the ALBA website

Image: Overall structure of P110. Two views, 90° apart from each other, of the extracellular region of P110 that is formed by a large N-domain, with a seven blade β-propeller (green), the crown (brown), and the C-domain (orange). In the right side panel the view is along the central axis of the β-propeller. The situation of the seven blades in the propeller is explicitly indicated showing that the two terminal blades I and VII are close to the C-terminal domain and opposite to the crown.


Targeting bacteria that cause meningitis and sepsis

The work provides molecular-level information about how the antibody confers broad immunity against a variable target and suggests strategies for further improvement of available vaccines.

Our central nervous systems (brain and spinal cord) are surrounded by three membranes called “meninges.” Meningitis is caused by the swelling of these membranes, resulting in headache, fever, and neck stiffness. Most cases of meningitis in the United States are the result of viral infections and are relatively mild. However, meningitis caused by bacterial infection, if left untreated, can be deadly or lead to serious complications, including hearing loss and neurologic damage.

The bacterium responsible for meningitis (Neisseria meningitidis) can also infect the bloodstream, causing another life-threatening condition known as sepsis. N. meningitidis is spread through close contact (coughing or kissing) or lengthy contact (e.g. in dorm rooms or military barracks). In this work, researchers were interested in understanding how humans develop immunity to bacterial meningitis and sepsis, collectively known as meningococcal disease, by vaccination with a new protein-based vaccine.

>Read more on the Advanced Light Source website

Image: The work provides molecular-level information about how the antibody confers broad immunity against a variable target and suggests strategies for further improvement of available vaccines.

Scientists unravel mechanism for body odour in armpits

British researchers from the University of York and the University of Oxford have shown the mechanism that leads to body odour in armpits by studying the molecular process at the ESRF and other lightsources.

Stepping into a cramped bus on a hot summer day can sometimes translate into having to hold your breath and a very unpleasant experience. Sweat production increases in hot weather, and, with it, body odour. Despite much research and antiperspirant deodorants, scientists still haven’t managed to selectively block body odour.

Researchers from the University of York and the University of Oxford have recently used the ESRF and Diamond Lightsource to find out what happens at a molecular level when we smell badly. They focused on the apocrine gland, which is found only in the armpit, genitalia and ear canal. It secrets an odourless lipid-rich viscous secretion, which is likely to play a role in scent generation, but it is not involved in thermoregulation.

It all comes down to bacteria. “The skin of our underarms provides a unique niche for bacteria,” explains investigator Gavin Thomas, professor in the department of biology at the University of York and co-leader of the study. “Through the secretions of various glands that open onto the skin or into hair follicles, this environment is nutrient-rich and hosts its own microbial community, the armpit microbiome, of many species of different microbes.”

>Read more on the European Synchrotron (ESRF) website

Image: Picture showing how body odour is produced in armpits.
Credit: University of York and Oxford. 

How legionella manipulates the host cell by means of molecular mimics

Using synchrotron light, researchers from CIC bioGUNE have solved the structure of RavN, a protein that Legionella pneumophila uses for stealing functions and resources of the host cell.

Mimicry is the ability of some animals to resemble others in their environment to ensure their survival. A classic example is the stick bug whose shape and colour make him unnoticed to possible predators. Many intracellular pathogens also use molecular mimicry to ensure their survival. A part of a protein of the pathogen resembles another protein totally different from the host and many intracellular microorganisms use this capability to interfere in cellular processes that enable their survival and replication.

The Membrane Trafficking laboratory of the CIC bioGUNE in the Basque Country, led by Aitor Hierro, in collaboration with other groups from the National Institutes of Health in the United States, have been working for several years in understanding how the infectious bacterium Legionella pneumhopila interacts with human cells. During this research, experiments have been carried out at the XALOC beamline of the ALBA Synchrotron and I04 beamline of Diamond Light Source (UK). The results enabled scientists to solve the structure of RavN, a protein of L. pneumophila that uses this molecular mimicry to trick the infected cell.

>Read more on the ALBA website

Figure: (extract) Schematic representation of the structure of RavN1-123 as ribbon diagram displayed in two orientations (rotated by 90° along the x axis). Secondary elements are indicated as spirals (helices) or arrows (beta strands), with the RING/U-box motif colored in orange and the C-terminal structure colored in slate. (Full image here)

UBC scientists break down tuberculosis structure

Scientists from the University of British Columbia have taken a crucial step towards starving out tuberculosis, following research into how the infection grows in the body.

Tuberculosis, a bacterial infection which generally affects the lungs, is a global threat; worldwide, it kills more people than HIV and malaria combined. In Canada, there are around 1,600 new cases of tuberculosis reported every year, with about 20 per cent of those cases affecting First Nations peoples, according to the Government of Canada. Researchers using the Canadian Light Source have investigated how the bacteria grow in lungs in an effort to better understand how tuberculosis can be treated.

Lindsay Eltis, a UBC professor of Microbiology and Immunology and Canada Research Chair in Microbial Catabolism and Biocatalysis, has spent the last 25 years studying bacteria and determining how they grow on different compounds. In 2007, Eltis’ group discovered that tuberculosis bacteria grow on cholesterol and that this is important for causing disease.

“Many bacteria, like humans, grow using glucose, a type of sugar. They derive energy from it, converting it to water and carbon dioxide, and use it to make building blocks essential to life. The tuberculosis bacterium is a bit unusual in that it can grow on cholesterol, deriving energy and essential building blocks from it,” explains Eltis. “This ability to grow on cholesterol helps the bacterium establish infection in our lungs.”

>Read more on the Canadian Light Source website

Image: Crystal structure of the newly imaged carbon-ring cleaving enzyme from the tuberculosis bacterium, IpdABMtb.
Credit: Lindsay Eltis

ALS passes the 7000-protein milestone

The eight structural biology beamlines at the ALS have now collectively deposited over 7000 proteins into the Protein Data Bank (PDB), a worldwide, open-access repository of protein structures. The 7000th ALS protein structure (entry no. 6C7C) is an enzyme from Mycobacterium ulcerans (strain Agy99), solved with data from Beamline 5.0.2. This bacterium produces a toxin that eats away at skin tissue, causing what’s known as Buruli ulcers (Google at your own risk!). The bacterium is antibiotic-resistant, and treatment involves the surgical removal of infected tissues, including amputation.

The enzyme structure was solved by a group from the Seattle Structural Genomics Center for Infectious Disease (SSGID), whose mission is to obtain crystal structures of potential drug targets on the priority pathogen list of the National Institute of Allergy and Infectious Diseases (NIAID). As of May 2018, SSGCID has deposited 1090 structures in the PDB, with data for more than a quarter of those collected at ALS beamlines.

>Read more on the Advanced Light Source website

Image: PDB 6C7C: Enoyl-CoA hydratase, an enzyme from M. ulcerans (strain Agy99).

How dolphins could potentially lead to new antibiotics

The world is currently living through a multidrug resistance problem, where antibiotics that traditionally work are not effective anymore. A European team of scientists at the University of Hamburg (Germany), University of Munich (Germany), University of Bordeaux (France), University of Trieste (Italy) and University of London (UK) have studied how some peptides in dolphins target bacterial ribosomes and hence, could provide clues about potential new antibiotics.

Proline-rich antimicrobial peptides (PrAMPs) are antibacterial components of the immune systems of animals such as honey bees, cows and, as this study proves, bottlenose dolphins. These peptides are a first response for the killing of bacteria. In humans, antimicrobial peptides (AMPs) mainly kill bacteria by disrupting the bacterial cell membrane, but so far no evidence of PrAMPs has been found. PrAMPs have a different mechanism of action to AMPs: they pass through the membrane of the cell without perturbing it and bind to ribosomes to inhibit protein synthesis.

The European team have been studying the mechanism of action of bacteria killing peptides in animals: “We want to compare PrAMPs from different organisms to mechanistically understand how these peptides inhibit bacteria”, Daniel Wilson explains.

>Read more on the European Synchrotron website

Illustration showing the mechanism of Tur1A. (entire image: here)
Credits: D. Wilson

The proteins that bind

Researchers reveal the structure of a protein that helps bacteria aggregate

Serine-rich repeat proteins (SRRPs), which help bacteria attach to surfaces, have been structurally characterised in pathogenic bacteria but not in beneficial bacteria such as those present in the gut. Dr Nathalie Juge’s team at the Quadram Institute Bioscience has previously identified SRRP as a main adhesin in Lactobacillus reuteri strains from pigs and mice. Now, together with colleagues at the University of East Anglia, they have described the structure and activity of the binding region of L. reuteri SRRPs in a paper published in PNAS. Using the Macromolecular Crystallography beamlines (I03 and I04) at Diamond Light Source, they discovered that the structure of these proteins is unique among characterised SRRPs and is surprisingly similar to pectin degrading enzymes. Molecular simulations and binding experiments revealed a pH-dependent binding to pectin and to proteins from the epithelium known as mucins. Altogether, these findings shed light on the activity of a key protein in these bacteria and may help guide the development of more targeted probiotic interventions.

>Read more on the Diamond Light Source website

Figure: (Left) Cartoon representation of crystal structures of the binding region of SRRP53608. (Right) Cartoon representation of crystal structures of the binding region of SRRP100-23. The N-terminus is shown with blue balls and the C-terminus is shown with red balls.

Scientists develop sugar-coated nanosheets to target pathogens

Molecular Foundry-designed 2-D sheets mimic the surface of cells

Researchers have developed a process for creating ultrathin, self-assembling sheets of synthetic materials that can function like designer flypaper in selectively binding with viruses, bacteria, and other pathogens.
In this way the new platform, developed by a team led by scientists at the U.S. Department of Energy’s Lawrence Berkeley National Laboratory (Berkeley Lab), could potentially be used to inactivate or detect pathogens.

The team, which also included researchers from New York University, created the synthesized nanosheets at Berkeley Lab’s Molecular Foundry, a nanoscale science center, out of self-assembling, bio-inspired polymers known as peptoids. The study was published earlier this month in the journal ACS Nano.
The sheets were designed to present simple sugars in a patterned way along their surfaces, and these sugars, in turn, were demonstrated to selectively bind with several proteins, including one associated with the Shiga toxin, which causes dysentery. Because the outside of our cells are flat and covered with sugars, these 2-D nanosheets can effectively mimic cell surfaces.

>Read more on the Advanced Light Source website

Image: A molecular model of a peptoid nanosheet shows loop structures in sugars (orange) that bind to the Shiga toxin (shown as a five-color bound structure at upper right).
Credit: Berkeley Lab

Scientists map important immune system enzyme for the first time

Biochemists from McGill University are getting a good look at just how a specific enzyme that is part of the human immune system interacts with a certain group of bacteria that are described as gram-negative.

Researchers around the world “have been studying the enzyme, known as AOAH, for more than 30 years. This is the first time anyone has been able to see exactly what it looks like,” according to Bhushan Nagar, an associate professor of biochemistry at McGill University in Montreal.

More than that, the 3D images captured a moment in time which shows just how AOAH inactivates a toxic molecule that is commonly part of various gram-negative bacteria. The research was conducted at the Canadian Light Source.

Numerous types of gram-negative bacteria exist throughout the environment. While some are harmless, many cause a variety of human illnesses, says Nagar. For example, several species such as E. coli and Salmonella, cause food borne illness. Others cause infections such as pneumonia, meningitis, bloodstream infections or gonorrhea.

>Read more on the Canadian Light Source

Image: Bhushan Nagar (principal investigator), Alexei Gorelik (first author of paper) and Katalin Illes (research assistant at Nagar lab) at their McGill University lab.
Credit: Bhushan Nagar.