The future of fighting infections

Scientists analyze 3D model of proteins from disease-causing bacteria at the CLS.

Millions of people are affected by the Streptococcus pneumoniae bacterium, which can cause sinus infections, middle ear infections and more serious life-threatening diseases, like pneumonia, bacteremia, and meningitis. Up to forty percent of the population are carriers of this bacterium.
Researchers from the University of Victoria (UVic) used the Canadian Light Source (CLS) at the University of Saskatchewan to study proteins that the pathogen uses to break down sugar chains (glycans) present in human tissue during infections. These proteins are key tools the bacterium uses to cause disease.

They used the Canadian Macromolecular Crystallography Facility (CMCF) at the CLS to determine the three-dimensional structure of a specific protein, an enzyme, that the bacterium produces to figure out how it interacts with and breaks down glycans.

>Read more on the Canadian Light Source website

Image: The 3D structure of an enzyme from the disease-causing bacterium Streptococcus pneumoniae.

Research on how light-harvesting bacteria toggle off and on

The results could have long-range implications for artificial photosynthesis and optogenetics—the use of light to selectively activate biological processes.

Cyanobacteria are water-dwelling microbes capable of absorbing sunlight and converting it into chemical energy through photosynthesis. Long ago, ancient versions of these bacteria were incorporated into plant cells, where they eventually evolved into chloroplasts, the organelles responsible for carrying out photosynthesis in green plants. Today, in seeking to develop artificial photosynthesis to harness the sun’s abundant energy, scientists look to cyanobacteria to better understand the nuts and bolts of how natural photosynthesis works.

Cyanobacterial “off switch”

One topic of interest is how cyanobacteria respond to too much light. If a sunlight-harvesting system becomes overloaded with absorbed solar energy, it most likely will suffer some form of damage. Nature has solved the problem in cyanobacteria through a protective mechanism—an energy-quenching “off switch” in which excess solar energy is safely dissipated as heat.

>Read more on the Advanced Light Source at BNL

Illustration: X-ray footprinting provides time-resolved information about where key conformational changes occur. On the left is the overall OCP structure. The two structures on the right highlight local areas with increasing protein packing over time (blue shading) and areas with decreasing protein packing over time (red shading). The changes in accessibility are initiated by the movement of the carotenoid molecule (magenta chain).

Study offers new target for antibiotic resistant bacteria

As antibiotic resistance rises, the search for new antibiotic strategies has become imperative. In 2013, the Centers for Disease Control estimated that antibiotic resistant bacteria cause at least 2 million infections and 23,000 deaths a year in the U.S.; a recent report raised the likely mortality rate to 162,044.
New Cornell research on an enzyme in bacteria essential to making DNA offers a new pathway for targeting pathogens. In “Convergent Allostery in Ribonucleotide Reductase,” published June 14 in Nature Communications, researchers used the MacCHESS research stations at the Cornell High Energy Synchrotron Source (CHESS) to reveal an unexpected mechanism of activation and inactivation in the protein ribonucleotide reductase (RNR).

Understanding the “switch” that turns RNR off provides a possible means to shut off the reproduction of harmful bacteria.
RNRs take ribonucleotides, the building blocks of RNA, and convert them to deoxyribonucleotides, the building blocks of DNA. In all organisms, the regulation of RNRs involves complex mechanisms, and for good reason: These mechanisms prevent errors and dangerous mutations.

>Read more on the CHESS website

Image: William Thomas, a graduate student in the field of chemistry and chemical biology, collects data on ribonucleotide reductase.

Winning the fight against influenza

Annual influenza epidemics and episodic pandemics continue to cause widespread illness and mortality. The World Health Organization estimates that annual influenza epidemics cause around 3–5 million cases of severe illness and up to 650,000 deaths worldwide. Seasonal influenza vaccination still remains the best strategy to prevent infection, but the vaccines that are available now offer a very limited breadth of protection. Human broadly neutralizing antibodies (bnAbs) that bind to the hemagglutinin (HA) stem region provide hope for a universal vaccine (Figure 1a)1,2. Binding of these bnAbs prevents the pH-induced conformational changes that are required for viral fusion in the endosomal compartments of target cells in the respiratory tract and, hence, viral entry in our cells.

>Read more on the SSRL at SLAC website

Image: Complex of Influenza virus HA with (a) Fab CR6261, (b) llama single domain antibody SD36, and (c) JNJ4796.

How stained glass can help in the battle against superbugs

Ancient skills meet cutting edge technology in the battle against antibiotic resistance

Bacteria can form colonies (known as biofilms) on the surface of objects. This is a particular problem when it occurs on medical devices implanted into the body, such as catheters, prosthetic cardiac valves and intrauterine devices, as biofilms can display resistance to both antibiotics and the body’s immune response. Any incision into the body risks a surgical infection, and if a biofilm takes hold it can be difficult to eradicate. With the rise in antibiotic resistance, scientists are seeking new ways to prevent infections, and there is increasing interest in impregnate medical devices with antimicrobial substances. In work recently published in ACS Biomaterials Science & Engineering, researchers from Aston University in Birmingham, led by Dr Richard Martin, explored the antimicrobial potential of phosphate glasses doped with cobalt, and found them to be effective against Escherichia coli, Staphylococcus aureus and Candida albicans when placed in direct contact, suggesting that cobalt-doped bioactive glasses could be developed with antimicrobial properties. The technique they discovered is similar to those used to make stained glass in medieval times.

>Read more on the Diamond Light Source website
Image: Images of the copper (left) and cobalt (right) doped bioactive glasses.
Credit: Dr Richard Martin

“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