Newly Discovered Reactions From An Old Drug May Lead To New Antibiotics

A mineral found at health food stores could be the key to developing a new line of antibiotics for bacteria that commonly cause diarrhea, tooth decay and, in some severe cases, death.The trace mineral selenium is found in a number of proteins in both bacterial cells and human cells called selenoproteins. University of Central Florida Associate Professor William Self's research shows that interrupting the way selenoproteins are made can halt the growth of the super bug Clostridium difficile and Treponema denticola, a major contributor to gum disease.

Infections of Clostridium difficile (commonly known as C-diff) lead to a spectrum of illnesses ranging from severe diarrhea to colitis, which can cause death. It's a life-threatening problem in hospitals and nursing homes worldwide, and the number of cases is on the rise. There are an estimated 500,000 cases per year in the United States alone. Between 15,000 to 20,000 people die each year while infected with this superbug. Treponema denticola is one of leading causes of gum disease and costs individuals thousands of dollars in dental care each year.

Self's findings are published in the May and June editions of the Journal of Biological Inorganic Chemistry and the Journal of Bacteriology. The National Institutes of Health and the Florida Department of Health funded the research, which was conducted at UCF during the past three years.

"It's the proof of principle that we are excited about," Self said from his research lab at UCF. "No one has ever tried this approach, and it could potentially be a source for new narrow spectrum antibiotics that block bacteria that require selenium to grow."

The key discovery occurred when the team found that the gold drug Auranofin, used to treat arthritis, impacted selenium's metabolism process. The chemical reaction changes the selenium, which prevents bacteria from using it to grow. Auranofin is an FDA-approved gold salt compound that is used to control inflammation and is already known to inhibit the activity of certain selenoproteins. Since certain bacteria, such as C. difficile, require selenoproteins for energy metabolism, the drug acts as a potent antimicrobial halting the growth of the bacteria.

The initial studies with C. difficile led to studies with T. denticola, known for several years to require selenium for growth. While testing the gold salt, Self's group also uncovered another surprise; the stannous salts found in many antimicrobial toothpastes in the form of stannous fluoride also inhibited the synthesis of selenoproteins. Previous independent research had already established that stannous salts are more effective at preventing tooth decay and inhibiting growth of T. denticola, but the mechanism of this inhibition of growth was not yet known. These findings could lead to new approaches to preventing gum disease.

"No one has tried to block the metabolism of selenium before as a therapeutic approach," Self said. "That's what's new and exciting and could lead to a whole host of other possibilities, including a better understanding of how the gold salt works for arthritis."

Self said more research is needed, and he already has another grant proposal before the NIH that would move his research forward.

First Broad-spectrum Anti-microbial Paint To Kill 'Superbugs'

Scientists in South Dakota are reporting development of the first broad-spectrum antimicrobial paint, a material that can simultaneously kill not just disease-causing bacteria but mold, fungi, and viruses. Designed to both decorate and disinfect homes, businesses, and health-care settings, the paint is the most powerful to date, according to their new study.The paint shows special promise for fighting so-called "superbugs," antibiotic-resistant microbes that infect hospital surfaces and cause an estimated 88,000 deaths annually in the United States, the researchers say.

In the study, Yuyu Sun and Zhengbing Cao note in the antimicrobial paints already on are store shelves. These paints, however, are only effective against a narrow range of disease-causing microorganisms, limiting their usefulness.

The scientists already were aware of research on the germ-killing effects of that N-halamines, bleach-like substances already in wide use. They developed a new antimicrobial polymer that includes a type of N-halamine. It has no undesirable effects on the quality of latex paints. Laboratory tests showed that the new polymer kills a wide range of disease-causing microbes including those resistant to multiple antibiotics. The paint retains an anti-microbial punch for extended periods, and it can be easily "recharged" with a simple chlorination process, the researchers note.

Mass Spec Technique Analyzes Defensive Chemicals On Seaweed Surfaces For Potential Drugs

A new analytical technique is helping scientists learn how organisms as simple as seaweed can mount complex chemical defenses to protect themselves from microbial threats such as fungus. Known as desorption electrospray ionization mass spectrometry (DESI-MS), the technique for the first time allows researchers to study unique chemical activity taking place on the surfaces of these organisms.Understanding this surface chemistry could one day allow scientists to borrow and adapt some of those defensive chemical compounds for use against cancer, HIV, malaria, drug-resistant bacteria and other diseases of humans. In a paper scheduled to be published online in the journal Proceedings of the National Academy of Sciences, researchers from the Georgia Institute of Technology describe a sophisticated chemical defense system that uses 28 different compounds to protect a species of seaweed against a single fungus.

"Plants and animals in the wild use chemistry as way to fight with one another," said Julia Kubanek, a professor in Georgia Tech's School of Biology. "Using this new technology, scientists can listen in on this fight to perhaps learn from what's going on and steal some of the strategies for human biomedical applications."

As part of a long-term project sponsored by the Natural Institutes of Health, Georgia Tech scientists have been cataloging and analyzing natural compounds from more than 800 species found in the waters surrounding the Fiji Islands. They have been particularly interested in Callophycus serratus, an abundant species of red seaweed that seems particularly successful – and adept at fighting off microbial infections.

Using the DESI-MS technique, the researchers analyzed recently-collected samples of the seaweed and found groups of potent anti-fungal compounds in light-colored microscopic surface patches covering what may be wounds on the surface of the seaweed. In laboratory testing, these bromophycolide compounds and callophycoic acids effectively inhibited the growth of Lindra thalassiae, a common marine fungus.

"It is possible that the alga is marshalling its defenses and displaying them in a way that blocks the entry points for microbes that might invade and cause disease," Kubanek said. "Seaweeds don't have B cells, T cells and immune responses like humans do. But instead they have some chemical compounds in their tissues to protect them."

Though all the seaweed they studied was from a single species, the researchers were surprised to find two distinct groups of anti-fungal chemicals. From one seaweed subpopulation, dubbed the "bushy" type for its appearance, 18 different anti-fungal compounds were identified. In a second group of seaweed, the researchers found 10 different anti-fungal compounds – all different from the ones seen in the first group.

"This species is producing some unique chemical compounds that other seaweeds don't produce, and it is producing a large number of compounds, each of which has a role to play in the overall defense against the fungus," Kubanek noted. "We think the compounds work together in an additive way."

Though chemically different, the compounds are structurally related and seem to arise from a similar metabolic pathway in the seaweed. Why one species of simple organism would produce 28 different anti-fungal compounds remains a mystery, though Kubanek believes the chemicals may also have other uses that are not yet understood.

The compounds have been tested for potential activity against drug-resistant bacteria, cancer, HIV, malaria and other human health threats. So far, preliminary testing suggests they have anti-malarial effects.

The DESI-MS technique allowed the researchers for the first time to analyze chemical activity occurring on the surface of the seaweed. Earlier techniques allowed identification of chemicals in the organism's tissue, but being able to confirm their location on the surface – the first line of defense against infection – confirms the role they play as defensive chemicals.

In DESI-MS, a charged stream of polar solvent is directed at the surface of a sample under study at ambient pressure and temperature. The spray desorbs molecules, which are then ionized and delivered to the mass spectrometer for analysis.

"This technique allows us to examine intact organisms and see how the chemical compounds are distributed," Kubanek explained. "For our research with seaweed, this is important because we'd like to understand how an organism distributes these compounds to protect itself from enemies."

In addition to Kubanek, others researchers contributing to the study included Leonard Nyadong, Asiri Galhena, Tonya Shearer, E. Paige Stout, R. Mitchell Parry, Mark Kwasnik, May Wang, Mark Hay, and Facundo Fernandez – all from Georgia Tech – and Amy Lane, now at Scripps Institution of Oceanography. Beyond the National Institutes of Health support, the research has also been sponsored by the National Science Foundation.

For the future, Kubanek and a graduate student are working to modify the most promising of the anti-malarial compounds, replacing some oxygen atoms for nitrogen atoms and bromine for chlorine and fluorine. The hope is to create a compound more potent against the malaria organism with less toxicity for humans.

"We are doing reaction chemistry using these 28 compounds as a starting point," she explained. "Learning about how other species avoid diseases may give us something we can use to avoid or treat our own diseases."

Improved Antibiotic: Genes For Synthesizing Thiostrepton Identified

Researchers at the Georgia Institute of Technology have identified the genetic machinery responsible for synthesizing thiostrepton, a powerful antibiotic produced by certain bacteria. Though effective against the dangerous MRSA (methicillin-resistant Staphylococcus aureus) and vancomycin-resistant enterococci, thiostrepton currently has only limited applications in humans because it is not water soluble.dentification of the gene cluster responsible for producing thiostrepton sets the stage for genetic manipulations that could make the drug more useful by improving its water solubility, potentially providing a new tool in the high-stakes battle against bacteria. Beyond the possible medical applications, the research produced a scientific surprise: thiostrepton is derived from a genetically encoded peptide that undergoes no fewer than 19 different modifications, one of the most complex such processes known – and a surprising capability for a single-celled bacterium.

"We are interested in making derivatives of this peptide drug that retain their potency and are efficiently processed by biochemical machinery," said Wendy L. Kelly, an assistant professor in Georgia Tech's School of Chemistry and Biochemistry and the Parker Petit Institute for Bioengineering and Bioscience. "We want to put in substitutions to the genetic machinery that may create a more water soluble analog and could potentially be used for development of a new class of antibacterial agent."

Kelly, graduate student Lisa Pan and postdoctoral fellow Chaoxuan Li began their study of thiostrepton by having the genome of one bacterium that produces it -- Streptomyces laurentii – sequenced by a commercial laboratory. They then studied different parts of the genome, searching for the genes responsible for producing the drug.

"It was a combination of DNA sequencing, bioinformatic analysis of the encoded proteins and biochemical characterization," said Kelly. "We didn't really know where on the chromosome this would be localized. Instead of taking a single shot and looking only at one location, we used a shotgun strategy that gave us insight into many different regions on the chromosome at the same time."

Fortunately, in simple organisms like bacteria, genes responsible for a particular task tend to be located close together, so when the researchers found one relevant gene, they knew the rest would be nearby. The researchers produced a knockout mutant to confirm that the genes they had identified were the correct ones.

The mechanism by which the bacterium produces thiostrepton turned out to be of considerable interest. Because peptides produced directly by ribosomal synthesis tend to be comparably simple, researchers had expected the complex thiostrepton molecule to be produced by a non-ribosomal route.

However, the Georgia Tech team showed that the drug results from a process controlled by the ribosome – which makes it a good target for genetic manipulation.

"The fact that we have a gene that produces a peptide that undergoes post-translational modification makes this a simpler target for biosynthetic engineering," Kelly noted. "Before this finding, we didn't know that such extensive modifications could be made to a peptide. Finding this mechanism completely changes how we look at this and similar systems, and changes the potential for biosynthetically engineering effective new systems."

Kelly's research team will next seek to understand the complex pathway used to synthesize the drug, then attempt to modify the right component of that machinery to create a variant of thiostrepton that is water soluble.

"You can think about this in terms of an assembly line for manufacturing cars, with the changes occurring in stages during construction," she said. "The same would be true of a microorganism building up a complex molecule. Some modifications that occur later in the process may require certain key elements to be present first. We need to understand what modifications are necessary and what features of the structure are important for recognition and processing down the line."

Produced by certain terrestrial and marine bacteria, thiostrepton was identified in the 1950s, and first synthesized in the laboratory in 2004. Thiostrepton and related thiopeptide antibiotics fight bacteria by disabling their protein biosynthesis, and also have promising anti-malarial and anti-cancer activity.

Though researchers face many challenges in attempting to modify the genes and enzymes required to produce the drug, the potential benefits are significant.

"With the development of resistance and pathogens such as MRSA, there's a crisis developing in anti-microbial treatments," she noted. "If they were to become resistant to the few drugs that are currently available to fight them, they would become untreatable. There is a big push to identify new drugs for clinical use in humans that are effective against these strains."

With an undergraduate degree in pharmacy and graduate degrees in chemistry, Kelly has focused on natural systems that produce useful drugs.

"I have a profound appreciation for nature's chemistry and how nature makes complex metabolites from very simple building blocks," she said. "If you compare the kinds of transformations that can be done inside a bacterium against what a synthetic chemist can do, you see the power of nature in its ability to catalyze highly specialized reactions under very mild conditions."

Details of the work were published online in the Journal of the American Chemical Society on March 5. The research was sponsored by the Camille and Henry Dreyfus Foundation, the American Society of Pharmacognosy and Georgia Tech.

Technique Tricks Bacteria Into Generating Their Own Vaccine

Scientists have developed a way to manipulate bacteria so they will grow mutant sugar molecules on their cell surfaces that could be used against them as the key component in potent vaccines.Any resulting vaccines, if proven safe, could be developed more quickly, easily and cheaply than many currently available vaccines used to prevent bacterial illnesses.

Most vaccines against bacteria are created with polysaccharides, or long strings of sugars found on the surface of bacterial cells. The most common way to develop these vaccines is to remove sugars from the cell surface and link them to proteins to give them more power to kill bacteria.

Polysaccharides alone typically do not generate a strong enough antibody response needed to kill bacteria. But this new technique would provide an easy approach to make a small alteration to the sugar structure and produce the polysaccharide by simple fermentation.

“We are showing for the first time that you don’t have to use complicated chemical reactions to make the alteration to the polysaccharide,” said Peng George Wang, Ohio Eminent Scholar and professor of biochemistry and chemistry at Ohio State University and senior author of the study. “All we need to do is ferment the bacteria, and then the polysaccharides that grow on the surface of the cell already incorporate the modification.”

The research is scheduled to appear in the online early edition of the Proceedings of the National Academy of Sciences.

In vaccines, polysaccharides linked with carrier proteins are injected into the body. That sets off a process that causes the release of antibodies that recognize the sugars as an unwanted foreign body. The antibodies then remain dormant but ready to attack if they ever see the same polysaccharides again – which would be a signal that bacteria have infected the body.

Polysaccharides are chains of sugars, or monosaccharides, and they are targeted for vaccine development because they are the portion of bacterial cells that interact with the rest of the body.

Escherichia coli was used as a model for the study. Wang and colleagues used one of the existing monosaccharides present on the E. coli cell surface polysaccharides, called fucose, to generate this new modification. They manipulated the structure of the fucose to create 10 different analogs, or forms of the sugar in which just one small component is changed.

The scientists then manually introduced these altered forms of fucose to a solution in which bacterial cells were growing, and the bacterial cells absorbed the altered fucose as they would normal forms of the sugar. The presence of these altered forms of fucose then altered the properties of the polysaccharides that grew on the surface of the cells.

“This way, we don’t have to do anything to modify the polysaccharides. We let bacteria do it for us,” Wang said.

“Bacteria grow lots of polysaccharides – it’s similar to the way humans grow hair. But for a vaccine, you need to make the molecules more active, or energetic,” he said. “In our method, we feed the bacteria these chemicals while they are growing, and those chemicals end up in the polysaccharides and that makes them more immunogenic. That’s the technology.”

Wang said the approach is likely to be applicable to many different kinds of bacteria. But each type of pathogen must be tested individually with the alteration of sugars unique to its surface.

“If you want to prevent one type of bacteria, you have to find something very unique for this bacteria because different microbes have different characteristics,” he said. “You have to find the oddest thing on the cell surface. It has to be on surface because what the body sees first is the surface.”

His lab will next be testing the method’s effectiveness on the pneumococcus bacteria under an exploratory $100,000 grant from the Bill & Melinda Gates Foundation. The current vaccine to prevent pneumonia in babies and the elderly combines 23 strains of bacteria, making it complex and expensive to produce. Each injection costs about $50 in the United States. A less expensive way to develop the vaccine would increase its availability in the developing world, Wang said.

This published research was supported by an endowed Ohio Eminent Scholar Professorship on Macromolecular Structure and Function in the Department of Biochemistry at Ohio State.

Co-authors of the work are Wen Yi, a recipient of a Ph.D. from the Ohio State Biochemistry Program who is now at the California Institute of Technology; Xi Chen of the University of California, Davis; Jianjun Li of the Institute for Biological Sciences at National Research Council of Canada; Chengfeng Xia, Guangyan Zhou and Wenpeng Zhang of Ohio State’s Departments of Biochemistry and Chemistry; Yanhong Li of the University of California, Davis; Xianwei Liu of Shandong University, China; and Wei Zhao of Nankai University, China.

MRSA’s 'Weak Point' Visualized By Scientists

An enzyme that lives in MRSA and helps the dangerous bacterium to grow and spread infection through the human body has been visualised for the first time, according to a new study.Now, armed with detailed information about the structure of this enzyme, researchers hope to design new drugs that will seek it out and disable it, providing a new way of combating MRSA and other bacterial infections.

The enzyme, a ‘worker-protein’ called LtaS, produces an important component of the protective outer-layer that surrounds all Staphylococcus aureus cells as well as many other bacteria that cause disease.

Staphylococcus aureus is a type of bacterium that causes a variety of infections in the human body, including skin infections and abscesses, sometimes leading to blood poisoning and life-threatening lung or brain infections. MRSA is a particular strain of Staphylococcus aureus, which has evolved to be resistant to the antibiotic methicillin and a large number of other antibiotics, and can be life threatening.

To counter this drug resistance and ensure that it is possible to treat MRSA infection in the future, new antibiotics are needed that work differently, for example by attacking parts of the pathogen that are not targeted by current drugs.

The team from Imperial College London behind today’s study, funded by the Medical Research Council, thinks that LtaS might be a good candidate target for a new antibiotic to which MRSA will not be resistant. This is because its job is to build a polymer called lipoteichoic acid (LTA), which is an important structure found on the surface of Staphylococcus aureus cells.

Although the role of the cell surface polymer LTA is not fully understood, lab tests carried out by the same researchers have shown that if the LtaS enzyme is depleted, production of LTA on the cell surface draws to a halt. As a result growth of the Staphylococcus aureus cell is blocked. So in a patient infected with MRSA, inhibiting this enzyme could clear up the infection because the bacterial cells would be unable to grow properly. Many existing antibiotics work in a similar way by inhibiting the production of other such important structures on the surface of bacterial cells.

The trick, according to one of the paper’s lead authors, Dr Angelika Grundling from Imperial College London’s Division of Investigative Science, is to now find a way of using the new knowledge to develop a drug for use in real world scenarios:

“We’re not quite sure how it works, but we know that this surface structure called LTA is involved in cell growth and cell division – we have shown that without it the cell cannot grow properly, and eventually dies. Because LtaS is the ‘machine’, which builds LTA, developing a drug that knocks out the machine will provide us with a new way to disable the growth of these cells, which would represent a novel new treatment for MRSA and other Staphylococcus aureus infections.”

Dr Grundling and her colleagues have produced a detailed image of the molecular structure of the LtaS enzyme using X-ray crystallography techniques. The image includes a map of LtaS’s active binding site: the part of the enzyme which plays a key role in building LTA. This is the very part that researchers now need to home in on with a drug, in order to prevent the LtaS enzyme from doing its job.

Professor Paul Freemont from Imperial’s Division of Molecular Biosciences, co-lead-author of the paper, explains the importance of the information they have gained about this particular part of the enzyme:

“If we’re to develop a drug which disables LtaS from doing its job, then we need to make sure the drug molecule is as perfectly matched as possible to the enzyme’s binding site, so it can trick the enzyme into taking it up. Once the drug is bound to the enzyme it will be able start its job of sabotage.

“So the more detailed information about the binding site we have, the better we’ll be able to develop an effective drug to match it,” he said.

The two Imperial teams led by Professor Freemont and Dr Grundling now hope to work with the College’s Drug Discovery Centre to search for a biological agent that interacts with the LtaS binding site, as the basis for a new antibiotic drug.

They hope that in the future such a drug could be used to treat not just MRSA, but a whole host of infections caused by bacterial pathogens.

Additional funding for the research was obtained through the US National Institute of Health.

New Family Of Antibacterial Agents Uncovered

As bacteria resistant to commonly used antibiotics continue to increase in number, scientists keep searching for new sources of drugs. One potential new bactericide has now been found in the tiny freshwater animal Hydra.

The protein identified by Joachim Grötzinger, Thomas Bosch and colleagues at the University of Kiel, hydramacin-1, is unusual (and also clinically valuable) as it shares virtually no similarity with any other known antibacterial proteins except for two antimicrobials found in another ancient animal, the leech.

Hydramacin proved to be extremely effective though; in a series of laboratory experiments, this protein could kill a wide range of both Gram-positive and Gram-negative bacteria, including clinically-isolated drug-resistant strains like Klebsiella oxytoca (a common cause of nosocomial infections). Hydramacin works by sticking to the bacterial surface, promoting the clumping of nearby bacteria, then disrupting the bacterial membrane.

Grötzinger and his team also determined the 3-D shape of hydramacin-1, which revealed that it most closely resembled a superfamily of proteins found in scorpion venom; within this large group, they propose that hydramacin and the two leech proteins are members of a newly designated family called the macins.