Avian Flu Viruses Which Are Transmissible Between Humans Could Evolve in Nature

It might be possible for human-to-human airborne transmissible avian H5N1 influenza viruses to evolve in nature, new research has found.

Colorized transmission electron micrograph of Avian influenza A H5N1 viruses (seen in gold) grown in MDCK cells (seen in green) [Credit: CDC/Courtesy of Cynthia Goldsmith; Jacqueline Katz; Sherif R. Zaki]

The findings, from research led by Professor Derek Smith and Dr Colin Russell at the University of Cambridge, were published June 22 in the journal Science.

Currently, avian H5N1 influenza, also known as bird flu, can be transmitted from birds to humans, but not (or only very rarely) from human to human. However, two recent papers by Herfst, Fouchier and colleagues in Science and Imai, Kawaoka and colleagues in Nature reveal that potentially with as few as five mutations (amino acid substitutions), or four mutations plus reassortment, avian H5N1 can become airborne transmissible between mammals, and thus potentially among humans. However, until now, it was not known whether these mutations might evolve in nature.

The Cambridge researchers first analysed all of the surveillance data available on avian H5N1 influenza viruses from the last 15 years, focusing on birds and humans. They discovered that two of the five mutations seen in the experimental viruses (from the Fouchier and Kawaoka labs) had occurred in numerous existing avian flu strains. Additionally, they found that a number of the viruses had both of the mutations.

Colin Russell, Royal Society University Research Fellow at the University of Cambridge, said: "Viruses that have two of these mutations are already common in birds, meaning that there are viruses that might have to acquire only three additional mutations in a human to become airborne transmissible. The next key question is 'is three a lot, or a little?' "

The scientists explored this key question using a mathematical model of how viruses replicate and evolve within a mammalian host and assessed the influence of various factors on whether the remaining three mutations could evolve in a single host or in a short chain of transmission between hosts

The factors that increased the likelihood of mutations evolving are:

1. Random mutation. The replication mechanisms of influenza viruses don't make perfect copies. On average, every time an influenza virus replicates itself it makes approximately one mutation somewhere in the genome of each new virus. In each infected human there will be billions of viruses, and thus with many viruses replicating, multiple mutations can accumulate within a single host.

2. Positive selection. If some of the remaining mutations help the avian virus to adapt to mammals, then those mutations will make the viruses more fit and thus will be positively selected and preferentially accumulate.

3. Long infection. The longer someone is infected and producing new viruses, the more time there is for mutations to accumulate.

4. Functionally equivalent substitutions. The sets of substitutions identified by Fouchier and Kawaoka are unlikely to be the only combinations of substitutions capable of producing an aerosol transmissible virus. The probability of emergence increases with the number of combinations.

5. Diversity in the within-bird virus population. Given all of the mutations there are likely to be within a host due to random mutation, it is possible that the viruses from a bird that infect a human might have a mutation that would not be detected by routine surveillance. For example, if 100 virus particles from a bird infect a human and one of those particles had a key mutation, it would increase the probability of the mutation reaching high levels within a host even though routine sequencing would not detect it.

6. Transmission between mammals. If mammals are capable of transmitting viruses that have some but not all of the necessary substitutions it could increase the probability of an airborne transmissible virus evolving.

The factors that decreased the likelihood of mutations evolving are:

1. An effective immune response. An effective immune response would shorten the length of an infection and thus decrease the time available to accumulate mutations.

2. Deleterious substitutions. If any of the substitutions necessary for airborne transmission were harmful to the virus it would, on average, slow the accumulation of mutations.

3. Order of acquiring mutations. It is not currently known if the mutations for airborne transmissibility need to be acquired in a specific order. If they do, it would, on average, slow the accumulation of mutations.

"With the information we have, it is impossible to say what the exact risk is of the virus becoming airborne transmissible among humans. However, the results suggest that the remaining three mutations could evolve in a single human host, making a virus evolving in nature a potentially serious threat," said Derek Smith, Professor of Infectious Disease Informatics at the University of Cambridge. "We now know that it is in the realm of possibility that these viruses can evolve in nature, and what needs to be done to assess the risk more accurately of these mutations evolving in nature."

The scientists recommend the following activities be considered high priority for estimating and ameliorating the risk of emergence of aerosol transmissible H5N1 viruses.

First, additional surveillance in regions where viruses with airborne transmission enabling substitutions have been observed and in regions connected to those regions by bird migration and trade. Also, increased surveillance for mutations that might have the same function as those found by the Fouchier and Kawaoka labs.

Second, related to surveillance, some targeted sequencing of H5N1 viruses should be done by "deep sequencing" where the lab sequences many viruses from an individual host to look for viruses that might have accumulated the critical mutations, even if those viruses are just a small proportion of the viruses within an animal.

Third, further investigations are needed to determine which substitutions and combinations of substitutions that are not the same as, but have the same function as, the substitutions identified by the Fouchier and Kawaoka labs are capable of making viruses airborne transmissible between mammals.

Fourth, further studies are needed to elucidate the changes in within-host fitness and between-host transmissibility associated with each airborne transmission enabling substitution and combination of substitutions.

Professor Smith added: "The situation is similar to assessing the risk of an earthquake or tsunami. We don't know exactly when and where, but by increasing monitoring and research -- some of which is already underway -- scientists and public health officials will be able to increase the accuracy with which the risk can be assessed and to minimise those risks."

The research was funded by multiple sources including the European Commission through framework 7 grants EMPERIE and ANTIGONE, the Royal Society, the Human Frontiers Science Program, the Wellcome Trust, and the National Institutes of Health.

Source: University of Cambridge [June 21, 2012]

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Mystery of Bacterial Growth and Resistance Solved

Scientists at The Scripps Research Institute have unraveled a complex chemical pathway that enables bacteria to form clusters called biofilms. Such improved understanding might eventually aid the development of new treatments targeting biofilms, which are involved in a wide variety of human infections and help bacteria resist antibiotics.

The report, published online ahead of print on April 26, 2012, by the journal Molecular Cell, explains how nitric oxide, a signaling molecule involved in the immune system, leads to biofilm formation.

"It is estimated that about 80 percent of human pathogens form biofilms during some part of their life cycle," said Scripps Research president and CEO Michael Marletta, PhD, who led the work. "In this study, we have detailed for the first time the signaling pathway from nitric oxide to the sensor through cellular regulators and on to the biological output, biofilm formation."

"There's a lot of interest right now in finding ways to influence biofilm formation in bacteria," said lead author Lars Plate, a graduate student in Marletta's team, which recently moved to Scripps Research from the University of California, Berkeley. "Figuring out the signaling pathway is a prerequisite for that."

Biofilm formation is a critical phenomenon that occurs when bacterial cells adhere to each other and to surfaces, at times as part of their growth stage and at other times to gird against attack. In such aggregations, cells on the outside of a biofilm might still be susceptible to natural or pharmaceutical antibiotics, but the interior cells are relatively protected. This can make them difficult to kill using conventional treatments.

Biofilms can form on surgical instruments such as heart valves or catheters, leading to potentially deadly infections. Likewise, difficult-to-eliminate biofilms also play key roles in a host of conditions from gum disease to cholera, and from cystic fibrosis to Legionnaires' disease.

For years, the Marletta lab and other groups have been studying how nitric oxide regulates everything from blood vessel dilation to nerve signals in humans and other vertebrates. Past research had also revealed that nitric oxide is involved in influencing bacterial biofilm formation.

Nitric oxide in sufficient quantity is toxic to bacteria, so it's logical that nitric oxide would trigger bacteria to enter the safety huddle of a biofilm. But nobody knew precisely how.

In the new study, the scientists set out to find what happens after the nitric oxide trigger is pulled. "The whole project was really a detective story in a way," said Plate.

In vertebrates, nitric oxide can bind to something called the Heme-Nitric Oxide/Oxygen (H-NOX) binding domain on a specific enzyme, activating that enzyme and beginning the chemical cascades that lead to physiological functions such as blood vessel dilation.

Many bacteria also have H-NOX domains, including key pathogens, so this seemed the best starting point for the investigation. From there, the team turned to genomic data.

Genes for proteins that interact are often found adjacent to one another. Based on this fact, the researchers were able to infer a connection between the bacterial H-NOX domain and an enzyme called histidine kinase, which transfers phosphate chemical groups to other molecules in signaling pathways. The question was where the phosphates were going.

To learn more, the researchers used a technique called phosphotransfer profiling. This involved activating the histidine kinase and then allowing them to react separately with about 20 potential targets. Those targets that the histidine kinase rapidly transferred phosphates to had to be part of the signaling pathway. "It's a neat method that we used to get an answer that was in fact very surprising," said Plate.

The experiments revealed that the histidine kinase phosphorylated three proteins called response regulators that work together to control biofilm formation for the project's primary study species, the bacterium Shewanella oneidensis, which is found in lake sediments.

Further work showed that each regulator plays a complementary role, making for an unusually complex system. One regulator activates gene expression, another controls the activity of an enzyme producing cyclic diguanosine monophosphate, an important bacterial messenger molecule that is critical in biofilm formation, and the third tunes the degree of activity of the second.

Since other bacterial species use the same chemical pathway uncovered in this study, the findings pave the way to further explore the potential for pharmaceutical application. As one example, researchers might be able to block biofilm formation with chemicals that interrupt the activity of one of the components of this nitric oxide cascade.

Marletta's group has already explored nitric oxide's role in controlling Legionnaires' disease and, among other goals, will focus now on understanding biofilm formation in the bacterium that causes cholera.

This research was supported by the National Institutes of Health and a Chang-Lin Tien Graduate Fellowship in the Environmental Sciences.

Source: The Scripps Research Institute [April 25, 2012]

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'Backpacking' bacteria help ferry nano-medicines inside humans

To the ranks of horses, donkeys, camels and other animals that have served humanity as pack animals or beasts of burden, scientists are now enlisting bacteria to ferry nano-medicine cargos throughout the human body. They reported on progress in developing these "backpacking" bacteria -- so small that a million would fit on the head of a pin -- in San Diego on March 29 at the 243rd National Meeting & Exposition of the American Chemical Society (ACS). 

Bacterial cells could deliver diagnostics, therapeutics or sensors to where they are needed most in the body ]Credit:Sean Parsons, ACS]

"Cargo-carrying bacteria may be an answer to a major roadblock in using nano-medicine to prevent, diagnose and treat disease," David H. Gracias, Ph.D., leader of the research team said. Gracias explained that nanotechnology is the engineering of ultra-small machines and other devices. These devices generally lack practical self-sustaining motors to move particles of medication, sensors and other material to diseased parts of the body. So why not attach such cargo to bacteria, which have self-propulsion systems, and have them hike around the human body? 

"Currently, it is hard to engineer microparticles or nanoparticles capable of self-propelled motion in well-defined trajectories under biologically relevant conditions," Gracias said. He is with Johns Hopkins University in Baltimore, Maryland. "Bacteria can do this easily, and we have established that bacteria can carry cargo." 

In addition, bacteria can respond to specific biochemical signals in ways that make it possible to steer them to desired parts of the body. Once there, bacteria can settle down, deposit their cargo and grow naturally. Bacteria already live all over the body, particularly in the large intestine, with bacterial cells outnumbering human cells 10-to-1. Despite their popular reputation as disease-causers, there are bacteria in the human body, especially in the intestinal tract, that are not harmful, and the backpackers fall into that category. 

Gracias' bacteria don't really carry little nylon or canvas backpacks. Their "backpacks" are micro- or nano-sized molecules or devices that have useful optical, electrical, magnetic, electrical or medicinal properties. The cargos that the team tested also varied in size, shape and material. So far, the team has loaded beads, nanowires and lithographically fabricated nanostructures onto bacteria. 

Other scientists are seeking to enlist bacteria in transporting nano-cargo. They already have established, for instance, that large numbers of bacteria -- so-called "bacterial carpets" -- can move tiny objects. Gracias' research focuses on attaching one piece of cargo to an individual bacterium, rather than many bacteria to much larger cargo. The bacteria, termed "biohybrid devices," can still move freely, even with the cargo stuck to them. 

"This is very early-stage exploratory research to try and enable new functionalities for medicine at the micro- and nanoscale by leveraging traits from bacteria," explained Gracias. "Our next steps would be to test the feasibility of the backpacking bacteria for diagnosing and treating disease in laboratory experiments. If that proves possible, we would move on to tests in laboratory mice. This could take a few years to complete." 

Source: American Chemical Society [March 29, 2012]

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With you in the room bacteria counts spike by about 37 million bacteria per hour

A person's mere presence in a room can add 37 million bacteria to the air every hour -- material largely left behind by previous occupants and stirred up from the floor -- according to new research by Yale University engineers. 

Rendering of bacteria. A person's mere presence in a room can add 37 million bacteria to the air every hour -- material largely left behind by previous occupants and stirred up from the floor -- according to new research by Yale University engineers [Credit: © Jezper/Fotolia]

"We live in this microbial soup, and a big ingredient is our own microorganisms," said Jordan Peccia, associate professor of environmental engineering at Yale and the principal investigator of a study recently published online in the journal Indoor Air. "Mostly people are re-suspending what's been deposited before. The floor dust turns out to be the major source of the bacteria that we breathe." 

Many previous studies have surveyed the variety of germs present in everyday spaces. But this is the first study that quantifies how much a lone human presence affects the level of indoor biological aerosols. 

Peccia and his research team measured and analyzed biological particles in a single, ground-floor university classroom over a period of eight days -- four days when the room was periodically occupied, and four days when the room was continuously vacant. At all times the windows and doors were kept closed. The HVAC system was operated at normal levels. Researchers sorted the particles by size. 

Overall, they found that "human occupancy was associated with substantially increased airborne concentrations" of bacteria and fungi of various sizes. Occupancy resulted in especially large spikes for larger-sized fungal particles and medium-sized bacterial particles. The size of bacteria- and fungi-bearing particles is important, because size affects the degree to which they are likely to be filtered from the air or linger and recirculate, the researchers note. 

"Size is the master variable," Peccia said. 

Researchers found that about 18 percent of all bacterial emissions in the room -- including both fresh and previously deposited bacteria -- came from humans, as opposed to plants and other sources. Of the 15 most abundant varieties of bacteria identified in the room studied, four are directly associated with humans, including the most abundant, Propionibacterineae, common on human skin. 

Peccia said carpeted rooms appear to retain especially high amounts of microorganisms, but noted that this does not necessarily mean rugs and carpets should be removed. Extremely few of the microorganisms commonly found indoors -- less than 0.1 percent -- are infectious, he said. 

Still, understanding the content and dynamics of indoor biological aerosols is helpful for devising new ways of improving air quality when necessary, he said. 

"All those infectious diseases we get, we get indoors," he said, adding that Americans spend more than 90 percent of their time inside. 

The researchers have begun a series of similar studies outside the United States. 

The paper's lead author is J. Qian of Yale. Other contributors are D. Hospodsky and N. Yamamoto, both of Yale, and W.W. Nazaroff of the University of California-Berkeley. 

The research was supported by the Alfred P. Sloan Foundation. 

Author: Eric Gershon | Source: Yale University [March 28, 2012]

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Honeycomb structure responsible for bacteria's extraordinary sense

Cornell researchers have peered into the complex molecular network of receptors that give one-celled organisms like bacteria the ability to sense their environment and respond to chemical changes as small as 1 part in 1,000. 

Honeycomb-like hexagonal lattice formed by the network of receptor molecules (pink), associated enzymes (blue) and coupling proteins (green) in motile bacteria. Such a cooperative lattice arranged across their cell membranes helps bacteria sense their environment with extreme sensitivity. Inset shows the structure of a unit cell of the lattice in detail (top left). [Credit: Crane Lab]

Just as humans use five senses to navigate through surroundings, bacteria employ an intricate structure of thousands of receptor molecules, associated enzymes and linking proteins straddling their cell membranes that trigger responses to external chemical changes. 

Researchers in the lab of Brian Crane, professor of chemistry and chemical biology, with collaborators in the lab of Grant Jensen at the California Institute of Technology, have mapped out the honeycomb-like hexagonal arrangement of these receptor complexes in unprecedented detail. 

They report their discovery in the Feb. 20 issue of the Proceedings of the National Academy of Sciences. 

"The highlight of the paper is that by using a combination of [techniques], we've been able to image these complex arrays in live cells and determine how they are structured -- they are a very unique biological assembly that is conserved across nearly all classes of motile bacteria," said Crane. 

The findings might have potential applications in engineering biologically inspired synthetic molecular devices to detect specific chemicals with high sensitivity over a wide dynamic range; they also could shed light on the pathogenesis of various bacterial diseases like syphilis, cholera and Lyme disease. They may also give some insight into the functioning of the human immune system, where special cells may be employing similar cooperative networks of receptors to recognize and fight against foreign antigens, Crane said. 

Scientists are trying to determine how one receptor -- on detecting nutrients, oxygen or acidity, for example -- triggers communication through multiple enzymes in the complex, setting off a chain reaction. This cooperation leads to a gain or amplification in the signal that is finally communicated to the tails (flagella) of the bacterium, affecting the way they spin. Such a response allows the bacterium to move toward food sources or away from toxic environments.  

The latest work takes a big step toward understanding this mechanism by demonstrating for the first time how the individual pieces of receptors, enzymes and coupling proteins fit together to generate an extended network. It builds on previous work in the Crane lab done in collaboration with Jack Freed, Cornell professor of chemistry and chemical biology. 

"We were able to see how the enzymes and proteins are linked together in the complex array and also their interactions with the receptors," said Xiaoxiao Li, graduate student and joint lead author on the study. 

High-resolution X-ray images of the bacterial membrane receptor complex were obtained after extraction, purification and crystallization at the Cornell High Energy Synchrotron Source (CHESS). The scientists reconstructed the complicated molecular structure of the complex with an accuracy that is within a nanometer (a billionth of a meter). Pictures of the structure were also captured inside living cells, by first "flash-freezing" the cells and then scattering electrons off them. 

The researchers deduced that trimers (groups of three) of receptor molecules are arranged at the vertices of hexagons surrounding rings of enzymes and coupling proteins. These rings linked by proteins form the backbone of the extended honeycomb-like network. 

Author: Vivek Venkataraman | Source: Cornell University [February 20, 2012]

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Understanding how bacteria come back from the dead

Salmonella remains a serious cause of food poisoning in the UK and throughout the EU, in part due to its ability to thrive and quickly adapt to the different environments in which it can grow. New research involving a team of IFR scientists, funded by BBSRC, has taken the first detailed look at what Salmonella does when it enters a new environment, which could provide clues to finding new ways of reducing transmission through the food chain and preventing human illness. 

Bacteria can multiply rapidly, potentially doubling every 20 minutes in ideal conditions. However, this exponential growth phase is preceded by a period known as lag phase, where no increase in cell number is seen. Lag phase was first described in the 19th Century, and was assumed to be needed by bacteria to prepare to exploit new environmental conditions. Beyond this, surprisingly little was known about lag phase, other than bacteria are metabolically active in this period. But exactly what are bacteria doing physiologically during this period? 

To fill in this knowledge gap researchers at IFR, along with colleagues at Campden BRI, a membership-based organisation carrying out research and development for the food and drinks industry, have developed a simple and robust system for studying the biology of Salmonella during lag phase. In this system, lag phase lasts about two hours, but the cells sense their new environment remarkably quickly, and within four minutes switch on a specific set of genes, including some that control the uptake of specific nutrients. 

For example, one nutrient accumulated is phosphate which is needed for many cellular processes, and a gene encoding a phosphate transporter was the most upregulated gene during the first four minutes of lag phase. The cellular uptake mechanisms for iron were also activated during lag phase, and are needed for key aspects of bacterial metabolism. This increase in iron leads to a short term sensitivity to oxidative damage. Manganese and calcium are also accumulated in lag phase, but are lost from the cell during exponential growth. 

This new understanding of Salmonella metabolism during lag phase show how rapidly Salmonella senses favourable conditions and builds up the materials needed for growth. This study was carried out by two BBSRC-CASE studentships, which were partially funded by Campden BRI. 

Future research to work out the regulatory mechanisms behind these processes and the switch from lag phase to exponential growth will tell us more about how Salmonella can flourish in different environments, and could point to new ways of controlling its transmission in the food chain.  

Source: Norwich BioScience Institutes [February 02, 2012]

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Permafrost bacteria may slow down aging

A hardy type of bacteria recently discovered in the permafrost of Siberia could help slow down the ageing process, Russian scientists claimed on Tuesday. 

The species of bacteria -- given the name Bacillius F -- was found in laboratory tests to have shown signs of slowing down the process of ageing on mice, the Russian Academy of Sciences (RAN) said. 

The Siberian branch of the RAN said Bacillius F lags 3 million years behind similar bacteria in evolutionary terms, according to the characteristics of proteins and some other factors. 

"Taking into consideration the unusual living environment, one can only marvel at the resilience of these bacteria," it said. 

It added that the organisms found in Russia's northern region of Yakutia -- home to the coldest inhabited area on the planet -- reproduce at just 5 degrees Celsius. 

"We just thought: since the bacteria were found in the permafrost where they were successfully preserved they will possibly have mechanisms of retaining viability," added Nadezhda Mironova, senior research scientist at the Institute of Chemical Biology and Fundamental Medicine of the Russian Academy of Sciences. 

"This is what happened," she was quoted as saying. 

Injections of the bacteria into mice have helped boost the natural defences of the animals as they grew older. 

"Bacillius F injections have favourably affected the quality of being of the aging animals," the Russian scientists said. 

"First and foremost, this concerns immunity and the speed of its activation." 

Experiments have shown that metabolism in the tested mice have increased by 20 to 30 percent, the scientists said, adding that the bacterium may also reduce instances of senile blindness but not the emergence of tumours. 

The Russian Academy of Sciences did not say how many mice were tested, adding more animals were needed for the experiments to be more reliable. The mice from a test group lived longer than those in a control group however, it said, calling the results "impressive." 

Source: AFP [January 17, 2012]

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Drugs used to overcome cancer may also combat antibiotic resistance

Drugs used to overcome cancer may also combat antibiotic resistance, finds a new study led by Gerry Wright, scientific director of the Michael G. DeGroote Institute for Infectious Disease Research at McMaster University. 

"Our study found that certain proteins, called kinases, that confer antibiotic resistance are structurally related to proteins important in cancer," says Wright about the study published in Chemistry & Biology. 

"The pharmaceutical sector has made a big investment in targeting these proteins, so there are a lot of compounds and drugs out there that, although they were designed to overcome cancer, they can in fact be looked at with fresh eyes and maybe repurposed to address the problem of antibiotic resistance." 

The large-scale study involved screening 14 antibiotic resistant molecules against 80 chemically diverse protein kinase inhibitors. 

Antibiotic resistance is a problem growing in global scope, as more viruses have overcome currently available antibiotics. 

"As a result, new drugs and antibiotic strategies are urgently needed to fill the gap in infectious disease control," says Wright, adding he hopes future studies in combination therapies will provide new insight into antibiotic resistance. 

"One of the challenges facing the drug discovery community is the lack of new chemical scaffolds with antibiotic activity. This has led to the open question of whether all easily implementable antibiotic chemical scaffolds have already been exploited over the last 50 years: the so-called ''low hanging fruit''." 

Source: McMaster University [December 21, 2011]

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How Bacteria Build Homes Inside Healthy Cells

Bacteria are able to build camouflaged homes for themselves inside healthy cells -- and cause disease -- by manipulating a natural cellular process. 

Purdue University biologists led a team that revealed how a pair of proteins from the bacteria Legionella pneumophila, which causes Legionnaires disease, alters a host protein in order to divert raw materials within the cell for use in building and disguising a large structure that houses the bacteria as it replicates. 

Zhao-Qing Luo, the associate professor of biological sciences who headed the study, said the modification of the host protein creates a dam, blocking proteins that would be used as bricks in cellular construction from reaching their destination. The protein "bricks" are then diverted and incorporated into a bacterial structure called a vacuole that houses bacteria as it replicates within the cell. Because the vacuole contains materials natural to the cell, it goes unrecognized as a foreign structure. 

"The bacterial proteins use the cellular membrane proteins to build their house, which is sort of like a balloon," Luo said. "It needs to stretch and grow bigger as more bacterial replication occurs. The membrane material helps the vacuole be more rubbery and stretchy, and it also camouflages the structure. The bacteria is stealing material from the cell to build their own house and then disguising it so it blends in with the neighborhood." 

The method by which the bacteria achieve this theft is what was most surprising to Luo. 

The bacterial proteins, named AnkX and Lem3, modify the host protein through a biochemical process called phosphorylcholination that is used by healthy cells to regulate immune response. Phosphorylcholination is known to happen in many organisms and involves adding a small chemical group, called the phosphorylcholine moiety, to a target molecule, he said. 

The team discovered that AnkX adds the phosphorylcholine moiety to a host protein involved in moving proteins from the cell's endoplasmic reticulum to their cellular destinations. The modification effectively shuts down this process and creates a dam that blocks the proteins from reaching their destination. 

The bacterial protein Lem3 is positioned outside the vacuole and reverses the modification of the host protein to ensure that the protein "bricks" are free to be used in creation of the bacterial structure. 

This study was the first to identify proteins that directly add and remove the phosphorylcholine moiety, Luo said. 

"We were surprised to find that the bacterial proteins use the phosphorylcholination process and to discover that this process is reversible," he said. "This is evidence of a new way signals are relayed within cells, and we are eager to investigate it." 

The team also found that the phosphorylcholination reaction is carried out at a specific site on the protein called the Fic domain. Previous studies had shown this site induced a different reaction called AMPylation. 

It is rare for a domain to catalyze more than one reaction, and it was thought this site's only responsibility was to transfer the chemical group necessary for AMPylation, Luo said. 

"Revealing that this domain has dual roles is very important to identify or screen for compounds to inhibit its activity and fight disease," he said. "This domain has a much broader involvement in biochemical reactions than we thought and may be a promising target for effective treatments." 

During infection bacteria deliver hundreds of proteins into healthy cells that alter cellular processes to turn the hostile environment into one hospitable to bacterial replication, but the specific roles of only about 20 proteins are known, Luo said. 

"In order to pinpoint proteins that would be good targets for new antibiotics, we need to determine their roles and importance to the success of infection," he said. "We need to understand at the biochemical level exactly what these proteins do and how they take over natural cellular processes. Then we can work on finding ways to block these activities, stop the infection and save lives." 

A paper detailing their National Institutes of Health-funded work is published in the current issue of the Proceedings of National Academy of Sciences. In addition to Luo, Purdue graduate student Yunhao Tan and Randy Ronald of Indiana University co-authored the paper. 

Luo next plans to use the bacterial proteins as a tool to learn more about the complex cellular processes controlled by phosphorylcholination and to determine the biochemical processes role in cell signaling. 

Source: Purdue University [December 20, 2011]

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Researchers create living 'neon signs' composed of millions of glowing bacteria

In an example of life imitating art, biologists and bioengineers at UC San Diego have created a living neon sign composed of millions of bacterial cells that periodically fluoresce in unison like blinking light bulbs. 

Thousands of fluorescent E. coli bacteria make up a biopixel [Credit: Hasty Lab, UC San Diego]

Their achievement, detailed in this week's advance online issue of the journal Nature, involved attaching a fluorescent protein to the biological clocks of the bacteria, synchronizing the clocks of the thousands of bacteria within a colony, then synchronizing thousands of the blinking bacterial colonies to glow on and off in unison. 

A little bit of art with a lot more bioengineering, the flashing bacterial signs are not only a visual display of how researchers in the new field of synthetic biology can engineer living cells like machines, but will likely lead to some real-life applications. 

Using the same method to create the flashing signs, the researchers engineered a simple bacterial sensor capable of detecting low levels of arsenic. In this biological sensor, decreases in the frequency of the oscillations of the cells' blinking pattern indicate the presence and amount of the arsenic poison. 

The smaller chips contain about 500 blinking bacterial colonies or biopixels [Credit: Hasty Lab, UC San Diego]

Because bacteria are sensitive to many kinds of environmental pollutants and organisms, the scientists believe this approach could be also used to design low cost bacterial biosensors capable of detecting an array of heavy metal pollutants and disease-causing organisms. And because the senor is composed of living organisms, it can respond to changes in the presence or amount of the toxins over time unlike many chemical sensors. 

"These kinds of living sensors are intriguing as they can serve to continuously monitor a given sample over long periods of time, whereas most detection kits are used for a one-time measurement," said Jeff Hasty, a professor of biology and bioengineering at UC San Diego who headed the research team in the university's Division of Biological Sciences and BioCircuits Institute. "Because the bacteria respond in different ways to different concentrations by varying the frequency of their blinking pattern, they can provide a continual update on how dangerous a toxin or pathogen is at any one time." 

"This development illustrates how basic, quantitative knowledge of cellular circuitry can be applied to the new discipline of synthetic biology," said James Anderson, who oversees synthetic biology grants at the National Institutes of Health's National Institute of General Medical Sciences, which partially funded the research. "By laying the foundation for the development of new devices for detecting harmful substances or pathogens, Dr. Hasty's new sensor points the way toward translation of synthetic biology research into technology for improving human health." 

The larger chips contain about 13,000 biopixels [Credit: Hasty Lab, UC San Diego]

The development of the techniques to make the sensor and the flashing display built on the work of scientists in the Division of Biological Sciences and School of Engineering, which they published in two previous Nature papers over the past four years. In the first paper, the scientists demonstrated how they had developed a way to construct a robust and tunable biological clock to produce flashing, glowing bacteria. In the second paper, published in 2010, the researchers showed how they designed and constructed a network, based on a communication mechanism employed by bacteria, that enabled them to synchronize all of the biological clocks within a bacterial colony so that thousands of bacteria would blink on and off in unison. 

"Many bacteria species are known to communicate by a mechanism known as quorum sensing, that is, relaying between them small molecules to trigger and coordinate various behaviors," said Hasty, explaining how the synchronization works within a bacterial colony. "Other bacteria are known to disrupt this communication mechanism by degrading these relay molecules." 

But the researchers found the same method couldn't be used to instantaneously synchronize millions of bacteria from thousands of colonies. 

Tiny microfluidic chips allow the researchers to synchronize the bacteria to fluoresce or blink in unison [Credit: Hasty Lab, UC San Diego]

"If you have a bunch of cells oscillating, the signal propagation time is too long to instantaneously synchronize 60 million other cells via quorum sensing," said Hasty. But the scientists discovered that each of the colonies emit gases that, when shared among the thousands of other colonies within a specially designed microfluidic chip, can synchronize all of the millions of bacteria in the chip. "The colonies are synchronized via the gas signal, but the cells are synchronized via quorum sensing. The coupling is synergistic in the sense that the large, yet local, quorum communication is necessary to generate a large enough signal to drive the coupling via gas exchange," added Hasty. 

Graduate students Arthur Prindle, Phillip Samayoa and Ivan Razinkov designed the microfluidic chips, which for the largest ones, contain 50 to 60 million bacterial cells and are about the size of a paper clip or a microscope cover slip. The smaller microfluidic chips, which contain approximately 2.5 million cells, are about a tenth of the size of the larger chips. 

Each of the blinking bacterial colonies comprise what the researchers call a "biopixel," an individual point of light much like the pixels on a computer monitor or television screen. The larger microfluidic chips contain about 13,000 biopixels, while the smaller chips contain about 500 pixels. 

Hasty said he believes that within five years, a small hand-held sensor could be developed that would take readings of the oscillations from the bacteria on disposable microfluidic chips to determine the presence and concentrations of various toxic substances and disease-causing organisms in the field.

Source: University of California - San Diego [December 18, 2011]

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Researcher determines how Legionnaires’ bacteria proliferate, cause disease

A University of Louisville scientist has determined for the first time how the bacterium that causes Legionnaires’ disease manipulates our cells to generate the amino acids it needs to grow and cause infection and inflammation in the lungs. The results are published online today (Nov. 17) in Science. 

Yousef Abu Kwaik, Ph.D., the Bumgardner Endowed Professor in Molecular Pathogenesis of Microbial Infections at UofL, and his team believe their work could help lead to development of new antibiotics and vaccines. 

“It is possible that the process we have identified presents a great target for new research in antibiotic and vaccine candidates, not only for Legionnaires’ disease but in other bacteria that cause illness,” he said. 

According to the Centers for Disease Control and Prevention, Legionnaires’ disease is a lung infection caused by the bacterium called Legionella. The bacterium got its name in 1976, when many people who went to a Philadelphia convention of the American Legion suffered from an outbreak of pneumonia of unknown causes that was later determined to be caused by the bacterium. Each year, between 8,000 and 18,000 people are hospitalized with Legionnaires’ disease in the U.S. There is no vaccine currently available for it. 

For two years, the researchers examined Legionella which is an intercellular bacterium that exists in amoebae in the water systems; it is transmitted to humans through inhalation of water droplets. Cooling towers and whirlpools are the major sources of transmission. The bacterium uses the amoeba’s cellular process to “tag” proteins, causing them to degrade into their basic elements of amino acids. These amino acids are used by the bacteria as the main source of energy to grow and cause disease. 

“The bacteria live on an ‘Atkins diet’ of low carbs and high protein, and they trick the host cell to provide that specialized diet,” Abu Kwaik said. 

The same process occurs in a host – animal or human – who inhales the bacterium and is diagnosed with Legionnaires’ disease. However, the bacteria do not tag the proteins, but rather trick the host into tagging the proteins for degradation to generate the amino acids. 

In the laboratory, Abu Kwaik and his team saw that by inactivating the bacterial virulence factor responsible for tricking the cell into tagging proteins for degradation in mice models, the pulmonary disease was totally prevented. This was totally due to disabling the bacteria from generating amino acids, he said. 

The process was then reversed, and the disease became evident when the mice, infected by the disabled bacteria, were injected with amino acids to compensate for the inability of the altered bacteria. 

“Bacteria need to live on high protein and amino acids as sources of nutrition and energy in order to replicate in a host. This is what causes pulmonary disease,” Abu Kwaik said. “No one has known how they generate sufficient sources of nutrients from the host to proliferate. Our work is the first to identify this process for any bacteria that cause disease.” 

He added that the type of host infected does not appear to affect the process. “Whether in a single-cell amoeba or a multi-cellular mammal, Legionella seems to know what to do; the process is the same, and is highly conserved through evolution.  By interfering with the bacterium’s sources of nutrients, we can stop it from thriving and causing disease.” 

Examining nutrient sources for organisms with the goal of stopping them from acquiring nutrients is a relatively new arena of basic research that deserves further study, he said. “We went after the basics – the food and energy source – which are prerequisite for the bacteria to grow and cause disease. It is not a process that is well understood yet, but by first discovering how an organism gets nutrients by tricking the host into degrading proteins, and then interfering with that process, we can, in effect, starve it to death and prevent or treat the disease.” 

Source: University of Louisville [November 17, 2011]

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Report answers questions about E. coli: The good, the bad and the deadly

It has been the cause of infamous international foodborne disease outbreaks and yet it is the most studied bacterium in science, an essential part of the human digestive tract, and a backbone of the biotech industry. To enhance public understanding of the bacterium Escherichia coli, the American Academy of Microbiology brought together the nation's leading experts to consider and answer some of the most frequently asked questions about this multifaceted microorganism. 

"The story of E. coli, what we are trying to tell in this report, is really much larger than just its role as a pathogen. It's been such a large component of research for so long – so much of what we know about biology has come from studying E. coli," says Michael Doyle of the University of Georgia Center for Food Safety, a member of the steering committee. 

The report, entitled FAQ – E.coli: Good, Bad and Deadly is based on the deliberations of 13 of the nation's leading experts who met for one day in September 2011 to develop clear answers to frequently asked questions regarding the role of E. coli in scientific research, human health and foodborne disease. 

Some of the questions considered by the report are: 

• What is E. coli anyway? 

• How has E. coli contributed to our understanding of biology? 

• What does naturally occurring E. coli in our GI tract do? 

• What is the difference between "good" E. coli that inhabits our GI tract and the "bad" E. coli that makes us sick? 

• Why does E. coli make some people sick and not others? 

• How does E. coli become pathogenic? 

• How does our food become contaminated with E. coli? 

• What steps are being taken to protect our food from contamination by pathogenic E. coli? 

• What types of food are most commonly associated with E. coli, and why do there seem to be more cases of contamination recently? 

Most answers begin with a simple paragraph summarizing what is known, followed by a more detailed explanation. In addition, spread throughout the report are sidebar boxes discussing issues related to the questions such as a list of Nobel Prizes awarded for work done on E. coli and a discussion of toxins created by the bacterium. 

FAQ – E. coli: Good, Bad and Deadly is the latest offering in a series of reports designed to provide a rapid response to emerging issues. Traditionally Academy reports are based on multi-day colloquia after which the final report can take up to a year to develop. The FAQ series are based on single-day meetings focused on specific questions after which a final report is published in 2-3 months. 

"The Academy FAQ reports explain complex microbiological problems in a timely, balanced format that is easily understandable by the public, the media and policy makers," says Stanley Maloy of San Diego State University, who moderated the colloquium. 

Source: American Society for Microbiology [November 15, 2011]

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