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HIV-1 contains a conical capsid built from ~1,500 copies of the viral CA protein, which pack together on a hexagonal lattice and enclose the RNA genome (left). Electron cryocrystallography was used to produce the first unambiguous pseudoatomic model of the hexamers from which the lattice is assembled (right). The hexamers are stabilized by close interactions between the two CA domains of adjacent subunits (shown in different colors). This newly visualized intermolecular interaction is a novel drug target to disrupt capsid assembly, which may prevent the formation of infectious particles.

by Jason Socrates Bardi, The Scripps Research Institute – Scientists at The Scripps Research Institute have published a detailed molecular model of the full-length HIV CA protein—a viral protein that forms a cone-shaped shell around the genome of HIV. This structure reveals a never-before-seen molecular interaction that may be a weakness at the core of the virus.

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Cell biologists Mark Yeager and Barbie Ganser-Pornillos show a model of the HIV capsid, in which the CA protein forms a cone-shaped shell around the genome of HIV.

CA plays a crucial role in the lifecycle of HIV because it forms a protein shell inside infectious particles, providing a scaffold that organizes important components of the virus. The new CA structure, published in the October 5 issue of the journal Cell, has clinical implications and may help scientists develop new drugs for treating HIV.

“AIDS is a bona fide pandemic,” says study author Mark Yeager. “There are several effective drugs and methods for treating and preventing HIV infections, but there is an ongoing need for new therapy due to the shear enormity of the disease and the emergence of drug resistance.”

Yeager is a professor in the Department of Cell Biology at The Scripps Research Institute and staff cardiologist and director of cardiovascular research at Scripps Clinic. He also has a joint appointment as Andrew P. Somlyo Professor and Chair of the Department of Molecular Physiology and Biological Physics at the University of Virginia. Yeager supervised the research, which was conducted by his postdoctoral fellow Barbie Ganser-Pornillos.

Since it was first reported more than 25 years ago, HIV has spread to every corner of the world. Globally, according to the latest figures available from the World Health Organization, some 40 million people were living with HIV in 2006. The Centers for Disease Control and Prevention (CDC) estimates that 40,000 people become infected with HIV every year in the United States.

HIV infections can be successfully managed for years with a variety of existing drugs known as antiretrovirals, which interfere with critical parts of the viral lifecycle. Interfering with some of these stages can prevent the virus from replicating, integrating its genome into the cell’s DNA, or processing new infectious viral particles.

Doctors often prescribe a regimen of several antiretrovirals from different classes for people living with HIV because AIDS drugs with different mechanisms of action are more effective in combination than when taken alone. Finding new drugs with new mechanisms of action is important because HIV constantly mutates and may become resistant to existing drugs.

In general, the capsid (the protein coat that covers the core of a virion) is an attractive target because it plays a crucial role in the viral lifecycle. It packages and organizes the HIV genome, and this is necessary for the virus to transmit and replicate efficiently. If chemical compounds could target the CA protein, scientists might be able to prevent the protein’s assembly into capsid shells and thereby block infectivity of HIV. Capsid inhibitors would be a novel class of drugs that would complement existing pharmaceuticals.

So Ganser-Pornillos and Yeager set out to find the complete structure of the HIV-1 CA.

But this was no easy task. Assembled capsid shells are large enough to be seen under the most powerful electron microscopes, but are too small and asymmetric to be studied in detail. For years, structural biologists attempted to solve the structure of the CA protein using other methods, but the protein is flexible, and the structure proved elusive. The problem was that CA has two rigid pieces or domains held together by a flexible linker. Think of them like two water balloons tied together with a short string. Scientists had successfully chopped the protein into pieces and solved the structures of the two domains, but despite many years of trying, nobody had visualized how the two domains fit together and how hundreds of copies of CA pack within the lattice of the capsid shell.

Ganser-Pornillos solved the problem by finding exact conditions that fixed assemblies of the full-length CA molecules into well-ordered, two-dimensional (2D) arrays. Normally, recombinant CA molecules form cylindrical shells in vitro, but a few years ago, a single mutation was identified that allowed the formation of alternative shapes—cylinders, cones, and spheres. By creating conditions in which CA formed large hollow spheres, Ganser-Pornillos was able to generate extended 2D crystalline sheets by flattening the spheres onto a thin layer of carbon. Ganser-Pornillos, assisted by Scripps Research Staff Scientist Anchi Cheng, solved the three-dimensional structure of the CA molecules in the sheets by computational analysis of images of tilted 2D crystals recorded in the electron microscope.

Even though the 2D crystals of CA were generated artificially, they were thought to recapitulate the lattice in the HIV capsid because the packing of the CA molecules had a hexagonal, honeycomb like pattern, similar to that seen in authentic viral particles. However, the hexagonal packing of the molecules in the 2D crystals was much more regular, so that a detailed structure could be determined. The clarity of the three-dimensional electron microscopy map was sufficient so that existing high-resolution structures of the two domains of CA could be “docked” into place. The resulting atomic model of the capsid lattice revealed three types of interactions that stabilize this inner core of HIV.

In particular, an interaction between the two pieces of CA had not been visualized previously. Interfering with this interaction could disrupt assembly of the capsid shell and block formation of infectious particles.

“The structure allows us to visualize the interface between the two domains of CA, which we think is the target for a set of experimental drugs,” says Ganser-Pornillos. She and Yeager are now working to improve the structure. They hope to find novel compounds that bind to CA to test if they interfere with the virus’s infectivity.

The article is entitled “Structure of Full-Length HIV-1 CA: A Model for the Mature Capsid Lattice,” and appears in the October 5, 2007 issue of the journal Cell.

Support for this work was provided by grants from the National Institutes of Health and through a postdoctoral fellowship from the George E. Hewitt Foundation for Medical Research.

image0026.jpgBy Thomas J. Donohue, President and CEO, U.S. Chamber of Commerce
November 27, 2007

Last week, in the first of three columns on U.S. health care, I examined some of the strengths of our system, including widespread coverage, excellent medical facilities, and tremendous new employment opportunities. Our health care system has achieved many outstanding results, including adding 30 years to Americans’ life spans in the last century. Despite its accomplishments, it has many significant shortcomings.

Costs. We pay more for health care than any other modern society. Yet on a national basis, we fall short on some key indices such as infant mortality and life expectancy. Costs are escalating with no end in sight–for businesses, families, and the government. Unless solutions are found–and soon–these spiraling costs will bankrupt companies, force businesses and individuals to drop coverage, destroy the long-term viability of Medicare and Medicaid, and erode America’s global competitiveness.

Medical Mistakes. Medical accidents are unacceptably high. An estimated 98,000 Americans die annually from preventable medical mistakes. According to the Institute of Medicine, medication errors harm at least 1.5 million people each year. In addition to the pain and heartbreak, these errors add incalculable costs to our health care system.

Medical Liability. Legal redress should be available for the victims of these mistakes, but that’s no excuse for all the frivolous liability claims that are driving up prices and driving health care providers out of the profession.

Health IT. Most providers lack the IT systems necessary to coordinate a patient’s care with other providers, share needed information, and monitor compliance with prevention and disease-management programs. This makes it impossible for doctors to provide the highest level of care and drives up costs by contributing to errors and redundant tests.

Consumer Responsibility. We need a far greater level of personal responsibility on the part of our citizens. Consumers need to understand the impact of their health care decisions and the cost of their treatments. And, they need to take better care of themselves.

The Uninsured. There are 47 million people in this country without health care coverage, but that’s only part of the story. In fact, nearly half of the 47 million uninsured remain so on average for just four months. In addition, if you subtract noncitizens, those making more than $75,000 who choose not to purchase insurance, and those who are eligible for government-provided care but don’t take it, the number of long-term uninsured Americans is probably in the range of 10 to 15 million. That’s still an unacceptably high number, but it’s nowhere near 47 million.

So how do we build on the positive aspects of our health care system while addressing its significant shortcomings, and without implementing an expensive, inefficient government-run program that would take us in the wrong direction? Stay tuned …

http://www.uschambermagazine.com/content/071127?n=w

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The surface represents the combination of protein stability and folding kinetics required for it to be exported from the endoplasmic reticulum with minimum efficiency. Located in this space defined by protein folding and misfolding kinetics and thermodynamics are two points, which represent two variants of a protein: a mutant, which is outside the surface, and is therefore not exported efficiently enough for it to function, and the wild type, which is inside the surface and is exported normally.

by Jason Bardi, Scripps Research Institute – A team of scientists at The Scripps Research Institute has come up with a simple but comprehensive way of probing the parts of our biological machinery that controls protein folding, packaging, and export from our cells.

The team has created a new model integrating the chemistry and biology of protein folding, called folding for export (FoldEx), which provides a general framework for understanding the causes and potential treatment strategies of diseases that arise when this protein homeostasis machinery malfunctions. These diseases include type 2 diabetes, Gaucher’s disease, cystic fibrosis, and Alzheimer’s disease.

The team describes its new mathematical model of the chemistry and biology of cellular protein homeostasis in the latest issue of the journal Cell, published on November 16.

“If we can begin to understand how the cellular folding, packaging, and export pathways work,” says Jeffery Kelly, who is the Lita Annenberg Hazen Professor of Chemistry and a member of the The Skaggs Institute for Chemical Biology at Scripps Research, “this should give us insight into how to ameliorate diseases that arise when these pathways get out of balance.”

“Our new model allows us to think about therapeutic intervention in a completely new way,” says Professor William Balch, a Scripps Research cell biology professor and a member of the Scripps Research Institute of Childhood and Neglected Diseases.

Balch and Kelly guided the creation of the FoldEx model with Professor Joel Buxbaum. The new approach began as a back-of-the-envelope idea and was made a reality by the combined efforts of Assistant Professor Evan Powers and Luke Wiseman, a recent graduate of the Kellogg School of Science and Technology at Scripps Research.
Biological Machinery in Health and Disease

In many heritable diseases, specific mutations within a particular gene simply cause the protein product of the gene to malfunction. But often a defect in the general biological machinery of the cell that interacts with the protein is also involved, causing a loss of activity because of this machinery’s crucial role in protein expression and function.

Gene expression starts when DNA is transcribed into RNA and is then translated into proteins, but the process does not end there. Proteins are unfolded when first expressed, and they usually must fold into a compact, three-dimensional structure in order to function properly. The biological machinery helps them achieve this by providing special molecules called chaperones and folding enzymes that help proteins fold.

Quite often, proteins also need help to get to where they are going. Some have to be transported and inserted into a cell’s membrane. Others are intended to be secreted into the bloodstream or surrounding tissues, and have to be transported outside the cell. The biological machinery of the cell accomplishes all this starting with a tubular labyrinth called the endoplasmic reticulum (ER). The ER is like a massive shipping facility that folds, packages, and exports proteins to destinations inside and outside of the cell. About a third of the proteins in the human body go through the ER.

The biological machinery of the ER also protects the body by degrading potentially dangerous proteins. Sometimes it is too efficient at doing so, and many diseases can be traced to this action. Cystic fibrosis, for instance, is caused by the loss of an essential protein called cystic fibrosis transmembrane conductance regulator (CFTR), a chloride channel, that regulates hydration of the lung surface. Without CFTR, the mucous in the lungs becomes thick, sticky, and prone to harboring bacterial infections—the classic symptoms of cystic fibrosis.

People with cystic fibrosis have mutations in their CFTR protein, and the biological machinery responds to these mutations by targeting mutated CFTR for degradation. Thus, it is not that people with cystic fibrosis cannot make CFTR, but that the protein they do make simply can never make it to the cell surface. A single mutation in CFTR is enough to cause it to misfold, become degraded, and cause cystic fibrosis.

Many other diseases result from a similar malfunction, including Gaucher disease, which is caused by the accumulation of a fatty substance in the spleen, liver, lungs, bone marrow, and sometimes the brain because of the loss of the lipid metabolizing enzyme that breaks it down. Gaucher disease is often treated, in fact, by administering a recombinant form of the missing enzyme.

A converse problem occurs when the biological machinery exports proteins out of the ER that then misfold and cause destruction once they get to their destination. A number of rare and common conditions termed “amyloid” diseases stem from this etiology. Familial amyloid polyneuropathy (FAP), for instance, results from the misfolding and deposition of one of 100 mutants of the protein transthyretin. An analogous disease called familial amyloid cardiomyopathy (FAC) causes fibril formation in the heart and leads to cardiac dysfunction. About one million African-Americans carry the gene that predisposes them to FAC. Another amyloid disease affecting the heart, Senile Systemic Amyloidosis (SSA), afflicts an estimated 10 to 15 percent of all Americans over the age of 60.

Moreover, the deposition of protein plaques in the brain is seen in diseases like Alzheimer’s, Parkinson’s, and Huntington’s, though it is not yet clear whether these plaques are the cause or a downstream effect of the disease.

For years, scientists have recognized that these diseases all in some way relate to the underlying biological machinery, which protects us when we are young. But how can scientists approach tinkering with this biology to treat these diseases? That is exactly the question that the FoldEx mathematical model addresses.
The Model and What It Predicts

FoldEx started with a conversation three years ago among Wiseman, Powers, and Buxbaum on how the biological machinery of the ER is related to misfolding diseases.

Soon the conversation grew to involve Kelly, Balch, and nearly everybody in their two laboratories. The product of this work, after many, many rounds of refinement, was a sophisticated analysis that treats rather complex competing pathways as if they were single enzymes, greatly simplifying the analysis without loosing the essence of the chemistry and biology of protein homeostasis.

Because of this simplicity, FoldEx can represent complicated processes with well-established biochemical principles that help explain how these pathways compete for various conformational ensembles of proteins and dictate the balance between folding, secretion, and degradation. But this simplification does not come at the expense of relevance. In the Cell paper, the Scripps Research team showed that FoldEx can predict concepts, principles, and results that have already been validated. The real value of the model, however, is that it can be used to predict how folding and misfolding diseases may be delayed or reversed by manipulating the innate biological machinery.

FoldEx shows that by adjusting the folding and export machinery, it may be possible to address misfolding diseases without replacing the problematic proteins. The model predicts how the system would respond if you tweak it here or there to normalize the physiology—for instance by increasing degradation to reduce export and therefore reduce the amount of amyloid protein that gets deposited in tissues like the brain.

Likewise, one can imagine adjusting the levels of chaperone helper molecules so that a fraction of the proteins that are normally degraded will be folded and exported to restore function in loss-of-function diseases. The idea is that if partially functional folded mutant proteins can be delivered to where they are needed, they will be active enough to avoid disease.

What this means, say Balch and Kelly, is that you aren’t necessarily destined to get a disease even if you’ve inherited a bad mutation if we can learn to adjust the fold or the biological folding environment. Here, a severe mutation could be viewed as a more benign polymorphism.

The article in Cell is titled”An Adaptable Standard for Protein Export from the Endoplasmic Reticulum.”

http://www.cell.com/content/article/abstract?uid=PIIS0092867407013438.

Support for this work was provided by the National Institutes of Health, the Skaggs Institute for Chemical Biology, the Lita Annenberg Hazen Foundation, the Cystic Fibrosis Foundation, a Norton B. Gilula Fellowship, and a Fletcher Jones Foundation Fellowship.

by Mark Schrope and Mika Ono Benedyk, Scripps Research Institute

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“Our approach sidesteps the [antibiotic] resistance problem,” says Professor Kim Janda, who led the new study. Staph bacteria magnified 4,780 times. Image courtesy of Janice Carr, Centers for Disease Control and Prevention.

In hopes of combating the growing scourge of antibiotic-resistant bacteria, in particular drug-resistant staph bacteria, a team of scientists from The Scripps Research Institute has designed a new type of vaccine that could one day be used in humans to block the onset of infection. The advantage of the new vaccine is that it would work not only on current bacterial resistant stains but also would not induce the potential for new bacterial resistance because, rather than killing bacterial cells, it blocks their communication system, preventing the shift from harmless to virulent, thus allowing the body’s natural defenses to combat the bacteria.

The work was published in the October 29 issue of the journal Chemistry and Biology.

Staph and other infections are becoming increasingly deadly because many strains of the bacteria that cause disease develop resistance to the array of antibiotics used to control them. A recent Centers for Disease Control (CDC) report released last week estimated that more than 94,000 Americans were infected in 1995 by a drug-resistant staph “superbug” called methicillin-resistant Staphylococcus aureus (MRSA), and more than 18,000 Americans died that year during hospital stays involving this type of infection.

The bacterial infection process is dependent on a sort of chemical conversation between individual bacterial cells, referred to as quorum sensing. In their free-living state, bacteria are typically easy to kill and non-virulent. The shift to virulence is dependent on small molecules emitted by bacteria known as autoinducers, because bacteria sense when concentrations of these autoinducers are high enough to suggest a large number of other bacteria are present.

“Bacteria basically sense they have enough of their buddies around to allow them to say, ‘OK, we’re in a favorable environment to start turning on certain genes,'” says team leader Professor Kim Janda, director of the Worm Institute for Research and Medicine at Scripps Research and a vaccine expert who has worked on the development of vaccines for obesity and drugs of addiction, among other problems.

The genes turned on by quorum sensing may encode proteins harmless to their hosts, but they can also code for the toxins and other products arising from bacterial infections that cause disease. Sequestering autoinducers in some way could therefore block quorum sensing and, hence, the establishment of infections. The scientists predict that such a strategy would not lead to resistance in bacteria because it wouldn’t kill the cells. Bacteria would simply remain in an inert form because they would be tricked into “thinking” not enough other cells were present to shift into their virulent mode.

Bacteria use a variety of genetic mechanisms in quorum sensing. The Scripps Research team focused on Gram-positive bacteria, whose quorum sensing is controlled by four basic types of autoinducers tied to a circuit known as the accessory gene regulator. Based on the known structure of one of these autoinducers, the team designed a molecule known as a hapten that, when conjugated with specific proteins using well-established procedures, induces the production of antibodies by the immune system.

The Janda group intentionally designed the hapten to be stable enough to work well as a potential treatment, and ultimately chose to pursue work with one of the haptens that proved the most stable. Past research by other groups has involved successfully blocking quorum sensing using molecules that essentially plug the keyholes on cell surfaces that allow bacteria to sense autoinducers, but such strategies have been hampered by the inherent instabilities of the molecules involved.

Next, the team isolated and studied the antibodies produced in mice injected with the hapten, called AP4. Subsequent experiments revealed that one of these antibodies in particular, when administered to mice infected with Staphylococcus aureus, was highly effective at binding with and sequestering the targeted autoinducer, and to a lesser extent with a second autoinducer. This activity proved to effectively block quorum sensing and infection in the mice.

Resistance to S. aureus, a common form of Staph infection has become a major concern in hospitals, and, as the recent CDC report indicates, outside of medical settings as well. As a result, says Janda, “I think the impact of this approach could be really huge, because our approach sidesteps the resistance problem with common antibiotic treatments.”

Janda says the antibody AP4-24H11 could one day be given to humans as a passive vaccine to block infections as it did in mice. The AP4 hapten could also be applied as an active vaccine that would induce production of antibodies to block quorum sensing. He says such vaccines could, for instance, be given to patients entering the hospital for surgery to prevent infection by Staph bacteria. This would not, however, probably be an effective treatment against infections that have already progressed, because in such cases the damage from quorum sensing would already have been done.

Janda and his colleagues, including Junguk Park, Gunnar Kaufmann and Richard Ulevitch, chairman of the Scripps Research Department of Immunology, are already working to design related haptens that will induce antibodies effective against all the autoinducers used by Gram-positive bacteria, which might one day be administered as a vaccine cocktail to prevent infection by a wide range of bacteria. The group is seeking a pharmaceutical partner to fund further tests with AP4 and AP4-24H11 in animal models and, if all goes well, to carry a vaccine through human clinical trials.

Bacteria can 1) ___ between species, and have evolved mechanisms to interfere with the communication. Probably this is but one of many cunning strategies bacteria have for manipulating chemical communication. Certain snippets of their chemical conversation are almost universally understood. Howard Hughes Medical Institute researchers have found that bacteria of different 2) ___ can talk to each other using a common language, and that some species can manipulate the conversation to confuse other bacteria. The interspecies crosstalk and misdirection could have important consequences for 3) ___ health. The ability of cells to communicate with one another and the ability to interfere with the communication process could have consequences in niches containing competing species of bacteria or in niches where bacteria associate with humans. In the gut, the normal 4) ___ might interfere with cell-cell communication to thwart bacterial invaders. Using a chemical communication process called 5) ___ ___, bacteria converse among themselves to count their numbers and to get the population to act in unison. A synchronized group of bacteria can mimic the power of a multi-cellular 6) ___, ready to face challenges too daunting for an individual microbe going it alone. Swelling populations trigger their quorum-sensing apparatuses, which have different effects in different types of bacteria. One species might respond by releasing a 7) ___, while another might cut loose from a biofilm and move on to another environment. Each species of bacteria has a private 8) ___, but most also share a molecular vernacular, discovered about 10 years ago. A chemical signal called autoinducer-2 (AI-2), originating from the same gene in all bacteria, is released outside the cell to announce the cell’s 9) ___. Nearby bacteria take a local census by monitoring AI-2 levels and conduct themselves as the circumstances warrant. Researchers have speculated that AI-2 is a universal language, and the new studies from Princeton are the first to show those conversations taking place – and producing consequences — between co-mingling species. But this common language does not guarantee that the correct 10) ___ gets through. In an earlier study, it was found, that E. coli both produce and consume AI-2. This study set up an experiment where multitudes of E. coli first produced then devoured enough AI-2 to confuse marine bacteria, essentially fooling the thriving oceanic group into thinking its members were few, thereby terminating its quorum-sensing behaviors. In a more realistic encounter, E. coli was mixed with V. cholerae , the cholera-causing bacteria that mixes with E. coli in human guts. When cholera bacteria sense a quorum, they turn off their toxins and excrete an enzyme to cut themselves loose from the intestine in order move out of the body where they can infect another person. Here, E. coli squelched much of the quorum-sensing response of the cholera bacteria, although the effect was not as dramatic as with the marine bacteria. Consumption of the signal could be a mechanism that allows one kind of bacteria to block another kind of bacteria from counting how many neighbors they have and, in turn, properly controlling its behavior. This study moves us closer to really understanding how these 11) ___ interactions happen in nature.

ANSWERS: 1) communicate; 2) species; 3) human; 4) microflora; 5) quorum sensing; 6) organism; 7) toxin; 8) language; 9) presence; 10) message; 11) chemical

This past week, Dr. Glen Park, Sr. Director, Clinical and Regulatory Affairs presided at a very successful, highly interactive Pre-CTA meeting in Ottawa. The weather was beautiful and the view of the Ottawa River from the meeting room was breathtaking. The project is already ongoing in Israel, South Africa, and the US and will be shortly starting in Europe. Target Health is monitoring in the US and Canada, and performing 1) the global EDC trial, 2) data management, 3) biostatistics and 4) medical writing. We will also submit the NDA at the completion of the program.

For more information, please contact Dr. Jules T. Mitchel or Joyce Hays. For new business opportunities, contact Adrian Pencak, (Vice President, Business Development).

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May all of our BLOG community, have a healthy Thanksgiving !

Here is an FDA link, to report any product that presents a health problem: Report a Problem with a Product


Food Protection Plan
An integrated strategy for protecting the nation’s food supply

November 2007

Department of Health and Human Services
Food and Drug Administration

“Americans enjoy unprecedented choice and convenience in filling the cupboard today, but we also face new challenges to ensuring that our food is safe. This Food Protection Plan will implement a strategy of prevention, intervention and response to build safety into every step of the food supply chain.”

Michael O. Leavitt
Secretary of Health and Human Services
U.S. Department of Health and Human Services

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A Message from the Commissioner

As a physician and the Commissioner of Food and Drugs, protecting America’s food supply is extremely important to me.

American consumers have one of the safest food supplies in the world, but the world is changing and we know it can be safer. New food sources, advances in production and distribution methods, and the growing volume of imports due to consumer demand call for a new approach to protecting our food from unintentional or deliberate contamination. The U.S. Food and Drug Administration (FDA) must keep pace with these changes so that the safety of the nation’s food supply remains second to none.

In the past few years, FDA has introduced several initiatives that address microbial and other food safety hazards with domestic or imported produce and that guide industry practices in the safe production of fresh-cut fruits and vegetables. FDA has also worked hard to raise awareness about food defense issues and preparedness. These are just a few things we are doing to improve food safety and food defense.

Recent nationwide recalls remind us how devastating foodborne illness can be. In the past year, contaminated peanut butter led to illnesses in more than 300 people and at least 50 hospitalizations. Contaminated spinach resulted in 206 illnesses, three deaths, and more than 100 people hospitalized. Reports of kidney failure and deaths in cats and dogs prompted a recall of more than 100 brands of pet food.

For every one of these emergencies, the FDA responded immediately to minimize harm. FDA investigators traced each problem’s source and worked without delay to remove the affected products from market shelves. FDA staff continue to work diligently to protect our food supply, by containing outbreaks and preventing further illnesses.

With this FDA Food Protection Plan we are going even further. It is a forward-oriented concept that uses science and modern information technology to identify potential hazards ahead of time. By preventing most harm before it can occur, enhancing our intervention methods at key points in the food production system, and strengthening our ability to respondimmediately when problems are identified, FDA can provide a food protection framework that keeps the American food supply safe.

Andrew C. von Eschenbach, M.D.
Commissioner of Food and Drugs


TABLE OF CONTENTS

I. Executive Summary

II. Introduction

III. Changes and Challenges

· Trends in Demographics and Consumption

o Shifting Demographics

o Convenience
Trends

o Consumption Patterns

· Global Food Supply

· New Threats

o New Foodborne Pathogens

o Intentional Contamination

· Communication

IV. An Overview of the Approach

· Core Elements

o Prevention – Build safety in from the start

o Intervention – Verify prevention and intervene when risks are identified

o Response – Respond rapidly and appropriately

· Cross-Cutting Principles

1. Focus on risks over a product’s life cycle from production to consumption

2. Target resources to achieve maximum risk reduction

3. Address both unintentional and deliberate contamination

4. Use science and modern technology systems

V. The Integrated Plan

· Core Element #1: Prevention

1.1 Promote Increased Corporate Responsibility to Prevent Foodborne Illnesses
1.2 Identify Food Vulnerabilities and Assess Risks
1.3 Expand the Understanding and Use of Effective Mitigation Measures

· Core Element #2: Intervention

2.1 Focus Inspections and Sampling Based on Risk
2.2 Enhance Risk-based Surveillance
2.3 Improve the Detection of Food System “Signals” that Indicate Contamination

· Core Element #3: Response

3.1 Improve Immediate Response
3.2 Improve Risk Communications to the Public, Industry and Other Stakeholders

VI. Enhance Information Technology

VII. Conclusion

To read more, go to http://www.fda.gov/oc/initiatives/advance/food.html

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AMERICAN COOKERY
BY AMELIA SIMMONS,
AN AMERICAN ORPHAN.

Published according to Act of Congress.

Printed for SIMEON BUTLER,
NORTHHAMPTON.
1798.

Pompkin.

No. 1. One quart stewed and strained, 3 pints cream, 9 beaten eggs, sugar, mace, nutmeg and ginger, laid into paste No. 7 or 3, and with a dough spur, cross and chequer it, and baked in dishes three quarters of an hour.

No. 2. One quart of milk, 1 pint pompkin, 4 eggs, molasses, allspice and ginger in a crust, bake 1 hour.

While we tend to think of bacteria as harmful, we all carry plenty of microbes that work to the good. Can we use them to prevent or treat diseases?
by Maya Pines

The night before having dental surgery in 1998, a 71-year-old Canadian woman was given antibiotics to prevent infection. The operation on her teeth went well, but a few days later she developed diarrhea so severe that she went into shock and was rushed to the hospital. Tests showed she had been hit with toxins produced by Clostridium difficile, a generally mild bug that resides naturally in the intestinal tract. Usually kept in check by the body’s “good” bacteria, C. difficile poses little threat unless something—like a course of antibiotics—kills off some of those protective bacteria.

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Jeffrey I. Gordon (left) and Eduardo A. Groisman, both at Washington University School of Medicine in St. Louis, are among investigators who study bacteria—both the “good” and the bad—that comprise what Gordon calls the “bacterial nation.”

After two months of intensive treatment and physical rehabilitation, the dental patient survived. Other people have not been so lucky. In a single Quebec hospital over the last 18 months, 100 patients died of C. difficile infection. Fatalities of this sort have been increasing rapidly not only in the province of Quebec, whose health minister suggested that “enthusiastic” prescribing of antibiotics might have caused the outbreaks, but in other parts of Canada and the United States as well.

We each carry two to five pounds of live bacteria in our bodies. Some, like C. difficile, are potentially harmful. Many bacteria, however, are quite useful—so useful, in fact, that we could not live without them.

Until recently, scientific research has focused on fighting “bad” bacteria—the ones that cause cholera, scarlet fever, typhoid, tuberculosis, and other major infectious diseases. Scientists pretty much ignored the good bacteria, which often outnumber the bad ones.

In the past few years, however, researchers have begun to recognize the enormous contributions made by this friendly “bacterial nation,” as Jeffrey I. Gordon, director of the Center for Genome Sciences at Washington University School of Medicine in St. Louis, calls it. Trillions upon trillions of microbes, representing some 1,000 species, are packed within us, especially in our guts. A single milliliter of the colon’s contents might harbor 100 billion of them. This “nation” functions like a kind of internal organ, says Gordon, affecting our well-being.

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Beneficial bacteria include (top) Lactococcus lactis, as well as (right) Lactobacillus

THE PROMISE OF PROBIOTICS?

Using good bacteria to promote health—a practice sometimes called probiotics—has a long history, but it never quite became an accepted therapy. About a century ago Elie Metchnikoff, director of the Pasteur Institute in Paris, France, started a yogurt craze when he announced that Bulgarian peasants who ate yogurt regularly tended to live to ripe old ages; yogurt contains live cultures of lactobacilli, one of the better-known strains of good bacteria. Several other substances that supposedly contained beneficial bacteria were also used to treat infections in the gut or vagina. This practice ended around the time of World War II, when the newly discovered, often life-saving, antibiotics proved to work more rapidly and effectively.

Now, however, “We are being forced to look at alternatives to antibiotics to combat the ever-increasing number of infections that occur because of excessive use of antibiotics,” Christopher J. Bulpitt and his colleagues at London’s Imperial College School of Medicine wrote in the British Medical Journal in 2002. Severe diarrhea, for instance, often results from treatment with antibiotics, which wipe out good bacteria along with the bad. Could this side effect be prevented by maintaining enough good bacteria in the patients’ intestines to act as guardians?

To find out, Bulpitt’s team analyzed nine randomized, double-blind, placebo-controlled trials in which all patients had been treated with antibiotics, but some also received various combinations of microbes that were believed to be good, while others received placebos. The team concluded that “probiotics are a possible solution,” but only for preventing antibiotic-associated diarrhea. They found little support for using probiotics as a cure.

the one child in 20 who comes down with these painful infections frequently—up to six times a year—despite repeated treatment with antibiotics, to which the child becomes increasingly resistant. So they started out by “harvesting” the microbes they found living in the eustachian tubes (which connect the nose and middle ear) of healthy children at a daycare center. Among these microorganisms they identified some 800 different strains of α-hemolytic streptococci. Next they tested each strain’s ability to stop the growth of otitis-causing bacteria in the lab. Finally, they chose the five most active strains, which they put into the nasal spray.

At the end of three months, 42 percent of the children who had been given this bacterial spray remained free from otitis, while only 22 percent of those who received a placebo escaped new ear infections. The scientists concluded that “recolonization” with selected bacteria does protect against recurrent attacks of otitis, at least to some extent.

THIN LINE

The British and Swedish efforts, and other clinical studies of this sort, generally paint an optimistic picture of probiotics. But many scientists remain skeptical, and few such treatments are currently in use. The real hurdle is still our lack of precise knowledge. What proportion of the bacteria in our bodies is good? How many are pathogenic? How many good bacteria sometimes become bad, and vice versa? Are many of them simply straddling the fence?

Nobody knows. An adult human has about 10 times more microbial cells than human cells, so “based on cell number, each of us is 90 percent microbial and 10 percent human,” says Gordon. “The genomes of our gut microbes probably contain 100 times more genes than our own genome, providing us with traits we haven’t needed to develop on our own.” Yet at least half of these bacteria cannot be grown outside the gut because “we haven’t learned how to reproduce their normal conditions in the lab,” he says, “so we don’t have an accurate view of them.” Together with some acquired viruses, yeast cells, archaea (single-celled microorganisms that live in geysers and other extreme environments), and occasional parasites, the bacteria form “a constantly open ecosystem,” Gordon says.

Some of the good bacteria have a symbiotic relationship with our intestines (they help us and we help them, usually by providing nutrients). Others have a commensal relationship (one partner benefits without harming the other). But in our guts, nothing is permanent. Bacteria take on shifting roles as they encounter changing circumstances. “If the formidable barrier produced by symbiotic bacteria is destroyed,” notes Gordon, “some previously minor bacteria can expand and produce disease. There’s also a lot of horizontal gene transfer (from one bacterium to another), creating new strains and spreading antibiotic resistance. It’s very dynamic!”

“Take Bacteroides fragilis, for instance. Usually it’s fairly innocent,” he says. “But after stomach surgery or some other insults, it can cause abscesses. A researcher at Harvard, Laurie F. Comstock, recently discovered that this happens when the bacterium’s outer capsule changes, making it more dangerous.”

Similarly, up to two-thirds of the world’s population carries Helicobacter pylori, and in most people it does no harm. In 10 percent of infected people, however, it leads to stomach ulcers or cancer (which may be either gastric lymphoma or adenocarcinoma of the stomach).

“The question one should ask is not how many bacteria in our guts are pathogenic,” says Gordon, “but how many of them have pathogenic potential.”

ORIGINS OF VIRULENCE

Bacterial virulence seems to involve what B. Brett Finlay, an HHMI international research scholar at the University of British Columbia in Vancouver, calls a kind of “cross-talk” between bacteria and their hosts.

Bacteria appeared in the world long before humans did. After we came on the scene, some bacteria “co-evolved” with us so they could take advantage of what is for them a wonderful environment—the human gut, where so many nutrients are concentrated. This meant the bacteria had to learn how to overcome the many physical, cellular, and molecular barriers the human body presented, wrote Howard Ochman, a biochemist at the University of Arizona, in a recent issue of Science. They may have added or subtracted certain genes.

Natural selection favored those bacteria that made the most effective changes. This happened “regardless of whether the ultimate outcome of the interaction is harmful, benign, or beneficial to the host,” said Ochman. “Only from the host’s perspective are these distinctions crucial.”

The first job of infectious bacteria is to attach themselves to specific receptors on human cells, says Finlay. And sometimes the host cell collaborates. As his team discovered while studying diarrhea-causing enteropathogenic Escherichia coli (EPEC), these bacteria use two different kinds of adhesive molecules to latch onto human cells. The first molecule somehow “rings a doorbell” on the host cell, telling it to produce a sort of pedestal, which almost immediately grows out of the cell surface. This pedestal then enables EPEC to attach itself securely to the cell with its second adhesive molecule.

Although most diseases are caused by the initial adherence of bacteria to cells, says Finlay, no drugs are yet available to derail this process. If scientists learn to block bacterial attachment, they may be able to prevent or stop infections, he suggests.

But why these particular bacteria tried to adhere to human cells in the first place remains a mystery. Were they previously good bacteria that somehow turned bad?

Eduardo A. Groisman, an HHMI investigator at Washington University’s School of Medicine in St. Louis, first tackled this problem 10 years ago in an article, “How to become a pathogen,” that he and Howard Ochman published in Trends in Microbiology. The idea was to find the genes responsible for producing virulence, focusing on differences between the activities of nonpathogenic E. coli bacteria and a strain of Salmonella that can cause typhoid fever. With the tools available at the time, and more recently with the help of sequenced microbe genomes, the two scientists worked to identify “pathogenicity islands”—groups of genes that are found only in pathogens and that contribute to disease.

In some cases, however, pathogenic and benign bacteria have almost the same genes, Groisman observes. One possible explanation for their different behavior is that these genes are regulated in alternative ways. “We’re now studying how this differential regulation may affect virulence,” he says.

ANIMAL MODELS

The complexity of the interactions between the huge bacterial nation and the human gut “defies imagination,” says Gordon. So his lab created animal models that could be analyzed more easily. All the animals—mice or zebrafish—were raised under germ-free conditions; for comparison, half were later “colonized” with specific strains of bacteria.

For the mouse experiments, Gordon chose a strain of Bacteroides thetaiotaomicron, good bacteria frequently found in the guts of both mice and humans; its main job is to provide the enzymes needed to process certain carbohydrates in plants. The B. thetaiotaomicron genome was sequenced in 2003 and the proteins it produces have been sorted out, making it possible to examine the microbe’s activities with some of the newest tools of genetics. The Gordon team has shown, for instance, that B. thetaiotaomicron stimulates production of an antibiotic protein that can kill infectious microbes such as Listeria monocytogenes, which causes food-borne gastroenteritis. Another of the researchers’ findings is that B. thetaiotaomicron promotes the development of small “networks of branched, interconnected blood vessels” in newborns, the scientists reported in the Proceedings of the National Academy of Sciences. This capillary network does not grow properly in germ-free mice.

Zebrafish offer several advantages to researchers. Because these small fish develop rapidly (the larvae hatch within three days of fertilization) and remain transparent through early adulthood, it is possible to observe the embryos’ growing digestive tracts and their resident bacteria. Using DNA microarrays, Gordon’s team recently examined the genes that were activated in zebrafish intestines after exposure to specific good bacteria. They found that 59 zebrafish genes responded to these bacteria in exactly the same way as do the corresponding genes in mice, even though the two species diverged millions of years ago. This implies that responses to these bacteria go back very far in evolution; most likely they were critical not only to mice and zebrafish but also to many other species.

OVERSOLD THERAPIES?

While researchers work step by step, accumulating information with the aid of animal models, the marketplace apparently is not waiting for final results. Various brands of probiotic food supplements are already being sold around the world with promises of fabulous benefits for cats, dogs, birds, horses, and farm animals: “improved growth,” “better health,” “establishment of beneficial gut microflora,” “better utilization of food,” “reduced intestinal upsets,” and “increased resistance to infections,” which should reduce the need to treat livestock with antibiotics.

Chickens in particular are frequently treated with Preempt, a product developed with help from U.S. Department of Agriculture scientists, which contains 29 kinds of good bacteria found in healthy chickens. At least 10 percent of chickens are infected with Salmonella bacteria, a leading cause of food-borne illnesses in humans. The idea is to spray newly hatched chicks with Preempt so that when they peck at their wet feathers they will swallow its bacteria. The reasoning is that the product’s good bacteria will grow in the chicks’ intestines, forming a protective barrier that cannot be breached. As a result, any ingested Salmonella will be unable to attach themselves to the chicks’ intestines and will be forced out of the animals’ bodies. This model seems to work to some extent.

When it comes to humans, however, the use of probiotics remains more controversial. There are strong commercial interests in its favor—yogurt marketed as “probiotic” is one example—but inconsistent experimental results. Much of the published research consists of reports on only a few patients, and many of these reports are contradictory. Skeptical scientists have called probiotics everything from “conbiotics” to “snake oil.” Even the manufacturers of probiotic compounds agree that more precise information is needed.

According to the Harvard Health Letter of March 2002, “Probiotics have been oversold. The claims are seductive: pills, powders, and solutions containing ‘friendly’ bacteria will boost the immune system, prevent cancer, and perform assorted other health miracles. …But that doesn’t mean it’s based upon total fiction. …The evidence suggests that probiotic therapy could be useful someday as a form of preventive medicine—and not just for diseases affecting the gut.”

As Gordon puts it, “Bacteria have learned to manipulate our biology in many ways that benefit themselves and us. We now have the tools to identify the pathways through which they operate, as well as the chemicals they synthesize.” This information could lead to new ways of diagnosing, treating, and ultimately preventing a variety of diseases. “Bacteria are fabulous teachers,” says Gordon. “They are pointing the way.”

Beneficial bacteria include (top) Lactococcus lactis, as well as (right) Lactobacillus bulgaricus (blue), Streptococcus thermophilus (orange), and a member of the Bifidobacterium family (magenta), all found in yogurt and cheese. Normally benign, Bacteroides fragilis (bottom) is an intestinal microbe that can wreak havoc under certain conditions, such as post-surgery. (The bacteria in these scanning electron micrographs have been color enhanced.)

One way to learn which bacteria are most useful in particular circumstances is to let nature be your guide, as a group of Swedish researchers did when they concocted a highly unusual nasal spray. In 2001, Kristian Roos and his associates at the Lundby Hospital in Gothenburg were seeking new treatments for infants and toddlers who have repeated bouts of ear infections (otitis media). They targeted the one child in 20 who comes down with these painful infections frequently—up to six times a year—despite repeated treatment with antibiotics, to which the child becomes increasingly resistant. So they started out by “harvesting” the microbes they found living in the eustachian tubes (which connect the nose and middle ear) of healthy children at a daycare center. Among these microorganisms they identified some 800 different strains of α-hemolytic streptococci. Next they tested each strain’s ability to stop the growth of otitis-causing bacteria in the lab. Finally, they chose the five most active strains, which they put into the nasal spray.

At the end of three months, 42 percent of the children who had been given this bacterial spray remained free from otitis, while only 22 percent of those who received a placebo escaped new ear infections. The scientists concluded that “recolonization” with selected bacteria does protect against recurrent attacks of otitis, at least to some extent.

THIN LINE

The British and Swedish efforts, and other clinical studies of this sort, generally paint an optimistic picture of probiotics. But many scientists remain skeptical, and few such treatments are currently in use. The real hurdle is still our lack of precise knowledge. What proportion of the bacteria in our bodies is good? How many are pathogenic? How many good bacteria sometimes become bad, and vice versa? Are many of them simply straddling the fence?

Nobody knows. An adult human has about 10 times more microbial cells than human cells, so “based on cell number, each of us is 90 percent microbial and 10 percent human,” says Gordon. “The genomes of our gut microbes probably contain 100 times more genes than our own genome, providing us with traits we haven’t needed to develop on our own.” Yet at least half of these bacteria cannot be grown outside the gut because “we haven’t learned how to reproduce their normal conditions in the lab,” he says, “so we don’t have an accurate view of them.” Together with some acquired viruses, yeast cells, archaea (single-celled microorganisms that live in geysers and other extreme environments), and occasional parasites, the bacteria form “a constantly open ecosystem,” Gordon says.

Some of the good bacteria have a symbiotic relationship with our intestines (they help us and we help them, usually by providing nutrients). Others have a commensal relationship (one partner benefits without harming the other). But in our guts, nothing is permanent. Bacteria take on shifting roles as they encounter changing circumstances. “If the formidable barrier produced by symbiotic bacteria is destroyed,” notes Gordon, “some previously minor bacteria can expand and produce disease. There’s also a lot of horizontal gene transfer (from one bacterium to another), creating new strains and spreading antibiotic resistance. It’s very dynamic!”

“Take Bacteroides fragilis, for instance. Usually it’s fairly innocent,” he says. “But after stomach surgery or some other insults, it can cause abscesses. A researcher at Harvard, Laurie F. Comstock, recently discovered that this happens when the bacterium’s outer capsule changes, making it more dangerous.”

Similarly, up to two-thirds of the world’s population carries Helicobacter pylori, and in most people it does no harm. In 10 percent of infected people, however, it leads to stomach ulcers or cancer (which may be either gastric lymphoma or adenocarcinoma of the stomach).

“The question one should ask is not how many bacteria in our guts are pathogenic,” says Gordon, “but how many of them have pathogenic potential.”

ORIGINS OF VIRULENCE

Bacterial virulence seems to involve what B. Brett Finlay, an HHMI international research scholar at the University of British Columbia in Vancouver, calls a kind of “cross-talk” between bacteria and their hosts.

Bacteria appeared in the world long before humans did. After we came on the scene, some bacteria “co-evolved” with us so they could take advantage of what is for them a wonderful environment—the human gut, where so many nutrients are concentrated. This meant the bacteria had to learn how to overcome the many physical, cellular, and molecular barriers the human body presented, wrote Howard Ochman, a biochemist at the University of Arizona, in a recent issue of Science. They may have added or subtracted certain genes.

Natural selection favored those bacteria that made the most effective changes. This happened “regardless of whether the ultimate outcome of the interaction is harmful, benign, or beneficial to the host,” said Ochman. “Only from the host’s perspective are these distinctions crucial.”

The first job of infectious bacteria is to attach themselves to specific receptors on human cells, says Finlay. And sometimes the host cell collaborates. As his team discovered while studying diarrhea-causing enteropathogenic Escherichia coli (EPEC), these bacteria use two different kinds of adhesive molecules to latch onto human cells. The first molecule somehow “rings a doorbell” on the host cell, telling it to produce a sort of pedestal, which almost immediately grows out of the cell surface. This pedestal then enables EPEC to attach itself securely to the cell with its second adhesive molecule.

Although most diseases are caused by the initial adherence of bacteria to cells, says Finlay, no drugs are yet available to derail this process. If scientists learn to block bacterial attachment, they may be able to prevent or stop infections, he suggests.

But why these particular bacteria tried to adhere to human cells in the first place remains a mystery. Were they previously good bacteria that somehow turned bad?

Eduardo A. Groisman, an HHMI investigator at Washington University’s School of Medicine in St. Louis, first tackled this problem 10 years ago in an article, “How to become a pathogen,” that he and Howard Ochman published in Trends in Microbiology. The idea was to find the genes responsible for producing virulence, focusing on differences between the activities of nonpathogenic E. coli bacteria and a strain of Salmonella that can cause typhoid fever. With the tools available at the time, and more recently with the help of sequenced microbe genomes, the two scientists worked to identify “pathogenicity islands”—groups of genes that are found only in pathogens and that contribute to disease.

In some cases, however, pathogenic and benign bacteria have almost the same genes, Groisman observes. One possible explanation for their different behavior is that these genes are regulated in alternative ways. “We’re now studying how this differential regulation may affect virulence,” he says.

ANIMAL MODELS

The complexity of the interactions between the huge bacterial nation and the human gut “defies imagination,” says Gordon. So his lab created animal models that could be analyzed more easily. All the animals—mice or zebrafish—were raised under germ-free conditions; for comparison, half were later “colonized” with specific strains of bacteria.

For the mouse experiments, Gordon chose a strain of Bacteroides thetaiotaomicron, good bacteria frequently found in the guts of both mice and humans; its main job is to provide the enzymes needed to process certain carbohydrates in plants. The B. thetaiotaomicron genome was sequenced in 2003 and the proteins it produces have been sorted out, making it possible to examine the microbe’s activities with some of the newest tools of genetics. The Gordon team has shown, for instance, that B. thetaiotaomicron stimulates production of an antibiotic protein that can kill infectious microbes such as Listeria monocytogenes, which causes food-borne gastroenteritis. Another of the researchers’ findings is that B. thetaiotaomicron promotes the development of small “networks of branched, interconnected blood vessels” in newborns, the scientists reported in the Proceedings of the National Academy of Sciences. This capillary network does not grow properly in germ-free mice.

Zebrafish offer several advantages to researchers. Because these small fish develop rapidly (the larvae hatch within three days of fertilization) and remain transparent through early adulthood, it is possible to observe the embryos’ growing digestive tracts and their resident bacteria. Using DNA microarrays, Gordon’s team recently examined the genes that were activated in zebrafish intestines after exposure to specific good bacteria. They found that 59 zebrafish genes responded to these bacteria in exactly the same way as do the corresponding genes in mice, even though the two species diverged millions of years ago. This implies that responses to these bacteria go back very far in evolution; most likely they were critical not only to mice and zebrafish but also to many other species.

OVERSOLD THERAPIES?

While researchers work step by step, accumulating information with the aid of animal models, the marketplace apparently is not waiting for final results. Various brands of probiotic food supplements are already being sold around the world with promises of fabulous benefits for cats, dogs, birds, horses, and farm animals: “improved growth,” “better health,” “establishment of beneficial gut microflora,” “better utilization of food,” “reduced intestinal upsets,” and “increased resistance to infections,” which should reduce the need to treat livestock with antibiotics.

Chickens in particular are frequently treated with Preempt, a product developed with help from U.S. Department of Agriculture scientists, which contains 29 kinds of good bacteria found in healthy chickens. At least 10 percent of chickens are infected with Salmonella bacteria, a leading cause of food-borne illnesses in humans. The idea is to spray newly hatched chicks with Preempt so that when they peck at their wet feathers they will swallow its bacteria. The reasoning is that the product’s good bacteria will grow in the chicks’ intestines, forming a protective barrier that cannot be breached. As a result, any ingested Salmonella will be unable to attach themselves to the chicks’ intestines and will be forced out of the animals’ bodies. This model seems to work to some extent.

When it comes to humans, however, the use of probiotics remains more controversial. There are strong commercial interests in its favor—yogurt marketed as “probiotic” is one example—but inconsistent experimental results. Much of the published research consists of reports on only a few patients, and many of these reports are contradictory. Skeptical scientists have called probiotics everything from “conbiotics” to “snake oil.” Even the manufacturers of probiotic compounds agree that more precise information is needed.

According to the Harvard Health Letter of March 2002, “Probiotics have been oversold. The claims are seductive: pills, powders, and solutions containing ‘friendly’ bacteria will boost the immune system, prevent cancer, and perform assorted other health miracles. …But that doesn’t mean it’s based upon total fiction. …The evidence suggests that probiotic therapy could be useful someday as a form of preventive medicine—and not just for diseases affecting the gut.”

As Gordon puts it, “Bacteria have learned to manipulate our biology in many ways that benefit themselves and us. We now have the tools to identify the pathways through which they operate, as well as the chemicals they synthesize.” This information could lead to new ways of diagnosing, treating, and ultimately preventing a variety of diseases. “Bacteria are fabulous teachers,” says Gordon. “They are pointing the way.”

Chemical Conversations With Bacteria

Howard Hughes Medical Institute, November 15, 2007

Bonnie L. Bassler PhD

Researchers have deciphered the molecular language that cholera bacteria use to coordinate their infectivity. The bacteria use this chemical communication to signal their presence to one another, so that they can plan as a group when to be most virulent and when to escape their host to find new victims.

Although cholera is rare in the U.S., it is epidemic in parts of Africa, Asia and Latin America, and the severe diarrhea it causes can lead to death if not treated. The researchers say that by interrupting the bacterium’s chemical conversation, they may be able to stop cholera virulence. Their findings also offer hope that similar approaches may form the basis of effective treatments for a wide range of other bacterial diseases.

“If our studies with cholera demonstrate that it is possible to trick bacteria into reducing virulence, they constitute the first demonstration that manipulating such bacterial conversations can be a useful treatment.”
Bonnie L. Bassler

Howard Hughes Medical Institute investigator Bonnie Bassler and her colleagues at Princeton University reported their findings in the November, 15, 2007, issue of the journal Nature.

Bassler and her colleagues have long studied a type of bacterial chemical conversation known as quorum sensing. This process depends on the bacteria releasing signaling chemicals called autoinducers into their environment, and subsequently detecting and responding to the build up of these molecules to coordinate with one another to ensure maximum infectivity and other group behaviors.

“We had shown that cholera had quorum sensing, and we had produced a mutant form of cholera that couldn’t perform quorum sensing properly, which affected virulence,” said Bassler. “This finding told us that there must be an autoinducer molecule that this mutant couldn’t make that had a role in virulence, but we had no idea what that molecule was.”

Bassler explained that the way the cholera bacteria use that molecule suggested it could make a useful treatment. “When people first get cholera, the bacteria immediately stick to the intestine in a structure called a biofilm and they release toxins,” she said. “During this time, they are multiplying rapidly and also releasing the autoinducer molecule. When the bacteria reach high cell numbers, the high concentration of the autoinducer molecule represses virulence and stops biofilm formation, enabling the bacteria to escape into the environment to spread to other people. So, if we could isolate and purify this molecule, and supply it to the bacteria to get them to prematurely terminate virulence, we thought it could be used as a treatment approach.”

Through their mutational studies, the researchers had identified the gene that codes for the enzyme that makes the unknown molecule. They inserted that gene into the gut bacterium E. coli, transforming the bacterium into a biological factory for large amounts of the chemical. That strategy allowed them to purify the chemical, which they called CAI-1, and analyze its molecular structure.

“This structure produced a real surprise,” said Bassler. “CAI-1 turned out to be a molecule brand new to biology. What’s more, it was a simple molecule, almost like one you could buy at a chemical supply house. Because there was no precedent for this molecule, we felt we had to go to a lot of effort to demonstrate that this really was the correct molecule.” To do so, the researchers created synthetic CAI-1 and introduced it into cultures of cholera bacteria. The synthetic CAI-1 repressed virulence in those cells exactly like the natural molecule did. Carrying their studies further, the researchers are now exploring how CAI-1 is made by analyzing the function of the enzyme that produces it.

CAI-1’s success in terminating virulence in cultures of cholera has encouraged Bassler and her colleagues to test the chemical as a treatment. “Next, we want to see whether we can cure a mouse of cholera using CAI-1,” she said. “These experiments also will enable us to answer some important questions about the properties of the molecule. For example, does it last in the gut? Is it stable? What should be the dose? Do we have to adjust the structure to make it more potent or less potent?”

The discovery of CAI-1 may also inspire efforts to control quorum sensing to treat other bacterial diseases, said Bassler. “Cholera uses quorum sensing in a different way than most other bacteria,” she said. “Cholera causes an acute infection; it gets into the host and then has to get out, so its strategy is to use quorum sensing to repress virulence when the bacterial cells reach high numbers. But other bacteria that cause persistent infections use quorum sensing to turn on virulence only when they reach high numbers—which makes biological sense because they want to hide from the immune system until they have successfully reproduced and then launch their attack en masse. Thus, treatments for other bacteria that target quorum sensing are focused on developing drugs that block autoinducers. These drugs are very hard to make, and such efforts have not yet been very successful.

“If our studies with cholera demonstrate that it is possible to trick bacteria into reducing virulence, they constitute the first demonstration that manipulating such bacterial conversations can be a useful treatment. It will give the field solid evidence that quorum sensing is a viable new therapeutic target, which is especially important given the failure of so many traditional antibiotics.”

Also, emphasized Bassler, whose team has solved the structures of other quorum sensing molecules, the discovery of diverse quorum-sensing molecules such as CAI-1 represents another step in a promising and productive effort to decipher and manipulate the chemical language of bacteria.

“We know that there are molecules analogous to CAI-1 that are very species-specific, and we also understand that there are molecules that are generic and enable inter-species communication. Together, they give bacteria a multicellular character. And the fact that we are coming to understand this communication and even learn how to manipulate it both for medical and industrial purposes makes this a very exciting time for this research field.”

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