Nitric Oxide modulates bacterial biofilm formation

Scripps Research Institutes Scientists Solve a Mystery of Bacterial Growth and Resistance
Findings Shed Light on How Bacteria Form Protective Biofilms

LA JOLLA, CA, April 26,2012:-
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 immue 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 lead 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."

Dangerous Get Togethers


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 time 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.

The Detective Story


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 transfer phosphate chemical groups to other molecules in signaling pathways. The question was where the phosphate were going.

To learn more, the researchers used a technique called Phosphotrasfer profiling. This involved activating the histidine kinase and then allowing them to react separately with about 20 potential tragets. 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 a primary study species, the bacterium Shewanells 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 di guanosine monophosphate, an important bacterial messenger molecule that is critical in biofilm formation, and the third tunes the degree of activity of the second.

Divide and Conquer


Since other bacterial species use the same chemical pathway uncovered in this study, the finidings 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.

The work for the paper, " Nitric Oxide modulates bacterial biofilm formation through a multi-component cyclic-di-GMP signaling network," was supported by the National Institutes of Health and a Chang-Lin Tien Graduate Fellowship in the Environmental Sciences.

press@scripps.edu

Effectively tipping the balance in favour of the Mycobacterium surviving in the cell.

Autophagy: when ‘self-eating’ is good for you

Broken pieces of internal structures, damaged organelles and harmful clumps of proteins are all examples of the molecular detritus that builds up continuously in our cells. Fortunately, we are equipped with an intracellular process called autophagy (literally ‘self-eating’) that gathers up the debris, wraps it in a double membrane and delivers it to an intracellular sac called the lysosome. Here, the material is ingested, digested and recycled, ready to be used again.
Recent work has suggested that autophagy may also be important in killing intracellular bacteria that are able to escape the normal processes that control infection within cells. Autophagy appears to be critical in controlling infections of Mycobacterium tuberculosis (MTB) and related species called non-tuberculous mycobacteria (NTM), which are able to block degradation by lysosomes and thereby replicate within cells.
Researchers working at the University’s Cambridge Institute for Medical Research (CIMR) and Papworth Hospital have noticed that patients with chronic lung diseases such as cystic fibrosis (CF) are becoming increasingly infected with a highly pathogenic, multi-drug-resistant (MDR) NTM called Mycobacterium abscessus. Their research, published recently in the Journal of Clinical Investigation, suggests that M. abscessus infection may be linked to long-term use of azithromycin, an antibiotic with anti-inflammatory properties. They propose that azithromycin blocks autophagy in a type of white blood cell called the macrophage, effectively tipping the balance in favour of the Mycobacterium surviving in the cell.
It’s an ominous outcome for patients, as lead researcher Dr Andres Floto explained: “Developing an infection with Mycobacterium abscessus is a big deal for patients with CF. It’s resistant to virtually all antibiotics, is very hard to treat, can accelerate lung damage and may rule out future lung transplantation. While the benefits of long-term azithromycin therapy in CF are clear, our data suggest that there may also be a downside to watch out for.”
He added: “Recent studies showing the benefit of azithromycin therapy in asthma and smoking-related chronic obstructive pulmonary disease (COPD) will no doubt increase the numbers of patients on long- term azithromycin therapy. Physicians will need to be aware of the potential for harm with this treatment, and carefully monitor patients for mycobacterial disease”.

‘Forced stoppage’ of the cleaning crew

Floto is a Wellcome Trust Senior Clinical Fellow in the Department of Medicine and Principal Investigator in the CIMR. He is also Director of Research at the Cambridge Centre for Lung Infection at Papworth Hospital. The Centre cares for over 260 adults with CF and almost 2,000 patients with recurrent or difficult lung infections.
“We’d been struck by the rising rates of M. abscessus infection in CF patients in centres around the world and wondered whether it could be connected to an increased use of long-term azithromycin,” explained Floto. “Azithromycin is a broad-spectrum antibiotic which paradoxically is used to treat some mycobacterial infections. In patients with CF and, more recently, other inflammatory lung diseases such as asthma and COPD, it’s being prescribed as a long-term anti-inflammatory therapy.”
The association between azithromycin use and M. abscessus infection was first suggested when Floto and colleagues carried out an epidemiological study of adult patients with CF at Papworth Hospital and found that those patients with NTM infection were much more likely to be taking long-term azithromycin. When they looked at cells in culture, they discovered that the antibiotic impaired autophagy, effectively causing a ‘forced stoppage’ of the waste-recycling unit and preventing cells from clearing infecting mycobacteria. Moreover, azithromycin had a profound effect on M. abscessus infection of a mouse model; whereas untreated mice were able to clear the infection rapidly, those given the drug developed persistent lung infection.

Exploiting autophagy

The interactions of mycobacteria with immune cells are extremely complex and poorly understood. In a paper published in 2006 in Science, Floto together with Professor Paul Lehner at the CIMR began to define how immune cells respond to specific proteins from mycobacteria which then control the immune response. Subsequent work has suggested that the ability of macrophages to kill mycobacteria can be enhanced through a number of pathways, including autophagy.
Working with Professor David Rubinsztein at the CIMR, who has been interested in enhancing autophagy to clear the build-up of damaging clumps of proteins in neurodegenerative diseases, Floto has begun to study whether autophagy might be exploited therapeutically to treat multi-drug- resistant tuberculosis (MDR-TB), for which there is an urgent need to find new treatments. In 2008, the World Health Organization estimated that of the 9.4 million new TB cases 440,000 were MDR-TB. “It’s a massive challenge”, explained Floto, “We believe that stimulating autophagy to kill TB will bypass the problem of multi-drug resistance and may lead to potential new treatments for MDR-TB.”
Meanwhile, Floto’s work on azithromycin has already begun to influence clinical practice in a number of major CF centres in the UK. Although at pains to stress that a larger, prospective multi-centre study is needed before universal guidance can be given for the management of patients on chronic azithromycin therapy, Floto explained how the Cambridge Centre for Lung Infection has adopted a policy that is already proving successful: “We give patients who are not doing as well as we’d expect a holiday off the medication for a month while we screen carefully for NTM. When we do this, we often find a mycobacterial infection brewing in the lungs and can treat it immediately, instead of allowing the infection to take hold covertly without being detected.”
Floto’s research exemplifies the importance of a seamless link between fundamental research and clinical translation, and will benefit still further from the planned move of Papworth Hospital to the Cambridge Biomedical Campus . “Papworth has a unique group of patients and an international reputation for the treatment of patients with difficult lung infections,” he explained. “Forging greater links between Papworth and the University will inevitably promote research into a number of clinically important areas and smooth the route from bench to bedside, and back again.”

 http://www.cam.ac.uk/research/features/autophagy-when-self-eating-is-good-for-you/?source=researchnews