mBiosphere

Resistance to last-line antibiotic also makes bacteria resistant to immune attacks

Bacterial resistance to antibiotics is bad enough, but what if drug resistance also gives bacteria the ability to fend off attacks by the immune system? In an Observation piece in mBio this week scientists identify a lose-lose situation with the antibiotic colistin and Acinetobacter baumannii: bacteria resistant to colistin are also commonly resistant to antimicrobial substances made by the human body. Cross-resistance to colistin and host antimicrobials LL-37 and lysozyme, which help defend the body against bacterial attack, could mean that patients with life-threatening multi-drug resistant infections are also saddled with a crippled immune response.

A Fifty Year Old Drug

Colistin is a last-line drug for treating several kinds of drug-resistant infections, but colistin resistance and the drug's newfound impacts on bacterial resistance to immune attack underscore the need for newer, better antibiotics, says corresponding author David Weiss of Emory University.

Weiss says the results show that colistin therapy can fail patients in two ways. "The way that the bacteria become resistant [to colistin] allows them to also become resistant to the antimicrobials made by our immune system. That is definitely not what doctors want to do when they're treating patients with this last line antibiotic," says Weiss.

Although it was developed fifty years ago, colistin remains in use today not so much because it's particularly safe or effective, but because the choices for treating multi-drug resistant A. baumannii and other resistant infections are few and dwindling. Colistin is used when all or almost all other drugs have failed, often representing a patient's last hope for survival.

Drug Action Works Like Immune Proteins

Weiss says he and his colleagues noted that colistin works by disrupting the inner and outer membranes that hold Gram-negative bacterial cells together, much the same way LL-37 and lysozyme do. LL-37 is a protein found at sites of inflammation, whereas lysozyme is found in numerous different immune cells and within secretions like tears, breast milk, and mucus, and both are important defenses against invading bacteria. Weiss and his collaborators from Emory, the CDC, Walter Reed Army Institute of Research, and Grady Memorial Hospital in Atlanta set out to find whether resistance to colistin could engender resistance to attack by LL-37 or lysozyme.

Looking at A. baumannii isolates from patients around the country, they noted that all the colistin-resistant strains harbored mutations in pmrB, a regulatory gene that leads to the modification of polysaccharides on the outside of the cell in response to antibiotic exposure. Tests showed a tight correlation between the ability of individual isolates to resist high concentrations of colistin and the ability to resist attacks by LL-37 or lysozyme.

Patient Isolates Provide Causative Evidence

This was very convincing, write the authors, that mutations in the pmrB gene were responsible for cross-resistance to LL-37 and lysozyme, but to get closer to a causative link between treatment and cross-resistance, they studied two pairs of A. baumannii isolates taken from two different patients before and after they were treated for three or six weeks with colistin. The results helped confirm the cross-resistance link: neither strain taken before treatment was resistant to colistin, LL-37, or lysozyme, but the strains taken after treatment showed significant resistance to colistin and lysozyme. (One post-colistin isolate was no more or less resistant to LL-37 than its paired pre-colistin isolate.) Like the resistant strains tested earlier, both post-colistin isolates harbored crucial mutations in the pmrB gene that apparently bestow the ability to resist treatment.

The authors point out that the apparent link between resistance to colistin and cross-resistance to antimicrobial agents of the immune system could well extend to other pathogens that are treated with colistin, including Pseudomonas aeruginosa and Klebsiella pneumoniae. Weiss says he plans to follow up with studies to determine whether this bears out.

More Funding Needed for Antibiotic R&D

For Weiss, the problems with colistin are symptomatic of a much larger trio of problems: increasing levels of drug resistance, cuts in federal funding for antibiotic research, and lack of incentives for pharmaceutical companies to invest in antibiotic R&D. "We don’t have enough antibiotics, and it's really important for the research community and the public to support increases in funding for research to develop new antibiotics," says Weiss.

"We got complacent for a while and the bugs are becoming resistant. This is something we can reverse - or make a lot better - if we have the resources."

Algae capture, store, and release nitrogen to feed reef-building coral

Symbiotic algae that live within reef-forming corals scoop up available nitrogen, store the excess in the form or uric acid crystals, and slowly feed it to the coral as needed, according to a study in mBio this week. Scientists have known for years that these symbiotic microorganisms serve up nitrogen to their coral hosts, but this new study sheds light on the dynamics of the process and reveals that the algae have the ability to store excess nitrogen, a capability that could help corals cope with the ups and downs of nitrogen concentrations in the environment.

"It was a great surprise to find the nitrogen-rich crystals inside the algae," writes corresponding author Anders Meibom of the École Polytechnique Fédérale de Lausanne, Switzerland, in an email interview. But, he writes, "it all makes perfect sense now. The algae suck up the ammonium and nitrate like a sponge when the concentrations of these molecules increases, then stores this nitrogen as uric acid crystals for later use."

Hard coral: a nitrogen-based economy

Like all reef-forming corals, the species Kopp et al. studied, cauliflower coral (Pocillopora damicornis), is actually a symbiosis of two different organisms: the coral provides protection to a species of photosynthetic dinoflagellates, which, in turn, provide sugars and nitrogen to the coral host. The symbiosis allows the coral to thrive in clear, tropical waters that are naturally nutrient-poor. In many places, however, coral reefs are suffering from an excess of nutrients - pollution from sewage and fertilizers that impacts the symbiotic relationship and the health of coral in unknown - but probably detrimental - ways.

To better understand these exchanges of materials and to determine how an excess of nutrients might affect the balance, Kopp et al. exposed pieces of coral (called "nubbins" - isn't that cute?) to varying concentrations of isotopically-labeled ammonium, nitrate, and aspartic acid and applied nano-scale secondary ion mass-spectrometry (NanoSIMS) to follow the path of the nitrogen. NanoSIMS enabled them to visualize and quantify the uptake, movement, and accumulation of this labeled nitrogen within the coral.

Dinoflagellates store excess nitrogen as crystals

When supplied with any of the nitrogen-rich compounds, the dinoflagellates responded by rapidly storing the nitrogen as crystals of uric acid within its cells. But the dinoflagellates don't hang onto the nitrogen for long. Starting at about six hours after exposure, the microbes begin translocating nitrogen-rich compounds to the coral host, where the nitrogen is used in specific cellular compartments all over the epithelium of the coral.

This storage and release process helps explain how these corals get through the ups and downs of nitrogen concentrations, says Meibom. "This gives the coral-algae symbiosis a very efficient way to deal with strong fluctuations in nitrogen availability," writes Meibom. "When the nitrogen availability suddenly becomes high, the algae can take-up large amounts of nitrogen on a timescale of a few hours, store it into crystals inside the algae cells and then release this stored nitrogen for metabolic processes and growth when the nitrogen levels become normal again."

To follow up on this work, Meibom says he and his colleagues are now studying how carbon-based nutrients are taken up and distributed in the same coral-algae symbiosis.

Protein as spork: Opportunistic pathogen uses 1 protein in 2 ways

To infect its host, the respiratory pathogen Pseudomonas aeruginosa takes an ordinary protein usually involved in protein synthesis and adds three small molecules to turn it into a key virulence factor according to a study in mBio this week, economically employing one protein in two different ways. Scientists at Emory University School of Medicine, the University of Virginia, and Universidad de las Islas Baleares in Mallorca, Spain, uncover this previously unknown virulence factor in P. aeruginosa, one of the most common causes of hospital-acquired pneumonia.

How is EF-Tu like a spork? P. aeruginosa uses it for two purposes: protein synthesis AND as a virulence factor.

Co-author Joanna Goldberg of Emory says scientists have long thought P. aeruginosa mostly uses elongation factor-Tu (EF-Tu) inside the cell, but she and her collaborators have learned that as a virulence factor, it could represent a vulnerability for the bacterium. "EF-Tu is presumed to be an essential protein, and it's performing these moonlighting functions as well. If we figured out how it was doing that, we could devise strategies to inhibit it," says Goldberg.

Environmental pathogen is a scourge in hospitals

P. aeruginosa pneumonia is, of course, a big problem in the hospital setting, where it is a frequent cause of hospital-acquired pneumonia and is the leading cause of death among critically ill patients whose airways have been damaged by ventilation, trauma, or other infections. The pathogen is also a contributor to disease in the lungs of cystic fibrosis patients and forms thick biofilms that are difficult or impossible to treat with antibiotics. Goldberg and her co-author Sebastian Alberti and their colleagues study the molecular events that enable the bacterium to infect human cells in the hopes of finding ways to prevent P. aeruginosa pneumonia.

In their earlier work, Goldberg and Alberti  found that P. aeruginosa takes the protein EF-Tu, which was generally thought to exist only inside the cell, and decorates the exterior of the cell with it, but in a modified form. This modified EF-Tu is recognized by antibodies to the common bacterial epitope phosphorylcholine (ChoP), indicating that the EF-Tu is modified somehow to mimic ChoP, allowing P. aeruginosa to enjoy the benefits of ChoP. By interacting with receptors on human cells, ChoP carries out a crucial step for setting up an infection for a number of different types of respiratory pathogens.

But how is EF-Tu modified, they wondered? And does it help P. aeruginosa establish an infection? This study answers those questions.

Small changes add up to a powerful mimic

Using a host of techniques, including mass spectrometry, site directed mutagenesis of key residues in the protein, and genetic loss of function/gain of function studies, they found that P. aeruginosa only makes small changes to EF-Tu to get it to mimic this powerful ligand. P. aeruginosa transfers three methyl groups to a lysine on EF-Tu, giving it a structure similar to ChoP and allowing it to fit in the PAFR receptor in the way ChoP does.

But the modified EF-Tu not only looks like ChoP, it many ways it works like ChoP: testing in cultures of human airway cells shows that the modification of EF-Tu enables the bacterium to adhere to human cells.

"It allows [P. aeruginosa] to adhere to the cells and invade," says Goldberg. "And it seems to be involved in virulence in mouse models. It might also impact persistence in the lung."

As an environmental pathogen, P. aeruginosa lives in soil, water, and other environments outside the body, a fact that may offer a clue why it uses this re-purposed protein as a virulence factor. Proteins that can be put to work in both worlds - in the environment and the in the human host -  would be useful to P. aeruginosa in much the way a spork can allow you to enjoy both the coleslaw and the pudding in your take out dinner.

"Its interaction with humans is accidental. It's an opportunist. The fact that it has this novel modification on this protein that is inherent in the bacterium that enables it to attach and persist and cause disease is exciting," says Goldberg.

Protein improves efficacy of tumor-killing enzyme

Researchers at NIAID have devised a method for delivering tumor cell-killing enzymes in a way that protects the enzyme until it can do its work inside the cell. In their study in mBio this week, researchers assembled microscopic protein packages that can deliver an enzyme called PEIII to the insides of cells. By attaching a protein called ubiquitin to the enzyme, they were able to protect the PEIII from degradation by the cell, allowing the PEIII to complete its mission of cell assassination. The results indicate that ubiquitin may be a useful addition to targeted toxins, therapies that specifically target and kill tumor cells.

 

Domain structures and sequences of ubiquitin fusion proteins.

Although researchers have been developing tumor-directed "targeted toxins" for decades, their success has been hindered by technical problems, including inadequate tumor specificity, low efficiency of delivery to the cytosol, and other issues. In their study in mBio, researchers from the National Institute of Allergy and Infectious Diseases (NIAID) sought to improve the persistence of the payload enzyme in the cytosol.  

The targeted toxin assemblies they created included two components the researchers have used before in targeted toxins: the "killing" enzyme PEIII, and a set of targeting proteins called LFn that deliver the PEIII enzyme via pores to the inside of the cell. The LFn delivery system was engineered to specifically target and attach to tumor cells.

The third component in the bundle was a new addition: ubiquitin, a small protein that is normally used by cells to target waste proteins for degradation. The researchers inserted ubiquitin in between the LFn and the PEIII, then tested the bundle on mice with tumors. The idea was to use the cell's own ubiquitin-cleaving enzymes to cut the ubiquitin off and free up the PEIII enzyme once it's inside the cell.

The system worked, write the authors. Tumor growth was inhibited in mice treated with targeted toxins that either carried the wild-type ubiquitin or engineered ubiquitin without lysine residues in it, a change that should prevent it from being degraded by the cell. The addition of ubiquitin enhanced the ability of the PEIII enzyme to persist inside the cell, they write, thereby enhancing its potency. And the ubiquitin didn't seem to hinder the efficiency of delivering the PEIII inside the cell.

As an added bonus, the addition of ubiquitin reduced the toxicity of the targeted toxin to non-tumor tissues.

The authors point out that the use of ubiquitin linkers shows considerable promise and could be an effective strategy for enhancing the potency of tumor-targeting toxins for use in patients. In research currently underway, they are attempting to improve on the system by making changes to the ubiquitin that allow it to unfold appropriately inside the cell.