NEW ANTIBIOTIC ATTACK S DRUG RESISTANT MICROBES

The multitude of microbes scientists have found populating the human body have good, bad and mostly mysterious implications for our health. But when something goes wrong, we defend ourselves with the undiscriminating brute force of traditional antibiotics, which wipe out everything at once, regardless of the consequences.

Researchers at Rockefeller University and their collaborators are working on a smarter antibiotic. And in research to be published October 5 in Nature Biotechnology, the team describes a 'programmable' antibiotic technique that selectively targets the bad bugs, particularly those harboring antibiotic resistance genes, while leaving other, more innocent microbes alone.

"In experiments, we succeeded in instructing a bacterial enzyme, known as Cas9, to target a particular DNA sequence and cut it up," says lead researcher Luciano Marraffini, head of the Laboratory of Bacteriology. "This selective approach leaves the healthy microbial community intact, and our experiments suggest that by doing so you can keep resistance in check and so prevent certain types of secondary infections, eliminating two serious hazards associated with treatment by classical antibiotics."

The new approach could, for instance, reduce the risk of C. diff, a severe infection of the colon, caused by the Clostridium difficile bacterium, that is associated with prolonged courses of harsh antibiotics and is a growing public health concern.

The Cas9 enzyme is part of a defense system that bacteria use to protect themselves against viruses. The team coopted this bacterial version of an immune system, known as a CRISPR (clustered regularly interspaced short palindromic repeats) system and turned it against some of the microbes. CRISPR systems contain unique genetic sequences called spacers that correspond to sequences in viruses. CRISPR-associated enzymes, including Cas9, use these spacer sequences as guides to identify and destroy viral invaders.

The researchers were able to direct Cas9 at targets of their choosing by engineering spacer sequences to match bacterial genes then inserting these sequences into a cell along with the Cas9 gene. The cell's own machinery then turns on the system. Depending on the location of the target in a bacterial cell, Cas9 may kill the cell or it may eradicate the target gene. In some cases, a treatment may prevent a cell from acquiring resistance, they found.

"We previously showed that if Cas9 is programmed with a target from a bacterial genome, it will kill the bacteria. Building on that work, we selected guide sequences that enabled us to selectively kill a particular strain of microbe from within a mixed population," says first author David Bikard, a former Rockefeller postdoc who is now at the Pasteur Institute in Paris.

In initial experiments, Bikard and colleagues targeted a strain of the common skin and respiratory bacteria Staphylococcus aureus that is resistant to the antibiotic kanamycin. Treatment by Cas9 programmed to target a part of the resistance gene killed most of the resistant Staph, but left behind the kanamycin-susceptible Staph.

Targeted bacterial genocide is only one option. Bacteria share genes, including those conferring drug resistance, in the form of rings of DNA known as plasmids. In a second series of experiments, researchers turned Cas9 on tetracycline resistance-harboring plasmids in a strain of the potentially deadly multidrug resistant bacteriaStaphylococcus aureus (MRSA). Not only did the resistant cells become sensitive to tetracycline after Cas9 destroyed the plasmids, but the arrival of Cas9 in other Staphcells acted as an immunization, preventing them from taking on resistance-carrying plasmids.

And, in a final set of experiments, conducted in collaboration with Vincent Fischetti's Laboratory of Bacterial Pathogenesis and Immunology, adjunct faculty member Chad Euler confirmed their test tube results on living skin, by using Cas9 to selectively kill kanamycin-resistant Staph infecting the shaved backs of mice.

In spite of the promising results, the delivery system needs improvement. The researchers used bacteria-infecting viruses to inject the programmed Cas9 enzymes into the bacterial cells, but these viruses only attack specific types of cells. Scientists need to devise a less discriminating method of delivery, before the technology can be used to develop a new class of antibiotics, Marraffini says.

In addition to its potential as a much-needed new weapon against drug-resistant microbes, the new system could also be used to advance research on the complex populations of microbes in the body, about which very little is known. "There are enormous microbial communities in the human body," Marraffini says. "Programmable Cas9 enzymes may make it possible to analyze these populations by eliminating their members, one by one, and studying the effects."

SEARCH FOR BETTER BIOFUELS MICROBES LEADS TO HUMAN GUT

Scientists have scoured cow rumens and termite guts for microbes that can efficiently break down plant cell walls for the production of next-generation biofuels, but some of the best microbial candidates actually may reside in the human lower intestine, researchers report.

Their study, reported in the Proceedings of the National Academy of Sciences, is the first to use biochemical approaches to confirm the hypothesis that microbes in the human gut can digest fiber, breaking it down into simple sugars in order to ferment them into nutrients that nourish human cells. These findings have significance for human health but also for biofuels production, since the same sugars can be fed to yeast to generate ethanol and other liquid fuels. The human microbes appear to be endowed with enzymes that break down a complex plant fiber component more efficiently than the most efficient microbes found in the cow rumen, the researchers report.

Their work in cows led the researchers to the human microbes, said University of Illinois animal sciences and Institute for Genomic Biology professor Isaac Cann, who led the new analysis with his colleagues, animal sciences professor Roderick Mackie and M.D./Ph.D. student Dylan Dodd. Cann also is a microbiology professor and a principal investigator at the Energy Biosciences Institute. Dodd is now at Stanford University.

"In looking for biofuels microbes in the cow rumen, we found that Prevotella bryantii, a bacterium that is known to efficiently break down (the plant fiber) hemicellulose, gears up production of one gene more than others when it is digesting plant matter," Cann said.

When searching a database for similar genes in other organisms, the researchers found them in microbes from the human gut. The team focused on two of these human microbes, Bacteroides intestinalis and Bacteroides ovatus, which belong to the same bacterial phylum as Prevotella from the cow.

"We expressed the human gut bacterial enzymes and found that for some related enzymes, the human ones actually were more active (in breaking down hemicellulose) than the enzymes from the cow," Cann said.

When the researchers looked more closely at the structure of the human enzymes, they saw something unusual: many single polypeptide (protein) chains actually contained two enzymes, one of which was embedded in the other. Further analysis of the most important protein revealed that the embedded component was a carbohydrate-binding module (CBM), which, as its name implies, latches onto carbohydrates such as hemicellulose. This enzyme shreds the plant fiber hemicellulose so that other enzymes can work on it to break it down into its unit sugars.

Working with U. of I. biochemistry professor Satish Nair, the researchers also noticed that the CBM "put a kink" in the fiber when it bound to it. This bending action may bring the fiber close to the other enzyme in the protein so it can get to work breaking the bonds between the sugars. Further research is needed to confirm this hypothesis, Cann said.

The study points to human microbes as a potentially potent source of microbes that can aid in biofuels production, Cann said.

"In addition to finding microbes in the cow rumen and termite gut, it looks like we can actually make some contributions ourselves," he said. "And our bugs seem to have some enzymes that are even better than those in the cow rumen."