Salmonella stays deadly with a 'beta' version of cell behavior

Salmonella cells have hijacked the protein-building process to maintain their ability to cause illness, new research suggests.

Scientists say that these bacteria have modified what has long been considered typical cell behavior by using a beta form of an amino acid – as opposed to an alpha form – during the act of making proteins.

Beta versions of amino acids occur in nature under rare and specific circumstances, but have never been observed as part of protein synthesis. Before this finding, in fact, researchers had determined that virtually all proteins were constructed with the alpha forms of amino acids.

This work has shown that when researchers delete any one of three genes from the process that makes use of the beta form of the amino acid, or if they insert the alpha form in the beta version's place, Salmonella cells are no longer able to cause disease. The amino acid in question is lysine, one of 22 genetically encoded amino acids that are strung together in cells to make proteins.

"When these genes were knocked out, the cells became sensitive to antibiotics. And if we put beta lysine into the medium where cells were growing, they became resistant to antibiotics," said Michael Ibba, professor of microbiology at Ohio State University and a senior author of the study. "So we could see the beta amino acid being taken up and used. The cells really do need the beta amino acid to be resistant to antibiotics, and for other aspects of their virulence."

This finding suggests that the process using this specific beta amino acid could be an attractive antibiotic target for this common pathogen, the researchers say.

The Centers for Disease Control and Prevention estimates that about 1.4 million people in the United States are infected with Salmonella each year, though only 40,000 cases are reported. Most people infected with Salmonella develop diarrhea, fever and abdominal cramps. Though recovery can occur within a week without treatment, some severe cases require antibiotic treatment and hospitalization.

The study is published in the Aug. 14 online edition of the journal Nature Chemical Biology.

This work began when University of Toronto scientists exploring the origins of Salmonella's virulence identified three genes that were clear players in the process. These three genes – called YjeK, PoxA and EF-P – were unusual in this context.

Genes that confer virulence in bacteria typically have a specific job, such as producing toxins or transporters. But these three virulence genes all looked like they should have a role in the protein synthesis machinery – which is Ibba's expertise.

Under normal circumstances in cells, an enzyme will select amino acids in the cell and place them on a molecule called transfer RNA, or tRNA, which leads to translation of the genetic code into proteins.

In Salmonella cells, these steps are similar, but with a few surprising twists, Ibba said. He and colleagues confirmed that the YjeK gene makes beta lysine, and showed that the PoxA gene takes that beta lysine and attaches it to EF-P – a protein that partially mimics the shape and function of tRNA.

"It's a really unexpected pathway," said Ibba, also an investigator in Ohio State's Center for RNA Biology. "It is a mimic of what normally makes protein in a cell. Where a cell would normally be expected to use an alpha amino acid, Salmonella puts on a beta amino acid. And it ends up making molecules that lead to the cells being virulent."

The research team first reconstructed this unusual protein synthesis process in test tube experiments, and then followed with studies in cell cultures. Even before they took on studying the mechanism, however, they knew that the effects of these virulence genes were powerful: In earlier animal studies, deleting any one of the three genes and then infecting mice with these altered Salmonella cellshad no effect on the animals. When the genes were left intact and cells were injected into mice, the resulting Salmonella infection killed the animals.

In addition, when the researchers tricked Salmonella cells into using alpha lysine for this pathway instead of beta lysine, the cells lost their ability to cause illness.

"This tells us the cell is not going to be able to easily replace the beta amino acid," Ibba said. "It is essential for virulence in Salmonella."

And that, he said, is why that amino acid might be such an effective drug target, especially as humans don't seem to make beta amino acids at all. "You have to make an antibiotic look like something natural, only different. If you have something that's already different like a beta amino acid, you've potentially got a much better drug target because it involves chemistry that's comparatively rare in the cell. It's harder for the cell to try to alter its own chemistry to develop resistance," Ibba said.

From here, the researchers are observing cell behavior later in the protein-building process to figure out how this hijacked system actually gives Salmonella its virulence.

Provided by The Ohio State University (news : web)

Click chemistry with copper -- a biocompatible version

Berkeley Lab researchers have found a way to make copper-catalyzed click chemistry biocompatible. By adding a ligand that minimizes the toxicity of copper but still allows it to catalyze the click chemistry reaction, the researchers can safely use their reaction in living cells.

Biomolecular imaging can reveal a great deal of information about the inner workings of cells and one of the most attractive targets for imaging are glycans – sugars that are ubiquitous to living organisms and abundant on cell surfaces. Imaging a glycan requires that it be tagged or labeled. One of the best techniques for doing this is a technique called click chemistry. The original version of click chemistry could only be used on cells in vitro, not in living organisms, because the technique involved catalysis with copper, which is toxic at high micromolar concentrations. A copper-free version of click chemistry that can safely be used in living organisms is available, but it is not always optimal in terms of reaction kinetics and target specificity. Now, a variation of click chemistry has been introduced that retains the copper catalyst of the original reaction - along with its speed and specificity – but is safe for cells in vivo.

Researchers with the Lawrence Berkeley National Laboratory (Berkeley Lab), in collaboration with researchers at the Albert Einstein College of Medicine at Yeshiva University in New York, have found a way to make copper-catalyzed click chemistry biocompatible. By adding a ligand that minimizes the toxicity of copper but still allows it to catalyze the click chemistry reaction, the researchers can safely use their reaction in living organisms. Compared to the copper-free click chemistry reaction, which can take up to an hour, the ligand-accelerated copper-catalyzed click chemistry reaction can achieve effective labeling within 3-5 minutes. The presence of the copper catalyst also enables this new formulation of click chemistry to be more target-specific with fewer background side reactions.

"The discovery of this new accelerating ligand for copper-catalyzed click chemistry should provide an effective complimentary tool to copper-free click chemistry," says Yi Liu, a chemist with Berkeley Lab's Molecular Foundry and the co-leader of this research with Peng Wu, of the Albert Einstein College of Medicine.

"While copper-free click chemistry may have advantages for whole animal imaging experiments such as imaging in mice," Liu says, "our ligand-accelerated copper reaction is better suited for enriching glycoproteins for their identification."

The ligand-accelerated copper-catalyzed reaction was used to label glycans in recombinant glycoproteins, glycoproteins in cell lysates, glycoproteins on live cell surfaces, and glycoconjugates in live zebrafish embryos. Because a zebrafish embryo is transparent in the first 24 hours of its development, it allows labeled glycans to be detected via molecular imaging techniques, making it a highly useful model for developmental biology studies.

"Based on our results," says Peng Wu, "we believe that ligand-accelerated copper-catalyzed click chemistry represents a powerful and highly adaptive bioconjugation tool that holds great promise for further improvement with the discovery of more versatile catalyst systems."

Click chemistry, which was introduced in 2002 by the Nobel laureate chemist Barry Sharpless of the Scripps Research Institute, utilizes a copper-catalyzed azide-alkyne cycloaddition (CuAAC) reaction that makes it possible for certain chemical building blocks to "click" together in an irreversible linkage, analogous to the snapping together of Lego blocks. While the technique immediately proved valuable for attaching small molecular probes to various biomolecules in a test tube or on fixed cells, it could not be used for biomolecule labeling in live cells or organisms because of the copper catalyst.

In 2007, Carolyn Bertozzi, a chemist who holds joint appointments with Berkeley Lab, the University of California (UC) Berkeley, and the Howard Hughes Medical Institute, led a research effort that produced a copper-free version of click chemistry. In this version, glycans were metabolically labeled with azides - a functional group featuring three nitrogen atoms - via reactions that were carried out through the use of cyclooctyne reagents that required no copper catalyst. With their latest reagent, biarylazacyclooctynone (BARAC), Bertozzi and her group have provided a copper-free click chemistry technique that delivers relatively fast reaction kinetics and the bioorthogonality needed for biomolecule labeling. However, the technique can only be used on biomolecules that can be tagged with azides.

"Our bio-benign ligand-accelerated copper-catalyzed click chemistry reaction liberates bioconjugation from the limitation where ligations could only be accomplished with azide-tagged biomolecules," Liu says. "Now terminal alkyne residues can also be incorporated into biomolecules and detected in vivo."

Provided by Lawrence Berkeley National Laboratory (news : web)

Click chemistry with copper: A biocompatible version

Biomolecular imaging can reveal a great deal of information about the inner workings of cells and one of the most attractive targets for imaging are glycans -- sugars that are ubiquitous to living organisms and abundant on cell surfaces. Imaging a glycan requires that it be tagged or labeled. One of the best techniques for doing this is a technique called click chemistry. The original version of click chemistry could only be used on cells in vitro, not in living organisms, because the technique involved catalysis with copper, which is toxic at high micromolar concentrations.

A copper-free version of click chemistry that can safely be used in living organisms is available, but it is not always optimal in terms of reaction kinetics and target specificity. Now, a variation of click chemistry has been introduced that retains the copper catalyst of the original reaction -- along with its speed and specificity -- but is safe for cells in vivo.

Researchers with the Lawrence Berkeley National Laboratory (Berkeley Lab), in collaboration with researchers at the Albert Einstein College of Medicine at Yeshiva University in New York, have found a way to make copper-catalyzed click chemistry biocompatible. By adding a ligand that minimizes the toxicity of copper but still allows it to catalyze the click chemistry reaction, the researchers can safely use their reaction in living organisms. Compared to the copper-free click chemistry reaction, which can take up to an hour, the ligand-accelerated copper-catalyzed click chemistry reaction can achieve effective labeling within 3-5 minutes. The presence of the copper catalyst also enables this new formulation of click chemistry to be more target-specific with fewer background side reactions.

"The discovery of this new accelerating ligand for copper-catalyzed click chemistry should provide an effective complimentary tool to copper-free click chemistry," says Yi Liu, a chemist with Berkeley Lab's Molecular Foundry and the co-leader of this research with Peng Wu, of the Albert Einstein College of Medicine.

"While copper-free click chemistry may have advantages for whole animal imaging experiments such as imaging in mice," Liu says, "our ligand-accelerated copper reaction is better suited for enriching glycoproteins for their identification."

The ligand-accelerated copper-catalyzed reaction was used to label glycans in recombinant glycoproteins, glycoproteins in cell lysates, glycoproteins on live cell surfaces, and glycoconjugates in live zebrafish embryos. Because a zebrafish embryo is transparent in the first 24 hours of its development, it allows labeled glycans to be detected via molecular imaging techniques, making it a highly useful model for developmental biology studies.

"Based on our results," says Peng Wu, "we believe that ligand-accelerated copper-catalyzed click chemistry represents a powerful and highly adaptive bioconjugation tool that holds great promise for further improvement with the discovery of more versatile catalyst systems."

Click chemistry, which was introduced in 2002 by the Nobel laureate chemist Barry Sharpless of the Scripps Research Institute, utilizes a copper-catalyzed azide-alkyne cycloaddition (CuAAC) reaction that makes it possible for certain chemical building blocks to "click" together in an irreversible linkage, analogous to the snapping together of Lego blocks. While the technique immediately proved valuable for attaching small molecular probes to various biomolecules in a test tube or on fixed cells, it could not be used for biomolecule labeling in live cells or organisms because of the copper catalyst.

In 2007, Carolyn Bertozzi, a chemist who holds joint appointments with Berkeley Lab, the University of California (UC) Berkeley, and the Howard Hughes Medical Institute, led a research effort that produced a copper-free version of click chemistry. In this version, glycans were metabolically labeled with azides -- a functional group featuring three nitrogen atoms -- via reactions that were carried out through the use of cyclooctyne reagents that required no copper catalyst. With their latest reagent, biarylazacyclooctynone (BARAC), Bertozzi and her group have provided a copper-free click chemistry technique that delivers relatively fast reaction kinetics and the bioorthogonality needed for biomolecule labeling. However, the technique can only be used on biomolecules that can be tagged with azides.

"Our bio-benign ligand-accelerated copper-catalyzed click chemistry reaction liberates bioconjugation from the limitation where ligations could only be accomplished with azide-tagged biomolecules," Liu says. "Now terminal alkyne residues can also be incorporated into biomolecules and detected in vivo."

This work was supported by a grant from the National Institutes of Health, and in part as a User Project at the Molecular Foundry, which is funded through DOE's Office of Science.

Story Source:

The above story is reprinted (with editorial adaptations ) from materials provided by DOE/Lawrence Berkeley National Laboratory.

Journal Reference:

Christen Besanceney-Webler, Hao Jiang, Tianqing Zheng, Lei Feng, David Soriano del Amo, Wei Wang, Liana M. Klivansky, Florence L. Marlow, Yi Liu, Peng Wu. Increasing the Efficacy of Bioorthogonal Click Reactions for Bioconjugation: A Comparative Study. Angewandte Chemie International Edition, 2011; DOI: 10.1002/anie.201101817

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