Team first to model atomic structures of three bacterial nanomachines

April 21, 2015
Hong Zhou runs the Electron Imaging Center for Nanomachines laboratory at CNSI, where a highly sophisticated cryo electron microscope made the research possible.

Researchers at UCLA's California NanoSystems Institute have become the first to produce images of the atomic structures of three specific biological nanomachines, each derived from a different potentially deadly bacterium—an achievement they hope will lead to antibiotics targeted toward specific pathogens.

The scientists used a leading-edge technology called cryo electron microscopy, or cryoEM, to reveal the form and function of these important structures. Papers on their findings were published in three top-tier journals: Nature, Cell, and Nature Structural and Molecular Biology.

Two of the nanomachines are structures called contractile ejection systems, which their bacteria use to transfer toxic molecules into healthy cells to usurp them for their own purposes, to attack rival bacteria by delivering toxins into them, and other functions. These structures have sheath–tube assemblies that create openings in the outer membranes of target cells through which they can insert toxic molecules.

The third nanomachine—different from the other two—is a pore structure that delivers deadly anthrax toxin into mammalian cells, once the is in the bloodstream. This mechanism is how anthrax bacteria activate the disease in an infected animal or person.

How the nanomachines work had been poorly understood, but the UCLA researchers used a cryoEM equipped with a special camera called a direct electron detector to produce highly detailed images. The scientists hope the new information about how they function will enable them to create antibiotics that target bacterial pathogens.

The team, led by Hong Zhou, professor of microbiology, immunology and , and of chemistry and biochemistry, runs the Electron Imaging Center for Nanomachines laboratory, which is based at CNSI and houses UCLA's Titan Krios electron microscope—a highly sophisticated and rare cryoEM.

"As the centerpiece of our electron microscopy core lab, the cryo electron microscope is enabling exploration of new territory in ," said Jeff Miller, director of the California NanoSystems Institute. "These unprecedented images enable us to understand the actual workings of these remarkable structures."

Anthrax toxin

In a paper published online by Nature, Professor Zhou and his team reported that they were the first to determine the atomic structure of the anthrax toxin pore, the major disease molecule of Bacillus anthracis, the bacterium that causes the disease anthrax in humans and animals. The anthrax toxin pore's atomic structure is mushroom-shaped with a gate inside the "shaft."

The finding confirms how the disease affects cells. When healthy cells encounter nanoscale objects in the body, they assume the objects are nutrients and absorb them. Like a Trojan horse, the toxin pore appears to the cells as something beneficial—in this case, a nutrient—and is taken in by the cell. But once inside the cell, the pore senses the change to a more acidic environment, which opens the pore's gate and releases the molecule into the cell.

"This is a very important step toward understanding this mechanism, and it is essential for any anthrax countermeasure," Zhou said. "It also informs our understanding of the mechanisms of other toxins that function like anthrax, which could lead to other targeted antibiotic drugs."

Tularemia type VI secretion system

Another nanomachine was described by Dr. Marcus Horwitz, a UCLA professor of medicine and of microbiology, immunology and molecular genetics, who worked with Zhou's team. In a study published in the journal Cell, the scientists reported the first atomic resolution model of any type VI secretion system, or T6SS, a nanomachine found in roughly 25 percent of .

Gram-negative bacteria are responsible for diseases such as cholera, salmonellosis, Legionnaires' disease and melioidosis, and severe infections including gastroenteritis, pneumonia and meningitis. For the new study, the scientists examined Francisella tularensis, a bacterium that causes tularemia and is of great concern as a potential bioterrorism agent.

Built from component proteins, the T6SS nanomachine has an atomic structure that resembles a piston. When F. tularensis is taken up into a type of white blood cell called a macrophage it is surrounded by a bubble-like membrane, a structure known as a phagosome. The T6SS nanomachine then assembles inside the bacterium, where it plunges a tube through the bacterial wall and the membrane of the phagosome into the cytoplasm, the substance inside the macrophage. This enables the bacterium to escape the phagosome into the cytoplasm, where it can complete its lifecycle and multiply. Soon, the macrophage fills with bacteria and ruptures, freeing the bacteria to infect other cells. Thus, the T6SS is a novel target for antibiotics against this bacterium, and against others that use it to survive within host cells or to combat rival bacteria.

"We are already identifying drug molecules that target the F. tularensis T6SS," Horwitz said. "Knowing how this structure works guides us in selecting drug molecules that block its assembly or function. The overall goal is to find new antibiotics that directly target this top-tier bioterrorism agent and other gram-negative bacteria with a T6SS such as Vibrio cholerae, Pseudomonas aeruginosa, Burkholderia pseudomallei, and pathogenic Escherichia coli."

Horwitz and his team could potentially also develop wider-spectrum drugs that work on many different gram-negative pathogens that have in common a T6SS.

Pseudomonas aeruginosa

In humans and animals, a bacterium called Pseudomonas aeruginosa causes infectious diseases that lead to generalized inflammation and sepsis, a dangerous infection of the blood. A team led by Zhou and Miller discovered the of R-type pyocins, contractile ejection systems of Pseudomonas aeruginosa. Their findings were published online by Nature Structural and Molecular Biology.

R-type pyocins are used by the bacterium to rapidly insert their nanotubes, like battering rams, into the cell membranes of competing bacteria to kill the competitors, giving Pseudomonas aeruginosa easier access to nutrients. These pyocins appear to create a channel in the outer envelope of the target bacteria, which essentially acts to weaken and kill it. This ability has made R-type pyocins the focus of research into possible antimicrobial and bioengineering applications, and scientists believe they could be engineered to give drugs a powerful antibacterial component.

"The R2 pyocin is an extraordinary molecular machine that uses energy from its own biological battery to function," said Miller, who also is a professor of microbiology, immunology and molecular genetics. "It is ideal for engineering targeted antibiotics that kill the bad bacteria without disrupting a patient's protective gut bacteria."

The scarcity of the technology and the expertise needed to use it make CNSI one of the world's few facilities capable of imaging atomic structures like these nanomachines at atomic-level resolution, which is why researchers from around the world come to UCLA to use the Electron Imaging Center for Nanomachines, a fee-for-service laboratory open to any scientist in academia or industry.

Other UCLA researchers who contributed to the three papers were Daniel Clemens, adjunct professor of medicine; Xuekui Yu, adjunct assistant professor of microbiology, immunology and molecular genetics; Peng Ge, a research associate; Bai-Yu Lee, an associate researcher; and Jiansen Jiang, a postdoctoral fellow. Bradley Pentelute of Massachusetts Institute of Technology, R. John Collier of Harvard University Medical School, Dean Scholl of AvidBiotics and Petr Leiman of the Ecole Polytechnique Federale de Lausanne's Institute of Physics of Biological Systems were the other co-authors.

Explore further: Pseudomonas deploys a toxin delivery machine to breach cell walls of rivals without hurting itself

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JVK
1 / 5 (1) Apr 23, 2015
Excerpt: "When healthy cells encounter nanoscale objects in the body, they assume the objects are nutrients and absorb them. Like a Trojan horse, the toxin pore appears to the cells as something beneficial—in this case, a nutrient—and is taken in by the cell. But once inside the cell, the pore senses the change to a more acidic environment, which opens the pore's gate and releases the anthrax toxin molecule into the cell."

My comment: Evolutionary theorists assume that mutations and natural selection lead to the differentiation of cell types that serious scientists like these know is nutrient-dependent. When others learn the differences between virus-driven mutations and amino acid substitutions, they will finally link amino acid substitutions to protein structure and function. At the same time they will link perturbed protein folding from mutations to pathology and convince theorists that mutations are not beneficial.
JVK
1 / 5 (1) Apr 23, 2015
Nutrient-dependent pheromone-controlled ecological adaptations: from atoms to ecosystems
http://figshare.c...s/994281

"This atoms to ecosystems model of ecological adaptations links nutrient-dependent epigenetic effects on base pairs and amino acid substitutions to pheromone-controlled changes in the microRNA / messenger RNA balance and chromosomal rearrangements. The nutrient-dependent pheromone-controlled changes are required for the thermodynamic regulation of intracellular signaling, which enables biophysically constrained nutrient-dependent protein folding; experience-dependent receptor-mediated behaviors, and organism-level thermoregulation in ever-changing ecological niches and social niches. Nutrient-dependent pheromone-controlled ecological, social, neurogenic and socio-cognitive niche construction are manifested in increasing organismal complexity in species..."
anonymous_9001
5 / 5 (1) Apr 24, 2015
And yet there's an entire subset of techniques used to deliberately cause mutations in order to alter and improve enzyme function...

Error-prone polymerases don't have precognition about what changes they need to make to DNA.

Edit: And you STILL cannot explain how Lenski saw the appearance of a new trait in only 1 of his 12 populations if they had the same environmental conditions. If you make the claim that citrate caused the promoter shift, then you must explain why it didn't happen in the other 11.
JVK
1 / 5 (1) Apr 24, 2015
http://www.scienc...4756.htm

Excerpt: "...an interdisciplinary team of researchers has created a computational model that enables one to examine how emerging metastatic tumors interact with the immune system."

Computational models are grossly inaccurate misrepresentations of biologically-based cause and effect. They fail to include links from physics and the chemistry of protein folding to the conserved molecular mechanisms of cell type differentiation in all genera. Ridiculous claims frustrate serious scientists who are combating evolution to fight disease at the same time theorists contribute to preventable pathologies.

Nutrient-dependent pheromone-controlled ecological adaptations: from atoms to ecosystems http://figshare.c...s/994281
anonymous_9001
5 / 5 (1) Apr 24, 2015
Computational models are grossly inaccurate ... They fail to include links from physics and the chemistry of protein folding


That's completely wrong. Are you aware of the computational biology that Stanford leads? Folding@home ring any bells? They deal fundamentally with the biophysics behind folding. You can't dismiss an entire field when one of the largest distributed computing projects ever does exactly what you say computational biology doesn't do.
JVK
1 / 5 (1) Apr 24, 2015
They deal fundamentally with the biophysics behind folding.


I deal with facts that link the biophysically constrained chemistry of nutrient-dependent protein biosynthesis and degradation to RNA-mediated amino acid substitutions and their fixation in the context of the physiology of reproduction.

Nutrient-dependent pheromone-controlled ecological adaptations: from atoms to ecosystems http://figshare.c...s/994281

See also: Inching toward the 3D genome http://www.scienc...10.short and my comment at http://comments.s....6217.10

Alternatively, keep taking what I claim out of context and regurgitating it along with your pseudoscientific nonsense based on mutagenesis experiments.
JVK
1 / 5 (1) Apr 25, 2015
"DNA study could shed light on how genetic faults trigger disease." April 24th, 2015. http://medicalxpr...ase.html

Excerpt: "Errors in protein production can result in a wide range of diseases in people, researchers say."

Biologically uninformed science idiots like Andrew Jones (aka anonymous_9001) claim that errors in protein folding (called mutations) can somehow lead to the evolution of morphological and behavioral diversity.

Excerpt: The new method could help researchers pinpoint the source of disease-causing mutations in enhancers. Until now, these genetic errors have been difficult to interpret as the link between enhancers and the genes they control was not clear.

Also, biologically uninformed science idiots have claimed that disease-causing mutations are sometimes beneficial because they believe what they have been taught about Lenski's experiments with E. coli.

JVK
1 / 5 (1) Apr 25, 2015
And you STILL cannot explain how Lenski saw the appearance of a new trait in only 1 of his 12 populations if they had the same environmental conditions.


I don't try to explain what Lenski or anyone else thinks he saw. I use examples of how viral microRNAs and nutrient-dependent microRNAs link RNA-mediated events to cell type differentiation in species from microbes to humans via amino acid substitutions.

See also: Viral proteins may regulate human embryonic development
http://phys.org/n...nic.html

Differences in HERV-K LTR insertions in orthologous loci of humans and great apes
http://www.scienc...00000627

Abstract excerpt: "Human-specific integrations for some very young LTRs were demonstrated. The possibility of LTRs and HERVs involvement in the evolution of primates is discussed."

See also: The Darwin Code by Greg Bear http://rna-mediat...eg-bear/

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