viernes, 20 de mayo de 2016

Antibiotics From Scratch (desde la línea de salida)

Researchers have devised an approach for synthesizing new macrolide antibiotics from simple chemical building blocks. Using this method,Andrew Myers of Harvard University and colleagues synthesized more than 300 new antibiotic candidates, several of which were effective against some of the most stubbornly drug-resistant bacterial strains, according to the study published today (May 18) in Nature.

Here they present a practical, fully synthetic route to macrolide antibiotics by the convergent assembly of simple chemical building blocks, enabling the synthesis of diverse structures not accessible by traditional semisynthetic approaches. 

“It’s a tour de force in synthetic chemistry,” Kim Lewis of Northeastern University in Boston, who was not involved in the study, told The Scientist. “This is the first time there is a relatively easy path to synthesize macrolide erythromycin-type antibiotics from scratch.”
For most of the field’s history, natural products have been the starting point for new antibiotics. Most of them have been made by chemically modifying natural products in a process known as semisynthesis. Every existing antibiotic in a class called macrolides—including the commonly prescribed drug azithromycin—has been made by modifying erythromycin, which was first discovered in a soil sample in 1949. But some bacteria are developing resistance to these drugs at a startling rate, and semisynthesis is limited by the difficulty of modifying such complex molecules.
In the present study, Myers and his colleagues have developed a method for synthesizing macrolides from eight simple chemical building blocks to produce a diverse collection of structures that would be practically impossible to make using semisynthetic methods. In the same way you might build any complex device, from a cell phone to an airplane, “you divide it into building blocks,” and assemble those blocks, Myers told The Scientist. This approach “allows for, theoretically, tens of thousands of compounds” or more, he added.
Macrolides consist of a macrolactone ring with one or two sugars. “Decorating” these rings with different chemical groups by combining the building blocks in various combinations, Myers’s team created more than 300 synthetic macrolides, including the US Food and Drug Administration-approved drug telithromycin and the candidate solithromycin, which is currently being tested in Phase 3 clinical trials.
Next, the researchers evaluated the newly synthesized compounds with a panel of different bacteria, including two strains of methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant Enterococcus (VRE) isolated from clinical samples. “Some of these are really scary bugs,” said Myers.
Most of the compounds were effective against garden-variety pneumonia bacteria, and a few of them were more potent than approved antibiotics against MRSA, VRE, and other highly resistant strains, the researchers found. One of the synthetic compounds, called FSM-100573, was especially effective against gram-negative bacteria, which are traditionally less susceptible to macrolides. (The technology for synthesizing these macrolides has been licensed to the Watertown, Massachusetts-based Macrolide Pharmaceuticals, which Myers cofounded.)
These compounds have yet to be tested preclinically. And, as with all antibiotics, bacteria will ultimately develop resistance to them. However, this approach allows scientists to develop an exponentially larger number of new drug candidates. “At the end of the day, it’s kind of a numbers game,” Myers said.
“The work represents a paradigm shift in the discovery of novel antibiotics from the macrolide class,”Rodrigo Andrade of Temple University in Philadelphia, Pennsylvania, who was not involved in the work, told The Scientist. Synthesis of complex molecules can be a lengthy and expensive process, but Myers has developed a route that is more efficient, Andrade added. “This opens the door to myriad macrolide drug candidates that can help offset the incessant and inevitable onset of antibiotic resistance.”
“I’m impressed by the scale of the work and elegance of approach,” said Gerry Wright of McMaster University in Canada, who was also not involved in the research. “At the end of the day, it’s impossible to know whether or not they’re going to generate anything in clinic,” he said. Even so, “this is a significant step forward to regain the upper hand” against drug-resistant bugs,” Wright added.
Myers and his colleagues have previously synthesized novel tetracycline antibiotics, one of which is now a Phase 3 clinical trial candidate. His team is also developing methods to synthesize other classes of antibiotics, he said.

miércoles, 4 de mayo de 2016

Structures of the nucleoid occlusion protein SlmA bound to DNA and the C-terminal domain of the cytoskeletal protein FtsZ

Cell division in most prokaryotes is mediated by FtsZ, which polymerizes to create the cytokinetic Z ring. Multiple FtsZ-binding proteins regulate FtsZ polymerization to ensure the proper spatiotemporal formation of the Z ring at the division site. The DNA-binding protein SlmA binds to FtsZ and prevents Z-ring formation through the nucleoid in a process called “nucleoid occlusion” (NO). As do most FtsZ-accessory proteins, SlmA interacts with the conserved C-terminal domain (CTD) that is connected to the FtsZ core by a long, flexible linker. However, SlmA is distinct from other regulatory factors in that it must be DNA-bound to interact with the FtsZ CTD. Few structures of FtsZ regulator–CTD complexes are available, but all reveal the CTD bound as a helix. To deduce the molecular basis for the unique SlmA–DNA–FtsZ CTD regulatory interaction and provide insight into FtsZ–regulator protein complex formation, we determined structures of Escherichia coli, Vibrio cholera, and Klebsiella pneumonia SlmA–DNA–FtsZ CTD ternary complexes. Strikingly, the FtsZ CTD does not interact with SlmA as a helix but binds as an extended conformation in a narrow, surface-exposed pocket formed only in the DNA-bound state of SlmA and located at the junction between the DNA-binding and C-terminal dimer domains. Binding studies are consistent with the structure and underscore key interactions in complex formation. Combined, these data reveal the molecular basis for the SlmA–DNA–FtsZ interaction with implications for SlmA’s NO function and underscore the ability of the FtsZ CTD to adopt a wide range of conformations, explaining its ability to bind diverse regulatory proteins.

The bacterial protein FtsZ polymerizes into protofilaments to create the cytokinetic ring responsible for directing cell division. Cellular levels of FtsZ are above the concentration required for Z-ring formation. Hence, FtsZ-binding proteins have evolved that control its spatiotemporal formation. The SlmA protein is one such factor that, when bound to specific chromosomal DNA, inhibits FtsZ polymerization to prevent Z rings from forming through the bacterial chromosome. This inhibition depends on complex formation between SlmA-DNA and the FtsZ C-terminal domain (CTD). Here we describe SlmA–DNA–FtsZ CTD structures. These structures and complementary biochemistry unveil the molecular basis for the unique requirement that SlmA be DNA-bound to interact with FtsZ, a mechanism that appears to be conserved among SlmA-containing bacteria.

miércoles, 27 de abril de 2016

A mobile genetic element profoundly increases heat resistance of bacterial spores

Bacterial endospores are among the most resilient forms of life on earth and are intrinsically resistant to extreme environments and antimicrobial treatments. Their resilience is explained by unique cellular structures formed by a complex developmental process often initiated in response to nutrient deprivation. Although the macromolecular structures of spores from different bacterial species are similar, their resistance to environmental insults differs widely. It is not known which of the factors attributed to spore resistance confer very high-level heat resistance. Here, we provide conclusive evidence that in Bacillus subtilis, this is due to the presence of a mobile genetic element (Tn1546-like) carrying five predicted operons, one of which contains genes that encode homologs of SpoVAC, SpoVAD and SpoVAEb and four other genes encoding proteins with unknown functions. This operon, named spoVA2mob, confers high-level heat resistance to spores. Deletion of spoVA2mob in a B. subtilis strain carrying Tn1546 renders heat-sensitive spores while transfer of spoVA2mob into B. subtilis 168 yields highly heat-resistant spores. On the basis of the genetic conservation of different spoVA operons among spore-forming species of Bacillaceae, we propose an evolutionary scenario for the emergence of extremely heat-resistant spores in B. subtilis, B. licheniformis and B. amyloliquefaciens. This discovery opens up avenues for improved detection and control of spore-forming bacteria able to produce highly heat-resistant spores.

sábado, 9 de abril de 2016

This Artist Paints With Bacteria, And It’s Strangely Beautiful

No es Ingeniería genética, pero sí ARTE con bacterias

Tomado de

Peñil’s piece “Cell to Cell,” which won the People’s Choice award in the 2015 ASM Agar Art contest.
You’ve never seen bacteria quite like this before.
Mixed media artist Maria Peñil Cobo, who was born in Spain and currently resides in Massachusetts, told The Huffington Post on Thursday that she has often turned to nature as inspiration for her artwork. But instead of looking to vast oceans or forest landscapes, it’s the much smaller ecosystems that fascinate her the most.
Peñil has spent the past five years growing colorful bacteria, with help from microbiologist Dr. Mehmet Berkmen, and then “painting” the microbes into stunning masterpieces.

Peñil’s “Neurons,” which won first place in the 2015 ASM Agar Art contest.

“It is very technically difficult,” Berkmen, a staff scientist at the Ipswich, Massachusetts-based company New England Biolabs, told HuffPost. “You have to imagine that these bacteria we’re using are all different species. ... Each one grows differently and eats differently. Some don’t become colorful immediately, while others become old and then get their color.”
Berkmen taught Peñil how to “paint” with bacteria on agar, a gelatinous substance in which jungles of bacteria can grow. The artist uses a petri dish as her canvas.
Now, watch as the bacteria grow below.
So far, Peñil has attempted to “paint” with bacteria found on her own lips — which she collected after kissing a petri dish — as well as the germs that grew when she put her own house key on the dish.
Peñil, who will be giving a TED Talk about her work in Chicago on April 9, said that she hopes her artwork will shift the public dialogue around bacteria from one of fear and disgust to one of appreciation and curiosity.
After all, bacteria are a normal part of human life — they live all around us and even inside of our own bodies.
“I’m a scientist, and I appreciate this project a lot,” Berkmen said. “When we do science, there is always an element of art, and while Maria is doing pure art, there is an element of science in what we are observing. We are observing scientific phenomena.”
Scroll down to see more of Peñil’s bacteria artwork below.

  • Maria Peñil Cobo/Mehmet Berkmen

  • Maria Peñil Cobo/Mehmet Berkmen

  • Maria Peñil Cobo/Mehmet Berkmen

  • Maria Peñil Cobo/Mehmet Berkmen

  • Maria Peñil Cobo/Mehmet Berkmen

  • Maria Peñil Cobo/Mehmet Berkmen

  • Maria Peñil Cobo/Mehmet Berkmen

  • Maria Peñil Cobo/Mehmet Berkmen

  • Maria Peñil Cobo/Mehmet Berkmen

  • Maria Peñil Cobo/Mehmet Berkmen

  • Maria Peñil Cobo/Mehmet Berkmen

  • Maria Peñil Cobo/Mehmet Berkmen

  • Maria Peñil Cobo/Mehmet Berkmen

  • Maria Peñil Cobo/Mehmet Berkmen

jueves, 7 de abril de 2016

Candidalysin is a fungal peptide toxin critical for mucosal infection

A protein fragment released by filaments of the fungus Candida albicans destroys host cells. This is the first demonstration that human fungal pathogens other than moulds can release toxic peptides. 

Interactions between C. albicans and epithelial cells, which line the body's cavities, occur during the early stages of mucosal infections such as thrush and vaginitis. Hyphae elicit several epithelial-cell responses, including the production of signalling molecules called cytokines that recruit cells of the immune system to defend tissues, and loss of cell integrity through cell-membrane deterioration. Moyes et al. discovered that a strain of C. albicans in which the geneECE1 was mutated could not elicit epithelial-cell responses, despite growing apparently normal hyphae. Moreover, the authors validated these tissue-culture observations in vivo — the ECE1mutant was unable to reliably infect mucosa in a zebrafish swimbladder model and a mouse model of thrush.
ECE1 was one of the first genes to be identified in hyphal-specific expression screens more than 20 years ago, yet until now it has been one of the most poorly understood genes in C. albicans. In fact, ECE1 is among the most highly expressed genes in hyphae, but its role has not previously been investigated thoroughly because mutants show no defects in hyphal morphology or cell proliferation. Thus, the function of the Ece1 protein has remained a puzzle.
How does Ece1 promote epithelial-cell responses? The protein's amino-acid sequence suggests that it is secreted from hyphae as a group of eight short protein fragments, or peptides, and so would be well positioned to interact with host cells. Moyes and colleagues confirmed that all eight Ece1 peptides are secreted from hyphae. Analysis of synthetic versions of each peptide revealed that one, Ece1-III, elicits the same responses from epithelial cells as do hyphae. Moreover, precise deletion of the genetic region that codes for only Ece1-III created a mutant C. albicansthat secreted the remaining seven peptides, but did not elicit epithelial-cell responses or cause mucosal disease in animal models. These results clearly demonstrate that Ece1-III mediates the pathogenic activity associated with ECE1.
By what mechanism does Ece1-III exert this activity? Certain chemical and structural features indicate that Ece1-III might function like peptide toxins, such as the bee-venom toxin melittin. Indeed, the authors show that the peptide causes rapid and transient permeabilization of artificial cell membranes in vitro. These activities are enhanced in the presence of cholesterol, a component of animal — but not fungal — membranes. The researchers conclude that Ece1-III acts as a peptide toxin, which they name Candidalysin.
Moyes and colleagues' study establishes that C. albicans hyphae evolved to damage host cells. When combined with our knowledge of hyphal adhesin proteins and enzymes, a simple program of tissue destruction emerges (Fig. 1). First, the hyphal-specific adhesin Hwp1 attaches to mucosal surfaces. Second, the hyphal-specific invasion protein Als3, acting with the protein Ssa1, binds to receptors on the surface of the host cell, promoting engulfment of the hypha by the host cell8. Finally, Candidalysin accumulates in the invasion pocket around the hypha, attacking the host's cholesterol-containing membrane.
Figure 1: A toxic relationship.
A toxic relationship.
The pathogenic fungus Candida albicans infects its host by forming filamentous structures called hyphae. Proteins on the hyphal surface — the adhesin Hwp1, the invasin Als3 and its partner Ssa1 — make contact with the host cell directly or through receptor proteins to promote adhesion and engulfment of the hypha by the host. Moyes et al.2 report that the protein Ece1 is secreted from the hypha as eight short peptides. One of these, Candidalysin, acts as a toxin that accumulates in the invasion pocket and attacks the host-cell membrane, leading to membrane permeabilization and the induction of host defences.
This attack leads to membrane permeabilization, leakage of cell contents and a defensive cytokine response, which serves to limit the size of the C. albicans population in healthy individuals. However, impaired defences in people with conditions such as AIDS, diabetes and some cancers permit C. albicans growth and consequent disease.
 Enlace a los  trabajos originales:

lunes, 4 de abril de 2016

Design and synthesis of a minimal bacterial genome

We used whole-genome design and complete chemical synthesis to minimize the 1079kilobase pair synthetic genome of Mycoplasma mycoides JCVI-syn1.0. An initial design, based on collective knowledge of molecular biology combined with limited transposon mutagenesis data, failed to produce a viable cell. Improved transposon mutagenesis methods revealed a class of quasi-essential genes that are needed for robust growth, explaining the failure of our initial design. Three cycles of design, synthesis, and testing, with retention of quasi-essential genes, produced JCVI-syn3.0 (531 kilobase pairs, 473 genes), which has a genome smaller than that of any autonomously replicating cell found in nature. JCVI-syn3.0 retains almost all genes involved in the synthesis and processing of macromolecules. Unexpectedly, it also contains 149 genes with unknown biological functions. JCVI-syn3.0 is a versatile platform for investigating the core functions of life and for exploring whole-genome design.

Cite this article as C. A. Hutchison III et al., Science 351, aad6253 (2016). DOI: 10.1126/science.aad6253

jueves, 3 de diciembre de 2015

Complete nitrification by a single microorganism (Comammox)

Nitrification is a two-step process where ammonia is first oxidized to nitrite by ammonia-oxidizing bacteria and/or archaea, and subsequently to nitrate by nitrite-oxidizing bacteria. Already described by Winogradsky in 1890, this division of labour between the two functional groups is a generally accepted characteristic of the biogeochemical nitrogen cycle. Complete oxidation of ammonia to nitrate in one organism (complete ammonia oxidation; comammox) is energetically feasible, and it was postulated that this process could occur under conditions selecting for species with lower growth rates but higher growth yields than canonical ammonia-oxidizing microorganisms. Still, organisms catalysing this process have not yet been discovered. Here we report the enrichment and initial characterization of two Nitrospira species that encode all the enzymes necessary for ammonia oxidation via nitrite to nitrate in their genomes, and indeed completely oxidize ammonium to nitrate to conserve energy. Their ammonia monooxygenase (AMO) enzymes are phylogenetically distinct from currently identified AMOs, rendering recent acquisition by horizontal gene transfer from known ammonia-oxidizing microorganisms unlikely. We also found highly similar amoA sequences (encoding the AMO subunit A) in public sequence databases, which were apparently misclassified as methane monooxygenases. This recognition of a novel amoA sequence group will lead to an improved understanding of the environmental abundance and distribution of ammonia-oxidizing microorganisms. Furthermore, the discovery of the long-sought-after comammox process will change our perception of the nitrogen cycle.

In situ detection of Nitrospira and their ammonia-oxidizing capacity.

a, Co-aggregation of Nitrospira and Brocadia in the enrichment. Cells are stained by FISH with probes for all bacteria (EUB338mix, blue), and specific for Nitrospira (Ntspa712, green, resulting in cyan) and anammox bacteria (Amx820, red, resulting in magenta). b, AMO labelling by FTCP (green). Nitrospira was counterstained by FISH (probes Ntspa662 (blue) and Ntspa476 (red), resulting in white). c, Ammonium-dependent CO2 fixation by Nitrospira shown by FISH-MAR. Silver grain deposition (black) above cell clusters indicates 14CO2 incorporation. Nitrospira was stained by FISH (probes Ntspa476 (red) and Ntspa662 (blue), resulting in magenta). Images in b and c are representative of two individual experiments, with three (b) or two (c) technical replicates each. Scale bars in all panels represent 10 μm.

Enlace al trabajo original