jueves, 22 de junio de 2017

Una cucharada de azucar puede acabar con las infecciones urigenitales

The mammalian intestine is a natural habitat for trillions of bacteria, including harmless species called commensals and potentially pathogenic species. However, long-term persistence is a challenge for these resident bacteria, which must resist expulsion by waves of peristalsis — the process that pushes the intestinal contents downstream. The ways in which pathogenic bacteria adhere to host cells at sites of infection have been extensively investigated, but little is known about how commensal bacteria cling to mucosal surfaces in the gut. Spaulding et al (2017) report that uropathogenic Escherichia coli (UPEC), a bacterium that is a commensal in the gut but pathogenic in the bladder, persists in the intestine thanks to filamentous protein complexes called pili that project from the bacterium and promote its adhesion to the gut wall. The authors also identify a sugar derivative that can combat pilus-mediated adhesion.

A small molecule clears pathogens from the gut. 
Fig. 1. Inhibición de la adherencia de  Escherichia coli (UPEC) en el intestino mediado por el manósido M4284.

Uropathogenic Escherichia coli (UPEC) strains colonize the gut and are shed in faeces. If, from faeces, they gain access to the area around the urethra, the bacteria can work their way up the urethra, causing urinary-tract infections (UTIs). Preventing the colonization of the gut by these bacteria could therefore be a way to stop UTIs (particularly recurrent infections) from arising. 

UPEC isolates encode up to 16 distinct chaperone-usher pathway pili, and each pilus type may enable colonization of a habitat in the host or environment. For example, the type 1 pilus adhesin FimH binds mannose on the bladder surface, and mediates colonization of the bladder. However, little is known about the mechanisms underlying UPEC persistence in the gut. 

Using a mouse model, authors show that F17-like and type 1 pili promote intestinal colonization and show distinct binding to epithelial cells distributed along colonic crypts. Phylogenomic and structural analyses reveal that F17-like pili are closely related to pilus types carried by intestinal pathogens, but are restricted to extra-intestinal pathogenic E. coli. Moreover, they show that targeting FimH with M4284, a high-affinity inhibitory mannoside, reduces intestinal colonization of genetically diverse UPEC isolates, while simultaneously treating UTI, without notably disrupting the structural configuration of the gut microbiota. By selectively depleting intestinal UPEC reservoirs, mannosides could markedly reduce the rate of UTIs and recurrent UTIs.


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Fig. 2. Estructura del manósido M4284

miércoles, 22 de marzo de 2017

Deep-sea vent phage DNA polymerase specifically initiates DNA synthesis in the absence of primers

Deep-sea vent phage DNA polymerase specifically initiates DNA synthesis in the absence of primers


Most DNA polymerases initiate DNA synthesis by extending a preexisting primer. Exceptions to this dogma are recently characterized bifunctional primase–polymerases (prim–pols) that resemble archaeal primases in their structure and initiate DNA synthesis de novo using only NTPs or dNTPs. We report here a DNA polymerase encoded by a phage NrS-1 from deep-sea vents. NrS-1 has a genome organization unlike any other known phage. Although this polymerase does not contain a zinc-binding motif typical for primases, it is nonetheless able to initiate DNA synthesis from a specific DNA sequence exclusively using dNTPs. Thus, it represents a unique de novo replicative DNA polymerase that possesses features found in DNA polymerases, primases, and RNA polymerases.


A DNA polymerase is encoded by the deep-sea vent phage NrS-1. NrS-1 has a unique genome organization containing genes that are predicted to encode a helicase and a single-stranded DNA (ssDNA)-binding protein. The gene for an unknown protein shares weak homology with the bifunctional primase–polymerases (prim–pols) from archaeal plasmids but is missing the zinc-binding domain typically found in primases. We show that this gene product has efficient DNA polymerase activity and is processive in DNA synthesis in the presence of the NrS-1 helicase and ssDNA-binding protein. Remarkably, this NrS-1 DNA polymerase initiates DNA synthesis from a specific template DNA sequence in the absence of any primer. The de novo DNA polymerase activity resides in the N-terminal domain of the protein, whereas the C-terminal domain enhances DNA binding.

jueves, 23 de junio de 2016

Rates and mechanisms of bacterial mutagenesis from maximum-depth sequencing

In 1943, Luria and Delbrück used a phage-resistance assay to establish spontaneous mutation as a driving force of microbial diversity1. Mutation rates are still studied using such assays, but these can only be used to examine the small minority of mutations conferring survival in a particular condition. Newer approaches, such as long-term evolution followed by whole-genome sequencing23, may be skewed by mutational ‘hot’ or ‘cold’ spots34. Both approaches are affected by numerous caveats567. Here we devise a method, maximum-depth sequencing (MDS), to detect extremely rare variants in a population of cells through error-corrected, high-throughput sequencing. We directly measure locus-specific mutation rates in Escherichia coli and show that they vary across the genome by at least an order of magnitude. Our data suggest that certain types of nucleotide misincorporation occur 104-fold more frequently than the basal rate of mutations, but are repaired in vivo. Our data also suggest specific mechanisms of antibiotic-induced mutagenesis, including downregulation of mismatch repair via oxidative stress, transcription–replication conflicts, and, in the case of fluoroquinolones, direct damage to DNA.

De novo mutations in bacteria remain a notoriously difficult target for high-throughput sequencing. Whereas E. coli mutate fewer than 1 in 109 bases per generation, high-fidelity polymerases used for library preparation polymerase chain reaction (PCR) cause errors in ~4 out of 106 bases8. Illumina machines misread ~1 in 103 bases9. Recent methods, such as barcoding of reads from the same original DNA molecule8, have lowered the error rate of sequencing. However, such methods can have low yields10 and do not address errors introduced by PCR. PCR errors can be overcome using duplex barcoding, which forms a consensus from both strands of a DNA template molecule11. However, even when a small region is targeted12, duplexing lowers yield even further. The mutational landscape of an RNA virus with mutation rate 104-fold greater than E. coli was recently mapped using ‘circle sequencing’. However, this technique is not designed for targeted coverage of a single locus, and its accuracy is limited by sequence read length1013.

jueves, 26 de mayo de 2016

Culturing of ‘unculturable’ human microbiota reveals novel taxa and extensive sporulation

Our intestinal microbiota harbours a diverse bacterial community required for our health, sustenance and wellbeing1, 2. Intestinal colonization begins at birth and climaxes with the acquisition of two dominant groups of strict anaerobic bacteria belonging to the Firmicutes and Bacteroidetes phyla2. Culture-independent, genomic approaches have transformed our understanding of the role of the human microbiome in health and many diseases1. However, owing to the prevailing perception that our indigenous bacteria are largely recalcitrant to culture, many of their functions and phenotypes remain unknown3

Here we describe a novel workflow based on targeted phenotypic culturing linked to large-scale whole-genome sequencing, phylogenetic analysis and computational modelling that demonstrates that a substantial proportion of the intestinal bacteria are culturable. 

Applying this approach to healthy individuals, we isolated 137 bacterial species from characterized and candidate novel families, genera and species that were archived as pure cultures. Whole-genome and metagenomic sequencing, combined with computational and phenotypic analysis, suggests that at least 50–60% of the bacterial genera from the intestinal microbiota of a healthy individual produce resilient spores, specialized for host-to-host transmission. Our approach unlocks the human intestinal microbiota for phenotypic analysis and reveals how a marked proportion of oxygen-sensitive intestinal bacteria can be transmitted between individuals, affecting microbiota heritability.

 a, Relative abundance of bacteria in faecal samples (x axis) compared with relative abundance of bacteria growing on YCFA agar plates (y axis) as determined by metagenomic sequencing. Bacteria grown on YCFA agar are representative of the complete faecal samples as indicated by Spearman ρ = 0.75 (n = 6). b, Principal component analysis plot of 16S rRNA gene sequences detected from six donor faecal samples (n = 6), representing bacteria in complete faecal samples (green), faecal bacterial colonies recovered from YCFA agar plates without ethanol pre-treatment (black) or with ethanol pre-treatment to select for ethanol-resistant spore-forming bacteria (red). Culturing without ethanol selection is representative of the complete faecal sample, ethanol treatment shifts the profile, enriching for ethanol-resistant spore-forming bacteria and allowing their subsequent isolation. c, Phylogenetic tree of bacteria cultured from the six donors constructed from full-length 16S rRNA gene sequences. Novel candidate species (red), genera (blue) and families (green) are shown by dot colours. Major phyla and family names are indicated. Proteobacteria were not cultured, but are included for context.

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miércoles, 25 de mayo de 2016

Making a decision: to stay or move

Glucose induces delocalization of a flagellar biosynthesis protein from the flagellated pole
Making a right decision to stay or move is critical for survival in a changing environment. Here we identify a novel mechanism for glucose-dependent on-off switching of flagellar synthesis in Vibrio vulnificus: When enzyme IIAGlc of the bacterial PEP:carbohydrate phosphotransferase system is dephosphorylated in the presence glucose, it delocalizes a protein required for flagellar biosynthesis from the flagellated pole. This leads to a loss of motility and enables bacteria to stay in a favorable habitat.

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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.