ISV RESEARCH HIGHLIGHT-16/06/2010
"Imaging the nanoscale organization of peptidoglycan in living Lactococcus lactis cells"
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The spatial organization of peptidoglycan, the major constituent of bacterial cell walls, is an important, yet still unsolved issue in microbiology. This is largely due to the lack of in vivo, high resolution imaging techniques. Unlike traditional methods which analyze peptidoglycan after isolation and purification, atomic force microscopy (AFM) is a unique technique for analysing the nanoscale organization of peptidoglycan directly in living cells, without using any fixation or staining procedures.
Two ISV teams headed by Pascal Hols and Yves Dufrêne in collaboration with INRA showed that the combined use of atomic force microscopy and cell wall mutants is a powerful approach for probing the nanoscale architecture of cell wall peptidoglycan in living Gram-positive bacteria. Using topographic imaging, they found that Lactococcus lactis wild-type cells display a smooth, featureless surface morphology, whereas mutant strains lacking cell wall exopolysaccharides feature 25-nm-wide periodic bands running parallel to the short axis of the cell. In addition, they used single-molecule recognition imaging to show that parallel bands are made of peptidoglycan. These data, obtained for the first time on living ovococci, argue for an architectural feature of the cell wall in the plane perpendicular to the long axis of the cell.
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Applicable to other microbes, the method presented here offers exciting possibilities for elucidating the nanoscale architecture and assembly of bacterial cell walls. Also, since peptidoglycan is targeted by many key antibiotics, this study opens up new avenues for understanding the molecular bases of peptidoglycan-drug interactions.
G. Andre, S. Kulakauskas, M.-P. Chapot-Chartier, B. Navet, M. Deghorain, E. Bernard, Pascal Hols, Y. F. Dufrêne
Nature Communications, 2010, 15th June
ISV RESEARCH HIGHLIGHT-23/04/10
“Efficient ER exit and vacuole targeting of yeast Sna2p require two tyrosine-based sorting motifs”
Secretory proteins posses specific targeting sequences facilitating their correct localization within the cell. Specific motifs are known for transport from endoplasmic reticulum (ER) to Golgi apparatus or for transport from Golgi apparatus to vacuole, endosomes or plasma membrane.
A research performed by Henri-François Renard and colleagues in the ISV team headed by Pierre Morsomme showed that the yeast vacuolar protein Sna2 has two functional tyrosine-based motifs (YXXØ) that are responsible of the targeting of the protein to the vacuole via two distinct Golgi to vacuole pathways. Moreover, these motifs also play a crucial role in the exit of Sna2p from the ER. Directed mutagenesis of these tyrosines led to a partial redirection of Sna2p to lipid bodies, probably due to a decrease in ER exit efficiency. Sna2p is the first yeast protein in which two YXXØ motifs have been identified and both were demonstrated to be functional at two different steps of the secretory pathway, ER exit and Golgi-to-vacuole transport.
Henri-François Renard, Didier Demaegd, Bérengère Guerriat and Pierre Morsomme, Traffic, Accepted Article Online: Apr 6 2010, DOI: 10.1111/j.1600-0854.2010.01070.x - article in pdf (web release) 
Co-localization of Sna2p with defective tyrosine motifs (in green) and lipid bodies (in red)
ISV RESEARCH HIGHLIGHT-23/09/08
"Control of acute, chronic, and constitutive hyperammonemia by wild-type and genetically engineered Lactobacillus plantarum in rodents"
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Hyperammonemia is a well-known complication of acute and chronic liver diseases and plays a central role in the pathogenesis of hepatic encephalopathy. This neurological dysfunction results, at least in part, from an increase in plasma ammonia level. Ammonia is produced by many tissues but its major external source results from deaminase and urease activities of the gut flora.
The ISV team headed by Pascal Hols in collaboration with Hospital Erasme (ULB, Prof. J. Devière) showed that hyperammonemia can be modulated in three rodent models by administration of a wild-type Lactobacillus plantarum, a genetically engineered ammonia hyper-consuming strain and a strain deficient for the ammonia transporter.
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Hyperammonemia occurring constitutively or induced by acute or chronic liver failure can be efficiently decreased by the administration of L. plantarum with a significant effect on survival. The absence of an ammonium transporter abolishes these effects while a genetically-modified ammonia hyper-consuming strain discloses similar effects which are maintained at a lower dose of administration. The mechanism involved direct ammonia consumption in the gut. This work opens the door to new opportunities in the treatment of hepatic encephalopathy.
Nicaise C, Prozzi D, Viaene E, Moreno C, Gustot T, Quertinmont E, Demetter P, Suain V, Goffin P, Devière J, Hols P.
Hepatology. 2008 Jun 9. [Epub ahead of print] Article PDF File (Web release)
ISV RESEARCH HIGHLIGHT-19/07/07
"FRET imaging in living maize cells reveals that plasma membrane aquaporins interact to regulate their subcellular localization"
The water permeability of cellular membranes is dependent on the presence of water channels or aquaporins. These proteins form tetramers in the membrane, each monomer being an independent water channel. Plant aquaporins constitute a large family of proteins that are highly regulated. The ISV team headed by F. Chaumont showed that the trafficking to the plasma membrane of several maize aquaporins is dependent on the formation of hetero-oligomers. This is a new mechanism of aquaporin regulation affecting the amount and activity of the proteins in the plasma membrane.
Enric Zelazny, Jan Willem Borst, Mélanie Muylaert, Henri Batoko, Marcus A. Hemminga, and François Chaumont
Proc. Natl. Acad. Sci. USA, 104, 12359-12364. Article PDF File (Web release)
Co-localization of ECFP-ZmPIP2;1 and EYFP-ZmPIP1;2 in the plasma membrane
ISV RESEARCH HIGHLIGHT-23/02/07
"New opportunities for understanding the binding forces of antibiotics at the single molecule level and for exploring the architecture and assembly of bacterial cell walls"
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Two ISV teams have succeeded in attaching antibiotics on AFM (Atomic Force Microscopy) tips for detecting their ligands on living bacteria.
The result of this work is now in press in Nano Letters and is entitled "Single-molecule force spectroscopy and imaging of the vancomycin/D-Ala-D-Ala interaction".
For the first time, vancomycin-modified tips were used to map individual vancomycin binding sites on living bacteria with unprecedented lateral resolution. This work opens the door to new opportunities for understanding the binding forces of antibiotics at the single molecule level and for exploring the architecture and assembly of bacterial cell walls.
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Yann Gilbert, Marie Deghorain, Ling Wang, Bing Xu, Philipp D. Pollheimer, Hermann J. Gruber, Jeff Errington, Bernard Hallet, Xavier Haulot, Claire Verbelen, Pascal Hols, and Yves F. Dufrêne*
Nano Lett.; 2007; (ASAP Web Release Date: 23-Feb-2007; (Letter) DOI: 10.1021/nl0700853)
