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1.  Novel Feature of Mycobacterium avium subsp. paratuberculosis, Highlighted by Characterization of the Heparin-Binding Hemagglutinin Adhesin 
Journal of Bacteriology  2013;195(21):4844-4853.
Mycobacterium avium subsp. paratuberculosis comprises two genotypically defined groups, known as the cattle (C) and sheep (S) groups. Recent studies have reported phenotypic differences between M. avium subsp. paratuberculosis groups C and S, including growth rates, infectivity for macrophages, and iron metabolism. In this study, we investigated the genotypes and biological properties of the virulence factor heparin-binding hemagglutinin adhesin (HBHA) for both groups. In Mycobacterium tuberculosis, HBHA is a major adhesin involved in mycobacterium-host interactions and extrapulmonary dissemination of infection. To investigate HBHA in M. avium subsp. paratuberculosis, we studied hbhA polymorphisms by fragment analysis using the GeneMapper technology across a large collection of isolates genotyped by mycobacterial interspersed repetitive-unit–variable-number tandem-repeat (MIRU-VNTR) and IS900 restriction fragment length polymorphism (RFLP-IS900) analyses. Furthermore, we analyzed the structure-function relationships of recombinant HBHA proteins of types C and S by heparin-Sepharose chromatography and surface plasmon resonance (SPR) analyses. In silico analysis revealed two forms of HBHA, corresponding to the prototype genomes for the C and S types of M. avium subsp. paratuberculosis. This observation was confirmed using GeneMapper on 85 M. avium subsp. paratuberculosis strains, including 67 strains of type C and 18 strains of type S. We found that HBHAs from all type C strains contain a short C-terminal domain, while those of type S present a long C-terminal domain, similar to that produced by Mycobacterium avium subsp. avium. The purification of recombinant HBHA from M. avium subsp. paratuberculosis of both types by heparin-Sepharose chromatography highlighted a correlation between their affinities for heparin and the lengths of their C-terminal domains, which was confirmed by SPR analysis. Thus, types C and S of M. avium subsp. paratuberculosis may be distinguished by the types of HBHA they produce, which differ in size and adherence properties, thereby providing new evidence that strengthens the genotypic differences between the C and S types of M. avium subsp. paratuberculosis.
PMCID: PMC3807500  PMID: 23974028
2.  A global benchmark study using affinity-based biosensors 
Rich, Rebecca L. | Papalia, Giuseppe A. | Flynn, Peter J. | Furneisen, Jamie | Quinn, John | Klein, Joshua S. | Katsamba, Phini S. | Waddell, M. Brent | Scott, Michael | Thompson, Joshua | Berlier, Judie | Corry, Schuyler | Baltzinger, Mireille | Zeder-Lutz, Gabrielle | Schoenemann, Andreas | Clabbers, Anca | Wieckowski, Sebastien | Murphy, Mary M. | Page, Phillip | Ryan, Thomas E. | Duffner, Jay | Ganguly, Tanmoy | Corbin, John | Gautam, Satyen | Anderluh, Gregor | Bavdek, Andrej | Reichmann, Dana | Yadav, Satya P. | Hommema, Eric | Pol, Ewa | Drake, Andrew | Klakamp, Scott | Chapman, Trevor | Kernaghan, Dawn | Miller, Ken | Schuman, Jason | Lindquist, Kevin | Herlihy, Kara | Murphy, Michael B. | Bohnsack, Richard | Andrien, Bruce | Brandani, Pietro | Terwey, Danny | Millican, Rohn | Darling, Ryan J. | Wang, Liann | Carter, Quincy | Dotzlaf, Joe | Lopez-Sagaseta, Jacinto | Campbell, Islay | Torreri, Paola | Hoos, Sylviane | England, Patrick | Liu, Yang | Abdiche, Yasmina | Malashock, Daniel | Pinkerton, Alanna | Wong, Melanie | Lafer, Eileen | Hinck, Cynthia | Thompson, Kevin | Primo, Carmelo Di | Joyce, Alison | Brooks, Jonathan | Torta, Federico | Bagge Hagel, Anne Birgitte | Krarup, Janus | Pass, Jesper | Ferreira, Monica | Shikov, Sergei | Mikolajczyk, Malgorzata | Abe, Yuki | Barbato, Gaetano | Giannetti, Anthony M. | Krishnamoorthy, Ganeshram | Beusink, Bianca | Satpaev, Daulet | Tsang, Tiffany | Fang, Eric | Partridge, James | Brohawn, Stephen | Horn, James | Pritsch, Otto | Obal, Gonzalo | Nilapwar, Sanjay | Busby, Ben | Gutierrez-Sanchez, Gerardo | Gupta, Ruchira Das | Canepa, Sylvie | Witte, Krista | Nikolovska-Coleska, Zaneta | Cho, Yun Hee | D’Agata, Roberta | Schlick, Kristian | Calvert, Rosy | Munoz, Eva M. | Hernaiz, Maria Jose | Bravman, Tsafir | Dines, Monica | Yang, Min-Hsiang | Puskas, Agnes | Boni, Erica | Li, Jiejin | Wear, Martin | Grinberg, Asya | Baardsnes, Jason | Dolezal, Olan | Gainey, Melicia | Anderson, Henrik | Peng, Jinlin | Lewis, Mark | Spies, Peter | Trinh, Quyhn | Bibikov, Sergei | Raymond, Jill | Yousef, Mohammed | Chandrasekaran, Vidya | Feng, Yuguo | Emerick, Anne | Mundodo, Suparna | Guimaraes, Rejane | McGirr, Katy | Li, Yue-Ji | Hughes, Heather | Mantz, Hubert | Skrabana, Rostislav | Witmer, Mark | Ballard, Joshua | Martin, Loic | Skladal, Petr | Korza, George | Laird-Offringa, Ite | Lee, Charlene S. | Khadir, Abdelkrim | Podlaski, Frank | Neuner, Phillippe | Rothacker, Julie | Rafique, Ashique | Dankbar, Nico | Kainz, Peter | Gedig, Erk | Vuyisich, Momchilo | Boozer, Christina | Ly, Nguyen | Toews, Mark | Uren, Aykut | Kalyuzhniy, Oleksandr | Lewis, Kenneth | Chomey, Eugene | Pak, Brian J. | Myszka, David G.
Analytical biochemistry  2008;386(2):194-216.
To explore the variability in biosensor studies, 150 participants from 20 countries were given the same protein samples and asked to determine kinetic rate constants for the interaction. We chose a protein system that was amenable to analysis using different biosensor platforms as well as by users of different expertise levels. The two proteins (a 50-kDa Fab and a 60-kDa glutathione S-transferase [GST] antigen) form a relatively high-affinity complex, so participants needed to optimize several experimental parameters, including ligand immobilization and regeneration conditions as well as analyte concentrations and injection/dissociation times. Although most participants collected binding responses that could be fit to yield kinetic parameters, the quality of a few data sets could have been improved by optimizing the assay design. Once these outliers were removed, the average reported affinity across the remaining panel of participants was 620 pM with a standard deviation of 980 pM. These results demonstrate that when this biosensor assay was designed and executed appropriately, the reported rate constants were consistent, and independent of which protein was immobilized and which biosensor was used.
PMCID: PMC3793259  PMID: 19133223
Biacore; Kinetics; Optical biosensor; Surface plasmon resonance
3.  Binding of the RamR Repressor to Wild-Type and Mutated Promoters of the ramA Gene Involved in Efflux-Mediated Multidrug Resistance in Salmonella enterica Serovar Typhimurium 
The transcriptional activator RamA is involved in multidrug resistance (MDR) by increasing expression of the AcrAB-TolC RND-type efflux system in several pathogenic Enterobacteriaceae. In Salmonella enterica serovar Typhimurium (S. Typhimurium), ramA expression is negatively regulated at the local level by RamR, a transcriptional repressor of the TetR family. We here studied the DNA-binding activity of the RamR repressor with the ramA promoter (PramA). As determined by high-resolution footprinting, the 28-bp-long RamR binding site covers essential features of PramA, including the −10 conserved region, the transcriptional start site of ramA, and two 7-bp inverted repeats. Based on the RamR footprint and on electrophoretic mobility shift assays (EMSAs), we propose that RamR interacts with PramA as a dimer of dimers, in a fashion that is structurally similar to the QacR-DNA binding model. Surface plasmon resonance (SPR) measurements indicated that RamR has a 3-fold-lower affinity (KD [equilibrium dissociation constant] = 191 nM) for the 2-bp-deleted PramA of an MDR S. Typhimurium clinical isolate than for the wild-type PramA (KD = 66 nM). These results confirm the direct regulatory role of RamR in the repression of ramA transcription and precisely define how an alteration of its binding site can give rise to an MDR phenotype.
PMCID: PMC3264254  PMID: 22123696
4.  The secretions of oviduct epithelial cells increase the equine in vitro fertilization rate: are osteopontin, atrial natriuretic peptide A and oviductin involved? 
Oviduct epithelial cells (OEC) co-culture promotes in vitro fertilization (IVF) in human, bovine and porcine species, but no data are available from equine species. Yet, despite numerous attempts, equine IVF rates remain low. Our first aim was to verify a beneficial effect of the OEC on equine IVF. In mammals, oviductal proteins have been shown to interact with gametes and play a role in fertilization. Thus, our second aim was to identify the proteins involved in fertilization in the horse.
Methods & results
In the first experiment, we co-incubated fresh equine spermatozoa treated with calcium ionophore and in vitro matured equine oocytes with or without porcine OEC. We showed that the presence of OEC increases the IVF rates. In the subsequent experiments, we co-incubated equine gametes with OEC and we showed that the IVF rates were not significantly different between 1) gametes co-incubated with equine vs porcine OEC, 2) intact cumulus-oocyte complexes vs denuded oocytes, 3) OEC previously stimulated with human Chorionic Gonadotropin, Luteinizing Hormone and/or oestradiol vs non stimulated OEC, 4) in vivo vs in vitro matured oocytes.
In order to identify the proteins responsible for the positive effect of OEC, we first searched for the presence of the genes encoding oviductin, osteopontin and atrial natriuretic peptide A (ANP A) in the equine genome. We showed that the genes coding for osteopontin and ANP A are present. But the one for oviductin either has become a pseudogene during evolution of horse genome or has been not well annotated in horse genome sequence. We then showed that osteopontin and ANP A proteins are present in the equine oviduct using a surface plasmon resonance biosensor, and we analyzed their expression during oestrus cycle by Western blot. Finally, we co-incubated equine gametes with or without purified osteopontin or synthesized ANP A. No significant effect of osteopontin or ANP A was observed, though osteopontin slightly increased the IVF rates.
Our study shows a beneficial effect of homologous and heterologous oviduct cells on equine IVF rates, though the rates remain low. Furthers studies are necessary to identify the proteins involved. We showed that the surface plasmon resonance technique is efficient and powerful to analyze molecular interactions during fertilization.
PMCID: PMC2785818  PMID: 19925651

Results 1-4 (4)