Table_2_Antimicrobial Susceptibility of Environmental Non-O1/Non-O139 Vibrio cholerae Isolates.pdf

<p>Vibrio cholerae serogroups O1 and O139 are the causative agents of cholera disease. There are more than 200 serogroups in this species that are termed V. cholerae non-O1/non-O139. Non-O1/non-O139 strains can cause gastroenteritis and cholera like diarrhea, wound infections, external otitis, and bacteraemia that may lead to mortality. Previous antimicrobial susceptibility studies were conducted mainly on O1/O139 serogroups and on clinical isolates. Our aim was to study and compare the antimicrobial susceptibilities of non-O1/non-O139 environmental strains isolated from chironomids, fish, and waterfowl. Significant differences were found in the antimicrobial susceptibilities between the environmental strains that were isolated from three different reservoir habitats. Significant increase in minimum inhibitory concentrations (MICs) of ampicillin and chloramphenicol was found in chironomid isolates from 2009 compared to those from 2005. V. cholerae isolates from different waterfowl species displayed the highest MIC values to chloramphenicol and trimethoprim-sulfamethoxazole (SXT), while chironomid isolates demonstrated the highest MIC values toward ampicillin. Isolates from fish and waterfowl showed high MIC values toward doxycycline. No significant differences were found between the MICs of isolates from the different waterfowl species. The percentage of antimicrobial resistance among V. cholerae isolates from waterfowl was the highest compared to the abundance of antimicrobial resistant isolates from chironomids or fish. The antimicrobial resistance genes can be carried on mobile genetic elements, thus, waterfowl may act as reservoirs for these elements and may spread them all over the globe. Data regarding treatment with antimicrobial agents toward V. cholerae non-O1/non-O139 serogroups is lacking and therefore further studies are needed.</p

Table_1_Antimicrobial Susceptibility of Environmental Non-O1/Non-O139 Vibrio cholerae Isolates.pdf

<p>Vibrio cholerae serogroups O1 and O139 are the causative agents of cholera disease. There are more than 200 serogroups in this species that are termed V. cholerae non-O1/non-O139. Non-O1/non-O139 strains can cause gastroenteritis and cholera like diarrhea, wound infections, external otitis, and bacteraemia that may lead to mortality. Previous antimicrobial susceptibility studies were conducted mainly on O1/O139 serogroups and on clinical isolates. Our aim was to study and compare the antimicrobial susceptibilities of non-O1/non-O139 environmental strains isolated from chironomids, fish, and waterfowl. Significant differences were found in the antimicrobial susceptibilities between the environmental strains that were isolated from three different reservoir habitats. Significant increase in minimum inhibitory concentrations (MICs) of ampicillin and chloramphenicol was found in chironomid isolates from 2009 compared to those from 2005. V. cholerae isolates from different waterfowl species displayed the highest MIC values to chloramphenicol and trimethoprim-sulfamethoxazole (SXT), while chironomid isolates demonstrated the highest MIC values toward ampicillin. Isolates from fish and waterfowl showed high MIC values toward doxycycline. No significant differences were found between the MICs of isolates from the different waterfowl species. The percentage of antimicrobial resistance among V. cholerae isolates from waterfowl was the highest compared to the abundance of antimicrobial resistant isolates from chironomids or fish. The antimicrobial resistance genes can be carried on mobile genetic elements, thus, waterfowl may act as reservoirs for these elements and may spread them all over the globe. Data regarding treatment with antimicrobial agents toward V. cholerae non-O1/non-O139 serogroups is lacking and therefore further studies are needed.</p

Table_3_Antimicrobial Susceptibility of Environmental Non-O1/Non-O139 Vibrio cholerae Isolates.pdf

<p>Vibrio cholerae serogroups O1 and O139 are the causative agents of cholera disease. There are more than 200 serogroups in this species that are termed V. cholerae non-O1/non-O139. Non-O1/non-O139 strains can cause gastroenteritis and cholera like diarrhea, wound infections, external otitis, and bacteraemia that may lead to mortality. Previous antimicrobial susceptibility studies were conducted mainly on O1/O139 serogroups and on clinical isolates. Our aim was to study and compare the antimicrobial susceptibilities of non-O1/non-O139 environmental strains isolated from chironomids, fish, and waterfowl. Significant differences were found in the antimicrobial susceptibilities between the environmental strains that were isolated from three different reservoir habitats. Significant increase in minimum inhibitory concentrations (MICs) of ampicillin and chloramphenicol was found in chironomid isolates from 2009 compared to those from 2005. V. cholerae isolates from different waterfowl species displayed the highest MIC values to chloramphenicol and trimethoprim-sulfamethoxazole (SXT), while chironomid isolates demonstrated the highest MIC values toward ampicillin. Isolates from fish and waterfowl showed high MIC values toward doxycycline. No significant differences were found between the MICs of isolates from the different waterfowl species. The percentage of antimicrobial resistance among V. cholerae isolates from waterfowl was the highest compared to the abundance of antimicrobial resistant isolates from chironomids or fish. The antimicrobial resistance genes can be carried on mobile genetic elements, thus, waterfowl may act as reservoirs for these elements and may spread them all over the globe. Data regarding treatment with antimicrobial agents toward V. cholerae non-O1/non-O139 serogroups is lacking and therefore further studies are needed.</p

Bioinformatic identification of two distinct domains within Csn6 (S6MPN and S6CD) in various fungal species.

<p>The Csn6-like proteins from fungal species were identified from multiple genome databases. Species were grouped according to their phyla, and their conserved (dark red/blue) or diverged (light red/blue) domains, MPN and S6CD. White boxes indicate the missing domains. The data is based on the “fungal genome program” of the “DOE Joint Genome Institute” (<a href="http://genome.jgi-psf.org/programs/fungi/index.jsf" target="_blank">http://genome.jgi-psf.org/programs/fungi/index.jsf</a>) and the “fungal genome initiative” of the Broad Institute (<a href="http://www.broadinstitute.org/scientific-community/science/projects/fungal-genome-initiative/fungal-genome-initiative" target="_blank">http://www.broadinstitute.org/scientific-community/science/projects/fungal-genome-initiative/fungal-genome-initiative</a>).</p

Only one copy of Csn6 is present in the CSN complex isolated via HA-S6FL or HA-S6CD.

<p>HA-tagged Csn6 constructs were co-expressed with a Flag-tagged full length Csn6, Flag-S6FL. Cell lysate (left 5 lanes) and HA (Bethyl S190-107) immunoprecipitated proteins were examined co-precipitation of Flag-S6FL or endogenous CSN subunits by immunoblotting using antibodies indicated on the right.</p

S6CD is necessary for the integrity of mammalian CSN complex.

<p><b>A:</b> Yeast-2-hybrid assay showing interaction of mouse Csn4 (EG-Csn4) with Csn6 (JG-S6-FL) and the Csn6 C-terminal region (JG-S6CD). (See supplementary <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0043980#pone-0043980-g002" target="_blank">figure 2</a> for further information). <b>B:</b> S6CD region is necessary for integration of the protein into the CSN complex. HA-tagged full length mouse Csn6 (HA-S6FL) or the truncation mutants (HA-S6MPN and HA-S6CD) were expressed in HeLa cells. Cell lysates (left 4 lanes) and the HA (Convance Inc.) immunoprecipitated proteins (right 4 lanes) were analyzed by immunoblotting using specified antibodies as indicated on the right. <b>C:</b> Flag-tagged two C-terminal fragments of mouse Csn6 (S6CD, S6CD2) as described in Fig. 2B, were expressed in HEK293 cells. Proteins co-precipitated with the Flag-tag were blotted using indicated antibodies. Asterisk indicates IgG background. (<b>B, C</b>).</p

Deneddylation by yeast and mammalian CSN complexes that lacked the MPN⁻ domain on yeast or mammalian cullin substrates.

<p>A: The rubylated Cdc53 in Δ<i>csn5</i> extract was used as a substrate to test the deneddylation activity of Flag-purified mammalian complexes. Flag-S6CD, Flag-S6MPN, or empty vector were expressed in HeLa cells, isolated via the Flag beads, and eluted with the Flag peptide. The proteins were immunoblotted with indicated antibodies. Yeast cell extracts of wild type, Δ<i>rub1</i> and Δ<i>csn5</i> strains served as size markers for Cdc53 rubylated and un-rubylated forms. Asterisk indicates IgG background. B: Purified CSN complexes from yeast (ScCSN) or mammalian cells (CSN^PS) were tested for the deneddylation activity on human Cul4a-Nedd8 conjugates in CSN-depleted HeLa cell extract. Δ<i>csn5</i> extract from yeast was used as a negative control. UT, untreated cell extract.</p

MPN⁻ domain of Csn6 is dispensable for CSN mediated cullin deneddylation.

<p>The deneddylation assays were performed using CSN-depleted cell extracts as a source of neddylated cullin substrates (lanes 2–7). The HA-purified complexes (S6FL, S6MPN, S6CD or mock) were tested for the deneddylation activity. Mock (Moc) was the HA purification from untransfected cells. Neddylation levels of Cul4a, Cul2, and Cul1 were examined by immunoblotting with respective antibodies. Normal extract (untreated) was taken before CSN-depletion. Biochemically purified porcine spleen CSN (CSN^PS) was used as a positive control. Relative intensities of neddylated and deneddylated cullin protein bands were quantified using densitometry and imaging software IMAGEJ, taking into consideration of the local background. The values were presented as percentages of deneddylated cullins (gray bar, Y axis) or neddylated cullins (black bar) of the total amount of cullin proteins. The bracket in the Cul1 blot indicates the multiple deneddylation products that were taken into account for the quantification.</p

Subunits comparison of related MPN-containing complexes in human and yeast.

<p>Human BRISC BRCA1-A, complexes are deubiquitinating enzymes. CSN is a deneddylase complex. The proteasome lid is a deubiquitinating enzyme when integrated into the proteasome. The eukaryotic translation initiation factor-3 (eIF3) complex has not been found to have a isopeptidase activity.</p

The Minimal Deneddylase Core of the COP9 Signalosome Excludes the Csn6 MPN⁻ Domain

<div><p>The COP9 signalosome (CSN) is a eukaryotic protein complex, which regulates a wide range of biological processes mainly through modulating the cullin ubiquitin E3 ligases in the ubiquitin-proteasome pathway. The CSN possesses a highly conserved deneddylase activity that centers at the JAMM motif of the Csn5 subunit but requires other subunits in a complex assembly. The classic CSN is composed of 8 subunits (Csn1–8), yet in several <em>Ascomycota</em>, the complex is smaller and lacks orthologs for a few CSN subunits, but nevertheless contains a conserved Csn5. This feature makes yeast a powerful model to determine the minimal assemblage required for deneddylation activity. Here we report, that Csi1, a diverged <em>S. cerevisiae</em> CSN subunit, displays significant homology with the carboxyl terminal domain of the canonical Csn6, but lacks the amino terminal MPN^- domain. Through the comparative and experimental analyses of the budding yeast and the mammalian CSNs, we demonstrate that the MPN⁻ domain of the canonical mouse Csn6 is not part of the CSN deneddylase core. We also show that the carboxyl domain of Csn6 has an indispensable role in maintaining the integrity of the CSN complex. The CSN complex assembled with the carboxyl fragment of Csn6, despite its lack of an MPN⁻ domain, is fully active in deneddylation of cullins. We propose that the budding yeast Csi1 is a functional equivalent of the canonical Csn6, and thus the composition of the CSN across phyla is more conserved than hitherto appreciated.</p> </div