Data_Sheet_1_Genetic Determinants Associated With in Vivo Survival of Burkholderia cenocepacia in the Caenorhabditis elegans Model.XLSX

<p>A Burkholderia cenocepacia infection usually leads to reduced survival and fatal cepacia syndrome in cystic fibrosis patients. The identification of B. cenocepacia essential genes for in vivo survival is key to designing new anti-infectives therapies. We used the Transposon-Directed Insertion Sequencing (TraDIS) approach to identify genes required for B. cenocepacia survival in the model infection host, Caenorhabditis elegans. A B. cenocepacia J2315 transposon pool of ∼500,000 mutants was used to infect C. elegans. We identified 178 genes as crucial for B. cenocepacia survival in the infected nematode. The majority of these genes code for proteins of unknown function, many of which are encoded by the genomic island BcenGI13, while other gene products are involved in nutrient acquisition, general stress responses and LPS O-antigen biosynthesis. Deletion of the glycosyltransferase gene wbxB and a histone-like nucleoid structuring (H-NS) protein-encoding gene (BCAL0154) reduced bacterial accumulation and attenuated virulence in C. elegans. Further analysis using quantitative RT-PCR indicated that BCAL0154 modulates B. cenocepacia pathogenesis via transcriptional regulation of motility-associated genes including fliC, fliG, flhD, and cheB1. This screen has successfully identified genes required for B. cenocepacia survival within the host-associated environment, many of which are potential targets for developing new antimicrobials.</p

Data_Sheet_2_Genetic Determinants Associated With in Vivo Survival of Burkholderia cenocepacia in the Caenorhabditis elegans Model.XLSX

<p>A Burkholderia cenocepacia infection usually leads to reduced survival and fatal cepacia syndrome in cystic fibrosis patients. The identification of B. cenocepacia essential genes for in vivo survival is key to designing new anti-infectives therapies. We used the Transposon-Directed Insertion Sequencing (TraDIS) approach to identify genes required for B. cenocepacia survival in the model infection host, Caenorhabditis elegans. A B. cenocepacia J2315 transposon pool of ∼500,000 mutants was used to infect C. elegans. We identified 178 genes as crucial for B. cenocepacia survival in the infected nematode. The majority of these genes code for proteins of unknown function, many of which are encoded by the genomic island BcenGI13, while other gene products are involved in nutrient acquisition, general stress responses and LPS O-antigen biosynthesis. Deletion of the glycosyltransferase gene wbxB and a histone-like nucleoid structuring (H-NS) protein-encoding gene (BCAL0154) reduced bacterial accumulation and attenuated virulence in C. elegans. Further analysis using quantitative RT-PCR indicated that BCAL0154 modulates B. cenocepacia pathogenesis via transcriptional regulation of motility-associated genes including fliC, fliG, flhD, and cheB1. This screen has successfully identified genes required for B. cenocepacia survival within the host-associated environment, many of which are potential targets for developing new antimicrobials.</p

Data_Sheet_3_Genetic Determinants Associated With in Vivo Survival of Burkholderia cenocepacia in the Caenorhabditis elegans Model.pdf

<p>A Burkholderia cenocepacia infection usually leads to reduced survival and fatal cepacia syndrome in cystic fibrosis patients. The identification of B. cenocepacia essential genes for in vivo survival is key to designing new anti-infectives therapies. We used the Transposon-Directed Insertion Sequencing (TraDIS) approach to identify genes required for B. cenocepacia survival in the model infection host, Caenorhabditis elegans. A B. cenocepacia J2315 transposon pool of ∼500,000 mutants was used to infect C. elegans. We identified 178 genes as crucial for B. cenocepacia survival in the infected nematode. The majority of these genes code for proteins of unknown function, many of which are encoded by the genomic island BcenGI13, while other gene products are involved in nutrient acquisition, general stress responses and LPS O-antigen biosynthesis. Deletion of the glycosyltransferase gene wbxB and a histone-like nucleoid structuring (H-NS) protein-encoding gene (BCAL0154) reduced bacterial accumulation and attenuated virulence in C. elegans. Further analysis using quantitative RT-PCR indicated that BCAL0154 modulates B. cenocepacia pathogenesis via transcriptional regulation of motility-associated genes including fliC, fliG, flhD, and cheB1. This screen has successfully identified genes required for B. cenocepacia survival within the host-associated environment, many of which are potential targets for developing new antimicrobials.</p

Characterization of <i>V. cholerae</i> isolates from Chandigarh.

<p>A: resistance to Streptomycin, Sulfisoxazole/Sulfamethoxazole and Trimethoprim and susceptible to Tetracycline, Chloramphenicol, and Ciprofloxacin. B: Susceptible to all antibiotics used in the study except Trimethoprim. NK = not known, ND = not done.</p

A SNP based maximum likelihood phylogeny of the Chandigarh <i>V. cholera</i>.

<p>The <i>V. cholerae</i> El Tor reference N16961 was used as the root of the tree. The colour key corresponds to location, clade, MLST type, VSP-II variants, <i>ctxB</i> alleles, SXT variants and the MLVA type. The scale of the tree is given as substitutions per variable site.</p

A SNP based maximum likelihood phylogeny of the seventh pandemic <i>V. cholerae</i> lineage.

<p>The phylogenetic tree shows the relation between the 2009 isolates from Chandigarh and the global collection of <i>V. cholera</i> genomes published by Mutreja et al <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002981#pntd.0002981-Mutreja1" target="_blank">[1]</a>. The positions of the isolates from Chandigarh are indicated with colour bars on the right representing the district they were isolated from (see <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002981#pntd-0002981-g001" target="_blank">Figure 1</a>). Other sub-clades that map close by on the tree to the Chandigarh isolates have been marked to highlight their global phylo-geographical context. The pre-seventh pandemic isolate M66 (2) was used as an out-group to root the tree. The scale given represents substitutions per variable site.</p

Structural variations in SXT among <i>V. cholerae</i> isolates.

<p>The structures of the SXT of <i>V. cholerae</i> isolates from Chandigarh were compared to published isolates from Nepal and Haiti. Reads were mapped to ICE<i>Vch</i>Hai1 (accession number JN648379). Plot showing presence (blue) or absence (white) of the genes of ICEVchHai1. The antibiotic resistance genes within the SXT cluster are highlighted in red.</p

A Map of the Chandigarh region of India.

<p>The locations of the clinics where the <i>V. cholerae</i> isolates were obtained are illustrated. Each district has been assigned a different colour (Chandigarh; orange, Yamuna Nagar; violet, Ambala; red, Patiala; blue and Morinda; green). Source of the map is ESRI ArcGIS geomapping software.</p

Essentiality of <i>eccC</i>_<i>5</i> and analysis of functional domains.

<p>* The <i>eccC</i>_<i>5</i> NBD mutants appear to have a dominant negative effect on the functioning of endogenous EccC₅.</p><p>^$ Colonies showed a strong growth defect, i.e. colonies were visible only after 17 days, compared to 10 days for the wild-type strain.</p><p>Replacement of pMV-<i>eccBC</i>_<i>5</i> by the input DNA was scored. Input DNA consisted of the pMV-361-<i>hyg</i> plasmid containing the indicated constructs. “+” indicates that more than 100 colonies were detected after electroporation with the indicated vector. “–” indicates between 0–20 colonies were found after electroporation. These latter colonies were shown by PCR to still contain the original vector, indicating illegitimate recombination or spontaneous antibiotic resistance. Results are representative data of three independent experiments.</p><p>Essentiality of <i>eccC</i>_<i>5</i> and analysis of functional domains.</p

Expression of MycP5 is essential for growth of <i>M</i>. <i>bovis</i> BCG.

<p>A, B) The BCG-Pasteur c-<i>mycP5</i>-tet-on (A) and c-<i>mycP5</i>-tet-off (B) mutants were grown for 21 days on Middlebrook 7H10 agar plates containing the indicated ATc concentrations. Full growth of c-<i>mycP5</i>-tet-on was only observed at 10 μg/ml ATc, whereas this concentration of ATc did not completely abolish colony growth of c-<i>mycP5</i>-tet-off. C, D) Resazurin reduction is dependent on ATc-induced expression/repression of <i>mycP5</i>. Cells of the BCG-Pasteur c-<i>mycP5</i>-tet-on (C), or c-<i>mycP5-</i>tet-off (D) mutants were grown as liquid cultures in 96-well microtiter plates for 6 days at 37°C at the indicated ATc concentrations, after which 10% Alamar Blue was added and fluorescence (585 nm) was measured after 16 h incubation to determine metabolic activity as a correlate of growth. Values are means of triplicates; error bars represent the standard deviation.</p