Novel Two-Step Hierarchical Screening of Mutant Pools Reveals Mutants under Selection in Chicks

Contaminated chicken/egg products are major sources of human salmonellosis, yet the strategies used by Salmonella to colonize chickens are poorly understood. We applied a novel two-step hierarchical procedure to identify new genes important for colonization and persistence of Salmonella enterica serotype Typhimurium in chickens. A library of 182 S. Typhimurium mutants each containing a targeted deletion of a group of contiguous genes (for a total of 2,069 genes deleted) was used to identify regions under selection at 1, 3, and 9 days postinfection in chicks. Mutants in 11 regions were under selection at all assayed times (colonization mutants), and mutants in 15 regions were under selection only at day 9 (persistence mutants). We assembled a pool of 92 mutants, each deleted for a single gene, representing nearly all genes in nine regions under selection. Twelve single gene deletion mutants were under selection in this assay, and we confirmed 6 of 9 of these candidate mutants via competitive infections and complementation analysis in chicks. STM0580, STM1295, STM1297, STM3612, STM3615, and STM3734 are needed for Salmonella to colonize and persist in chicks and were not previously associated with this ability. One of these key genes, STM1297 (selD), is required for anaerobic growth and supports the ability to utilize formate under these conditions, suggesting that metabolism of formate is important during infection. We report a hierarchical screening strategy to interrogate large portions of the genome during infection of animals using pools of mutants of low complexity. Using this strategy, we identified six genes not previously known to be needed during infection in chicks, and one of these (STM1297) suggests an important role for formate metabolism during infection

Defined Single-Gene and Multi-Gene Deletion Mutant Collections in Salmonella enterica sv Typhimurium

Artículo de publicación ISIWe constructed two collections of targeted single gene deletion (SGD) mutants and two collections of targeted multi-gene deletion (MGD) mutants in Salmonella enterica sv Typhimurium 14028s. The SGD mutant collections contain (1), 3517 mutants in which a single gene is replaced by a cassette containing a kanamycin resistance (KanR) gene oriented in the sense direction (SGD-K), and (2), 3376 mutants with a chloramphenicol resistance gene (CamR) oriented in the antisense direction (SGD-C). A combined total of 3773 individual genes were deleted across these SGD collections. The MGD collections contain mutants bearing deletions of contiguous regions of three or more genes and include (3), 198 mutants spanning 2543 genes replaced by a KanR cassette (MGD-K), and (4), 251 mutants spanning 2799 genes replaced by a CamR cassette (MGD-C). Overall, 3476 genes were deleted in at least one MGD collection. The collections with different antibiotic markers permit construction of all viable combinations of mutants in the same background. Together, the libraries allow hierarchical screening of MGDs for different phenotypic followed by screening of SGDs within the target MGD regions. The mutants of these collections are stored at BEI Resources (www.beiresources.org) and publicly available

Identification of Novel Salmonella Typhimurium Genes Required For Survival During Intestinal Inflammation

Head of Department: James SamuelBovine ligated ileal loops provide the best model to examine Salmonella Typhimurium genes required for survival during the early stages of infection, as humans and cattle develop very similar intestinal pathogenesis in response to the organism. Utilizing pools of mutants, both single gene and multi-gene deletion mutants, much of the S. Typhimurium genome can be screened simultaneously. A library consisting of multi-gene deletion mutants was screened in ligated ileal loops in calves. Regions of the genome required for survival in this model were identified in mucus and tissue samples using microarray analysis. STM1187-90 was one such region identified as under selection in both mucus and tissue. A ASTM1188 mutant was confirmed to poorly colonize the intestinal mucus and tissue in the presence of inflammation in competitive infections with the wild type. Complementation in trans reversed the phenotype of the ASTM1188 mutant. The phenotype of the ASTM1188 mutant was replicated and complemented in trans in the murine colitis model. STM1188 is a Salmonella specific gene whose protein product we show to be located in the inner membrane. This gene is absent in host-adapted serovars S. Typhi and S. Paratyphi A. Mutation of the putative lipobox cysteine to alanine resulted in mis- localization of STM1188C24A to the cytoplasm, and the inability to complement ASTM1188 in trans in mice. A second screen of a single gene deletion pool (SGD) identified many novel genes with potential roles during inflammation as well as many predicted genes with defined roles during the early stages of infection. Several novel gene phenotypes were confirmed and subsequently complemented in competitive infections with wild type. AhilE was identified during SGD screening and confirmed in bovine ligated ileal loops as being selected against during competitive infections with extensive inflammation, however, it was not selected against when the inflammatory immune response was limited. HilE has been previously shown to negatively regulate SPI-1 expression. Utilizing the murine colitis model, the AhilE phenotype was confirmed and complemented during competitive infection with wild type. By using (3-galactosidase assays, AhilE was confirmed to overexpress SPI-1, however, surprisingly AhilE also overexpressed SPI-2 during SPI-1 inducing conditions. In the current studies we performed Salmonella screens in bovine ligated ileal loops and confirmed novel virulence genes required for survival during inflammation

Novel Two-Step Hierarchical Screening of Mutant Pools Reveals Mutants under Selection in Chicks

Contaminated chicken/egg products are major sources of human salmonellosis, yet the strategies used by Salmonella to colonize chickens are poorly understood. We applied a novel two-step hierarchical procedure to identify new genes important for colonization and persistence of Salmonella enterica serotype Typhimurium in chickens. A library of 182 S. Typhimurium mutants each containing a targeted deletion of a group of contiguous genes (for a total of 2,069 genes deleted) was used to identify regions under selection at 1, 3, and 9 days postinfection in chicks. Mutants in 11 regions were under selection at all assayed times (colonization mutants), and mutants in 15 regions were under selection only at day 9 (persistence mutants). We assembled a pool of 92 mutants, each deleted for a single gene, representing nearly all genes in nine regions under selection. Twelve single gene deletion mutants were under selection in this assay, and we confirmed 6 of 9 of these candidate mutants via competitive infections and complementation analysis in chicks. STM0580, STM1295, STM1297, STM3612, STM3615, and STM3734 are needed for Salmonella to colonize and persist in chicks and were not previously associated with this ability. One of these key genes, STM1297 (selD), is required for anaerobic growth and supports the ability to utilize formate under these conditions, suggesting that metabolism of formate is important during infection. We report a hierarchical screening strategy to interrogate large portions of the genome during infection of animals using pools of mutants of low complexity. Using this strategy, we identified six genes not previously known to be needed during infection in chicks, and one of these (STM1297) suggests an important role for formate metabolism during infection

Novel determinants of intestinal colonization of Salmonella enterica serotype typhimurium identified in bovine enteric infection.

Cattle are naturally infected with Salmonella enterica serotype Typhimurium and exhibit pathological features of enteric salmonellosis that closely resemble those in humans. Cattle are the most relevant model of gastrointestinal disease resulting from nontyphoidal Salmonella infection in an animal with an intact microbiota. We utilized this model to screen a library of targeted single-gene deletion mutants to identify novel genes of Salmonella Typhimurium required for survival during enteric infection. Fifty-four candidate mutants were strongly selected, including numerous mutations in genes known to be important for gastrointestinal survival of salmonellae. Three genes with previously unproven phenotypes in gastrointestinal infection were tested in bovine ligated ileal loops. Two of these mutants, STM3602 and STM3846, recapitulated the phenotype observed in the mutant pool. Complementation experiments successfully reversed the observed phenotypes, directly linking these genes to the colonization defects of the corresponding mutant strains. STM3602 encodes a putative transcriptional regulator that may be involved in phosphonate utilization, and STM3846 encodes a retron reverse transcriptase that produces a unique RNA-DNA hybrid molecule called multicopy single-stranded DNA. The genes identified in this study represent an exciting new class of virulence determinants for further mechanistic study to elucidate the strategies employed by Salmonella to survive within the small intestines of cattle

Novel Determinants of Intestinal Colonization of Salmonella enterica Serotype Typhimurium Identified in Bovine Enteric Infection

Cattle are naturally infected with Salmonella enterica serotype Typhimurium and exhibit pathological features of enteric salmonellosis that closely resemble those in humans. Cattle are the most relevant model of gastrointestinal disease resulting from nontyphoidal Salmonella infection in an animal with an intact microbiota. We utilized this model to screen a library of targeted single-gene deletion mutants to identify novel genes of Salmonella Typhimurium required for survival during enteric infection. Fifty-four candidate mutants were strongly selected, including numerous mutations in genes known to be important for gastrointestinal survival of salmonellae. Three genes with previously unproven phenotypes in gastrointestinal infection were tested in bovine ligated ileal loops. Two of these mutants, STM3602 and STM3846, recapitulated the phenotype observed in the mutant pool. Complementation experiments successfully reversed the observed phenotypes, directly linking these genes to the colonization defects of the corresponding mutant strains. STM3602 encodes a putative transcriptional regulator that may be involved in phosphonate utilization, and STM3846 encodes a retron reverse transcriptase that produces a unique RNA-DNA hybrid molecule called multicopy single-stranded DNA. The genes identified in this study represent an exciting new class of virulence determinants for further mechanistic study to elucidate the strategies employed by Salmonella to survive within the small intestines of cattle

Novel Determinants of Intestinal Colonization of Salmonella enterica Serotype Typhimurium Identified in Bovine Enteric Infection

Cattle are naturally infected with Salmonella enterica serotype Typhimurium and exhibit pathological features of enteric salmonellosis that closely resemble those in humans. Cattle are the most relevant model of gastrointestinal disease resulting from nontyphoidal Salmonella infection in an animal with an intact microbiota. We utilized this model to screen a library of targeted single-gene deletion mutants to identify novel genes of Salmonella Typhimurium required for survival during enteric infection. Fifty-four candidate mutants were strongly selected, including numerous mutations in genes known to be important for gastrointestinal survival of salmonellae. Three genes with previously unproven phenotypes in gastrointestinal infection were tested in bovine ligated ileal loops. Two of these mutants, STM3602 and STM3846, recapitulated the phenotype observed in the mutant pool. Complementation experiments successfully reversed the observed phenotypes, directly linking these genes to the colonization defects of the corresponding mutant strains. STM3602 encodes a putative transcriptional regulator that may be involved in phosphonate utilization, and STM3846 encodes a retron reverse transcriptase that produces a unique RNA-DNA hybrid molecule called multicopy single-stranded DNA. The genes identified in this study represent an exciting new class of virulence determinants for further mechanistic study to elucidate the strategies employed by Salmonella to survive within the small intestines of cattle

Workflow of collection construction and verification.

<p>A. General procedure and logistics of mutant generation, storage and characterization. B. Identification of obtained mutants and mapping of mutants to specific wells by a “Hypercube” Combinatorial Pooling approach, essentially as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0099820#pone.0099820-Goodman1" target="_blank">[30]</a>.</p

Summary of collection statistics.

a<p>CDS where no transposon insertion was found in a high complexity random transposon library <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0099820#pone.0099820-Canals1" target="_blank">[29]</a>. List of mutants present in our collections. All <i>S</i>. Typhimurium strain 14028s annotated features <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0099820#pone.0099820-Jarvik1" target="_blank">[12]</a> are presented along with their status in our single-gene deletion (SGD) mutant collections. Corresponding genes in <i>S</i>. Typhimurium strain LT2 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0099820#pone.0099820-McClelland1" target="_blank">[27]</a> that have at least 70% DNA identity and at least 70% coverage are annotated. The SGD collections are listed in genome order, followed by the multi-gene deletion (MGD) collections in genome order. For MGDs, only those primer pairs that resulted in successful deletion in at least one of the two varieties (Kan or Cam) are shown. Primer sequences, their genome locations, and the results of mutant generation, verification, and mapping to 96-well plates are shown. Empty wells are not listed.</p