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

Optimal management of GIST tumors located near the gastroesophageal junction: Case report and review of the literature

Introduction: The safety and oncologic outcome of laparoscopic gastric GIST resection is well established especially for lesions <5 cm in diameter. The optimal management of GIST tumors near the GE junction remains unclear. Methods: We present a case-report of a 4.7 cm GIST tumor near the GE junction managed by endoscopically-assisted laparoscopic wedge resection (EAWR). We present a review of the literature highlighting the various combined laparo-endoscopic techniques available. Results: We used the non-touch lesion-lifting method to laparoscopically resect the GIST tumor under endoscopic guidance. There were no complications and the patient was discharged on postoperative day 3. Conclusions: Endoscopically-assisted laparoscopic wedge resections are feasible and safe for GIST tumors near the GE junction

Spontaneous endometriosis associated with an umbilical hernia: A case report and review of the literature

Introduction: Umbilical endometriosis occurring in the presence of an underlying hernia is extremely rare and presents a diagnostic challenge for the general surgeon. We present an interesting case and perform a comprehensive review of the literature. Methods: Medline and PubMed were queried for all cases of spontaneous umbilical endometriosis associated with an umbilical hernia. Data was analyzed and is presented along with an interesting case. Results: Only 7 cases have been reported in the literature. Median age was 38 years. Time to presentation was long (up to 5 years) and the majority had cyclical symptoms related to menstruation. All patients, including our case, were treated surgically. Discussion: Spontaneous umbilical endometriosis with an underlying hernia is often missed preoperatively. Preoperative suspicion warrants axial imaging for better operative planning and patient counseling. Surgery consists of enbloc excision of the umbilicus, implant and the hernia sac to avoid residual disease and reduce recurrence. The hernia defect can be repaired primarily or using mesh and the umbilicus reconstructed using skin flaps if necessary. Conclusions: Surgery is the mainstay of therapy for umbilical endometriosis associated with an underlying hernia. Clinical suspicion warrants preoperative imaging, and follow-up with a gynecologist is essential to address any pelvic disease

The ABC-Type Efflux Pump MacAB Protects Salmonella enterica serovar Typhimurium from Oxidative Stress

Multidrug efflux pumps are integral membrane proteins known to actively excrete antibiotics. The macrolide-specific pump MacAB, the only ABC-type drug efflux pump in Salmonella, has previously been linked to virulence in mice. The molecular mechanism of this link between macAB and infection is unclear. We demonstrate that macAB plays a role in the detoxification of reactive oxygen species (ROS), compounds that salmonellae are exposed to at various stages of infection. macAB is induced upon exposure to H2O2 and is critical for survival of Salmonella enterica serovar Typhimurium in the presence of peroxide. Furthermore, we determined that macAB is required for intracellular replication inside J774.A1 murine macrophages but is not required for survival in ROS-deficient J774.D9 macrophages. macAB mutants also had reduced survival in the intestine in the mouse colitis model, a model characterized by a strong neutrophilic intestinal infiltrate where bacteria may experience the cytotoxic actions of ROS. Using an Amplex red-coupled assay, macAB mutants appear to be unable to induce protection against exogenous H2O2 in vitro, in contrast to the isogenic wild type. In mixed cultures, the presence of the wild-type organism, or media preconditioned by the growth of the wild-type organism, was sufficient to rescue the macAB mutant from peroxide-mediated killing. Our data indicate that the MacAB drug efflux pump has functions beyond resistance to antibiotics and plays a role in the protection of Salmonella against oxidative stress. Intriguingly, our data also suggest the presence of a soluble anti-H2O2 compound secreted by Salmonella cells through a MacAB-dependent mechanism.The open access fee for this work was funded through the Texas A&M University Open Access to Knowledge (OAK) Fund

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

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

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