Signature-Tagged Mutagenesis of
            <i>Edwardsiella ictaluri</i>
            Identifies Virulence-Related Genes, Including a
            <i>Salmonella</i>
            Pathogenicity Island 2 Class of Type III Secretion Systems

Thune, Ronald L.; Fernandez, Denise H.; Benoit, Jennifer L.; Kelly-Smith, Maria; Rogge, Matthew L.; Booth, Natha J.; Landry, C.; Bologna, Rachel A.

doi:10.1128/aem.01115-07

Cited by 55 publications

(76 citation statements)

References 78 publications

(90 reference statements)

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“…Although state of the art at the time, it also allowed the incidental introduction of a functional Mu prophage into these strains and their respective pir derivatives, which have since been widely used for conjugation transfer of exogenous DNA, including suicide transposon-carrying vectors, to a large number of bacterial species (E. coli [4,23,29], Klebsiella pneumoniae [7], Edwardsiella ictaluri [46], Rhizobium meliloti [33], Vibrio cholerae [28], and Erwinia carotovora [32]). In some cases, it was reported that transconjugant transposon mutants obtained using S17-1 and SM10 donor strains did not display the expected genetic linkage between the screened phenotype and insertion events or phenotype instability in transconjugants, but the reasons for these inconsistencies were not investigated (26,38).…”

Section: Discussionmentioning

confidence: 99%

Silent Mischief: Bacteriophage Mu Insertions Contaminate Products of Escherichia coli Random Mutagenesis Performed Using Suicidal Transposon Delivery Plasmids Mobilized by Broad-Host-Range RP4 Conjugative Machinery

et al. 2010

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Random transposon mutagenesis is the strategy of choice for associating a phenotype with its unknown genetic determinants. It is generally performed by mobilization of a conditionally replicating vector delivering transposons to recipient cells using broad-host-range RP4 conjugative machinery carried by the donor strain. In the present study, we demonstrate that bacteriophage Mu, which was deliberately introduced during the original construction of the widely used donor strains SM10 pir and S17-1 pir, is silently transferred to Escherichia coli recipient cells at high frequency, both by hfr and by release of Mu particles by the donor strain. Our findings suggest that bacteriophage Mu could have contaminated many random-mutagenesis experiments performed on Mu-sensitive species with these popular donor strains, leading to potential misinterpretation of the transposon mutant phenotype and therefore perturbing analysis of mutant screens. To circumvent this problem, we precisely mapped Mu insertions in SM10 pir and S17-1 pir and constructed a new Mu-free donor strain, MFDpir, harboring stable hfr-deficient RP4 conjugative functions and sustaining replication of ⌸-dependent suicide vectors. This strain can therefore be used with most of the available transposon-delivering plasmids and should enable more efficient and easy-to-analyze mutant hunts in E. coli and other Mu-sensitive RP4 host bacteria.

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Section: Discussionmentioning

confidence: 99%

Silent Mischief: Bacteriophage Mu Insertions Contaminate Products of Escherichia coli Random Mutagenesis Performed Using Suicidal Transposon Delivery Plasmids Mobilized by Broad-Host-Range RP4 Conjugative Machinery

et al. 2010

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“…ABC60092). orf29/30 from E. ictaluri was also reported to be a single open reading frame based on a 6-fold sequencing coverage genome project of strain 93-146 (30).…”

Section: Identification Of Esejmentioning

confidence: 99%

Identification and Functional Characterization of the Novel Edwardsiella tarda Effector EseJ

et al. 2015

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e Edwardsiella tarda is a Gram-negative enteric pathogen that causes hemorrhagic septicemia in fish and gastro-and extraintestinal infections in humans. The type III secretion system (T3SS) of E. tarda has been identified as a key virulence factor that contributes to pathogenesis in fish. However, little is known about the associated effectors translocated by this T3SS. In this study, by comparing the profile of secreted proteins of the wild-type PPD130/91 and its T3SS ATPase ⌬esaN mutant, we identified a new effector by matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry. This effector consists of 1,359 amino acids, sharing high sequence similarity with Orf29/30 of E. tarda strain EIB202, and is renamed EseJ. The secretion and translocation of EseJ depend on the T3SS. A ⌬eseJ mutant strain adheres to epithelioma papillosum of carp (EPC) cells 3 to 5 times more extensively than the wild-type strain does. EseJ inhibits bacterial adhesion to EPC cells from within bacterial cells. Importantly, the ⌬eseJ mutant strain does not replicate efficiently in EPC cells and fails to replicate in J774A.1 macrophages. In infected J774A.1 macrophages, the ⌬eseJ mutant elicits higher production of reactive oxygen species than wild-type E. tarda. The replication defect is consistent with the attenuation of the ⌬eseJ mutant in the blue gourami fish model: the 50% lethal dose (LD 50 ) of the ⌬eseJ mutant is 2.34 times greater than that of the wild type, and the ⌬eseJ mutant is less competitive than the wild type in mixed infection. Thus, EseJ represents a novel effector that contributes to virulence by reducing bacterial adhesion to EPC cells and facilitating intracellular bacterial replication.

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“…E. ictaluri crosses the intestinal mucosa of channel catfish in 15 min after oral inoculation with 10 9 CFU (6). Although there are substantial descriptive data relative to the invasion, spread, and persistence of E. ictaluri in channel catfish (6)(7)(8), little is known about the molecular mechanisms of E. ictaluri fish intestinal pathogenicity and the pathogen-associated molecular patterns (PAMPs) recognized by fish.…”

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confidence: 99%

Inflammatory Effects of Edwardsiella ictaluri Lipopolysaccharide Modifications in Catfish Gut

et al. 2014

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e Bacterial lipopolysaccharides (LPS) are structural components of the outer membranes of Gram-negative bacteria and also are potent inducers of inflammation in mammals. Higher vertebrates are extremely sensitive to LPS, but lower vertebrates, like fish, are resistant to their systemic toxic effects. However, the effects of LPS on the fish intestinal mucosa remain unknown. Edwardsiella ictaluri is a primitive member of the Enterobacteriaceae family that causes enteric septicemia in channel catfish (Ictalurus punctatus). E. ictaluri infects and colonizes deep lymphoid tissues upon oral or immersion infection. Both gut and olfactory organs are the primary sites of invasion. At the systemic level, E. ictaluri pathogenesis is relatively well characterized, but our knowledge about E. ictaluri intestinal interaction is limited. Recently, we observed that E. ictaluri oligo-polysaccharide (O-PS) LPS mutants have differential effects on the intestinal epithelia of orally inoculated catfish. Here we evaluate the effects of E. ictaluri O-PS LPS mutants by using a novel catfish intestinal loop model and compare it to the rabbit ileal loop model inoculated with Salmonella enterica serovar Typhimurium LPS. We found evident differences in rabbit ileal loop and catfish ileal loop responses to E. ictaluri and S. Typhimurium LPS. We determined that catfish respond to E. ictaluri LPS but not to S. Typhimurium LPS. We also determined that E. ictaluri inhibits cytokine production and induces disruption of the intestinal fish epithelia in an O-PS-dependent fashion. The E. ictaluri wild type and ⌬wibT LPS mutant caused intestinal tissue damage and inhibited proinflammatory cytokine synthesis, in contrast to E. ictaluri ⌬gne and ⌬ugd LPS mutants. We concluded that the E. ictaluri O-PS subunits play a major role during pathogenesis, since they influence the recognition of the LPS by the intestinal mucosal immune system of the catfish. The LPS structure of E. ictaluri mutants is needed to understand the mechanism of interaction.

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Signature-Tagged Mutagenesis of Edwardsiella ictaluri Identifies Virulence-Related Genes, Including a Salmonella Pathogenicity Island 2 Class of Type III Secretion Systems

Cited by 55 publications

References 78 publications

Silent Mischief: Bacteriophage Mu Insertions Contaminate Products of Escherichia coli Random Mutagenesis Performed Using Suicidal Transposon Delivery Plasmids Mobilized by Broad-Host-Range RP4 Conjugative Machinery

Silent Mischief: Bacteriophage Mu Insertions Contaminate Products of Escherichia coli Random Mutagenesis Performed Using Suicidal Transposon Delivery Plasmids Mobilized by Broad-Host-Range RP4 Conjugative Machinery

Identification and Functional Characterization of the Novel Edwardsiella tarda Effector EseJ

Inflammatory Effects of Edwardsiella ictaluri Lipopolysaccharide Modifications in Catfish Gut

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