Sequencing and Analysis of the Edwardsiella ictaluri Plasmids

Fernandez, Denise H.; Pittman-Cooley, Lynn; Thune, Ronald L.

doi:10.1006/plas.2000.1499

Cited by 36 publications

(52 citation statements)

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“…E. ictaluri contains two native plasmids, pEI1 and pEI2, that are consistently present in channel catfish isolates (36). The sequences of both plasmids are available (18), but most of these plasmids' gene functions are poorly defined. Our results agree with previously reported findings that pEI1 is important in E. ictaluri virulence (46).…”

Section: Discussionmentioning

confidence: 99%

High-Throughput Bioluminescence-Based Mutant Screening Strategy for Identification of Bacterial Virulence Genes

Karsi

Gülsoy

Corb³

et al. 2009

Appl Environ Microbiol

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A high-throughput bioluminescence screening procedure for identification of virulence genes in bacteria was developed and applied to the fish pathogen Edwardsiella ictaluri. A random transposon mutant library expressing bioluminescence was constructed and robotically arrayed on 384-well plates. Mutants were cultivated and mixed with catfish serum and neutrophils in 96-well plates, and bioluminescence was used to detect mutants that are more susceptible to killing by these host factors. The virulence and vaccine efficacy of selected mutants were determined in channel catfish. Transposon insertion sites in 13 mutants attenuated in the natural host were mapped to the E. ictaluri genome. Ten unique genes were mutated, including genes encoding a negative regulator of sigmaE activity, a glycine cleavage system protein, tricarboxylic acid cycle enzymes, an O polysaccharide biosynthesis enzyme, proteins encoded on the native plasmid pEI1, and a fimbrial chaperon protein.Three of these mutants were found to have potential as live attenuated vaccines. This study demonstrates a novel application of bioluminescence to identify bacterial genes required for host resistance; as a result, efficacious and genetically defined live attenuated vaccine candidates were developed.

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Section: Discussionmentioning

confidence: 99%

High-Throughput Bioluminescence-Based Mutant Screening Strategy for Identification of Bacterial Virulence Genes

Karsi

Gülsoy

Corb³

et al. 2009

Appl Environ Microbiol

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“…IpaA exhibits sequence similarity with Sip/SspA from Salmonella serovar Typhimurium (35,40). In contrast to other type III chaperones which exhibit little sequence similarity, invB/spa15-like genes are present in a wide variety of bacteria, including Yersinia enterocolitica (ysaK [29]) and Sodalis glossinidius (invB [10]), and also on an Edwardsiella ictaluri plasmid (16). Based on this wide distribution, an unusual gene locus embedded between functional genes of the TTSS apparatus, and the ability to interact with multiple unrelated effector proteins, it has been proposed that InvB/Spa15 may represent a new family of chaperones (54,55).…”

Section: Discussionmentioning

confidence: 99%

Role of theSalmonellaPathogenicity Island 1 (SPI-1) Protein InvB in Type III Secretion of SopE and SopE2, TwoSalmonellaEffector Proteins Encoded Outside of SPI-1

et al. 2003

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Salmonella enterica subspecies 1 serovar Typhimurium encodes a type III secretion system (TTSS) within Salmonella pathogenicity island 1 (SPI-1). This TTSS injects effector proteins into host cells to trigger invasion and inflammatory responses. Effector proteins are recognized by the TTSS via signals encoded in their N termini. Specific chaperones can be involved in this process. The chaperones InvB, SicA, and SicP are encoded in SPI-1 and are required for transport of SPI-1-encoded effectors. Several key effector proteins, like SopE and SopE2, are located outside of SPI-1 but are secreted in an SPI-1-dependent manner. It has not been clear how these effector proteins are recognized by the SPI-1 TTSS. Using pull-down and coimmunoprecipitation assays, we found that SopE is copurified with InvB, the known chaperone for the SPI-1-encoded effector protein Sip/SspA. We also found that InvB is required for secretion and translocation of SopE and SopE2 and for stabilization of SopE2 in the bacterial cytosol. Our data demonstrate that effector proteins encoded within and outside of SPI-1 use the same chaperone for secretion via the SPI-1 TTSS.

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“…Following phagocytosis, the SPI-2 system translocates effector proteins that are involved in intracellular replication across the phagosomal membrane (11,24). In addition to the T3SS PAI, the E. ictaluri plasmids, pEI1 and pEI2, encode proteins with similarity to T3SS effector proteins of other pathogens (20,52). These two plasmids are found in all channel catfish isolates of E. ictaluri (31,37,42,47), suggesting that they are required for virulence.…”

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confidence: 99%

Regulation of the Edwardsiella ictaluri Type III Secretion System by pH and Phosphate Concentration through EsrA, EsrB, and EsrC

Rogge

Thune

2011

Appl Environ Microbiol

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A recently described Edwardsiella ictaluri type III secretion system (T3SS) with functional similarity to the Salmonella pathogenicity island 2 T3SS is required for replication in channel catfish head-kidney-derived macrophages (HKDM) and virulence in channel catfish. Quantitative PCR and Western blotting identified low pH and phosphate limitation as conducive to expression of the E. ictaluri T3SS, growth conditions that mimic the phagosomal environment. Mutagenesis studies demonstrated that expression is under the control of the EsrAB two-component regulatory system. EsrB also induces upregulation of the AraC-type regulatory protein EsrC, which enhances expression of the EscB/EseG chaperone/effector operon in concert with EsrB and induces expression of the pEI1-encoded effector, EseH. EsrC also induces expression of a putative type VI secretion system translocon protein, EvpC, which is secreted under the same low-pH conditions as the T3SS translocon proteins. The pEI2-encoded effector, EseI, was upregulated under low-pH and low-phosphate conditions but not in an EsrB-or EsrC-dependent manner. Mutations of EsrA and EsrB both resulted in loss of the ability to replicate in HKDM and full attenuation in the channel catfish host. Mutation of EsrC did not affect intracellular replication but did result in attenuation in catfish. Although EsrB is the primary transcriptional regulator for E. ictaluri genes within the T3SS pathogenicity island, EsrC regulates expression of the plasmidcarried effector eseH and appears to mediate coordinated expression of the T6SS with the T3SS.Edwardsiella ictaluri, the etiological agent of enteric septicemia of catfish (ESC), was isolated, named, and characterized by Hawke (22) and Hawke et al. (23). The disease is found throughout channel catfish (Ictalurus punctatus) growing regions and is the leading cause of disease-related mortality in channel catfish production facilities (2, 3). Recent reports have expanded the host and geographic ranges to the culture of striped catfish (Pangasianodon hypophthalmus) in Vietnam (14, 51) and yellow catfish (Pelteobagrus fulvidraco) in China (29).Using signature-tagged mutagenesis, Thune et al. (52) identified mutations in 50 E. ictaluri genes that led to an attenuated phenotype in channel catfish, including a type III secretion system (T3SS) apparatus gene. Further sequencing and genome analysis led to the identification of an E. ictaluri pathogenicity island (PAI) encoding a complete T3SS, including Edwardsiella secretion apparatus (esa), chaperone (esc), effector (ese), and regulatory (esr) genes. Mutation of the apparatus gene resulted in an inability to replicate in catfish cells and loss of virulence, indicating that the T3SS is essential to the pathogenesis of E. ictaluri. These systems are widespread in Gram-negative bacteria and function to translocate effector molecules directly from the bacterial cytosol to the host cell cytoplasm.The Edwardsiella tarda T3SS, which is involved in replication within fish macrophages (38,49,50,57), is hom...

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Sequencing and Analysis of the Edwardsiella ictaluri Plasmids

Cited by 36 publications

References 10 publications

High-Throughput Bioluminescence-Based Mutant Screening Strategy for Identification of Bacterial Virulence Genes

High-Throughput Bioluminescence-Based Mutant Screening Strategy for Identification of Bacterial Virulence Genes

Role of theSalmonellaPathogenicity Island 1 (SPI-1) Protein InvB in Type III Secretion of SopE and SopE2, TwoSalmonellaEffector Proteins Encoded Outside of SPI-1

Regulation of the Edwardsiella ictaluri Type III Secretion System by pH and Phosphate Concentration through EsrA, EsrB, and EsrC

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