Phenotype and Virulence of a Transposon-Derived Lipopolysaccharide O Side-Chain Mutant Strain of<i>Edwardsiella ictaluri</i>

Lawrence, Mark L.; Banes, Michelle M.; Williams, Michele L.

doi:10.1577/1548-8667(2001)013<0291:pavoat>2.0.co;2

Cited by 31 publications

(28 citation statements)

References 30 publications

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“…Attenuation of mutants EiAKMut01 to EiAKMut13 was confirmed in catfish by using the intraperitoneal injection route of exposure (29,30). Each 40-liter flowthrough tank contained 20 fish, and four tanks were used for each mutant.…”

Section: Methodsmentioning

confidence: 92%

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: Methodsmentioning

confidence: 92%

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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“…LPS is an important virulence factor for E. ictaluri (16)(17)(18). The E. ictaluri LPS gene cluster has been identified by transposon mutagenesis (16,17) and was recently fully described (18).…”

mentioning

confidence: 99%

“…The E. ictaluri LPS gene cluster has been identified by transposon mutagenesis (16,17) and was recently fully described (18). E. ictaluri LPS O-PS mutants exhibited different levels of virulence, tissue colonization, and intestinal gut inflammation in orally inoculated catfish (18).…”

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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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“…These vaccines have been shown to possess high immunogenicity and protect the catfish from ESC (Bader et al, 2004;Panangala et al, 2009b). The antigenicity of E. ictaluri has been studied (Baldwin et al, 1997;Dumpala et al, 2009) and has revealed many antigenic components, including flagella (Panangala et al, 2009a), extracellular capsular polysaccharides (Stanley et al, 1994), lipopolysaccharide (LPS) (Lawrence et al, 2001Williams et al, 2003), outer membrane proteins (OMPs) (Bader et al, 2004;Moore et al, 2002;Newton et al, 1990;Vinitnantharat et al, 1993), hemolysins (Williams and Lawrence, 2005), and chondroitinase (Cooper et al, 1996). However, little is known about the virulence factors of E. ictaluri responsible for antibody effectiveness (Panangala et al, 2009b).…”

Section: Vaccine Efficacy Of Glyceraldehyde-3-phosphate Dehydrogenasementioning

confidence: 99%

Vaccine efficacy of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) from Edwardsiella ictaluri against E. tarda in tilapia

Cao¹,

Tsai²,

Yang

et al. 2014

J. Gen. Appl. Microbiol.

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Glyceraldehyde-3-phosphate dehydrogenase (GAP-DH), derived from the outer-membrane protein (OMP) fraction, has been used as a potential candidate for vaccine development. The gene-encoding 37 kDa GAPDH outer membrane protein (OMP) from Edwardsiella ictaluri was amplified using polymerase chain reaction (PCR) and was cloned and expressed in Escherichia coli BL21 (DE3). Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), Western blotting, and nucleotide and amino acid sequencing were used to analyze the expressed antigenic protein and gene encoding this protein. Comparative DNA and protein sequence analysis of GAPDH from E. ictaluri GAPDHs from several Gram-negative bacterial species within the Enterobacteriaceae family revealed that the GAPDHs within this group are highly conserved and share a sequence similarity of 75 100% with E. ictaluri GDPDH. Rabbit antiserum raised against the E. ictaluri recombinant GAPDH (rGAPDH) protein recognized purified GADPH, indicating that it has a strong immunogenicity. Tilapia fish were intraperitoneally immunized with formalin-killed E. ictaluri whole cells, and rGAPDH (30 µg fish 1 ) from E. ictaluri, both of which were emulsified in ISA 763A adjuvant. At 3 months after immunization, fish were challenged with the E. tarda strain to assess vaccine efficacy; the relative percent survival (RPS) values were found to exceed 71.4%. The specific mean antibody titer log 2 level of groups vaccinated with rGAPDH at 3 months was significantly higher than that of non-vaccinated fish (control group). Therefore, this recombinant protein can be considered a multi-purpose candidate vaccine against several pathogenic bacteria.Key words: channel catfish; Edwardsiella ictaluri; enteric septicemia of catfish; glyceraldehyde-3-phosphate dehydrogenase (GAPDH) IntroductionEnteric septicemia of catfish (ESC), caused by the bacterial pathogen Edwardsiella ictaluri, was first identified in channel catfish (Ictalurus punctatus) by Hawke (1979). E. ictaluri has become the most lethal infectious disease of channel catfish and other ictalurids in the USA (Plumb, 1999). More than 50% of American catfish fry/fingerling losses are due to ESC (Williams and Lawrence, 2010). E. ictaluri was also isolated from cultured fish species in Indonesia, Vietnam, Japan, Turkey (Crumlish et al., 2002;Keskün et al., 2004;Nagai et al., 2008; Yuas et al., 2003) and China (Ye et al., 2009). ESC is currently controlled using antibiotics; however, extensive use of antimicrobial com pounds reduces their effectiveness by increasing the number of antibiotic-resistant bacterial strains, resulting in the need for higher dosages for effective treatment as well as higher residual amounts of drugs in the harvested fish Thompson and Adams, 2004). Vaccines that protect fish against bacterial diseases are an important alternative to antibiotics and are a safer strategy for disease prevention. Most vaccines for controlling ESC have been tested using Full Paper Vaccine efficacy of glyceraldehyde-3-phosphate dehydrogenase (G...

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Phenotype and Virulence of a Transposon-Derived Lipopolysaccharide O Side-Chain Mutant Strain ofEdwardsiella ictaluri

Cited by 31 publications

References 30 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

Inflammatory Effects of Edwardsiella ictaluri Lipopolysaccharide Modifications in Catfish Gut

Vaccine efficacy of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) from Edwardsiella ictaluri against E. tarda in tilapia

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