Phenotype, Virulence and Immunogenicity of Edwardsiella piscicida Cyclic AMP Receptor Protein (Crp) Mutants in Catfish Host

Abstract

Edwardsiella piscicida, a facultative aerobic pathogen belonging to the Enterobacteriaceae family, is the etiological agent of edwardsiellosis that causes significant economic loses in the aquaculture industry. cAMP receptor protein (CRP) is one of the most important transcriptional regulators, which can regulate large quantities of operons in different bacteria. Here we characterize the crp gene and report the effect of a crp deletion in E. piscicida. The crp-deficient mutant lost the capacity to utilize maltose, and showed significantly reduced motility due to the lack of flagella synthesis. We further constructed a ΔPcrp mutant to support that the phenotype above was caused by the crp deletion. Evidence obtained in fish serum killing assay and competitive infection assay strongly indicated that the inactivation of crp impaired the ability of E. piscicida to evade host immune clearance. More importantly, the virulence of the crp mutant was attenuated in both zebrafish and channel catfish, with reductions in mortality rates. In the end, we found that crp mutant could confer immune protection against E. piscicida infection to zebrafish and channel catfish, indicating its potential as a live attenuated vaccine.

Construction and Evaluation of Recombinant Attenuated Edwardsiella piscicida Vaccine (RAEV) Vector System Encoding Ichthyophthirius multifiliis (Ich) Antigen IAG52B

“…Deletion of either fur or crp attenuated E. piscicida ( 5 , 6 ) and E. ictaluri ( 37 , 38 ). Strains were constructed with deletion insertion mutations that conferred a phenotype of regulated-delayed attenuation in vivo ( 5 ).…”

“…To construct the E. piscicida ΔasdA10 strain (χ16000) with a regulated-delayed attenuation phenotype, Δ P fur and Δ P crp deletion insertion mutations were added sequentially to χ16000 by using suicide plasmids pG8R8024 and pG8R8009 as described previously ( 5 , 6 ). The resultant strain had a genotype Δ asdA10 ΔP fur170 ::TT araC P araBAD fur ΔP crp68 ::TT araC P araBAD crp and numbered as χ16022.…”

“…Conventional in-frame gene deletion can over attenuate the strain, making it more susceptible to host immune defenses and limit its ability to colonize internal tissue ( 4 ). To overcome these problems, our lab has previously demonstrated the ability to effectively attenuate E. piscicida through the use of regulated-delayed attenuation systems ( 5 , 6 ). The promoters of the fur and crp genes were replaced by a tightly-regulated araC P araBAD cassette such that expression was arabinose dependent during growth in vitro.…”

“…The safety and control of these strains can be further enhanced by replacing the promoters of several virulence factors with externally-regulated promoters. Additionally, these recombinant attenuated Edwardsiella vaccines (RAEVs) are capable of stimulating both innate and adaptive immune responses in fish ( 5 , 6 ). Zebrafish vaccinated with an RAEV showed up-regulation in the transcription of genes for several pro-inflammatory cytokines, including TNF-α and IL-1β ( 5 , 7 ).…”

Delivering the pain: an overview of the type III secretion system with special consideration for aquatic pathogens

“…Edwardsiella piscicida , has been described more recently and has been associated with diseases in aquatic animals, e.g. catfish and zebrafish [ 107 ]. It possesses both a T3SS and a T6SS, which are responsible to transport 12 effectors with synergistic activity [ 108 ].…”