The Role of TonB Gene in Edwardsiella ictaluri Virulence

Abstract

Edwardsiella ictaluri is a Gram-negative facultative intracellular pathogen that causes enteric septicemia in catfish (ESC). Stress factors including poor water quality, poor diet, rough handling, overcrowding, and water temperature fluctuations increase fish susceptibility to ESC. The TonB energy transducing system (TonB-ExbB-ExbD) and TonB-dependent transporters of Gram-negative bacteria support active transport of scarce resources including iron, an essential micronutrient for bacterial virulence. Deletion of the tonB gene attenuates virulence in several pathogenic bacteria. In the current study, the role of TonB (NT01EI_RS07425) in iron acquisition and E. ictaluri virulence were investigated. To accomplish this, the E. ictaluri tonB gene was in-frame deleted. Growth kinetics, iron utilization, and virulence of the EiΔtonB mutant were determined. Loss of TonB caused a significant reduction in bacterial growth in iron-depleted medium (p > 0.05). The EiΔtonB mutant grew similarly to wild-type E. ictaluri when ferric iron was added to the iron-depleted medium. The EiΔtonB mutant was significantly attenuated in catfish compared with the parent strain (21.69 vs. 46.91% mortality). Catfish surviving infection with EiΔtonB had significant protection against ESC compared with naïve fish (100 vs. 40.47% survival). These findings indicate that TonB participates in pathogenesis of ESC and is an important E. ictaluri virulence factor.

Keywords: ESC; channel catfish; iron; tonB; virulence.

Figure 1

Agarose gel picture showing genotypic confirmation of the EiΔtonB strain by PCR. Lane one is KB Ladder. Lane two is PCR product amplified from wild-type E. ictaluri. Lane three is PCR product amplified from genomic DNA of the EiΔtonB strain.

Figure 2

Nucleotide sequence alignment of tonB genes in E. ictaluri and EiΔtonB. The matching region is shadowed in black. “---” indicates deleted gene region in EiΔtonB. For clarity, the deleted gene region was cropped, which is shown by “…”. The green box and arrow indicate start codon, and the red box and arrow indicate stop codon of the tonB gene.

Figure 3

Growth of EiΔtonB and 93–146 in BHI broth and BHI broth supplemented with 100 μM 2′2-dipyridyl (DPD). These data represent the mean ± SE of 12 replicates from two experiments. Standard error bars are shown.

Figure 4

Growth of EiΔtonB and 93–146 in BHI broth containing 100 μM DPD and different ferric iron sources. Data represents the mean ± SE of four replicates.

Figure 5

Mean percent mortalities resulting from immersion challenge of EiΔtonB and 93–146 in channel catfish fingerlings (A). Mean percent survival of catfish fingerlings surviving infection with EiΔtonB and re-challenged with 93–146 at 21days post-immunization (B).