Thioredoxin H (TrxH) contributes to adversity adaptation and pathogenicity of Edwardsiella piscicida

Abstract

Thioredoxins (Trxs) play an important role in defending against oxidative stress and keeping disulfide bonding correct to maintain protein function. Edwardsiella piscicida, a severe fish pathogen, has been shown to encode several thioredoxins including TrxA, TrxC, and TrxH, but their biological roles remain unknown. In this study, we characterized TrxH of E. piscicida (named TrxH_Ep) and examined its expression and function. TrxH_Ep is composed of 125 residues and possesses typical thioredoxin H motifs. Expression of trxH_Ep was upregulated under conditions of oxidative stress, iron starvation, low pH, and during infection of host cells. trxH_Ep expression was also regulated by ferric uptake regulator (Fur), an important global regulatory of E. piscicida. Compared to the wild type TX01, a markerless trxH_Ep in-frame mutant strain TX01∆trxH exhibited markedly compromised tolerance of the pathogen to hydrogen peroxide, acid stress, and iron deficiency. Deletion of trxH_Ep significantly retarded bacterial biofilm growth and decreased resistance against serum killing. Pathogenicity analysis shows that the inactivation of trxH_Ep significantly impaired the ability of E. piscicida to invade host cells, reproduce in macrophages, and infect host tissues. Introduction of a trans-expressed trxH gene restored the lost virulence of TX01∆trxH. There is likely to be a complex relationship of functional complementation or expression regulation between TrxH and another two thioredoxins, TrxA and TrxC, of E. piscicida. This is the first functional report of TrxH in fish pathogens, and the findings suggest that TrxH_Ep is essential for coping with adverse circumstances and contributes to host infection of E. piscicida.

Figure 1

Alignment of the amino acid sequences of TrxH_Ep homologues. Dots denote gaps introduced for maximum matching. The consensus residues are in black, the residues that are ≥ 75% identical among the aligned sequences are in pink. Conserved WCXXC catalytic motif and QSTL/M motif are boxed. The characteristic elements of the thioredoxin fold (α1, α2, α3, α4, α5, β1, β2, β3, and β4) are underlined.

Figure 2

Sensitivity of Edwardsiella piscicida to oxidative stress, iron deficiency, and acid stress. TX01, TX01∆trxH, and TX01∆trxHC were cultured in LB medium and LB agar plates with pH = 7 (normal A), with pH = 5 (B), with 80 μM 2,2′dipyridyl (Dp, C), or with 1 mM H₂O₂ (D) at 28 °C for 24–48 h. The populations of cultivated bacteria were counted by diluted plate. E TX01 and TX01∆trxH were cultured to early logarithmic phase in normal LB medium, then 5 mM H₂O₂ were added to the medium, and cell density was measured at different time points by determining absorbance at OD₆₀₀. Data are the means of three independent experiments and presented as mean ± SEM (N = 3). N, the number of times the experiment was performed. *P < 0.05, **P < 0.01.

Figure 3

Effects of trxH_Ep mutation on resistance to serum and biofilm growth. A Survival of E. piscicida in fish serum. TX01, TX01∆trxH, and TX01∆trxHC were incubated with non-immune flounder serum or PBS (control). After incubation, the survival of the bacteria was determined by plate counting. B The biofilm forming capacity of E. piscicida. TX01, TX01∆trxH, and TX01∆trxHC were incubated in polystyrene plate and biofilm formation was determined by measuring the the A₅₇₀ of final eluates. Data are presented as mean ± SEM (N = 3). N, the number of times the experiment was performed. **P < 0.01.

Figure 4

Effect of trxH_Ep mutation on cellular infection and replication. A The invasion of Edwardsiella piscicida to flounder gill cells (FG cells). FG cells were infected with the same dose of TX01, TX01∆trxH, and TX01∆trxHC for various hours and washed with PBS. Then FG cells were lysed and the bacterial associated with and invaded into the host cells were determined. B FG cells were incubated with FITC-labeled E. piscicida TX01∆trxH or TX01, and the fluorescence of extracellular bacteria was quenched by adding trypan blue, then cells were observed with a confocal microscope. C Replication of E. piscicida in mouse macrophage cell RAW264.7. RAW264.7 cells were hatched with TX01, TX01∆trxH, and TX01∆trxHC for 2 h, then extracellular bacteria were killed. The cells were then incubated further for various hours, and the number of intracellular bacteria was determined by plate counting. Data are the means of three independent experiments and presented as mean ± SEM (N = 3). N, the number of times the experiment was performed. **P < 0.01.

Figure 5

In vivo infection of Edwardsiella piscicida in Japanese flounder. A Bacterial dissemination in the fish tissues. Flounder were infected with the same dose of TX01, TX01∆trxH, or TX01∆trxHC, bacterial recoveries from spleen and kidney were determined by plate count at 24 and 48 hpi. B Host mortality induced by E. piscicida. Flounder were infected with equivalent doses of TX01, TX01∆trxH, and TX01∆trxHC, and accumulated mortality were monitored for a period of 20 days (only 15 days are shown since no more deaths occurred after 15 days). Significance between the survivals of wild type and mutant infected fish was determined with logrank test. Data are presented as means ± SEM (N = 3). N, the number of times the experiment was performed. *P < 0.05, **P < 0.01.

Figure 6

Expression analysis of trxH_Ep. A Expression of trxH_Ep under different conditions. RT-qPCR was performed with total RNA extracted from Edwardsiella piscicida TX01 cultured in normal LB medium (control, pH = 7), in hydrogen peroxide condition (normal LB medium with H₂O₂), in acid condition (pH = 5), in iron limitation condition (with the iron chelator 2,2′dipyridyl, Dp), Expression level of trxH_Ep in normal LB medium was set as 1. B RT-qPCR was performed with total RNA extracted from TX01 incubated with or without (control) FG cells. Expression level of trxH_Ep without FG cells was set as 1. C RT-qPCR was performed with total RNA extracted from wild type TX01 and fur mutant strain cultured in normal LB medium. Expression level of trxH_Ep in wild type TX01 strain was set as 1. D DH5α/pSH510/pTFur and DH5α/pSH510/pT were streaked and cultured on LB plate with X-gal, kanamycin, and ampicillin. Data are presented as mean ± SEM (N = 3). N, the number of times the experiment was performed. **P < 0.01.

Figure 7

Expression of trxC and trxA in wild type Edwardsiella piscicida TX01 strain and TX01∆trxH strain. Total RNA was extracted from E. piscicida TX01 and TX01∆trxH cultured in normal LB medium (control, pH = 7), in hydrogen peroxide condition (with H₂O₂), in acid condition (pH = 5), or incubating with FG cells, respectively. Expressions of trxC and trxA were analyzed by RT-qPCR. Data are presented as mean ± SEM (N = 3). N, the number of times the experiment was performed. **P < 0.01.