The Thioredoxin System in Edwardsiella piscicida Contributes to Oxidative Stress Tolerance, Motility, and Virulence

Abstract

Edwardsiella piscicida is an important fish pathogen that causes substantial economic losses. In order to understand its pathogenic mechanism, additional new virulence factors need to be identified. The bacterial thioredoxin system is a major disulfide reductase system, but its function is largely unknown in E. piscicida. In this study, we investigated the roles of the thioredoxin system in E. piscicida (named TrxB_Ep, TrxA_Ep, and TrxC_Ep, respectively) by constructing a correspondingly markerless in-frame mutant strain: ΔtrxB, ΔtrxA, and ΔtrxC, respectively. We found that (i) TrxB_Ep is confirmed as an intracellular protein, which is different from the prediction made by the Protter illustration; (ii) compared to the wild-type strain, ΔtrxB exhibits resistance against H₂O₂ stress but high sensitivity to thiol-specific diamide stress, while ΔtrxA and ΔtrxC are moderately sensitive to both H₂O₂ and diamide conditions; (iii) the deletions of trxB_Ep, trxA_Ep, and trxC_Ep damage E. piscicida's flagella formation and motility, and trxB_Ep plays a decisive role; (iv) deletions of trxB_Ep, trxA_Ep, and trxC_Ep substantially abate bacterial resistance against host serum, especially trxB_Ep deletion; (v) trxA_Ep and trxC_Ep, but not trxB_Ep, are involved in bacterial survival and replication in phagocytes; (vi) the thioredoxin system participates in bacterial dissemination in host immune tissues. These findings indicate that the thioredoxin system of E. piscicida plays an important role in stress resistance and virulence, which provides insight into the pathogenic mechanism of E. piscicida.

Keywords: Edwardsiella piscicida; motility; oxidative stress; thioredoxin system; virulence.

Figure 1

Multiple sequence alignments of TrxB (A), TrxA (B), and TrxC (C) with their homologues. The consensus amino acid residues are shown in blue, and the amino acid residues with conservative degree higher than 75% are shown in light blue. A consensus sequence logo has been created using Jalview. The GenBank accession numbers of the TrxB homologues are as follows: Edwardsiella piscicida, WP_012849033.1; Hafnia alvei, WP_149226215.1; Escherichia coli, NP_415408.1; Salmonella enterica, AGK67561.1; Leminorella richardii, SQI39560.1; Listeria monocytogenes, WP_003722610.1. The GenBank accession numbers of the TrxA homologues are as follows: E. piscicida, WP_012846980.1; H. alvei, WP_115349310.1; Escherichia coli, HAW3242924.1; S. enterica, AGK68906.1; L. richardii, WP_111741836.1; L. monocytogenes, NP_464758.1. The GenBank accession numbers of the TrxC homologues are as follows: E. piscicida, WP_012847430.1; H. alvei, WP_130998671.1; E. coli, NP_417077.1; S. enterica, NP_461584.1; L. richardii, WP_111739434.1; L. monocytogenes, WP_003723835.1.

Figure 1

Multiple sequence alignments of TrxB (A), TrxA (B), and TrxC (C) with their homologues. The consensus amino acid residues are shown in blue, and the amino acid residues with conservative degree higher than 75% are shown in light blue. A consensus sequence logo has been created using Jalview. The GenBank accession numbers of the TrxB homologues are as follows: Edwardsiella piscicida, WP_012849033.1; Hafnia alvei, WP_149226215.1; Escherichia coli, NP_415408.1; Salmonella enterica, AGK67561.1; Leminorella richardii, SQI39560.1; Listeria monocytogenes, WP_003722610.1. The GenBank accession numbers of the TrxA homologues are as follows: E. piscicida, WP_012846980.1; H. alvei, WP_115349310.1; Escherichia coli, HAW3242924.1; S. enterica, AGK68906.1; L. richardii, WP_111741836.1; L. monocytogenes, NP_464758.1. The GenBank accession numbers of the TrxC homologues are as follows: E. piscicida, WP_012847430.1; H. alvei, WP_130998671.1; E. coli, NP_417077.1; S. enterica, NP_461584.1; L. richardii, WP_111739434.1; L. monocytogenes, WP_003723835.1.

Figure 2

Cellular localization of TrxB_Ep. (A) Protter illustration of TrxB_Ep, TrxA_Ep and TrxC_Ep. (B) protein components of Edwardsiella piscicida cells. (C) The distribution of TrxB_Ep tested by Western blot.

Figure 3

The roles of the Trx system in bacterial adversity resistance. (A) WT, ΔtrxB, ΔtrxA, and ΔtrxC were cultured in LB broth, and then the cell density was measured at OD_600, and the intracellular reductive capacity was detected. (B) Bacterial growths on LB agar plates (1) and in liquid LB (3) with 300 μM H₂O₂; bacteria in the logarithmic growth phase were challenged with 30 mM H₂O₂ for 5 h, and the intracellular reductive capacity was determined (2). Bacteria in the logarithmic growth phase were diluted to 1:1000 and treated with PBS containing 300 mM H₂O₂ for 1 h, then the amount of viable bacteria was determined (4). (C) bacterial growths on LB agar plates (1) and in liquid LB (3) with 550 mM diamide; bacteria in the logarithmic growth phase were challenged with 50 mM diamide for 5 h, and intracellular reductive capacity was detected (2). Bacteria in the logarithmic growth phase were diluted to 1:1000 and treated with PBS containing 550 mM diamide for 1 h, then the content of viable bacteria was determined (4). (D) Strains were cultured in LB broth containing acid pressure (pH = 5) (1) or iron deficiency stress (100 μM Dp) (2) and incubated at 28 °C for 48 h. Data are expressed as means ± SEM (N = 3). N, the number of experiments performed. p values were obtained by analysis of variance using SPSS 23.

Figure 3

The roles of the Trx system in bacterial adversity resistance. (A) WT, ΔtrxB, ΔtrxA, and ΔtrxC were cultured in LB broth, and then the cell density was measured at OD_600, and the intracellular reductive capacity was detected. (B) Bacterial growths on LB agar plates (1) and in liquid LB (3) with 300 μM H₂O₂; bacteria in the logarithmic growth phase were challenged with 30 mM H₂O₂ for 5 h, and the intracellular reductive capacity was determined (2). Bacteria in the logarithmic growth phase were diluted to 1:1000 and treated with PBS containing 300 mM H₂O₂ for 1 h, then the amount of viable bacteria was determined (4). (C) bacterial growths on LB agar plates (1) and in liquid LB (3) with 550 mM diamide; bacteria in the logarithmic growth phase were challenged with 50 mM diamide for 5 h, and intracellular reductive capacity was detected (2). Bacteria in the logarithmic growth phase were diluted to 1:1000 and treated with PBS containing 550 mM diamide for 1 h, then the content of viable bacteria was determined (4). (D) Strains were cultured in LB broth containing acid pressure (pH = 5) (1) or iron deficiency stress (100 μM Dp) (2) and incubated at 28 °C for 48 h. Data are expressed as means ± SEM (N = 3). N, the number of experiments performed. p values were obtained by analysis of variance using SPSS 23.

Figure 4

The effects of the thioredoxin system mutations on bacterial motility and flagellum formation. (A) The swimming of Edwardsiella piscicida. WT, ΔtrxB, ΔtrxA, and ΔtrxC were cultured in LB medium to an OD₆₀₀ of 0.6, and 1 μL cell suspensions were spotted onto the center of swimming plates containing LB medium plus 0.3% (w/v) agar. The plates were incubated at 28 °C for 24 h, and the motility zone diameter was measured. (B) The swarming of E. piscicida. Bacteria as described above were spotted onto the center of swimming plates containing LB medium plus 0.6% (w/v) agar and were incubated at 28 °C for 24 h. (C), the flagellum observation of E. piscicida. WT, ΔtrxB, ΔtrxA and ΔtrxC were grown in LB medium, and the flagella were observed using the TEM. Data are presented as the means ± SEM (N = 3). N, the number of times the experiments were performed. p values were obtained by analysis of variance using SPSS 23.

Figure 5

The effects of thioredoxin system mutations on bacterial survival in host serum and host macrophages. (A) The survival rate of WT, ΔtrxB, ΔtrxA and ΔtrxC against non-immune fish serum. Strains in the early logarithmic phase were incubated with non-immune tilapia serum or PBS (control) for 1 h. The number of viable bacteria was determined. (B) Edwardsiella piscicida replication in macrophages. The murine macrophage cell line RAW264.7 was incubated with WT, ΔtrxB, ΔtrxA and ΔtrxC for 2 h. After killing and washing extracellular bacteria, the macrophages were cultured for different lengths of time. At each time point, the viable intracellular bacteria were determined. Data are presented as the means ± SEM (N = 3). N, the number of times the experiments were performed. p values were obtained by analysis of variance using SPSS 23.

Figure 6

Effects of the thioredoxin system mutations on bacterial virulence. (A) Edwardsiella piscicida strains (WT, ΔtrxB, ΔtrxA, and ΔtrxC) were used to infect tilapias, then, after 24 h, bacteria recovery from immune tissues (spleen and kidney) was analyzed by plate counting. (B) 48 h post-infection, bacteria were counted by plate counting. Data are presented as the means ± SEM (N = 3). N, the number of times the experiments were performed. p values were obtained by analysis of variance using SPSS 23.