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Abstract <p><i>Aeromonas dhakensis</i>, an emerging opportunistic pathogen prevalent in tropical regions, poses significant threats to both public health and aquaculture industries. This study elucidates the synergistic effects of dual deletion of the two-component systems PhoBR and KdpDE on virulence attenuation. Using homologous recombination and seamless cloning techniques, we constructed a Δ<i>phoBR</i>Δ<i>kdpE</i> double mutant based on our laboratory's existing Δ<i>phoBR</i> mutant. Notably, phenotypic characterization revealed that the double mutant maintained comparable growth kinetics to wild-type, Δ<i>phoBR</i> mutant, and complemented strains under high-potassium conditions. However, under potassium-limiting conditions, Δ<i>phoBR</i>Δ<i>kdpE</i> exhibited significantly impaired growth, compromised antioxidant capacity, diminished biofilm formation, reduced extracellular protease activity, and attenuated cell adhesion compared to controls. Furthermore, the mutant demonstrated enhanced susceptibility to antimicrobial peptides and marked virulence attenuation in zebrafish models, as evidenced by a substantially higher LD<sub>50</sub> (6.53 × 10<sup>8</sup>&#xa0;CFU/mL) compared to Δ<i>phoBR</i> (1.15 × 10<sup>8</sup>&#xa0;CFU/mL) and wild-type strains (2.91 × 10<sup>7</sup>&#xa0;CFU/mL). Intriguingly, immunization trials showed moderate protective efficacy (43.3% survival) for the double mutant, though not statistically different from Δ<i>phoBR</i> (50% survival). This study provides the first evidence that Δ<i>phoBR</i>Δ<i>kdp</i>E mutant strain reduces virulence while maintaining partial immunogenicity, laying crucial groundwork for developing crocodile-targeted live attenuated vaccines against <i>A. dhakensis</i>.</p> Key points <p><i>• PhoBR/KdpE deletion</i><i> attenuates A. dhakensis virulence via K+ uptake and virulence factors.</i></p> <p><i>• ΔphoBRΔkdpE shows enhanced stress sensitivity but retains immunogenicity in zebrafish.</i></p> <p><i>• ΔphoBRΔkdpE is a promising live vaccine candidate against A. dhakensis in aquaculture.</i></p>

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Synergistic attenuation of Aeromonas dhakensis virulence by dual deletion of PhoBR and KdpE two-component systems and its protective immunity in zebrafish

  • 期刊论文

  • 出版模式：

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  • 发表日期：

    2026年5月15日

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  • Xuesong Li,
  • Yukun Wu,
  • Mengqi Wang,
  • Roushan Li,
  • Haibo Gao,
  • Chenlu Yang,
  • Aodi Yi,
  • Xueqian Wang,
  • Shuai Ye,
  • Guiying Guo,
  • Lixia Fan,
  • Nuo Yang,
  • Jifeng Zeng,
  • Jiping Zheng

摘要

Abstract

Aeromonas dhakensis, an emerging opportunistic pathogen prevalent in tropical regions, poses significant threats to both public health and aquaculture industries. This study elucidates the synergistic effects of dual deletion of the two-component systems PhoBR and KdpDE on virulence attenuation. Using homologous recombination and seamless cloning techniques, we constructed a ΔphoBRΔkdpE double mutant based on our laboratory's existing ΔphoBR mutant. Notably, phenotypic characterization revealed that the double mutant maintained comparable growth kinetics to wild-type, ΔphoBR mutant, and complemented strains under high-potassium conditions. However, under potassium-limiting conditions, ΔphoBRΔkdpE exhibited significantly impaired growth, compromised antioxidant capacity, diminished biofilm formation, reduced extracellular protease activity, and attenuated cell adhesion compared to controls. Furthermore, the mutant demonstrated enhanced susceptibility to antimicrobial peptides and marked virulence attenuation in zebrafish models, as evidenced by a substantially higher LD50 (6.53 × 108 CFU/mL) compared to ΔphoBR (1.15 × 108 CFU/mL) and wild-type strains (2.91 × 107 CFU/mL). Intriguingly, immunization trials showed moderate protective efficacy (43.3% survival) for the double mutant, though not statistically different from ΔphoBR (50% survival). This study provides the first evidence that ΔphoBRΔkdpE mutant strain reduces virulence while maintaining partial immunogenicity, laying crucial groundwork for developing crocodile-targeted live attenuated vaccines against A. dhakensis.

Key points

• PhoBR/KdpE deletion attenuates A. dhakensis virulence via K+ uptake and virulence factors.

• ΔphoBRΔkdpE shows enhanced stress sensitivity but retains immunogenicity in zebrafish.

• ΔphoBRΔkdpE is a promising live vaccine candidate against A. dhakensis in aquaculture.