Abstract

Aeromonas hydrophila is an important aquatic microorganism that can cause fish hemorrhagic septicemia. In this study, we identified a novel LysR family transcriptional regulator (LahS) in the A. hydrophila Chinese epidemic strain NJ-35 from a library of 947 mutant strains. The deletion of lahS caused bacteria to exhibit significantly decreased hemolytic activity, motility, biofilm formation, protease production, and anti-bacterial competition ability when compared to the wild-type strain. In addition, the determination of the fifty percent lethal dose (LD50) in zebrafish demonstrated that the lahS deletion mutant (ΔlahS) was highly attenuated in virulence, with an approximately 200-fold increase in LD50 observed as compared with that of the wild-type strain. However, the ΔlahS strain exhibited significantly increased antioxidant activity (six-fold). Label-free quantitative proteome analysis resulted in the identification of 34 differentially expressed proteins in the ΔlahS strain. The differentially expressed proteins were involved in flagellum assembly, metabolism, redox reactions, and cell density induction. The data indicated that LahS might act as a global regulator to directly or indirectly regulate various biological processes in A. hydrophila NJ-35, contributing to a greater understanding the pathogenic mechanisms of A. hydrophila.

Highlights

  • Aeromonas hydrophila is a gram-negative bacterium that is widely distributed in various aquatic environments, such as rivers, lakes, and swamps

  • Hemolytic molecules may contribute to the reddening skin and systemic hemorrhagic septicemia; symptoms that are observed in diseased fish infected with A. hydrophila, and have been defined as one of the major virulence markers of this bacterium [5]

  • In addition to hemolytic activity, LahS positively regulates a variety of virulence factors, including motility, biofilm formation, and protease activity, and it is involved in the anti-bacterial competition ability and virulence of this bacterium

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Summary

Introduction

Aeromonas hydrophila is a gram-negative bacterium that is widely distributed in various aquatic environments, such as rivers, lakes, and swamps. In addition to hemolytic activity, LahS positively regulates a variety of virulence factors, including motility, biofilm formation, and protease activity, and it is involved in the anti-bacterial competition ability and virulence of this bacterium. This is the first study to evaluate the role of LahS in virulence and environmental adaptation in A. hydrophila, the results of which are of great importance in gaining a better understanding of the pathogenesis of this bacterium

Isolation of Transposon Mutants with Reduced Hemolytic Activity
Materials and Methods
Screening Transposon Insertion Mutants for Hemolytic Activity
Identification of Insertion Sites by Tail-PCR
Construction of a lahS Mutant and Complemented Strains
Biofilm Formation Assay
Motility Assay
Anti-Bacterial Competition Assay
Oxidative Stress Resistance Test
Protease Activity
4.10. Determination of LD50 in Zebrafish
4.11. Comparative Proteomic Analysis
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