Identification of Aeromonas hydrophila Genes Preferentially Expressed after Phagocytosis by Tetrahymena and Involvement of Methionine Sulfoxide Reductases.

Maoda Pang,Yongjie Liu,Hechao Du,Chengping Lu,Changming Guo,Jin Liu,Xiaoqin Lin,Shanshan Gao

doi:10.3389/fcimb.2016.00199

Abstract

Free-living protozoa affect the survival and virulence evolution of pathogens in the environment. In this study, we explored the fate of Aeromonas hydrophila when co-cultured with the bacteriovorous ciliate Tetrahymena thermophila and investigated bacterial gene expression associated with the co-culture. Virulent A. hydrophila strains were found to have ability to evade digestion in the vacuoles of this protozoan. In A. hydrophila, a total of 116 genes were identified as up-regulated following co-culture with T. thermophila by selective capture of transcribed sequences (SCOTS) and comparative dot-blot analysis. A large proportion of these genes (42/116) play a role in metabolism, and some of the genes have previously been characterized as required for bacterial survival and replication within macrophages. Then, we inactivated the genes encoding methionine sulfoxide reductases, msrA, and msrB, in A. hydrophila. Compared to the wild-type, the mutants ΔmsrA and ΔmsrAB displayed significantly reduced resistance to predation by T. thermophila, and 50% lethal dose (LD50) determinations in zebrafish demonstrated that both mutants were highly attenuated. This study forms a solid foundation for the study of mechanisms and implications of bacterial defenses.

Highlights

Aeromonas hydrophila, a Gram-negative ubiquitous bacterium with diverse host specificity, is distributed widely in aquatic environments (Daskalov, 2006; Janda and Abbott, 2010)
We investigated the fate of A. hydrophila strains after co-culture with T. thermophila and used selective capture of transcribed sequences (SCOTS) to identify the genes that were preferentially expressed by A. hydrophila upon interaction with this protozoan
To investigate the fate of A. hydrophila in response to phagocytosis by T. thermophila, 12 A. hydrophila strains of different virulence were intrinsically labeled with green fluorescent protein (GFP) by transformation with the plasmid pWSK129-gfp

Summary

Introduction

A Gram-negative ubiquitous bacterium with diverse host specificity, is distributed widely in aquatic environments (Daskalov, 2006; Janda and Abbott, 2010). Aeromonas infection has been linked to major die-offs and fish kills and has resulted in significant economic losses around the world for decades (Pang et al, 2015) This bacterium has been proposed to cause a variety of serious illnesses in other cold-blooded species and humans (Janda and Abbott, 2010). The pathogenesis of A. hydrophila is multifactorial and is likely mediated by virulence factors such as adhesins, exotoxins, extracellular enzymes, secretion systems, iron acquisition systems, and quorum-sensing systems (Tomas, 2012). Environmental factors, such as predation by heterotrophic protists, have a dramatic effect on the virulence evolution of pathogens (Erken et al, 2013). The mechanism underlying this has not been investigated in A. hydrophila

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Conclusion