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In vitro Edwardsiella piscicida CK108 Transcriptome Profiles with Subinhibitory Concentrations of Phenol and Formalin Reveal New Insights into Bacterial Pathogenesis Mechanisms.

ABSTRACT: Phenol and formalin are major water pollutants that are frequently discharged into the aquatic milieu. These chemicals can affect broad domains of life, including microorganisms. Aquatic pollutants, unlike terrestrial pollutants, are easily diluted in water environments and exist at a sub-inhibitory concentration (sub-IC), thus not directly inhibiting bacterial growth. However, they can modulate gene expression profiles. The sub-IC values of phenol and formalin were measured by minimal inhibitory concentration (MIC) assay to be 0.146% (1.3 mM) and 0.0039% (0.38 mM), respectively, in Edwardsiella piscicida CK108, a Gram-negative fish pathogen. We investigated the differentially expressed genes (DEG) by RNA-seq when the cells were exposed to the sub-ICs of phenol and formalin. DEG analyses revealed that genes involved in major virulence factors (type I fimbriae, flagella, type III and type VI secretion system) and various cellular pathways (energy production, amino acid synthesis, carbohydrate metabolism and two-component regulatory systems) were up- or downregulated by both chemicals. The genome-wide gene expression data corresponded to the results of a quantitative reverse complementary-PCR and motility assay. This study not only provides insight into how a representative fish pathogen, E. piscicida CK108, responds to the sub-ICs of phenol and formalin but also shows the importance of controlling chemical pollutants in aquatic environments.

SUBMITTER: Yoon JB

PROVIDER: S-EPMC7409036 | biostudies-literature | 2020 Jul

REPOSITORIES: biostudies-literature

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In vitro <i>Edwardsiella piscicida</i> CK108 Transcriptome Profiles with Subinhibitory Concentrations of Phenol and Formalin Reveal New Insights into Bacterial Pathogenesis Mechanisms.

Yoon Ju Bin JB Hwang Sungmin S Baek Se-Won SW Lee Seungki S Bang Woo Young WY Moon Ki Hwan KH

Microorganisms 20200717 7

Phenol and formalin are major water pollutants that are frequently discharged into the aquatic milieu. These chemicals can affect broad domains of life, including microorganisms. Aquatic pollutants, unlike terrestrial pollutants, are easily diluted in water environments and exist at a sub-inhibitory concentration (sub-IC), thus not directly inhibiting bacterial growth. However, they can modulate gene expression profiles. The sub-IC values of phenol and formalin were measured by minimal inhibitor ...[more]

PMID: 32709101

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Project description:Edwardsiella piscicida is found to be an important facultative intracellular pathogen with a broad host range. These organisms can replicate and survive within host macrophages to escape from the subversion of the immune defense. E. piscicida-macrophage interaction is very important in determining the outcome of edwardsiellasis. As an effector protein of E. piscicida T6SS, EvpP has been determined to be a very important virulence factor for E. piscicida, although its precise role in E. piscicida-macrophage interactions is not yet clear. In this study, the roles of EvpP in E. piscicida-macrophage interactions were characterized. Here, we constructed the deletion mutants of evpP (?evpP) and complementation (?evpP-C) by the allelic exchange method. Compared to wild type strain (WT), ?evpP was found to be attenuated for growth within macrophages. In line with this observation, we found its survival capacity was lower than WT under oxidative and acid stress in vitro, which simulate conditions encountered in host macrophages. Attenuation of ?evpP also correlated with enhanced activation of macrophages, as reflected by augmented NO production in ?evpP-treated macrophages. Moreover, compared to WT, ?evpP induced markedly increased apoptosis of macrophages, characterized by increased Annexin V binding and the activation of cleaved caspase-3. These findings provided strong evidence that EvpP is involved in the process of E. piscicida-macrophage interactions and is required for its survival and replication in macrophages. Thus, we propose that EvpP might be an important factor that controlling the fate of E. piscicida inside macrophages. To further exploring the underlying mechanism of EvpP action, the cDNA library was constructed from E. piscicida-infected macrophages and a yeast two-hybrid screen was performed to search for cellular proteins interacting with EvpP. Ribosomal protein S5 (RPS5) was identified as a target of EvpP. Furthermore, the interaction was validated with co-immunoprecipitation assay. This result implies that the observed effect of EvpP on macrophages might be related to RPS5-mediated regulation, contributing to a better understanding of the mechanisms of EvpP involved in E. piscicida-macrophage interactions.

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Project description:Edwardsiella piscicida is an important pathogen that infects a wide range of hosts from fish to human. Recent studies demonstrated that E. piscicida can invade and survive within multiple nonphagocytic cells, but the internalization mechanism remains poorly understood. Here, we used HeLa cells as a nonphagocytic cell model to investigate the endocytic strategy used by the pathogenic E. piscicida isolate EIB202. Using a combination of optical and electron microscopy, we observed obvious membrane ruffles and F-actin rearrangements in HeLa cells after EIB202 infection. We also revealed that EIB202 internalization significantly depended on the activity of Na+/H+ exchangers and multiple intracellular signaling events related to macropinocytosis, suggesting that E. piscicida utilizes the host macropinocytosis pathway to enter HeLa cells. Further, using inhibitory drugs and shRNAs to block specific endocytic pathways, we found that a caveolin-dependent but not clathrin-dependent pathway is involved in E. piscicida entry and that its entry requires dynamin and membrane cholesterol. Together, these data suggest that E. piscicida enters nonphagocytic cells via macropinocytosis and caveolin-dependent endocytosis involving cholesterol and dynamin, improving the understanding of how E. piscicida interacts with nonphagocytic cells.IMPORTANCE Bacterial internalization is the first step in breaking through the host cell defense. Therefore, studying the mechanism of bacterial internalization improves the understanding of the pathogenic mechanism of bacteria. In this study, the internalization process on nonphagocytic cells by Edwardsiella piscicida was evaluated. Our results showed that E. piscicida can be internalized into nonphagocytic cells via macropinocytosis and caveolin-mediated endocytosis, and that cholesterol and dynamin are involved in this process. These results reveal a new method for inhibiting E. piscicida infection, providing a foundation for further studies of bacterial pathogenicity.

Dataset Information

In vitro Edwardsiella piscicida CK108 Transcriptome Profiles with Subinhibitory Concentrations of Phenol and Formalin Reveal New Insights into Bacterial Pathogenesis Mechanisms.

Publications

In vitro <i>Edwardsiella piscicida</i> CK108 Transcriptome Profiles with Subinhibitory Concentrations of Phenol and Formalin Reveal New Insights into Bacterial Pathogenesis Mechanisms.

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