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Comparative transcriptomic and proteomic analyses reveal upregulated expression of virulence and iron transport factors of Aeromonas hydrophila under iron limitation.

ABSTRACT: BACKGROUND:Iron plays important roles in the growth, reproduction and pathogenicity of Aeromonas hydrophila. In this study, we detected and compared the mRNA and protein expression profiles of A. hydrophila under normal and iron restricted medium with 200 ?M 2,2-Dipyridyl using RNA Sequencing (RNA-seq) and isobaric tags for relative and absolute quantification (iTRAQ) analyses. RESULTS:There were 1204 genes (601 up- and 603 down-regulated) and 236 proteins (90 up- and 146 down-regulated) shown to be differentially expressed, and 167 genes and proteins that showed consistent expression. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses revealed that the differentially expressed genes and proteins were mainly involved in iron ion transport, protein activity, energy metabolism and virulence processes. Further validation of the RNA-seq and iTRAQ results by quantitative real-time PCR (qPCR) revealed that 18 of the 20 selected genes were consistently expressed. The iron-ion absorption and concentration of A. hydrophila under iron-limited conditions were enhanced, and most virulence factors (protease activity, hemolytic activity, lipase activity, and swimming ability) were also increased. Artificial A. hydrophila infection caused higher mortality in cyprinid Megalobrama amblycephala under iron-limited conditions. CONCLUSION:Understanding the responses of pathogenic Aeromonas hydrophila within the hostile environment of the fish host, devoid of free iron, is important to reveal bacterial infection and pathogenesis. This study further confirmed the previous finding that iron-limitation efficiently enhanced the virulence of A. hydrophila using multi-omics analyses. We identified differentially expressed genes and proteins, related to enterobactin synthesis and virulence establishment, that play important roles in addressing iron scarcity.

SUBMITTER: Teng T

PROVIDER: S-EPMC5987420 | biostudies-literature | 2018 Jun

REPOSITORIES: biostudies-literature

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Comparative transcriptomic and proteomic analyses reveal upregulated expression of virulence and iron transport factors of Aeromonas hydrophila under iron limitation.

Teng Tao T Xi Bingwen B Chen Kai K Pan Liangkun L Xie Jun J Xu Pao P

BMC microbiology 20180604 1

<h4>Background</h4>Iron plays important roles in the growth, reproduction and pathogenicity of Aeromonas hydrophila. In this study, we detected and compared the mRNA and protein expression profiles of A. hydrophila under normal and iron restricted medium with 200 μM 2,2-Dipyridyl using RNA Sequencing (RNA-seq) and isobaric tags for relative and absolute quantification (iTRAQ) analyses.<h4>Results</h4>There were 1204 genes (601 up- and 603 down-regulated) and 236 proteins (90 up- and 146 down-reg ...[more]

PMID: 29866030

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Project description:Among the various virulence factors produced by Aeromonas hydrophila, a type II secretion system (T2SS)-secreted cytotoxic enterotoxin (Act) and the T3SS are crucial in the pathogenesis of Aeromonas-associated infections. Our laboratory molecularly characterized both Act and the T3SS from a diarrheal isolate, SSU of A. hydrophila, and defined the role of some regulatory genes in modulating the biological effects of Act. In this study, we cloned, sequenced, and expressed the DNA adenine methyltransferase gene of A. hydrophila SSU (dam(AhSSU)) in a T7 promoter-based vector system using Escherichia coli ER2566 as a host strain, which could alter the virulence potential of A. hydrophila. Recombinant Dam, designated as M.AhySSUDam, was produced as a histidine-tagged fusion protein and purified from an E. coli cell lysate using nickel affinity chromatography. The purified Dam had methyltransferase activity, based on its ability to transfer a methyl group from S-adenosyl-l-methionine to N(6)-methyladenine-free lambda DNA and to protect methylated lambda DNA from digestion with DpnII but not against the DpnI restriction enzyme. The dam gene was essential for the viability of the bacterium, and overproduction of Dam in A. hydrophila SSU, using an arabinose-inducible, P(BAD) promoter-based system, reduced the virulence of this pathogen. Specifically, overproduction of M.AhySSUDam decreased the motility of the bacterium by 58%. Likewise, the T3SS-associated cytotoxicity, as measured by the release of lactate dehydrogenase enzyme in murine macrophages infected with the Dam-overproducing strain, was diminished by 55% compared to that of a control A. hydrophila SSU strain harboring the pBAD vector alone. On the contrary, cytotoxic and hemolytic activities associated with Act as well as the protease activity in the culture supernatant of a Dam-overproducing strain were increased by 10-, 3-, and 2.4-fold, respectively, compared to those of the control A. hydrophila SSU strain. The Dam-overproducing strain was not lethal to mice (100% survival) when given by the intraperitoneal route at a dose twice that of the 50% lethal dose, which within 2 to 3 days killed 100% of the animals inoculated with the A. hydrophila control strain. Taken together, our data indicated alteration of A. hydrophila virulence by overproduction of Dam.

Project description:Many gram-negative bacteria use a type III secretion system (TTSS) to deliver effector proteins into host cells. Here we report the characterization of a TTSS chromosomal operon from the diarrheal isolate SSU of Aeromonas hydrophila. We deleted the gene encoding Aeromonas outer membrane protein B (AopB), which is predicted to be involved in the formation of the TTSS translocon, from wild-type (WT) A. hydrophila as well as from a previously characterized cytotoxic enterotoxin gene (act)-minus strain of A. hydrophila, thus generating aopB and act/aopB isogenic mutants. The act gene encodes a type II-secreted cytotoxic enterotoxin (Act) that has hemolytic, cytotoxic, and enterotoxic activities and induces lethality in a mouse model. These isogenic mutants (aopB, act, and act/aopB) were highly attenuated in their ability to induce cytotoxicity in RAW 264.7 murine macrophages and HT-29 human colonic epithelial cells. The act/aopB mutant demonstrated the greatest reduction in cytotoxicity to cultured cells after 4 h of infection, as measured by the release of lactate dehydrogenase enzyme, and was avirulent in mice, with a 90% survival rate compared to that of animals infected with Act and AopB mutants, which caused 50 to 60% of the animals to die at a dose of three 50% lethal doses. In contrast, WT A. hydrophila killed 100% of the mice within 48 h. The effects of these mutations on cytotoxicity could be complemented with the native genes. Our studies further revealed that the production of lactones, which are involved in quorum sensing (QS), was decreased in the act (32%) and aopB (64%) mutants and was minimal (only 8%) in the act/aopB mutant, compared to that of WT A. hydrophila SSU. The effects of act and aopB gene deletions on lactone production could also be complemented with the native genes, indicating specific effects of Act and the TTSS on lactone production. Although recent studies with other bacteria have indicated TTSS regulation by QS, this is the first report describing a correlation between the TTSS and Act of A. hydrophila and the production of lactones.

Dataset Information

Comparative transcriptomic and proteomic analyses reveal upregulated expression of virulence and iron transport factors of Aeromonas hydrophila under iron limitation.

Publications

Comparative transcriptomic and proteomic analyses reveal upregulated expression of virulence and iron transport factors of Aeromonas hydrophila under iron limitation.

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