Dual RNA-Seq Unveils the Role of the  Pseudomonas plecoglossicida fliA  Gene in Pathogen-Host Interaction with  Larimichthys crocea

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Abstract

In the present study, Larimichthys crocea and Pseudomonas plecoglossicida were selected as a host-pathogen interaction model for teleosts and prokaryotic pathogens. Five shRNAs were designed and synthesized to silence the fliA gene, all of which resulted in pronounced reductions in fliA mRNA; the mutant strain with the best silencing efficiency of 92.16% was chosen for subsequent analysis. A significant decrease in motility, intracellular survival and escape was observed for the fliA-RNAi strain of P. plecoglossicida, whereby silencing of the fliA gene led to a 30% decrease in mortality and a four-day delay in the onset of infection in L. crocea. Moreover, silencing of P. plecoglossicida fliA resulted in a significant change in both the pathogen and host transcriptome in the spleens of infected L. crocea. Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis of pathogen transcriptome data showed that silencing fliA resulted in downregulation of 18 flagellum-related genes; KEGG analysis of host transcriptome data revealed that infection with the fliA-RNAi strain caused upregulation of 47 and downregulation of 106 immune-related genes. These pathogen-host interactions might facilitate clearance of P. plecoglossicida by L. crocea, with a significant decrease in fliA-RNAi P. plecoglossicida strain virulence in L. crocea.

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Most cited references 49

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Flagellar and twitching motility are necessary for Pseudomonas aeruginosa biofilm development.

G. O'toole, R. Kolter (1998)

The formation of complex bacterial communities known as biofilms begins with the interaction of planktonic cells with a surface in response to appropriate environmental signals. We report the isolation and characterization of mutants of Pseudomonas aeruginosa PA14 defective in the initiation of biofilm formation on an abiotic surface, polyvinylchloride (PVC) plastic. These mutants are designated surface attachment defective (sad ). Two classes of sad mutants were analysed: (i) mutants defective in flagellar-mediated motility and (ii) mutants defective in biogenesis of the polar-localized type IV pili. We followed the development of the biofilm formed by the wild type over 8 h using phase-contrast microscopy. The wild-type strain first formed a monolayer of cells on the abiotic surface, followed by the appearance of microcolonies that were dispersed throughout the monolayer of cells. Using time-lapse microscopy, we present evidence that microcolonies form by aggregation of cells present in the monolayer. As observed with the wild type, strains with mutations in genes required for the synthesis of type IV pili formed a monolayer of cells on the PVC plastic. However, in contrast to the wild-type strain, the type IV pili mutants did not develop microcolonies over the course of the experiments, suggesting that these structures play an important role in microcolony formation. Very few cells of a non-motile strain (carrying a mutation in flgK) attached to PVC even after 8 h of incubation, suggesting a role for flagella and/or motility in the initial cell-to-surface interactions. The phenotype of these mutants thus allows us to initiate the dissection of the developmental pathway leading to biofilm formation.

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Dual RNA-seq unveils noncoding RNA functions in host-pathogen interactions.

Alexander Westermann, Konrad U Förstner, Fabian Amman … (2016)

Bacteria express many small RNAs for which the regulatory roles in pathogenesis have remained poorly understood due to a paucity of robust phenotypes in standard virulence assays. Here we use a generic 'dual RNA-seq' approach to profile RNA expression simultaneously in pathogen and host during Salmonella enterica serovar Typhimurium infection and reveal the molecular impact of bacterial riboregulators. We identify a PhoP-activated small RNA, PinT, which upon bacterial internalization temporally controls the expression of both invasion-associated effectors and virulence genes required for intracellular survival. This riboregulatory activity causes pervasive changes in coding and noncoding transcripts of the host. Interspecies correlation analysis links PinT to host cell JAK-STAT signalling, and we identify infection-specific alterations in multiple long noncoding RNAs. Our study provides a paradigm for a sensitive RNA-based analysis of intracellular bacterial pathogens and their hosts without physical separation, as well as a new discovery route for hidden functions of pathogen genes.

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Coupling of flagellar gene expression to flagellar assembly in Salmonella enterica serovar typhimurium and Escherichia coli.

G S Chilcott, K Hughes (2000)

How do organisms assess the degree of completion of a large structure, especially an extracellular structure such as a flagellum? Bacteria can do this. Mutants that lack key components needed early in assembly fail to express proteins that would normally be added at later assembly stages. In some cases, the regulatory circuitry is able to sense completion of structures beyond the cell surface, such as completion of the external hook structure. In Salmonella and Escherichia coli, regulation occurs at both transcriptional and posttranscriptional levels. One transcriptional regulatory mechanism involves a regulatory protein, FlgM, that escapes from the cell (and thus can no longer act) through a complete flagellum and is held inside when the structure has not reached a later stage of completion. FlgM prevents late flagellar gene transcription by binding the flagellum-specific transcription factor sigma(28). FlgM is itself regulated in response to the assembly of an incomplete flagellum known as the hook-basal body intermediate structure. Upon completion of the hook-basal body structure, FlgM is exported through this structure out of the cell. Inhibition of sigma(28)-dependent transcription is relieved, and genes required for the later assembly stages are expressed, allowing completion of the flagellar organelle. Distinct posttranscriptional regulatory mechanisms occur in response to assembly of the flagellar type III secretion apparatus and of ring structures in the peptidoglycan and lipopolysaccharide layers. The entire flagellar regulatory pathway is regulated in response to environmental cues. Cell cycle control and flagellar development are codependent. We discuss how all these levels of regulation ensure efficient assembly of the flagellum in response to environmental stimuli.

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Author and article information

Journal

Journal ID (nlm-ta): Microorganisms

Journal ID (iso-abbrev): Microorganisms

Journal ID (publisher-id): microorganisms

Title: Microorganisms

Publisher: MDPI

ISSN (Electronic): 2076-2607

Publication date (Electronic): 11 October 2019

Publication date Collection: October 2019

Volume: 7

Issue: 10

Electronic Location Identifier: 443

Affiliations

[1 ]Fisheries College, Key Laboratory of Healthy Mariculture for the East China Sea, Ministry of Agriculture, Jimei University, Xiamen 361021, China; sunyj37@ 123456mail2.sysu.edu.cn (Y.S.); 200561000132@ 123456jmu.edu.cn (L.Z.); lixinghuang@ 123456jmu.edu.cn (L.H.); yxqin@ 123456jmu.edu.cn (Y.Q.); xiaojinxu@ 123456jmu.edu.cn (X.X.)

[2 ]School of Marine Science and Engineering, Qingdao Agricultural University, Qingdao 266109, China; pinnie@ 123456qau.edu.cn

[3 ]Key Laboratory of Special Aquatic Feed for Fujian, Fujian Tianma Technology Company Limited, Fuzhou 350308, China; jolma66@ 123456163.com

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[* ]Correspondence: yanqp@ 123456jmu.edu.cn

Author information

Pin Nie https://orcid.org/0000-0002-9492-2940

Qingpi Yan https://orcid.org/0000-0001-8698-3121

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Publisher ID: microorganisms-07-00443

DOI: 10.3390/microorganisms7100443

PMC ID: 6843279

PubMed ID: 31614635

SO-VID: b3f45779-4ba2-4644-a9c4-bfb3d8997e60

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Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license ( http://creativecommons.org/licenses/by/4.0/).

Dual RNA-Seq Unveils the Role of the Pseudomonas plecoglossicida fliA Gene in Pathogen-Host Interaction with Larimichthys crocea

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Abstract

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Most cited references 49

Flagellar and twitching motility are necessary for Pseudomonas aeruginosa biofilm development.

Dual RNA-seq unveils noncoding RNA functions in host-pathogen interactions.

Coupling of flagellar gene expression to flagellar assembly in Salmonella enterica serovar typhimurium and Escherichia coli.

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