J Vet Sci.  2018 Mar;19(2):260-270. 10.4142/jvs.2018.19.2.260.

A two-component signal transduction system contributes to the virulence of Riemerella anatipestifer

• Wang Q
• Chen M
• Zhang W

• KMID: 2407625
• DOI: http://doi.org/10.4142/jvs.2018.19.2.260

Abstract

Similar to other studies of bacterial pathogens, current studies of the pathogenesis of Riemerella anatipestifer (RA) are focused mainly on in vitro culture conditions. To elucidate further the pathogenesis of RA in vivo, bacterial RNA was extracted from overnight tryptic soy broth cultures (in vitro) and from the blood of infected ducks (in vivo) for comparative RNA sequencing analysis. In total, 682 upregulated genes were identified in vivo. Among the upregulated genes, a signal transduction response regulator (ArsR) and a signal transduction histidine kinase (SthK) were predicted to be located on the same operon. A mutant was constructed by deletion of both of these genes. Duck infection tests showed that genes ArsR and SthK were related to the virulence of the pathogen in vivo. Differentially expressed genes identified by comparison of in vitro and in vivo conditions provided an insight into the physiological process of RA infection and provided an opportunity to identify additional virulence factors.

Keyword

Riemerella anatipestifer; comparative RNA sequencing; pathogenesis; virulence factors

MeSH Terms

Ducks
Genes, vif
Histidine
In Vitro Techniques
Operon
Phosphotransferases
Physiological Processes
Riemerella*
RNA, Bacterial
Sequence Analysis, RNA
Signal Transduction*
Virulence Factors
Virulence*
Histidine
Phosphotransferases
RNA, Bacterial
Virulence Factors

Figure

• 

  Fig. 1 (A) Giemsa staining of Riemerella anatipestifer (RA). RA in duck blood after challenge with vRAf153 (left). Purified RA in vivo (middle). RA cultured in vitro conditions (right). 100× (left to right). (B) Polymerase chain reaction identification of RA isolated from blood. M, DL5,000 DNA marker; Lane 1, RAf153 isolated from blood; Lane 2, vRAf153 cultured in vitro; Lane 3, negative control with distilled H2O as template.

• 

  Fig. 2 Differentially expressed genes (DEGs) between RAf153 cultured in vivo and in vitro. Among the 2,157 genes of RAf153, a total of 2,101 genes were identified by RNA sequencing. Among those, 853 genes were differentially expressed (682 upregulated [red] and 121 downregulated [green]) in bacteria cultured under in vivo conditions. FDR, false discovery rate; RPKM, Reads Per Kilobase per Million mapped reads.

• 

  Fig. 3 Gene ontology classification of differentially expressed genes (DEGs). In the biological processes category, upregulated DEGs were identified in relation to anatomical structure formation, cellular component organization, developmental process, immune system process, multi-organism process, and multicellular organismal process. In the molecular function processes category, DEGs related to transcription regulator activity were also upregulated in Riemerella anatipestifer cultured under in vivo conditions. Upregulated DEGs are shown in red and downregulated DEGs are shown in green.

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  Fig. 4 Heatmap of differentially expressed genes (DEGs). The heatmap illustrates expression levels for all DEGs. Red indicates high expression and green indicates low expression. Good repeatability was observed for the results of the in vivo and in vitro culture conditions.

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  Fig. 5 Validation of differentially expressed genes (DEGs) selected from RNA sequencing (RNA-seq) by quantitative real-time polymerase chain reaction (qRT-PCR). Transcriptional changes of six selected DEGs based on RNA-seq results (A) were validated by qRT-PCR (B). *p<0.05 and **p<0.01 were considered significant differences.

• 

  Fig. 6 Characterization of ΔArsR-SthK. (A) Sequence alignment of RAf153 and ΔArsR-SthK (upper). Sequence alignment of ΔArsR-SthK and spcR cassette (lower). (B) Construction of ΔArsR-SthK-deleted strain. M, DL5,000; Lane 1, ΔArsR-SthK-deleted strain was amplified by detected primers of deleted genes; Lane 2, vRAf153 was amplified by detected primers of deleted genes; Lane 3, ΔArsR-SthK-deleted strain was amplified by detected primers of spcR cassette; Lane 4, vRAf153 was amplified by detected primers of spcR cassette; Lane 5, 16S rRNA fragment was amplified from ΔArsR-SthK-deleted strain; Lane 6, 16S rRNA fragment was amplified from vRAf153 strain. (C) Polar effect identification of ΔArsR-SthK. M, DL2,000 DNA marker; Lane 1, amplification fragment of CG09_1826 with ΔArsR-SthK as template; Lane 2, vRAf153 control; Lane 3, amplification fragment of CG09_1830 with ΔArsR-SthK as template; Lane 4, vRAf153 control; Lane 5, quantitative real-time polymerase chain reaction amplification of reference gene with ΔArsR-SthK as template; Lane 6, vRAf153 control.

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  Fig. 7 Adherence and invasion assays of vRAf153 and ΔArsR-SthK. (A) Adherence assay. (B) Invasion assay. CFU, colonyforming unit. *p<0.05; ***p<0.001.

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