Effects of Avian Pathogenic Escherichia coli Hcp2b on the Expression Profile of Spleen mRNA in Chicken Spleen

doi:10.7668/hbnxb.20192557

Abstract

Abstract:

In order to study the effect of the effector protein Hcp2b on the transcriptomics of the spleen during the infection of Avian pathogenic E.coli(APEC)in chicks.The hcp2b-deletion strain Δhcp2b and the reverted strain Chcp2b(with AE17 strain as a positive control)constructed and preserved in the Anhui Province Key Laboratory of Veterinary Pathobiology and Disease Control were used to inject 7-day-old chicks intramuscularly.After 24 h,the spleen of depressed chicks was collected to detect the tissue load and make pathological sections.The deletion strain Δhcp2b and the wild strain AE17 were selected to infect spleen tissue for transcriptomics sequencing.Also,Real-time PCR was used to identify the sequencing results.Moreover,GO analysis and KEGG pathway enrichment analysis were performed on the differentially expressed genes.The results illustrated that the wild strain AE17,the deleted strain Δhcp2b and the reverted strain Chcp2b had no significant difference in the tissue load of the spleen.Observation of tissue sections that the spleens of wild strain AE17 and deletion strain Δhcp2b both suggested enlarged interstitial spaces and hyperemia.Transcriptomics analysis revealed that the deletion strain Δhcp2b induced the differential expression of spleen gene mRNA in chicks.515 differentially expressed genes were screened(185 genes were up-regulated and 330 genes were down-regulated).Real-time PCR confirmed that the expression of IL22,TNFRSF6B,and TNFRSF8 genes in the spleen was down-regulated after the deletion strain Δhcp2b infected chicks,which was consistent with the trend of the sequencing results.At 24 h,GO analysis found that the chick spleen differential genes were significantly enriched in items such as membrane,membrane composition,and extracellular space.KEGG analysis found that differential genes in the spleen were significantly enriched in the pathway of endoplasmic reticulum protein processing.The hcp2b gene had no effect on APEC colonization of spleen,the hcp2b participates in the pathogenic process through the signal pathway that affects the endoplasmic reticulum protein processing of the spleen in the body's immune organs.

Key words: Chicken, Avian pathogenic E.coli, Hcp, Spleen, Endoplasmic reticulum protein processing pathway

SONG Xiangjun, SONG Zichao, SHEN Xiao, CHEN Zhe, JIANG Huyan, HOU Manman, SHAO Ying, TU Jian, QI Kezong. Effects of Avian Pathogenic Escherichia coli Hcp2b on the Expression Profile of Spleen mRNA in Chicken Spleen[J]. Acta Agriculturae Boreali-Sinica, 2022, 37(1): 232-238. doi: 10.7668/hbnxb.20192557.

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Figures/Tables 8

Tab.1 Primer sequence of Real time quality PCR

引物名称 Prime	引物序列(5'—3') Prime sequences	大小/bp Size
IL22-F	GGAGCTTCTCAGGATGGGTTG	112
IL22-R	CAGGCTTGATGGGCATTGG
TNFRSF8-F	CACCCCAGACACCTCCTCCAT	162
TNFRSF8-R	GGAGAAGTGCCAGCCAGAGC
TNFRSF6B-F	GGAAACCAATACCACGACACC	111
TNFRSF6B-R	GAAGTCGATCAGGGCTTGCT
β-actin-F	TGAACTCCCTGATGGTCAGGTC	100
β-actin-R	ACCACAGGACTCCATACCCAAG

Fig.1 HE staining of the spleen tissue of chickens in the positive control group and the deleted strain Δhcp2b test group(400×)
A.Positive control group;B.Deletion strain Δhcp2b test group.

Fig.2 The infections in spleen of Δhcp2b strain

Fig.3 Volcano map of the differentially expressed gene in spleen Δhcp2b and AE17 infected
Red indicates up-regulation of differentially expressed genes;Blue indicates down-regulation of differentially expressed genes;Gray is unchanged differentially expressed genes.

Tab.2 Differentially expressed genes of hcp2b deleted strains partially expressed genes

基因 Gene	基因描述 Gene description	差异倍数 Log₂(FC)
RPN2	核糖蛋白Ⅱ	-1.20
EDEM3	ER 降解增强α-甘露糖苷酶样蛋白 3	-1.35
XBP1	X-box结合蛋白1	-1.24
DERL3	derlin 3	-2.81
IL1R2	白细胞间受体2	-2.06
IL-22	白细胞介素22	-1.86
SEL1L	ERAD E3 连接酶接头亚基	-1.23
IL1R1	白细胞间受体1	-1.93

Fig.4 Real-time quality PCR verification

Fig.5 The most significantly enriched GO pathway after hcp2b deletion

Fig.6 KEGG functional enrichment of differentially expressed genes of hcp2b deletion strain

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