AbstractHemolysin-coregulated protein (Hcp) of Salmonella enteritidis is known to be a structural and effector protein of the T6SS, but little is known about the role of Hcp in host cells. In this study, Hcp was expressed by plasmid pEGFP-N1-hcp in BHK-21 cells and the results showed that the subcellular localization of Hcp was predominantly in the cytoplasm of BHK-21 cells. When Hcp was over-expressed by transfecting plasmid pCI-neo-hcp in BHK-21 cells and mRNA sequencing was performed to analyze differentially expressed genes, the results showed a change in the expression levels of 307 mRNAs (fold change > 2, and p < 0.01). Amongst these, 125 mRNAs were up-regulated and 182 mRNAs were down-regulated. Kyoto encyclopedia of genes and genomes (KEGG) enrichment analysis showed that differentially expressed genes were enriched in tumor necrosis factor (TNF) signaling pathway, IL-17 signaling pathway, and cytokine–cytokine receptor interaction. Subsequently, we selected differentially expressed genes of TNF signaling pathway and verified the changes by real-time PCR. The results were consistent with the trend observed for the sequencing results. In conclusion, we demonstrated that Hcp of Salmonella enteritidis caused the change of mRNAs expression of TNF signaling pathway in the cytoplasm of BHK-21 cells.
Salmonella is a Gram-negative bacterium belonging to the family Enterobacteriaceae. The genus comprises two spe-cies, Salmonella bongori and Salmonella enterica (Brenner et al. 2000). Salmonella enterica can infect birds, rodents, and humans (Galan 1996). Type VI secretion system (T6SS) found within Salmonella pathogenicity island 6 (SPI-6) can be activated during infection and is thought to contribute to its pathogenesis (Wang et al. 2019). In Salmonella enteritidis strain P125109, a gene SEN1005 downstream of a trimmed T6SS region plays an important role in altering the expres-sion of genes, and the genes are not only involved in the invasion of bacteria into non-phagocytic cells, but also in
bacterial engulfment by macrophages and acute inflamma-tion in C57BL/6 mice (Das et al. 2018; Silva et al. 2012). A previous study has shown that in Salmonella enterica, the icmF gene cluster is associated with eukaryotic cell invasion (Folkesson et al. 2002).
T6SS is found to be wide spread in Gram-negative bac-teria (Pukatzki et al. 2007). T6SS deliver cytotoxic proteins (Ray et al. 2017) and are shown to be widely distributed in translocation pathways and are able to directly target bacte-rial and eukaryotic cells (Silverman et al. 2012). Hemoly-sin-coregulated protein (Hcp) is a secreted component of the T6SS with significant structural homology to T4 bac-teriophage tail tube (Kanamaru 2009; Kapitein and Mogk 2013). Moreover, Hcp is a central component of the bacteri-ophage-like model of T6SS-dependent intercellular effector transport. Studies have demonstrated as a universally and abundantly T6SS-exported protein, and Hcp is essential for both the assembly of the T6SS apparatus and export of its effectors (Hood et al. 2010; Mougous et al. 2006). Never-theless, the precise function of Hcp in the T6SS is unclear.
To evaluate the role of Hcp on the pathogenicity of Salmonella Enteritidis, we investigated the subcellular
Electronic supplementary material The online version of this article (https ://doi.org/10.1007/s1320 5-020-02296 -0) contains supplementary material, which is available to authorized users.
localization of Hcp and the impact of Hcp mRNAs’ expres-sion on host cellular.
Methods
BHK‑21 cells
BHK-21 cells were obtained from American Type Culture Collection (ATCC) and cultured in Dulbecco’s Minimal Essential Medium (DMEM) supplemented with 10% fetal bovine serum (Gibco BRL, Gaithersburg, MD, US), 100 IU of penicillin, and 100 mg of streptomycin per ml, at 37 °C in a 5% CO2 atmosphere incubator.
Hcp subcellular localization
The hcp gene of Salmonella enteritidis was generated by biosynthesis (S1), and then, the hcp gene was cloned into the corresponding sites in the eukaryotic expression vec-tor pEGFP-N1. According to enzyme digestion and DNA sequencing, the recombinant plasmid pEGFP-N1-hcp was confirmed. BHK-21 cells were seeded into 12-well dishes for transfections, and the treatment group was transfected pEGFP-N1-hcp (1000 ng) with Lipofectamine 2000 (Inv-itrogen, Carlsbad, CA, US) and the negative control group was transfected pEGFP-N1 (1000 ng) with the same opera-tion. After transfection of 48 h, the cells expressing Hcp or negative control (the cells expressing GFP) were washed with PBS and incubated with 4′,6-diamidino-2-phenylindole (DAPI) for 10 min, and then washed twice with phosphate-buffered saline (PBS).
Hcp gene transient transfection
The hcp gene of Salmonella enteritidis was PCR amplified. The amplified product was cloned into the corresponding sites in the pCI-neo expression vector. The pCI-neo-hcp were verified by DNA sequencing. BHK-21 cells were seeded into 6-well dishes for transfections. We transfected pCI-neo-hcp (2500 ng) into BHK-21 cells and pCI-neo (2500 ng) as negative control with the same operation. After incubation at 37 °C with 5% CO2 for 48 h, the cells were washed with PBS for twice and extracted RNA for following real-time PCR and mRNA sequencing, there are three cell samples for RNA sequencing in the treatment of Hcp gene transient transfection and negative control.
mRNA library construction and sequencing
Total RNA was extracted using Trizol reagent (Invitrogen, CA, USA) following the manufacturer’s procedure. The total RNA quantity and purity were analysis of Bioanalyzer 2100
and RNA 1000 Nano LabChip Kit (Agilent, CA, USA) with RIN number > 7.0. Poly(A). We performed the paired-end sequencing on an Illumina Novaseq™ 6000 at the (LC Sci-ences, USA) following the vendor’s recommended protocol. The raw sequencing data have been submitted to the National Center for Biotechnology Information (NCBI) Short Read Archive. The differentially expressed mRNAs were selected with log2 (fold change) ≥ 1 or log2 (fold change) ≤ − 1 and with statistical significance (– value < 0.05). The differen-tially expressed mRNAs were analyzed by gene ontology (GO) and KEGG.
The quantification of mRNAs by real‑time PCR
Total RNA was extracted using Trizol reagent (Invitrogen, Carlsbad, CA, USA) from BHK-21 cells according to the manufacturer’s instructions. The real-time PCR was used to validate the expression levels for selected genes. Primers are listed in Table S1 in the supplemental material. Treatment of total RNA with DNase (Fermentas, St. Leon-Rot, Germany) was performed at 37 °C for 30 min, followed by 15 min at 72 °C. One microgram of total RNA was reverse transcribed using random hexamers and M-MLV (Invitrogen, Carlsbad, CA, US). The real-time PCR was carried out using the Accu-Power 2 × Greenstar qPCR Master mix (Bioneer, Daejeon, Korea) in a 25 μL reaction volume on Step One Plus Real-Time PCR System (Applied Biosystems, USA). The reli-ability and amplification efficiency were calculated from the formula of previous methods (Rodríguez et al. 2015). The relative quantification of mRNAs was normalized to β-actin using the ∆∆Ct method (Livak and Schmittgen 2001).
Statistical analysis
All of the assays were performed at least in triplicate. All data were evaluated with a Student’s t test using SPSS ver. 17.0 software (SPSS Inc., Chicago, IL, USA). A p value less than 0.05 was considered significant.
Results
Hcp protein subcellular localization
To investigate subcellular localization of Hcp protein, the fusion protein of Hcp and enhanced green fluorescent pro-tein (EGFP) was expressed in BHK-21 cells and the nucleus was stained by DAPI. Meanwhile, a vector with EGFP was expressed in BHK-21 cells as negative control. Fluorescence microscope was used to investigate the subcellular localiza-tion of Hcp protein by observing the positioning of green fluorescent protein (GFP). The results showed that the fusion protein of Hcp and EGFP distributes predominantly in the
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cytoplasm of BHK-21 cells, while the GFP of negative con-trol was located in the whole cell (Fig. 1).
Cellular mRNAs profiling in response to Hcp transfection
To gain the overview of the impact of Hcp on host cel-lular mRNAs expression, the BHK-21 cells were trans-fected with hcp gene. The sequencing was performed with the total RNA extracted from BHK-21 cells transfected with pCI-neo-hcp vector or pCI-neo at 48 h. The hcp
gene expression at mRNA expression level was verified by real-time PCR. BHK-21 cells were transfected with pEGFP-N1 and pEGFP-N1-Hcp. Cells transfected with pEGFP-N1 were treatment group; cells transfected with pEGFP-N1-Hcp were negative control (Fig. 2a). The dif-ferential expression of mRNAs in hcp gene-transfected cells compared to negative control cells was observed in Fig. 2b. The expression levels of 307 mRNAs were changed remarkably (fold change > 2, and p < 0.01). Amongst these, 125 mRNAs were up-regulated and 182 mRNAs were down-regulated (Table S2).
Fig. 1 Subcellular localiza-tion of Hcp protein in BHK-21 cells. BHK-21 cells were transfected with pEGFP-N1 and pEGFP-N1-Hcp for 2 days. Cells transfected with pEGFP-N1 were treatment group; cells transfected with pEGFP-N1-Hcp were negative control. The localization of Hcp protein was showed with GFP (green); the cell nuclei were stained with DAPI (blue). Scale bar = 200 µm
pEGFP-N1
GFP DAPI Merged
pEGFP-N1-Hcp
Fig. 2 Identification of Hcp and differentially expressed genes. a The verify of the expression of Hcp gene at mRNA level, real-time PCR was used to analyze the mRNA expression level of BHK-21 cells transfected with pEGFP-N1 and pEGFP-N1-Hcp. Cells transfected with pEGFP-N1 were treatment group; cells transfected with pEGFP-N1-Hcp were negative control (M is for Marker, 1 is for Hcp, and 2 is for negative control). b Comparison of expression levels of mRNA.
RNA sequencing was used to analyze the differentially expressed genes of BHK-21 cells transfected with pEGFP-N1 and pEGFP-N1-Hcp. Cells transfected with pEGFP-N1 were treatment group; cells transfected with pEGFP-N1-Hcp were negative control. Red and blue points represent up- regulated and down-regulated mRNAs, respec-tively
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GO analysis and KEGG pathway annotation of the differentially expressed genes
The differentially expressed mRNAs were analyzed by bioinformatics methods. GO analysis revealed that some important GO terms found to be significantly enriched in positive regulation of inflammatory response, extracellular region, and cytokine activity. The 20 most common GO cat-egories were enriched as shown in Fig. 3a. To distinguish the biological pathways that become active in the BHK-21 cells in response to Hcp transfection, we studied the dif-ferentially expressed mRNAs by term enrichment analysis to identify their possible targets using the KEGG annota-tion and the results showed that several important pathways were enriched in TNF signaling pathway, IL-17 signaling pathway, and cytokine–cytokine receptor interaction. The 20 most common pathways were enriched, as shown in Fig. 3b.
Verification of sequencing results
The differentially expressed genes were found to be involved in several important pathways as shown in Table 1. The results showed that Fos, Il6, and Lif gene of TNF signaling pathway were up-regulated. Ccl20, Ccl2, and Map3k8 of TNF signaling pathway were down-regulated. To validate the sequencing results of differential-expressed regulated genes, we selected gene of TNF signaling pathway to ver-ify their expression level by real-time PCR and the results
showed that at mRNA level, the expressions of IL6, IL11, IL1r11, Fos, and Fosb are up-regulated and the expression of Cc120 was down-regulated, which data were consistent with the trend of sequencing results (Fig. 4).
Discussion
Secretion systems has been showed to provide a selective advantage to pathogenic bacteria by enhancing their viru-lence potential and environmental adaptation, respectively (Aschtgen et al. 2008; Schwarz et al. 2010). The T6SS act like a nanomachine, injecting bacterial proteins/toxins into the host cell and into the environment to affect virulence (Lertpiriyapong et al. 2012). Hcp in different bacteria has been showed to be associated with eukaryotic cell lysis and death (Lim et al. 2015; Pukatzki et al. 2007). While the pathogenic effect of Salmonella enteritidis on host cells has not been studied. Identifying the subcellular localiza-tion of a protein provides a better understanding of its func-tion (Parras-Molto et al. 2013). Until now, the subcellular localization of salmonella enteritidis secreted protein Hcp is unclear. Our study demonstrated that Hcp of Salmonella enteritidis was located in the cytoplasm of BHK-21 cells. NleF has been showed to be secreted by the Type III secre-tion system (T3SS) and subcellular localization is also in the host cytoplasm (Echtenkamp et al. 2008).
Fig. 3 Bioinformatic analysis of differentially expressed genes. a Functional annotation of DEGs based on GO analysis. Cells trans-fected with pEGFP-N1 were treatment group, cells transfected with pEGFP-N1-Hcp were negative control. b Functional annotation of
differentially expressed mRNAs based on KEGG pathway analysis. Cells transfected with pEGFP-N1 were treatment group; cells trans-fected with pEGFP-N1-Hcp were negative control
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The results showed that subcellular localization of Hcp was predominantly in the cytoplasm. The mechanisms of T6SS involved in Enteritidis pathogenicity are poorly under-stood. Most research focus on T3SS. Salmonella typhimu-rium utilized a T3SS to induce intestinal inflammation by delivering specific effector proteins that stimulate signal transduction pathways resulting in the production of pro-inflammatory cytokines (Sun et al. 2016). To research the
role of the Hcp on host signal pathways, we analyzed the mRNAs expression profiles of BHK-21. The overexpression of hcp gene caused the change of mRNAs profile of BHK-21cells. Moreover, differentially expressed genes associ-ated with the pathogenicity of Salmonella enteritidis which caused by Hcp were enriched in TNF signaling pathway.
Salmonella enteritidis infection can activate immune sys-tem functions by TNF signaling pathway (Polansky et al. 2018). Hcp regulated the gene (Fos, Il6, Lif, Ccl20, and Ccl2) expression of TNF signaling pathway at mRNA level. Previous studies found that Salmonella enterica infection up-regulated IL6 expression (Laptev et al. 2019; Swaggerty et al. 2004). The function of IL6 has been implicated in a wide variety of inflammation-associated disease. Leukemia inhibitory factor (LIF) has been showed to be interleukin 6 family cytokine (Jones and Jenkins 2018). TLR4-FOS/JUN signaling pathway can play a key role in Salmonella LPS stimulation. Fos encode leucine zipper proteins can dimerize with proteins of the JUN family. (Huang et al. 2016). Our results showed that Hcp up-regulated the Fos gene expres-sion at mRNA level. Upon infection with Salmonella enter-ica, mucosal expression of CCL2 was rapidly up-regulated, followed by systemic expression in the spleen (DePaolo et al. 2005). Ccl20 and Ccl2 are C–C motif chemokine ligand. Salmonella enterica infection has been showed to be induced CCL20 chemokine expression in human intesti-nal Caco-2 cells (Yim et al. 2014). These results indicated that Hcp-induced differentially expressed genes may play an important role in regulating inflammatory processes caused by Salmonella enterica infection.
Conclusions
In our studies, the confirm of Hcp subcellular localization and cellular mRNAs profiling in response to Hcp transfec-tion contributed to a deeper understanding of the pathogenic mechanisms of Salmonella T6SS. Our results indicated that Hcp affected gene expression of TNF signaling pathway by
Table 1 Differentially expressed gene of KEGG pathways
Fig. 4 Verification of mRNAs sequencing by real-time PCR. The genes IL6, IL11, IL1r11, Fos, Fosb, and Cc120 were ana-lyzed by PCR. The fold change in expression was determined using the 2−ΔΔCt method, and gene expression values were normalized against the β-actin internal reference gene. Data from real-time PCR were shown as means ± standard deviation (S.D.) from six independent experiments R
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IL6 IL11 IL1rl1 Fos Fosb Ccl20
Sequence Real-time PCR
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distributing in the cytoplasm cellular. Altogether, we pro-vided new insights for future studies regarding the interac-tion of Hcp with host cell.
Acknowledgements This research was funded by grants from Natural Science Foundation of China (Grant No. 31902225) and Anhui Prov-ince Natural Science Fund Project (Grant No. 1908085QC92).
Author contributions LZ and SL conceived and designed the experi-ments; SW and ML performed the experiments; ZL analyzed the data. LZ and SL wrote the paper. All authors critically read and contributed to the manuscript and approved the final version. All authors critically read and contributed to the manuscript and approved the final version.
Compliance with ethical standards
Conflict of interest The authors have declared that no competing inter-est exists.
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