Salmonella Enteritidis activates inflammatory storm via SPI-1 and SPI-2 to promote intracellular proliferation and bacterial virulence

Abstract

Salmonella Enteritidis is an important intracellular pathogen, which can cause gastroenteritis in humans and animals and threaten life and health. S. Enteritidis proliferates in host macrophages to establish systemic infection. In this study, we evaluated the effects of Salmonella pathogenicity island-1 (SPI-1) and SPI-2 to S. Enteritidis virulence in vitro and in vivo, as well as the host inflammatory pathways affected by SPI-1 and SPI-2. Our results show that S. Enteritidis SPI-1 and SPI-2 contributed to bacterial invasion and proliferation in RAW264.7 macrophages, and induced cytotoxicity and cellular apoptosis of these cells. S. Enteritidis infection induced multiple inflammatory responses, including mitogen-activated protein kinase (ERK-mediated) and Janus kinase-signal transducer and activator of transcript (STAT) (STAT2-mediated) pathways. Both SPI-1 and SPI-2 were necessary to induce robust inflammatory responses and ERK/STAT2 phosphorylation in macrophages. In a mouse infection model, both SPIs, especially SPI-2, resulted in significant production of inflammatory cytokines and various interferon-stimulated genes in the liver and spleen. Activation of the ERK- and STAT2-mediated cytokine storm was largely affected by SPI-2. S. Enteritidis ΔSPI-1-infected mice displayed moderate histopathological damage and drastically reduced bacterial loads in tissues, whereas only slight damage and no bacteria were observed in ΔSPI-2- and ΔSPI-1/SPI-2-infected mice. A survival assay showed that ΔSPI-1 mutant mice maintained a medium level of virulence, while SPI-2 plays a decisive role in bacterial virulence. Collectively, our findings indicate that both SPIs, especially SPI-2, profoundly contributed to S. Enteritidis intracellular localization and virulence by activating multiple inflammatory pathways.

Keywords: Inflammatory pathway; Salmonella Pathogenicity Island; bacterial colonization; macrophage; mouse infection model.

Figure 1

Construction of S. Enteritidis ΔSPI-1, ΔSPI-2 and ΔSPI-1/SPI-2 deficient mutants and biochemical identification of the deficient strains. (A) Schematic representation of the construction for S. Enteritidis ΔSPI-1, ΔSPI-2 and ΔSPI-1/SPI-2 mutants. The Salmonella deficient mutants were produced by using the λ-Red recombinase gene replacement method. (B) All gene deletions were verified by PCR analysis and by sequencing. The stn gene was used as the reference control. (C) Growth curves of S. Enteritidis C50041 wild-type, ΔSPI-1, ΔSPI-2 and ΔSPI-1/SPI-2 deletion strains. Bacteria were cultured in LB medium at 37°C with 180 rpm, and the OD₆₀₀ values of bacterial cultures were determined in 0.5 h intervals. (D) Biochemical tests of S. Enteritidis WT, ΔSPI-1, ΔSPI-2 and ΔSPI-1/SPI-2 strains using the API 20E identification kit. Sterile water was used as control.

Figure 2

Intracellular localization and virulence evaluation of S. Enteritidis ΔSPI-1, ΔSPI-2 and ΔSPI-1/SPI-2 deficient strains in macrophages. (A) The invasion was determined in RAW264.7 following the infection of S. Enteritidis WT, ΔSPI-1, ΔSPI-2 and ΔSPI-1/SPI-2 deficient strains. (B) The proliferation was determined at different time points in RAW264.7 macrophages following the infection of S. Enteritidis WT, ΔSPI-1, ΔSPI-2 and ΔSPI-1/SPI-2 deficient strains. (C) After Salmonella infection for 2 h, the supernatants were harvested for the quantification of LDH levels. LDH levels were determined with the LDH Cytotoxicity Assay Kit. (D) Apoptosis analysis with the Annexin V-FITC/PI staining and flow cytometry. The cell apoptosis among different groups was determined with the Annexin V-FITC/PI Apoptosis Detection Kit. P < 0.05 (*), P < 0.01 (**) and P < 0.001 (***) were considered statistically significant.

Figure 3

The SPI-1 and SPI-2 promoted S. Enteritidis to trigger inflammation storm via two different inflammatory pathways in macrophages. (A) Salmonella SPI-1 and SPI-2 induced the robust activation of MAPK and STAT signaling pathways. Macrophages were infected with S. Enteritidis WT, ΔSPI-1, ΔSPI-2 and ΔSPI-1/SPI-2 deficient strains, and the phosphorylation of ERK and STAT2 was determined. (B) Relative intensity of p-ERK and p-STAT2 were semi-quantified using ImageJ and presented as bar graphs. (C) Salmonella SPI-1 and SPI-2 contributed to significant increase of inflammatory cytokines and ISGs. The RAW264.7 macrophage was infected with S. Enteritidis WT, ΔSPI-1, ΔSPI-2 and ΔSPI-1/SPI-2 deficient strains, and total mRNA was extracted. The expression levels of inflammatory cytokines and various ISGs were determined by using qRT-PCR. Cells cultured in DMEM alone were served as the control group. P < 0.05 (*), P < 0.01 (**) and P < 0.001 (***) were considered statistically significant. ns, not significant.

Figure 4

The Salmonella SPI-1 and SPI-2 resulted in the cytokine storm in livers and spleens following infection in mice. Inflammatory cytokines and ISGs were determined in livers (A) and spleens (B) of mice using qRT-PCR. On 4 day following infection, the tissues were harvested and homogenized in RLT buffer for the mRNA extraction. The cytokine expression levels in the organs were measured using qRT-PCR. (C) Western blotting analysis of the phosphorylation of ERK- and STAT2-mediated inflammatory pathways in the spleen following Salmonella infection. On 4 day following infection, the spleens were homogenized and centrifuged to harvest the supernatant for the immunoblotting analysis. (D) Relative intensity of p-ERK and p-STAT2 were semi-quantified using ImageJ and presented as bar graphs. P < 0.05 (*), P < 0.01 (**) and P < 0.001 (***) were considered statistically significant. ns, not significant.

Figure 5

S. Enteritidis WT induced the pronounced activation of p-ERK1&2 and p-STAT2 in the livers and spleens of the infected mice. The activation of p-ERK1&2 (A) and p-STAT2 (B) in mouse liver and spleen were stained using the corresponding antibodies. The nuclear DNA was stained with DAPI. The sections were mounted with the coverslips and Prolong Diamond Antifade Mountant. Images were captured using the Leica confocal microscope at 400 × magnification.

Figure 6

S. Enteritidis SPI-1 and SPI-2 contributed to inflammation storm and tissue damages in the mouse infection model. (A) Histopathological assessment of the livers and spleens from mice infected with S. Enteritidis WT, ΔSPI-1, ΔSPI-2, and ΔSPI-1/SPI-2. Mice were infected with different bacteria, and the livers and spleens were stained with H&E after paraformaldehyde fixation at 4 dpi. Images were captured using a microscope at 400 × magnification. Pathological scores of livers (B) and spleens (C) were evaluated from mice infected with Salmonella C50041, ΔSPI-1, ΔSPI-2, and ΔSPI-1/SPI-2. Pathological evaluations were performed using criteria described in Supplementary Table 4 . P < 0.05 (*) and P < 0.01 (**) were considered statistically significant.

Figure 7

SPI-1 and SPI-2 promoted S. Enteritidis localization in tissues and the bacterial virulence in the mouse infection model. SPI-1 and SPI-2 promoted Salmonella localization in mouse liver (A), spleen (B) and cecum (C). The bacterial burden in tissues were determined on day 4 post infection. Mice were sacrificed, and the livers, spleens and cecums were aseptically collected, homogenized with sterile ice-cold PBS, serially 10‐fold diluted, and plated on LB plates for the determination of CFU. (D) Percentage survival of mice after infection with C50041, ΔSPI-1, ΔSPI-2 and ΔSPI-1/SPI-2. Mice were fasted for 4 h and given 106 CFU of bacteria in 0.1 mL PBS intragastrically. The mortality of mice in different infection groups was recorded daily and monitored for two weeks. P < 0.01 (**) was considered statistically significant. ND, not detected.

Figure 8

Schematic of S. Enteritidis pathopoiesis by SPI-1 and SPI-2 through inducing cytokine storm via multi-inflammatory signaling pathways. SPI-1 and SPI-2 activate the ERK- and STAT-mediated inflammatory responses and contribute to severe tissue damages, thus promoting the transmission, intracellular localization and bacterial virulence of S. Enteritidis.