Immunization with recombinant Salmonella expressing SspH2-EscI protects mice against wild type Salmonella infection

Abstract

Background: Enhancing caspase-1 activation in macrophages is helpful for the clearance of intracellular bacteria in mice. Our previous studies have shown that EscI, an inner rod protein of type III system in E. coli can enhance caspase-1 activation. The purpose of this study was to further analyze the prospect of EscI in the vaccine design.

Results: A recombinant Salmonella expressing SspH2-EscI fusion protein using the promotor of Salmonella effector SspH2, X4550(pYA3334-P-SspH2-EscI), was constructed. A control recombinant Salmonella expressing SspH2 only X4550(pYA3334-P-SspH2) was also constructed. In the early stage of in vitro infection of mouse peritoneal macrophages, X4550(pYA3334-P-SspH2-EscI) could significantly (P < 0.05) enhance intracellular caspase-1 activation and pyroptotic cell death of macrophages, when compared with X4550(pYA3334-P-SspH2). Except for the intracellular pH value, the levels of reactive oxygen species, intracellular concentration of calcium ions, nitric oxide and mitochondrial membrane potential in macrophages were not significantly different between the cells infected with X4550(pYA3334-P-SspH2-EscI) and those infected with X4550(pYA3334-P-SspH2). Besides, only lower inflammatory cytokines secretion was induced by X4550(pYA3334-P-SspH2-EscI) than X4550(pYA3334-P-SspH2). After intravenous immunization of mice (1 × 10⁶ cfu/mouse), the colonization of X4550(pYA3334-P-SspH2-EscI) in mice was significantly limited at one week post immunization (wpi), when compared with X4550(pYA3334-P-SspH2) (P < 0.05). The population of activated CD8⁺T lymphocytes in mouse spleens induced by X4550(pYA3334-P-SspH2-EscI) was lower than that induced by X4550(pYA3334-P-SspH2) at 2-3 wpi, and the ratio of CD4⁺T cells to CD8⁺T cells decreased. The blood coagulation assay indicated that no significant difference was found between X4550(pYA3334-P-SspH2-EscI) and uninfected control, while X4550(pYA3334-P-SspH2) could induce the quick coagulation. Notably, immunization of X4550(pYA3334-P-SspH2-EscI) could limit the colonization of challenged Salmonella strains in the early stage of infection and provide more effective protection.

Conclusion: The activation of caspase-1 in macrophages by EscI can be used in the design of live attenuated Salmonella vaccine candidate.

Keywords: Caspase-1; Mice; Protection; Salmonella; SspH2-EscI.

Fig. 1

In vitro infection of mouse peritoneal macrophages. Six-week-old female C57BL/6 mouse peritoneal macrophages were seeded on 96-well plates for culturing. Three hours later, non-adherent cells were removed and cell density was adjusted to 20,000 cells per well with RPMI 1640 complete medium without antibiotics and the freshly cultured X4550(pYA3334), X4550(pYA3334-P-SspH2) or X4550(pYA3334-P-SspH2-EscI) were added with MOI 100. The cell plate was centrifuged to enhance the contact of bacteria with the cells and the infected cells were then incubated for 30 min. The supernatants containing uninfected bacteria were replaced with RPMI 1640 complete medium (100 μl/well) containing 100 U/ml penicillin and 100 μg/ml streptomycin prior to the start of the subsequent incubation. The uninfected cells were used as control. a Activation of intracellular caspase-1 at different hours post-infection (hpi) using FLICA staining; b Cytotoxicity assay by lactate dehydrogenase (LDH) release at 5 hpi; c Cell morphology observation at 5 hpi. a, uninfected control; b, X4550(pYA3334-P-SspH2) infection; c, X4550(pYA3334-P-SspH2-EscI) infection. d Cell function including reactive oxygen species (ROS), nitric oxide (NO), intracellular Ca²⁺ concentration ([Ca²⁺]_i), and mitochondrial membrane potential (MMP) at 1, 3, 5 hpi stained with DCFH-DA, DAF-FM DA, Fluo-3 AM and rhodamine 123 (Rh123) respectively. e Intracellular pH value at 0, 1, 3, 5 hpi stained with BCECF-AM; f Supernatant cytokines levels at 1, 3, 5 hpi using cytometric bead array system kit. Results are representative of at least three independent experiments (c, d). *P < 0.05; X4550(pYA3334-P-SspH2-EscI) infection vs X4550(pYA3334-P-SspH2) or X4550(pYA3334) infection (e, f)

Fig. 2

In vivo infection of mice. Six-week-old C57BL/6 mice were intravenously injected with either freshly collected X4550(pYA3334), X4550(pYA3334-P-SspH2) or X4550(pYA3334-P-SspH2-EscI), 1× 10⁶ cfu/mouse. The uninfected mice were used as control. Several weeks later, the bacterial counts (a) in mouse spleens and livers, the activation of splenic T lymphocyte subsets (b), the ratio of splenic CD4⁺/CD8⁺ T cells (c) were analyzed. Five mice were used in each treatment. One-week post immunization, the immunized mice were challenged intraperitoneally with wild-type Salmonella strain D6. The bacterial colonization (d) in mouse spleens and livers challenged with 1× 10⁵ cfu/mouse and the survival of mice challenged with 5 × 10⁶ cfu/mouse were counted (e) for immune protection assay. Nine mice were used for each treatment