Identification of salmonella pathogenicity island-2 type III secretion system effectors involved in intramacrophage replication of S. enterica serovar typhimurium: implications for rational vaccine design

Abstract

Salmonella enterica serovars cause severe diseases in humans, such as gastroenteritis and typhoid fever. The development of systemic disease is dependent on a type III secretion system (T3SS) encoded by Salmonella pathogenicity island-2 (SPI-2). Translocation of effector proteins across the Salmonella-containing vacuole, via the SPI-2 T3SS, enables bacterial replication within host cells, including macrophages. Here, we investigated the contribution of these effectors to intramacrophage replication of Salmonella enterica serovar Typhimurium using Fluorescence Dilution, a dual-fluorescence tool which allows direct measurement of bacterial replication. Of 32 strains, each carrying single mutations in genes encoding effectors, 10 (lacking sifA, sseJ, sopD2, sseG, sseF, srfH, sseL, spvD, cigR, or steD) were attenuated in replication in mouse bone marrow-derived macrophages. The replication profiles of strains combining deletions in effector genes were also investigated: a strain lacking the genes sseG, sopD2, and srfH showed an increased replication defect compared to single-mutation strains and was very similar to SPI-2 T3SS-deficient bacteria with respect to its replication defect. This strain was substantially attenuated in virulence in vivo and yet retained intracellular vacuole integrity and a functional SPI-2 T3SS. Moreover, this strain was capable of SPI-2 T3SS-mediated delivery of a model antigen for major histocompatibility complex (MHC) class I-dependent T-cell activation. This work establishes a basis for the use of a poly-effector mutant strain as an attenuated vaccine carrier for delivery of heterologous antigens directly into the cytoplasm of host cells.

Importance: Live attenuated strains of Salmonella enterica serotype Typhi have generated much interest in the search for improved vaccines against typhoid fever and as vaccine vectors for the delivery of heterologous antigens. A promising vaccine candidate is the ΔaroC ΔssaV S. Typhi strain, which owes its attenuation mainly to lack of a type III secretion system (SPI-2 T3SS). The SPI-2 T3SS is important for bacterial proliferation inside macrophages, but not all of the effectors involved in this process have been identified. Here, we show that 10 effectors of the related strain S. Typhimurium contribute to intracellular replication in macrophages. Moreover, we establish that a poly-effector mutant strain of S. Typhimurium can have a severe replication defect and maintain a functional SPI-2 T3SS, which can be exploited for delivery of heterologous antigens.

FIG 1

Use of Fluorescence Dilution to measure the impact of the SPI-2 T3SS on S. Typhimurium replication in bone marrow-derived macrophages. (A) Expression of fluorescent proteins from the pFCcGi plasmid is constitutive for mCherry and under the control of an arabinose-inducible promoter for gfp. (B) In the absence of arabinose, levels of GFP fluorescence in preinduced bacteria become diluted as successive rounds of cell division occur. (Ci and Cii) Macrophages were infected with S. Typhimurium strains carrying pFCcGi that were grown with arabinose prior to infection. Infections were carried out in the absence of arabinose, and the cells were lysed at 2 h and 17 h postinoculation. Replication of recovered intracellular bacteria was measured by flow cytometry analysis of GFP Fluorescence Dilution (n = 10,000 events analyzed). (Ci) Representative histograms of GFP fluorescence in intracellular populations of wild-type, ΔssaV, and ΔsseB mutant strains. (Cii) Fold replication values were calculated from the geometric means of GFP fluorescence as the ratio of replication at 17 h to replication at 2 h. Data are expressed as means ± standard errors of the means (SEM) of the results of five independent experiments. P values were obtained by Student’s t test (***, P < 0.001) relative to the wild-type strain results.

FIG 2

Contribution of individual SPI-2 T3SS effectors to S. Typhimurium intracellular replication. Fluorescence Dilution analysis of the replication of S. Typhimurium strains carrying deletions in genes of effectors of the SPI-2 T3SS was done in bone marrow-derived macrophages over 17 h. The color code used to group SPI-2 T3SS effectors according to their function is as follows: SCV membrane dynamics, green; SCV localization, yellow; cell migration, red; ubiquitin pathways, purple; unknown function, dark gray; actin cytoskeleton, brown; host immune signaling, pink; SPI-1 translocated effectors, turquoise. Data are expressed as the means ± SEM of the results of 3 to 30 independent experiments (N) and were normalized to wild-type values. Statistical analysis was performed with one-way analysis of variance (ANOVA) and a post hoc Dunnett test (*, P < 0.05; **, P < 0.01) relative to the wild-type strain results.

FIG 3

Replication of S. Typhimurium poly-effector mutant strains. Fluorescence Dilution analysis of intracellular replication was carried out over 17 h in bone marrow-derived macrophages. (A) Fold replication of strains combining deletions in genes involved in similar functions. Data are expressed as the means ± SEM of the results of 3 to 8 independent experiments and were normalized to wild-type values. (B) Fold replication of strains combining deletions in the genes associated with different activities within host cells. Data are expressed as the means ± SEM of the results of four independent experiments and were normalized to wild-type values. Statistical analysis was performed with a one-way ANOVA and post hoc Dunnett test (**, P < 0.01) relative to the wild-type strain results. Statistical differences (**, P < 0.01) between ΔsseG ΔsopD2 or ΔsseG ΔsopD2 ΔsrfH and each of the corresponding single-mutation strains (ΔsseG, ΔsopD2, and ΔsrfH) were also confirmed. The color code used is as follows: SPI-2 T3SS effectors involved in SCV membrane dynamics, green; SCV localization, yellow; cell migration, red; ubiquitin pathways, purple; unknown function, dark gray.

FIG 4

Competitive index (CI) analysis of ΔsseG ΔsopD2 and ΔsseG ΔsopD2 ΔsrfH strains. C57 BL/6 mice were inoculated by intraperitoneal (i.p.) injection (5 × 10⁵ CFU) or intragastric (i.g.) inoculation (3 × 10⁸ CFU) with equal amounts of wild-type and ΔssaV, ΔsseG ΔsopD2, or ΔsseG ΔsopD2 ΔsrfH mutant strains. Bacteria were recovered from infected spleens 3 days (i.p.) or 5 days (i.g.) postinoculation. CI values were calculated as the ratio of wild-type strain to mutant strain recovered (Output) divided by the ratio of wild-type strain to mutant strain present in the inoculum (Input). The scatter plot displays values obtained for individual mice, and the means are indicated (long horizontal lines). P values obtained by Student’s t test confirmed that all CI values are statistically different from 1.0 (**, P < 0.01).

FIG 5

Vacuolar integrity and SPI-2 T3SS functionality in intracellular S. Typhimurium ΔsseG ΔsopD2 ΔsrfH. (Ai and Aii) Intravacuolar localization of ΔsseG ΔsopD2 ΔsrfH in epithelial cells. (Ai) Confocal microscopy images of LAMP-1 labeling of HeLa cells infected with S. Typhimurium wild-type, ΔssaV, or ΔsseG ΔsopD2 ΔsrfH strains for 14 h. Cells were fixed and immunolabeled with anti-Salmonella (green in merged images) and anti-LAMP-1 (red in merged images) antibodies. Scale bars represent 5 µm. (Aii) HeLa cells infected for 10 h with GFP-expressing S. Typhimurium wild-type, ΔsifA, or ΔsseG ΔsopD2 ΔsrfH strains were treated with digitonin to selectively permeabilize the plasma membrane and labeled with anti-Salmonella antibody. Percentages of intravacuolar and cytosolic bacteria were calculated based on differential labeling. (Bi and Bii) Translocation of an SPI-2 T3SS effector from S. Typhimurium ΔsseG ΔsopD2 ΔsrfH. Bone marrow-derived macrophages or HeLa cells were infected for 17 h or 14 h, respectively, with the indicated S. Typhimurium strains expressing HA-tagged SseJ from the plasmid pWSK29. (Bi) Confocal microscopy images of SseJ-2HA translocation in macrophages. Cells were fixed and immunolabeled with anti-Salmonella (green in merged images) and anti-HA (red in merged images) antibodies. Scale bars represent 5 µm. (Bii) Quantification of translocated SseJ-2HA by flow cytometry analysis of infected cells immunolabeled with anti-Salmonella and anti-HA antibodies. Data were calculated from the geometric means of fluorescence associated with anti-HA labeling and are expressed as means ± SEM of the results of three independent experiments. The ΔssaV strain was included as a negative control for effector translocation in each experiment (P < 0.001). P values were obtained by Student’s t test (*, P < 0.05) relative to the wild-type (WT) strain results.

FIG 6

T-cell stimulation by SPI-2-mediated delivery of a model antigen from S. Typhimurium ΔsseG ΔsopD2 ΔsrfH. Bone marrow-derived dendritic cells (BM-DC) were infected with indicated S. Typhimurium strains harboring the plasmid pWSK29 P_sseA sseJ::OVA::HA (p3631). Cells infected with wild-type S. Typhimurium, in the absence of the plasmid, were used as a negative control. Stimulation with the SIINFEKL peptide was used as a positive control. Infected cells were cocultured for 22 h with B3Z T-cell hybridoma reporter cells (ratio of BM-DC to T cells of 1:4) followed by a 6-h incubation in the presence of the β-galactosidase substrate chlorophenyl red β-galactopyranoside. β-Galactosidase activity was measured colormetrically by the amount of substrate converted at 595 nm. Results were normalized to values obtained with the negative control and are expressed as means ± SEM of the results of four independent experiments, each performed in triplicate. P values were obtained by Student’s t test relative to the wild-type negative-control results (*, P < 0.05; **, P < 0.01).