Mutations in the Salmonella enterica serovar Choleraesuis cAMP-receptor protein gene lead to functional defects in the SPI-1 Type III secretion system

Abstract

Salmonella enterica serovar Choleraesuis (Salmonella Choleraesuis) causes a lethal systemic infection (salmonellosis) in swine. Live attenuated Salmonella Choleraesuis vaccines are effective in preventing the disease, and isolates of Salmonella Choleraesuis with mutations in the cAMP-receptor protein (CRP) gene (Salmonella Choleraesuis Deltacrp) are the most widely used, although the basis of the attenuation remains unclear. The objective of this study was to determine if the attenuated phenotype of Salmonella Choleraesuis Deltacrp was due to alterations in susceptibility to gastrointestinal factors such as pH and bile salts, ability to colonize or invade the intestine, or cytotoxicity for macrophages. Compared with the parental strain, the survival rate of Salmonella Choleraesuis Deltacrp at low pH or in the presence of bile salts was higher, while the ability of the mutant to invade intestinal epithelia was significantly decreased. In examining the role of CRP on the secretory function of the Salmonella pathogenicity island 1 (SPI-1) encoded type III secretion system (T3SS), it was shown that Salmonella Choleraesuis Deltacrp was unable to secrete the SPI-1 T3SS effector proteins, SopB and SipB, which play a role in Salmonella intestinal invasiveness and macrophage cytotoxicity, respectively. In addition, caspase-1 dependent cytotoxicity for macrophages was significantly reduced in Salmonella Choleraesuis Deltacrp. Collectively, this study demonstrates that the CRP affects the secretory function of SPI-1 T3SS and the resulting ability to invade the host intestinal epithelium, which is a critical element in the pathogenesis of Salmonella Choleraesuis.

Figure 1.

Salmonella Choleraesuis ∆crp is attenuated in BALB/c mice. Survival of mice was recorded up to 3 weeks after peroral (p.o.) or intraperitoneal (i.p.) administration of varying doses of Salmonella Choleraesuis (WT) or the CP327 strain. Data are representative of two independent experiments (n = 5 in each group).

Figure 2.

Salmonella Choleraesuis ∆crp is more resistant to low pH or bile salts. Bacteria grown to stationary phase in LB broth were incubated for 1 h in M9 medium at pH 3.0 or 7.0 (A) or in saline, 15% bile salts, or 1% sodium deoxycholate (B). Survival of bacteria before and after incubation was determined on MacConkey agar plates. Percent survival was normalized to the initial inoculum. WT, wild type Salmonella Choleraesuis; CP327 and CP359, crp gene mutants; Comp., CP327 complemented with pTCRP plasmid; BS, bile salts; DOC, sodium deoxycholate. The results are from three independent experiments of at least triplicate determinations. Error bars are SEM. *p < 0.05; ***p < 0.001; n.s.: no significant difference from each other (ANOVA).

Figure 3.

Salmonella Choleraesuis ∆crp was less invasive in porcine ligated ileal loops and in macrophages. The degree of intestinal invasion by wild type Salmonella Choleraesuis (WT) and CP327 was determined at 3 h after inoculation of the loops (A). To recover bacteria, portions of the ileal wall with Peyer patches (PP) and associated mesenteric lymph nodes (LN) were harvested, homogenized, and plated onto BG agar. The results are from at least triplicate determinations. For in vitro invasion (B) and intracellular survival assays (C), porcine alveolar macrophages were co-cultured with WT and CP327 of Salmonella or E. coli (K12) strains at an MOI of ~ 20 for 2 h and intracellular survived bacteria were assessed. Relative invasion was determined at 4 hpi while intracellular survival rates were at 8 and 16 hpi. The results are representatives of three independent experiments of at least triplicate determinations. Error bars are SEM. *p < 0.05 and ***p < 0.001 compared to WT; n.s.: no significant difference from each other (ANOVA).

Figure 4.

Secretion of effector proteins SopB and SipB via SPI-1 T3SS was impaired in ∆crp. Wild type (WT), CP327 or PQ518 strains transformed with pUC19 (v1), pUC19 encoding sopB-myc (SopB), sipB-myc (SipB), p705 M (v2) or p705 M encoding crp-myc (CRP) were grown under conditions that induced SPI-1 T3SS function. Bacterial culture supernatants (sup.) and pellets (bac.) were harvested, normalized and subjected to Western blot analysis with an antibody to Myc-tag. The results of the SopB secretion assay in WT, CP327 and PQ518 strains (A), SipB secretion assay in WT and CP327 (B) and SopB secretion assay in CP327 complemented with p705CRP (C). Immunoblots were also probed with an anti-DnaK antibody as a loading control and to detect signals resulting from bacterial lysis. The average intensity of secreted proteins was quantified and normalized to the intensity of DnaK for WT, CP327 and PQ518 strains (A, lower panel).

Figure 5.

Salmonella Choleraesuis induced caspase-1 dependent cytotoxicity in RAW 264.7 cells was impaired in ∆crp mutants. Bacteria grown under conditions that induce SPI-1 T3SS were co-cultured at a MOI of ~ 50 with RAW 264.7 cells treated with 0.5% DMSO carrier (Vehicle) or with 50 μM caspase-1 inhibitor Ac-YVAD-CMK (Casp-1). Cell death was assessed at 6 hpi by measuring the release of lactate dehydrogenase (LDH). WT, wild type Salmonella Choleraesuis; CP327 and CP359, crp gene mutants; Comp., CP327 complemented with pTCRP plasmid. The results are from three independent experiments of at least triplicate determinations. Error bars are SEM. **p < 0.01; ***p < 0.001; n.s.: no significant difference from each other (ANOVA).