Oxygen-dependent regulation of SPI1 type three secretion system by small RNAs in Salmonella enterica serovar Typhimurium

Abstract

Salmonella Typhimurium induces inflammatory diarrhea and uptake into intestinal epithelial cells using the Salmonella pathogenicity island 1 (SPI1) type III secretion system (T3SS). Three AraC-like regulators, HilD, HilC and RtsA, form a feed-forward regulatory loop that activates transcription of hilA, encoding the activator of the T3SS structural genes. Many environmental signals and regulatory systems are integrated into this circuit to precisely regulate SPI1 expression. A subset of these regulatory factors affects translation of hilD, but the mechanisms are poorly understood. Here, we identified two sRNAs, FnrS and ArcZ, which repress hilD translation, leading to decreased production of HilA. FnrS and ArcZ are oppositely regulated in response to oxygen, one of the key environmental signals affecting expression of SPI1. Mutational analysis demonstrates that FnrS and ArcZ bind to the hilD mRNA 5' UTR, resulting in translational repression. Deletion of fnrS led to increased HilD production under low-aeration conditions, whereas deletion of arcZ abolished the regulatory effect on hilD translation aerobically. The fnrS arcZ double mutant has phenotypes in a mouse oral infection model consistent with increased expression of SPI1. Together, these results suggest that coordinated regulation by these two sRNAs maximizes HilD production at an intermediate level of oxygen.

Figure 1.. Simplified model of the SPI1 T3SS regulatory circuit.

Blue lines indicate transcriptional regulation, green lines indicate regulation of HilD at the protein level, and red lines indicate regulation of hilD translation. Dotted lines indicate that the exact mechanism of regulation is not known and is likely indirect.

Figure 2.. FnrS and ArcZ downregulate SPI1 expression by repressing hilD translation.

(A) β-galactosidase activity in E. coli strains containing the hilD’-’lacZ translational fusion and plasmids overexpressing FnrS or ArcZ from either E. coli (EC) and Salmonella (SM) grown in the presence of 100 μM IPTG and 0.001% arabinose to induce the sRNA expression and the fusion lacZ protein expression, respectively. β-galactosidase activity in Salmonella strains containing (B) a hilD’-’lacZ translational fusion, or (C) a hilA’-lacZ⁺ transcriptional fusion and plasmids overexpressing FnrS or ArcZ from either E. coli (EC) or Salmonella (SM) grown in SPI1 inducing conditions. β-galactosidase activity units are defined as (μmol of ONP formed min⁻¹) x 10⁶/(OD₆₀₀ x ml of cell suspension) and are reported as mean ± standard deviation where n=3. Strains used: JMS6500, JS892, or JS749, each with plasmid pBRplac, pFnrS-EC, pFnrS-SM, pArcZ-EC, or pArcZ-SM.

Figure 3.. FnrS and ArcZ regulate hilD translation by direct base-pairing interactions.

(A) Predicted base-pairing interactions between each sRNA and hilD mRNA. For hilD, nucleotides are numbered from the translational start site. The ribosome binding site of hilD mRNA is highlighted in green, and the translational start site of hilD is highlighted in red. Boxes mark nucleotides for which complementary mutations were created in hilD and each sRNA. (B) Relative β-galactosidase activity in E. coli strains containing the wild type or mutated hilD’-’lacZ translational fusion and plasmids overexpressing either the wild type (pFnrS) or mutated (pFnrS mt) sRNA grown as indicated in Figure 2A. (C) Relative β-galactosidase activity in E. coli strains containing the wild type or mutated hilD’-’lacZ translational fusion and plasmids overexpressing either the wild type (pArcZ) or mutated (pArcZ mt) sRNA grown as indicated in Figure 2A. β-galactosidase activity units are defined as (μmol of ONP formed min⁻¹) × 10⁶/(OD₆₀₀ × ml of cell suspension) and are reported as mean ± standard deviation where n=3. Strains used: JMS6500, JMS6501, and JMS6502 with plasmid pBRplac, pFnrS-SM, pFnrS-mt, pArcZ-SM, or pArcZ-mt.

Figure 4.. Loss of FnrS or ArcZ results in the increased level of hilA expression due to the abolished repression of hilD translation.

β-galactosidase activity in Salmonella strains containing a (A) hilD’-’lacZ translational or (B) hilA’-lacZ⁺ transcriptional fusion in the wildtype, ΔarcZ, or ΔfnrS background grown under either high aeration or low aeration conditions as described in Experimental procedures. β-galactosidase activity units are defined as (μmol of ONP formed min⁻¹) × 10⁶/(OD₆₀₀ × ml of cell suspension) and are reported as mean ± standard deviation where n=3. Strains used: JS892, JS2123, JS2124, JS749, JS2125 and JS2126.

Figure 5.. Mechanism of FnrS and ArcZ regulation of hilD mRNA translation in Salmonella.

β-galactosidase activity in Salmonella strains containing the hilD’-’lacZ translational fusion and either the empty vector or plasmids overexpressing FnrS or ArcZ from Salmonella in a (A) wildtype or (B) rne131 background grown in SPI1 inducing conditions. β-galactosidase activity units are defined as (μmol of ONP formed min⁻¹) × 10⁶/(OD₆₀₀ × ml of cell suspension) and are reported as mean ± standard deviation where n=3. Strains used: JS892, JS2118 or JS2119, each with plasmid pBRplac, pFnrS-SM, or pArcZ-SM.

Figure 6.. The half-life of hilD mRNA.

Cells were grown under (A) low aeration or (B) high aeration conditions and RNA was isolated at various time points after addition of rifampicin, and processed as described in Experimental procedures. (“C” indicates a sample from a ΔhilD strain.) The northern blots are representative of two independent experiments. The intensities of the hilD mRNA bands were quantified and normalized to the 5S bands. The WT bands at 0 min was considered 100%. mRNA decay curves represent the means and the standard errors (SEM) for the two experiments Strains used: 14028, JS481, JS2117, JS2121, JS2165, JS2166, and JS2167.

Figure 7.. FnrS regulation of SPI1 expression is independent of Fur.

β-galactosidase activity in Salmonella strains containing an fnrS’-lacZ⁺ transcriptional fusion in the wild type, Δfnr, or Δfur background grown in (A) high aeration or (B) low aeration conditions in the presence or absence of 200 μM dipyridyl (DPP). β-galactosidase activity in Salmonella strains containing (C) hilA’-lacZ⁺ transcriptional fusion or (D) sodB’-’lacZ translational fusion in the wild type or the indicated mutant background grown in SPI1 inducing conditions. β-galactosidase activity units are defined as (μmol of ONP formed min⁻¹) × 10⁶/(OD₆₀₀ × ml of cell suspension) and are reported as mean ± standard deviation where n=3. Strains used: JS2129, JS2130, JS2131, JS749, JS2125, JS583, JS2132, JS619, JS2133, JS620, and JS2134.

Figure 8.. Fnr regulates SPI1 expression independent of FnrS.

(A) β-galactosidase activity in Salmonella strains containing the hilA’-lacZ⁺ transcriptional fusion in the wild type, ΔfnrS, or Δfnr background in the presence or absence of HilD grown in low aeration conditions. (B) β-galactosidase activity in Salmonella strains containing hilA’-lacZ⁺ transcriptional fusion and the indicated mutations in the ΔhilD background with RtsA protein produced under a tetracycline regulated promoter grown under low aeration conditions with the indicated a-tetracycline concentrations. β-galactosidase activity units are defined as (μmol of ONP formed min⁻¹) × 10⁶/(OD₆₀₀ × ml of cell suspension) and are reported as mean ± standard deviation where n=3. Strains used: JS749, JS2125, JS2135, JS2136, JS2137, JS2138, JS2139, and JS2140.

Figure 9.. ArcA regulates SPI1 expression independent of ArcZ.

β-galactosidase activity in Salmonella strains containing the (A) hilD’-’lacZ translational fusion or the (B) hilA’-lacZ transcriptional fusion in a wild type, ΔarcZ, and/or ΔarcA background grown in high aeration conditions. (C) β-galactosidase activity in Salmonella strains containing hilA’-lacZ⁺ transcriptional fusion and the indicated mutations in the spi1 (Δspi1 ΔrtsA) background with RtsA protein produced under a tetracycline regulated promoter grown under high aeration conditions with the indicated a-tetracycline concentrations. β-galactosidase activity units are defined as (μmol of ONP formed min⁻¹) × 10⁶/(OD₆₀₀ × ml of cell suspension) and are reported as mean ± standard deviation where n=3. Strains used: JS892, JS2124, JS2143, JS2144, JS749, JS2126, JS2145, JS2146, JS2147, JS2148, JS2149, JS2150, JS2077, JS2151, JS2152, and JS2153.

Figure 10.. Loss of both FnrS and ArcZ abolishes oxygen-mediated regulation of hilA expression.

β-galactosidase activity in Salmonella strains containing the hilA’-lacZ⁺ transcriptional fusion in the wild type, ΔfnrS, ΔarcZ, or ΔfnrS ΔarcZ background grown in either high aeration or low aeration conditions. β-galactosidase activity units are defined as (μmol of ONP formed min⁻¹) × 10⁶/(OD₆₀₀ × ml of cell suspension) and are reported as mean ± standard deviation where n=3. Strains used: JS749, JS2125, JS2126. and JS2160.

Figure 11.. Mouse competition assays.

Mice were infected (intraperitoneally) IP or orally (as noted) with a 50:50 mix of the indicated strains. Competition assays were performed in streptomycin-treated (+Strep) or untreated (−Strep) mice (see Experimental procedures). Bacteria were recovered from the spleen (designated SP) in the case of IP competition assays or from the spleen (SP), small intestine (SI), and large intestine (LI) in oral competitions. The competitive index (CI) was calculated as described in experimental procedures and is shown for each mouse. The line indicates the geometric mean for each set. The Student t test was used to compare the CIs to the inocula or between groups. p=<0.05 (*), p=<0.005 (**), p=<0.0005 (***). The strains used were JS749, JS2125, JS2164, JS2160, JS2162, JS2075.