Fig 1.
SsrB represses the expression of the SPI-1-encoded regulator InvF.
Expression of InvF-FLAG and SsrB-FLAG in the WT S. Typhimurium strain containing the plasmid pK3-SsrB expressing SsrB from a constitutive promoter, or the vector pMPM-K3, was analyzed by Western blot using monoclonal anti-FLAG antibodies. Whole cell lysates were prepared from samples of bacterial cultures grown in LB at 37°C, at the OD600 or the time indicated, representing exponential, early stationary or late stationary phases of growth. As a loading control, the expression of DnaK was also determined using monoclonal anti-DnaK antibodies.
Fig 2.
SsrB represses the secretion of SPI-1-encoded proteins and Salmonella invasion of HeLa cells.
(A) Secretion profiles of the SPI-1-encoded proteins SipA, SipB, SipC and SipD were examined in the WT S. Typhimurium strain and its isogenic ΔSPI-2 mutant containing the plasmid pK3-SsrB that constitutively expresses SsrB, or the vector pMPM-K3, grown for 9 h in LB at 37°C. As a control, the secretion profile for the ΔhilD mutant that lacks the SipA-D proteins is also shown. FliC is the major subunit of the flagellar filament. (B) HeLa cells were infected with WT S. Typhimurium or isogenic ΔhilD and ΔflhDC mutants containing either pK3-SsrB or vector control pMPM-K3, and intracellular bacteria enumerated after 1 hr. White and black columns indicate the number of bacteria from the starting inoculum and from intracellular bacteria recovered from the HeLa cells, respectively. Data represents the mean with standard deviation of three independent experiments. *Statistically different values with respect to the WT strain with or without the vector pMPM-K3, P < 0.005.
Fig 3.
SsrB directly represses the hilD and hilA SPI-1 regulatory genes.
Expression of the hilD-cat-364+88 (A), hilA-cat-410+446 (B), invF-cat (C) and ssaG-cat (D) transcriptional fusions was tested in the WT S. Typhimurium strain containing the vector pMPM-K3 or the plasmid, pK3-SsrB, which expresses SsrB from a constitutive promoter. The CAT-specific activity was determined from samples collected of bacterial cultures grown for 4 and 9 h in LB at 37°C. Data represents the mean with standard deviation of three independent experiments. *Statistically different values with respect to the WT strain containing the vector pMPM-K3, P < 0.0005. SsrB binding to the DNA fragments contained in the hilD-cat-364+88 (E), hilA-cat-410+446 (F), invF-cat (G) and ssaG-cat (H) fusions were analyzed using EMSAs. The respective PCR-amplified and purified DNA fragments were incubated with increasing concentrations of purified 6H-SsrBc (0, 0.5, 1, 1.5 and 2 μM). DNA-protein complexes are indicated by an asterisk.
Fig 4.
Schematic representation of the hilD and hilA genes and their regulatory elements.
(A) Sequence logo for the PSSM used to predict the SsrB binding sites. (B) The locus containing hilD and hilA. The transcriptional start site (+1) of hilD and hilA is indicated by a bent arrow and red boxes represent their promoters. The SsrB-binding sites involved in repression of hilD or hilA are displayed as blue boxes below or above the respective regulatory region, which indicates the sense and anti-sense strand of DNA, respectively; their respective 18-bp sequence is shown. The HilD-binding sites on hilD and hilA are displayed as green boxes. The different hilD-cat and hilA-cat transcriptional fusions assessed in this study are also shown. All of the positions indicated are relative to the transcriptional start site of hilD or hilA.
Fig 5.
SsrB represses hilD by directly acting on its promoter.
Expression of the hilD-cat-108+88 (A), hilD-cat-48+88 (B) and hilD-cat-37+6 (C) transcriptional fusions was tested in the WT S. Typhimurium strain with the vector pMPM-K3, or the plasmid pK3-SsrB, which expresses SsrB from a constitutive promoter. The CAT-specific activity was determined from bacterial cultures grown for 9 h in LB at 37°C. Data represents the mean with standard deviation of three independent experiments. *Statistically different values with respect to the WT strain with pMPM-K3, P < 0.0005. EMSAs were performed to analyze whether SsrB binds to the hilD DNA fragments in the hilD-cat-108+88 (D), hilD-cat-48+88 (E) and hilD-cat-37+6 (F) fusions. The DNA fragments were incubated with increasing concentrations of purified 6H-SsrBc (0, 0.5, 1, 1.5 and 2 μM). DNA-protein complexes are indicated by an asterisk.
Fig 6.
SsrB represses hilA by binding to the regulatory region between positions -100 to -35.
Expression of the hilA-cat-410+66 (A), hilA-cat-100+6 (B), hilA-cat-35+6 (C) and hilA-cat-35+446 (D) transcriptional fusions was tested in the WT S. Typhimurium strain with the vector pMPM-K3, or the plasmid pK3-SsrB, which expresses SsrB from a constitutive promoter. The CAT-specific activity was determined from bacterial cultures grown for 9 h in LB at 37°C. Data represents the mean with standard deviation of three independent experiments. *Statistically different values with respect to the WT strain with pMPM-K3, P < 0.005. EMSAs were performed to determine whether SsrB binds to the hilA DNA fragments in the hilA-cat-410+66 (E), hilA-cat-100+6 (F), hilA-cat-35+6 (G) and hilA-cat-35+446 (H) fusions. The DNA fragments were incubated with increasing concentrations of purified 6H-SsrBc (0, 0.5, 1, 1.5 and 2 μM). DNA-protein complexes are indicated by an asterisk.
Fig 7.
SsrB represses HilD-mediated expression of hilA by binding to a sequence overlapping the HilD-binding sequence upstream of the hilA promoter.
Expression of the hilA-cat-100+6 WT (wt SsrB binding site) (A and B) and hilA-cat-100+6 Mut (mutated SsrB binding site) (C and D) fusions was determined in the WT S. Typhimurium strain (A and B) and its isogenic ΔSPI-1 ΔrtsA ΔCthns mutant (C and D) that lacks HilD, HilC, RtsA and other regulators encoded in SPI-1, as well as H-NS. The CAT-specific activity was determined from bacterial cultures grown for 9 h in LB at 37°C. Data represents the mean with standard deviation of three independent experiments. *Statistically different values relative to the WT strain containing the pMPM-K3 vector, P < 0.0005. The WT and mutated SsrB-binding sequence are indicated; the nucleotides that were changed in the mutated sequence are underlined. EMSAs were performed to analyze the interaction of SsrB with the hilA DNA fragments carried by the hilA-cat-100+6 WT (E) and hilA-cat-100+6 Mut (F) fusions. The DNA fragments were incubated with increasing concentrations of purified 6H-SsrBc (0, 0.5, 1, 1.5 and 2 μM). DNA-protein complexes are indicated by an asterisk.
Fig 8.
SsrB inversely regulates the expression of the invF (SPI-1) and ssaG (SPI-2) genes inside macrophages.
Expression of the invF-lux (A) and ssaG-lux (B) transcriptional fusions was analyzed in the WT S. Typhimurium strain and its isogenic ΔSPI-2 mutant (lacking SsrB) inside RAW264.7 murine macrophage-like cells. Monolayers of macrophages were infected with an equal number of the respective Salmonella strain. At the indicated times post-infection the cells were lysed and luminescence and CFU counts were determined. Data represents the mean with standard deviation of three independent experiments. *Statistically different values with respect to those shown by the invF-lux fusion in the WT strain at the same post-infection times in panel A or with respect to that shown by the ssaG-lux fusion in the WT strain at 1 h post-infection in panel B, P < 0.05. (C) Data used in panel A were graphed to show the fold change in the expression of invF-lux in the ΔSPI-2 mutant with respect to the WT strain at the different post-infection times. *Statistically different values with respect to those obtained for 1 h post-infection, P < 0.05. (D) Data used in panels A and B were graphed to show the expression of the invF-lux and ssaG-lux fusions in the WT strain at the different post-infection times. Positive (indicated by an arrow) and negative (denoted by a blunt-end line) SsrB-mediated regulation of ssaG and invF, respectively, is depicted.
Fig 9.
Mice were orally gavaged with the indicated strains and luciferase activity expressed from the hilA-lux-740+350 fusion was measured by live animal imaging. Images are representative of three experiments and data is shown as the mean with standard error at each time point from three separate animals.
Fig 10.
SsrB is involved in a molecular regulatory switch that aids in Salmonella transition to an intracellular lifestyle.
(A) HilD directly or indirectly activates the expression of the SPI-1 genes and several other genes located outside SPI-1, including the flagellar regulatory operon flhDC required for the invasion of host cells. (B) Following its uptake into macrophages, Salmonella resides inside vacuoles, where SsrB induces the expression of the SPI-2 genes and other genes located outside SPI-2, which are required for survival and replication, while simultaneously repressing the expression of the hilD and hilA SPI-1 regulatory genes, and the flagellar-based motility genes. Green arrows and red blunt-end lines indicate positive and negative control, respectively, whereas gray dashed arrows denote expression of the respective genes.
Table 1.
Bacterial strains and plasmids.
Table 2.
Oligonucleotides.