PhoP-Mediated Repression of the SPI1 Type 3 Secretion System in Salmonella enterica Serovar Typhimurium

Abstract

Salmonella must rapidly adapt to various niches in the host during infection. Relevant virulence factors must be appropriately induced, and systems that are detrimental in a particular environment must be turned off. Salmonella infects intestinal epithelial cells using a type 3 secretion system (T3SS) encoded on Salmonella pathogenicity island 1 (SPI1). The system is controlled by three AraC-like regulators, HilD, HilC, and RtsA, which form a complex feed-forward loop to activate expression of hilA, encoding the main transcriptional regulator of T3SS structural genes. This system is tightly regulated, with many of the activating signals acting at the level of hilD translation or HilD protein activity. Once inside the phagosomes of epithelial cells, or in macrophages during systemic stages of disease, the SPI1 T3SS is no longer required or expressed. Here, we show that the PhoPQ two-component system, critical for intracellular survival, appears to be the primary mechanism by which Salmonella shuts down the SPI1 T3SS. PhoP negatively regulates hilA through multiple distinct mechanisms: direct transcriptional repression of the hilA promoter, indirect transcriptional repression of both the hilD and rtsA promoters, and activation of the small RNA (sRNA) PinT. Genetic analyses and electrophoretic mobility shift assays suggest that PhoP specifically binds the hilA promoter to block binding of activators HilD, HilC, and RtsA as a mechanism of repression.IMPORTANCESalmonella is one of the most common foodborne pathogens, causing an estimated 1.2 million illnesses per year in the United States. A key step in infection is the activation of the bacterial invasion machinery, which induces uptake of the bacterium into epithelial cells and leads to induction of inflammatory diarrhea. Upon entering the vacuolar compartments of host cells, Salmonella senses an environmental transition and represses the invasion machinery with a two-component system relevant for survival within the vacuole. This adaptation to specific host niches is an important example of how signals are integrated for survival of the pathogen.

Keywords: PhoPQ; Salmonella; virulence regulation.

FIG 1

SPI1 T3SS regulatory circuit. Simplified regulatory model of the SPI1 T3SS and related regulators. Blue lines indicate transcriptional regulation, green lines indicate regulation at the protein level, and red lines indicate regulation at the posttranscriptional level. Arrows indicate activation and blunt ends indicate repression. Dotted lines indicate that the exact mechanism of regulation is not known and is likely indirect.

FIG 2

PhoP represses hilA expression. β-Galactosidase activity was measured from cells containing a hilA′-lacZ⁺ transcriptional fusion in the indicated background. Complementation of phoP was performed by integrating a wild-type copy of phoP elsewhere in the chromosome. Cells were grown overnight, subcultured 1:100, and then grown for 6 h with minimal aeration at 37°C in HSLB medium. β-Galactosidase activity units are defined as (micromoles of o-nitrophenol [ONP] formed per minute) × 10⁶/(optical density at 600 nm [OD₆₀₀] × milliliters of cell suspension) and are reported as means ± standard deviations, where n = 4. ***, P < 0.001 by unpaired t tests. n.s., not significant. Strains used: JS749, JS1055, JS2172, and JS2173.

FIG 3

Putative mediators of PhoP repression do not alleviate PhoP effects. β-Galactosidase activity was measured from cells containing a hilA′-lacZ⁺ transcriptional fusion in the presence or absence of possible mediators of PhoP repression. Cells were grown overnight, subcultured 1:100, and then grown for 6 h with minimal aeration at 37°C in HSLB medium. β-Galactosidase activity units are defined as micromoles of ONP formed per minute × 10⁶/(OD₆₀₀ × milliliters of cell suspension) and are reported as means ± standard deviations, where n = 4. ***, P < 0.001 by unpaired t tests. Strains used: JS749, JS1055, JS2103, JS2183, JS2174, JS2175, JS2176, JS2177, JS2178, JS2179, JS2181, and JS2182.

FIG 4

PhoP represses hilD and rtsA transcription. β-Galactosidase activity was measured from cells containing the indicated lacZ transcriptional fusions, in the presence or absence of the phoQ24 allele, with and without other activators. Cells were grown overnight, and subcultured 1:100, then grown for 6 h with minimal aeration at 37˚C in HSLB medium. β-Galactosidase activity units are defined as micromoles of ONP formed per minute × 10⁶/(OD₆₀₀ × milliliters of cell suspension) and are reported as means ± standard deviations, where n = 4. ***, P < 0.001 by unpaired t tests. Strains used: JS2196, JS2197, JS2198, JS2199, JS2187, JS2189, JS2191, JS2193, JS2184, JS2200, JS2201, and JS2202.

FIG 5

PhoP additionally represses hilA independently of hilD, rtsA, and pinT. β-Galactosidase activity was measured from cells containing a hilA′-lacZ⁺ transcriptional fusion and with rtsA controlled by the tetracycline-inducible promoter in the presence or absence of the phoQ24 allele and hilD (A) or with hilD controlled by the tetracycline-inducible promoter in the presence or absence of the phoQ24 allele and fliZ (B). Anhydrotetracycline (aTc) was added at the levels indicated. Cells were grown overnight, subcultured 1:100, and then grown statically for 20 h at 37°C in HSLB medium. β-Galactosidase activity units are defined as micromoles of ONP formed per minute × 10⁶/(OD₆₀₀ × milliliters of cell suspension) and are reported as means ± standard deviations, where n = 4. **, P < 0.01; ***, P < 0.001 by unpaired t tests. Strains used: JS953, JS955, JS1057, JS1058, JS2203, JS2204, JS2205, and JS2206.

FIG 6

PhoP-P repression of hilA. (A) PhoP-P was added at designated concentrations. The indicated fragments were PCR amplified and used at a final concentration of 20 nM. Electrophoretic mobility shift assays of PhoP-P binding to the hilA promoter. Negative-control DNA was a random section of the PDX1 plasmid, and the positive control was phoP, containing a known PhoP binding site. Gel shifts were stained with SYBR green. (B) A schematic diagram of the hilA promoter. Included are relevant HilD, HilC, and RtsA binding sites as well as putative PhoP binding sites. The figure is not drawn to scale. β-Galactosidase activity was measured from cells containing a hilA′-lacZ⁺ transcriptional fusion with the designated end points relative to the transcriptional start site. Backgrounds include the presence or absence of the phoQ24 allele. Cells were grown overnight, subcultured 1:100, and then grown with mild aeration for 6 h at 37°C in HSLB medium. β-Galactosidase activity units are defined as micromoles of ONP formed per minute × 10⁶/(OD₆₀₀ × milliliters of cell suspension) and are reported as means ± standard deviations, where n = 4. Strains used: JS2207, JS2208, JS2209, JS2210, JS2211, JS2212, JS2213, JS2214, JS2215, and JS2216.

FIG 7

PhoP may block binding of activators HilD, HilC, and RtsA. β-Galactosidase activity was measured from cells containing a hilA′-lacZ⁺ transcriptional fusion with the designated end points relative to the transcriptional start site in the designated background. Cells were grown overnight, subcultured 1:100, and then grown with mild aeration for 6 h at 37°C in HSLB medium. β-Galactosidase activity units are defined as micromoles of ONP formed per minute × 10⁶/(OD₆₀₀ × milliliters of cell suspension) and are reported as means ± standard deviations, where n = 4. *, P < 0.05; **, P < 0.01; ***, P < 0.001 by unpaired t tests. Strains used: JS2217, JS2218, JS2219, JS2220, JS2213, JS22134, JS2221, JS2222, JS2215, JS2216, JS2223, and JS2224.

FIG 8

HilD, HilC, and RtsA activate hilA expression independently of H-NS. β-Galactosidase activity was measured from cells containing hilA′-lacZ⁺ transcriptional fusions with the designated end points relative to the transcriptional start site. Backgrounds include the presence or absence of the phoQ24 allele and spi1 and rtsA. Cells were grown overnight, subcultured 1:100, and then grown with mild aeration at 37°C in HSLB medium. After 1.5 h, 10 mM arabinose was added and cells were grown for an additional 1 h, at which point β-galactosidase activity was measured. β-Galactosidase activity units are defined as micromoles of ONP formed per minute × 10⁶/(OD₆₀₀ × milliliters of cell suspension) and are reported as means ± standard deviations, where n = 4. *, P < 0.05; **, P < 0.01; ***, P < 0.001 by unpaired t tests. Strains used: JS2217, JS2219, JS2227, JS2228, JS2213, JS2221, JS2215, JS2223, JS2225, JS2226, JS749, JS2238, JS2236, JS2237, JS2207, JS2235, JS2233, JS2234, JS2210, JS2232, JS2211, JS2231, JS2229, and JS2230 containing plasmids pBAD30, pMK1, and pMK2.