Coordinate intracellular expression of Salmonella genes induced during infection

Abstract

Salmonella typhimurium in vivo-induced (ivi) genes were grouped by their coordinate behavior in response to a wide variety of environmental and genetic signals, including pH, Mg2+, Fe2+, and PhoPQ. All of the seven ivi fusions that are induced by both low pH and low Mg2+ (e.g., iviVI-A) are activated by the PhoPQ regulatory system. Iron-responsive ivi fusions include those induced under iron limitation (e.g., entF) as well as one induced by iron excess but only in the absence of PhoP (pdu). Intracellular expression studies showed that each of the pH- and Mg2+-responsive fusions is induced upon entry into and growth within three distinct mammalian cell lines: RAW 264.7 murine macrophages and two cultured human epithelial cell lines: HEp-2 and Henle-407. Each ivi fusion has a characteristic level of induction consistent within all three cell types, suggesting that this class of coordinately expressed ivi genes responds to general intracellular signals that are present both in initial and in progressive stages of infection and may reflect their responses to similar vacuolar microenvironments in these cell types. Investigation of ivi expression patterns reveals not only the inherent versatility of pathogens to express a given gene(s) at various host sites but also the ability to modify their expression within the context of different animal hosts, tissues, cell types, or subcellular compartments.

FIG. 1

Expression profiles of ivi fusions in response to pH and Mg²⁺ concentration. pH values are represented on the x axis, β-galactosidase activities [(micromoles of ONP formed per minute per A₆₀₀ unit per milliliter of cell suspension] [10³]) are plotted on the y axis, and Mg²⁺ concentrations are plotted on the z axis. Values represent the averages of three independent cultures with a standard deviation of <10% of the mean. (A) Seven PhoP-activated ivi fusions. (B) PhoP-repressed fusion iviXVII (pdu). In panels A and B, the columns represent, from left to right, expression profiles in a PhoP⁺ background, in a PhoP⁻ (phoP102::Tn10d-Cm) background, and in a phoQ24 background. A very similar induction profile was obtained for exponentially growing cells of the wild type under all 15 conditions tested (data not shown). The pH of each wild-type culture was quantitated under the 15 conditions tested before and after incubation. After 16 h of growth, the pH in MOPS minimal medium typically decreased 1 to 1.8 pH units; the pH in MOPS-buffered LB increased 1.0 to 1.5 pH units. The pH of exponentially grown cultures in MOPS minimal medium typically decreased 0.4 to 0.6 pH units in MOPS minimal medium or remained within 0.4 pH units in MOPS-buffered LB.

FIG. 1

Expression profiles of ivi fusions in response to pH and Mg²⁺ concentration. pH values are represented on the x axis, β-galactosidase activities [(micromoles of ONP formed per minute per A₆₀₀ unit per milliliter of cell suspension] [10³]) are plotted on the y axis, and Mg²⁺ concentrations are plotted on the z axis. Values represent the averages of three independent cultures with a standard deviation of <10% of the mean. (A) Seven PhoP-activated ivi fusions. (B) PhoP-repressed fusion iviXVII (pdu). In panels A and B, the columns represent, from left to right, expression profiles in a PhoP⁺ background, in a PhoP⁻ (phoP102::Tn10d-Cm) background, and in a phoQ24 background. A very similar induction profile was obtained for exponentially growing cells of the wild type under all 15 conditions tested (data not shown). The pH of each wild-type culture was quantitated under the 15 conditions tested before and after incubation. After 16 h of growth, the pH in MOPS minimal medium typically decreased 1 to 1.8 pH units; the pH in MOPS-buffered LB increased 1.0 to 1.5 pH units. The pH of exponentially grown cultures in MOPS minimal medium typically decreased 0.4 to 0.6 pH units in MOPS minimal medium or remained within 0.4 pH units in MOPS-buffered LB.

FIG. 2

iviXVII (pdu) and entF expression as a function of iron concentration in the medium. Cultures were grown overnight in iron-free MOPS minimal medium at pH 7.0. The cultures were diluted to OD₆₀₀ of 0.1 in the same medium grown to an OD₆₀₀ of 0.4 (mid-log phase). FeSO₄ was then added at the given concentration, and β-galactosidase activities were determined after 5 h. Values represent the averages of three independent cultures with a standard deviation of <10% of the mean.

FIG. 3

Intracellular expression of ivi genes in cultured macrophages and cultured epithelial cells. Individual ivi fusions were incubated with cultured RAW 264.7 murine macrophages and cultured human epithelial cell lines HEp-2 (larynx carcinoma) and Henle-407 (embryonic small intestine). The coculture was incubated for 30 min, washed, treated with gentamicin to kill extracellular bacteria, washed, and incubated for 4 h. The cultured mammalian cells were lysed with Triton X-100, and β-galactosidase assays were performed on the recovered intracellular bacteria. For comparison, β-galactosidase assays were also performed on individual ivi fusions grown for 4 h in LB and in MEM supplemented with 10% FCS. Preselected Lac⁻ and Lac⁺ strains were obtained from the initial nonselected pool of integrated IVET fusions. Error bars represent 1 standard deviation of the measured value.

FIG. 4

Intracellular expression of ivi genes in PhoP⁺ and PhoP⁻ (phoP102::Tn10d-Cm) backgrounds in cultured macrophages. Individual ivi fusions were incubated with cultured RAW 264.7 murine macrophages. The coculture was incubated for 30 min, washed, treated with gentamicin to kill extracellular bacteria, washed, and incubated for 4 h. The cultured macrophages were lysed with Triton X-100. β-Galactosidase assays were performed on the recovered intracellular bacteria and also on individual ivi fusions grown for 4 h in LB. Error bars represent 1 standard deviation of the measured value.