A FRET-Based DNA Biosensor Tracks OmpR-Dependent Acidification of Salmonella during Macrophage Infection

10.1371/journal.pbio.1002116PLoS Biology134e100211

Model for SPI-2 secretion in macrophages.

<p><b>(A)</b> Immediately post-infection, the bacterial cytosol is approximately pH 6.8 and the SPI-2 secretion apparatus is beginning to be assembled at the cell pole. <b>(B)</b> Within 30 min inside the SCV, the bacterial cytoplasm becomes acidic and the translocon complex SseBCD begins to assemble on the bacterial surface. The “<b>plug” model (C)</b> After 3.5 h, SseB is detached from the <i>Salmonella</i> surface into the macrophage cytoplasm, triggering the onset of SseJ effector secretion. <b>(D)</b> The bacterial cytoplasm remains acidified during the effector secretion process. <b>(E) The “SsaG needle elongation” model</b>: steps 1 and 2 are the same as in <b>(A, B)</b>. The SseBCD translocon complex begins to associate with the vacuolar membrane and “pulls” the SsaG needle, elongating the needle and driving the translocon away from the bacterial surface. <b>(F)</b> The pore opens and SseJ and other effectors are secreted. <b>(G)</b> At low external pH levels in the macrophage vacuole, protons (H⁺) cross the <i>Salmonella</i> outer and inner membrane and lower the pH of the cytoplasm. In WT <i>Salmonella</i>, EnvZ is activated by excess protons in the cytoplasm, activating OmpR. OmpR~P represses <i>cadC/BA</i> in the macrophage vacuole and the cytoplasm remains acidified. OmpR~P goes on to activate expression of SsrA/B, and SsrB~P activates SPI-2 transcription. <b>(H)</b> In the <i>ompR</i> null strain, CadC/BA is expressed and is responsible for amino acid decarboxylation. This process consumes intracellular protons, restoring cytoplasmic pH and maintaining intracellular pH homeostasis. Image credit: Chun Xi Wong.</p

Functional integrity of the I-switch in <i>Salmonella</i>.

<p>Wild-type (WT) <i>Salmonella</i> was electroporated with 6 μM I-switch and recovered in SOC for 1 h at 37°C, followed by three washes with PBS. Bacteria were then clamped at various pH values in K⁺-rich buffer containing 40 μM nigericin for 1 h at room temperature (RT) and imaged on an Applied Precision Delta Vision inverted wide-field fluorescence microscope. The fluorescence intensity of both donor and acceptor channels of 50 cells were measured for every pH value indicated in the figure. The ratio of D/A intensities were obtained and the normalized D/A ratios were plotted as a function of pH, which yielded a sigmoidal intracellular calibration curve (<b>A</b>). The intracellular curve is overlaid on the in vitro calibration curve. Error bars represent mean ± standard deviation (SD). <b>(B)</b> Representative Donor (D), FRET (A) and D/A ratio images of WT <i>Salmonella</i> clamped at various pH values. Scale bar, 3 μm, color scale, bottom, right. Pseudocolor images were generated by calculating the D/A ratio per pixel. Using ImageJ software, the pixels were then color-coded using 16 colors with blue (D/A = 0.1) to red (D/A = 1) to indicate the transition from acidic to neutral pH.</p

The <i>Salmonella</i> cytoplasm is rapidly acidified upon macrophage infection and requires OmpR.

<p>RAW264.7 macrophages infected with WT <i>Salmonella</i> and the <i>ΔompR</i> null mutant containing I_A488/I_A647 were imaged over time on a Nikon A1R confocal microscope. The D/A ratio in the <i>ΔompR</i> strain showed minimal FRET, indicating <i>ompR</i> was required for cytoplasmic acidification. RAW264.7 macrophages were pre-treated with 25 nM of the V-ATPase inhibitor bafilomycin A1 (BAF) for 30 min prior to infection, and macrophages were infected with I_A488/I_A647-electroporated <i>Salmonella</i> in the presence/absence of BAF for 3 h and imaged as before. BAF neutralized the SCV and prevented <i>Salmonella</i> acidification. <b>(A)</b> Representative images of D/A ratios are shown at 30 and 180 min post-infection and compared to WT <i>Salmonella</i> prior to infection. Infection was carried out as described in Materials and Methods. Scale bar, 3 μm. <b>(B)</b> Prior to infection, the D/A ratios of <i>Salmonella</i> were determined and the intracellular pH was approximately 6.8. The intracellular pH of <i>Salmonella</i> remained fairly constant in macrophages upon BAF treatment (approximately 6.8) or in the <i>ΔompR</i> null strain in macrophages, unlike untreated WT <i>Salmonella</i>. It showed a remarkable intracellular pH drop to 5.65, similar to the <i>ompR</i> null mutant complemented with <i>ompR</i> supplied in <i>trans</i>. Dataset is represented as the mean ± SEM (<i>n</i> = 3).</p

The <i>Salmonella</i> cytoplasm is acidified upon acid stress and requires OmpR.

<p><b>(A)</b><i>Salmonella</i> cultures of I_A488/I_A647-incorporated WT, <i>ompR</i> null mutant, and <i>ompR</i> null complemented with pWSK29-<i>ompR</i> (encoding <i>ompR</i>) were incubated at either acidic pH_e (5.6) or neutral pH_e (7.2) at indicated time points. Representative epifluorescence of the D/A ratio images are shown for WT <i>Salmonella</i>, the <i>ΔompR</i> mutant, and the <i>ΔompR</i> mutant with <i>ompR</i> provided in <i>trans</i>. Scale bar, 3 μm. <b>(B)</b> A plot of the intracellular pH of <i>Salmonella</i> WT at pH_e 5.6 and pH_e 7.2, an <i>ompR</i> null mutant at pH_e 5.6 and pH_e 7.2, and the <i>ompR</i> null mutant complemented with <i>ompR</i> supplied in <i>trans</i> at pH_e 5.6 and pH_e 7.2 over time. The D/A ratios of 50 cells were analyzed at each time point and the pH values were determined from the intracellular standard curve. Error bars represent mean ± standard error of the mean (SEM) (<i>n</i> = 3). (<b>C</b>) To demonstrate the reversibility of the I-switch and mimic vacuolar pH conditions in vitro, WT <i>Salmonella</i> cultures were incubated at pH_e (4.5) for 1 h, and samples were collected at 30 and 60 min for imaging. The cultures were then washed and incubated for an additional 1 h at pH_e 7.2. The D/A ratios of 50 cells were analyzed at the indicated time points. Representative epifluorescence of the D/A ratio images are shown. Scale bar, 3 μm. The average pH_i from 50 cells is listed under the image.</p

As SseB moves away from the <i>Salmonella</i> surface, it is associated with the vacuolar membrane.

<p><b>(A)</b> RAW264.7 macrophages were infected with WT <i>Salmonella</i> harboring pFPV25.1 for the constitutive expression of GFP (green). Cells were fixed, permeabilized, and immunostained for mouse monoclonal anti-LAMP-1 (blue) and rabbit polyclonal anti-SseB (red) followed by Alexa568- or Alexa647-labeled secondary antibodies, respectively, for various times as indicated. The merged image indicates that once SseB was detached from the bacterial cell surface, it was co-localized with the host endosomal membrane (i.e., LAMP-1–positive membranes). Images were obtained using the Nikon A1R confocal microscope. Localization resulted in magenta staining of <i>Salmonella</i> SseB as analyzed by Image J software. Scale bar = 3μm. <b>(B)</b> Results of three independent experiments in which macrophages were infected with WT GFP-expressing <i>Salmonella</i> and immunostained with LAMP-1 and SseB at 3.5 h, 4 h, 5 h, and 6 h post-infection. The values represent the mean ± SEM.</p

OmpR directly represses <i>cadC/BA</i> to prevent recovery from acidification in the macrophage vacuole.

<p><b>(A)</b> mRNA levels of <i>cadA</i>, <i>cadB</i>, <i>cadC</i>, <i>ssrA</i>, and <i>ompR</i> genes were determined by quantitative real-time polymerase chain reaction (qRT-PCR) from WT and <i>ompR</i> null strains as described in Materials and Methods. The error bars represent the mean ± SD (<i>n</i> = 6). The <i>ompR</i> null strain showed increased expression of <i>cadC/BA</i> genes, indicating OmpR repression. <b>(B)</b> mRNA levels of <i>cadA</i>, <i>cadB</i>, <i>cadC</i>, and <i>ssrA</i> genes were determined by qRT-PCR from WT, <i>envZ</i> null mutant and the <i>envZ</i> null mutant complemented with <i>envZc</i> as described in Materials and Methods. The error bars represent the mean ± SD (<i>n</i> = 6). The <i>envZ</i> null strain showed increased expression of <i>cadC/BA</i> genes, indicating a role for EnvZ in OmpR-mediated repression. The cytoplasmic domain of <i>envZ</i> alone could restore <i>ssrA</i> expression levels when provided in <i>trans</i>, indicating that it was responding to cytoplasmic signals. <b>(C–F)</b> Electrophoretic mobility shift assays (EMSA) experiments were conducted to examine the interaction between OmpR and <i>cadC/BA</i>. OmpR was incubated with 10 fmol of biotin end-labeled <i>cadA</i><b>(C)</b>, <i>cadB</i><b>(D)</b>, <i>cadC</i><b>(E)</b>, along with 60 bp biotin end-labeled EBNA DNA <b>(F)</b> as a negative control. Addition of 50-fold excess unlabeled DNA is indicated by an asterisk. <b>(G)</b> Representative images of D/A ratios are shown at 30 and 180 min post-infection of WT, the <i>cadBA</i> double null mutant, an <i>ompR/cadBA</i> triple null strain and a <i>cadBA</i> over-expressed strain of <i>Salmonella</i>. Infection is described in Materials and Methods. Scale bar, 3 μm. <b>(H)</b> Plot of the D/A ratios of various mutants of <i>Salmonella</i>. The D/A ratio of approximately 30 cells were calculated at the indicated time points post-infection. Symbols represent the mean ± SEM (<i>n</i> = 3).</p

The cytoplasmic domain of EnvZ is required for intracellular acidification upon acid stress.

<p><b>(A)</b><i>Salmonella</i> cultures of I_A488/I_A647-incorporated WT, <i>envZ</i> null mutant, and <i>envZ</i> null complemented with pMPM-<i>envZc</i> (encoding cytoplasmic domain of <i>envZ</i>) were incubated at acidic pH_e (5.6) at indicated time points. Representative epifluorescence of the D/A ratio images are shown. Scale bar, 3 μm. <b>(B)</b> A plot of the intracellular pH at pH_e 5.6 of <i>Salmonella</i> WT, an <i>envZ</i> null mutant and the <i>envZ</i> null mutant complemented with <i>envZc</i> supplied in <i>trans</i> over time. The D/A ratios of 50 cells were analyzed at each time point and the pH values were determined from the intracellular standard curve. Symbols represent the mean ± SEM (<i>n</i> = 3).</p