Engineering Transcriptional Regulation to Control Pdu Microcompartment Formation

<div><p>Bacterial microcompartments (MCPs) show great promise for the organization of engineered metabolic pathways within the bacterial cytoplasm. This subcellular organelle is composed of a protein shell of 100–200 nm diameter that natively encapsulates multi-enzyme pathways. The high energy cost of synthesizing the thousands of protein subunits required for each MCP demands precise regulation of MCP formation for both native and engineered systems. Here, we study the regulation of the propanediol utilization (Pdu) MCP, for which growth on 1,2-propanediol induces expression of the Pdu operon for the catabolism of 1,2-propanediol. We construct a fluorescence-based transcriptional reporter to investigate the activation of the P_pdu promoter, which drives the transcription of 21 <i>pdu</i> genes. Guided by this reporter, we find that MCPs can be expressed in strains grown in rich media, provided that glucose is not present. We also characterize the response of the P_pdu promoter to a transcriptional activator of the <i>pdu</i> operon, PocR, and find PocR to be a necessary component of Pdu MCP formation. Furthermore, we find that MCPs form normally upon the heterologous expression of PocR even in the absence of the natural inducer 1,2-propanediol and in the presence of glucose, and that Pdu MCPs formed in response to heterologous PocR expression can metabolize 1,2-propanediol <i>in vivo</i>. We anticipate that this technique of overexpressing a key transcription factor may be used to study and engineer the formation, size, and/or number of MCPs for the Pdu and related MCP systems.</p></div

Coomassie-stained gel and western blot of purified MCPs.

<p>4%–20% SDS-PAGE gel stained with Coomassie (top) and anti-GFP western blot (bottom) of a molecular mass marker (lane 1), and purified MCPs from <i>S. enterica</i> grown in NCE 1,2-PD (lane 2), <i>S. enterica</i> expressing PduP^1-18-GFP grown in NCE 1,2-PD (lane 3), <i>S. enterica</i> expressing PduP^1-18-GFP grown in LB 1,2-PD (lane 4), <i>S. enterica</i> expressing PduP^1-18-GFP and PocR grown in LB (lane 5), <i>S. enterica</i> Δ<i>pocR</i> expressing PduP^1-18-GFP and PocR grown in LB (lane 6), and cell lysate from <i>S. enterica</i> expressing PduP^1-18-GFP grown in LB (lane 7). Lanes with purified MCPs were loaded with 6 µg of total protein.</p

Bright field and fluorescence microscopy of <i>S. enterica</i> expressing PduP^1-18-GFP.

<p>Representative images of are shown for <i>S. enterica</i> expressing fluorescent encapsulation reporter PduP^1-18-GFP grown in (A) NCE, (B) NCE 1,2-PD, (C) LB, and (D) LB 1,2-PD. <i>S. enterica</i> expressing PduP^1-18-GFP are grown in LB carrying a secondary plasmid, either (E) the control vector pTET MBP without aTC, or (F) pTET PocR induced with 1 ng/mL aTc. <i>S. enterica</i> Δ<i>pocR</i> expressing PduP^1-18-GFP are grown in LB carrying a secondary plasmid containing either (G) the control vector pTET MBP without aTc, or (H) pTET PocR induced with 1 ng/mL aTc. Scale bars represent 1 µm.</p

Flow cytometry fluorescence time course of <i>S. enterica</i> harboring plasmid P_pdu–GFP.

<p>Time is indicated as hours after OD₆₀₀ = 0.4, at which point cultures continued to grow without 1,2-PD (open symbols) or with the addition of 1,2-PD (solid symbols). (A) Wild-type <i>S. enterica</i> grown in NCE minimal media. (B) Wild-type <i>S. enterica</i> grown in LB media. (C) Wild-type <i>S. enterica</i> grown in LB carrying a secondary plasmid containing either the control vector pTET MBP without aTc (squares), or pTET pocR induced with 1 ng/mL aTc (triangles). (D) <i>S. enterica</i> ΔpocR grown in LB carrying a secondary plasmid containing either the control vector pTET MBP without aTc (squares), or pTET pocR induced with 1 ng/mL aTc (triangles).</p

FcRγ deficiency results in an increased accumulation of DN T cells.

<p><b>A.</b> B6.LPR.FcRγ^+/+ and B6.LPR.FcRγ^−/− mice were given 4×10⁷ CB6F1 splenocytes intravenously. After 7 days, varying numbers of DN T cells were purified and incubated for a further 3 days with irradiated CB6F1 splenocytes (10⁵/well), after which 1 µCi ³H-thymidine was added to each culture. Thymidine uptake, reflecting live proliferated cell number, was determined by scintillation counting. Two-way ANOVA p<0.0001; **Bonferroni post tests p<0.001. <b>B.</b> B6.SCID mice (FcRγ^+/+) received 10⁷ B6.LPR.FcRγ^+/+ (n = 3, upright triangles) or B6.LPR.FcRγ^−/− (n = 3, inverted triangles) DN T cells. On days 1, 5, 7, 10, and 14 blood samples were obtained and peripheral blood mononuclear cells (PBMCs) were stained for TCRβ, CD4, CD8, and NK1.1 expression. The percentage of DN T cells in the PBMC compartment was then determined by flow cytometry. Two-way repeated measures ANOVA p = 0.0279 for the effect of FcRγ genotype; Bonferroni post test p<0.001 at day 14. <b>C.</b> At day 7 and day 14, the splenocytes of the DN T cell recipients were counted and stained for TCRβ, CD4, CD8, and NK1.1 and examined by flow cytometry. The number of splenic DN T cells in each type of recipient was then determined. Two-way ANOVA p = 0.0005 for the effect of FcRγ genotype; Bonferroni post test p<0.001 at day 14.</p

Fc receptor γ-expressing DN T cells are lost with disease progression in LPR mice and have an increased rate of apoptosis <i>ex vivo</i>.

<p><b>A.</b> Splenocytes of young (≤12 weeks, n = 4) and older (≥16 weeks, n = 4) female LPR mice were counted and stained for expression of CD4, CD8, NK1.1, TCRβ, and CD16 and then examined by flow cytometry. The percentage of DN T cells expressing CD16 was examined as a function of total spleen cell count; linear regression r² = 0.91, p = 0.0002. <b>B.</b> LPR FcRγ^+/+ (n = 5) and LPR FcRγ^−/− mice (n = 5) aged 8 weeks were fed BrdU for 6 days. Their splenocytes were then stained for expression of CD4, CD8, NK1.1, TCRβ, annexin V and CD16, fixed and stained for BrdU incorporation. They were then examined by flow cytometry. Left panels show expression of CD16 versus side light scatter in LPR FcRγ^+/+ (top) and LPR FcRγ^−/− DN T cells (bottom); CD16^hi and CD16^lo gates are indicated, and the numbers above each gate indicate the percentage of DN T cells falling into the indicated gates. Right panels show representative BrdU and annexin V staining in LPR FcRγ^−/− DN T cells (bottom) and in the CD16^lo and CD16^hi subsets of LPR FcRγ^+/+ DN T cells (middle and top panels, respectively). Numbers inside contour plots reflect the percentage of gated cells falling into each quadrant. <b>C.</b> The percentages of DN T cells staining with annexin V in LPR FcRγ^−/− mice and in the CD16^hi and CD16^lo subsets of LPR FcRγ^+/+ mice are presented with respect to BrdU incorporation. Two-way ANOVA p<0.0001; **Bonferroni post tests p<0.01 compared with either CD16^lo or LPR FcRγ^−/− DN T cells amongst both BrdU⁺ and BrdU⁻ DN T cells.</p

FcRγ-expressing DN T cells are a distinct effector-memory subset in both B6 and LPR mice.

<p><b>A.</b> Freshly isolated B6 (n = 3, left column) and LPR (n = 4, right column) splenocytes were stained for TCRβ, CD4, CD8, NK1.1, CD16/32, CD44, and CD62L expression and examined by flow cytometry. Within the DN T cell gate (TCRβ⁺, CD4⁻, CD8⁻, NK1.1⁻), expression of CD44 (top row) and CD62L (bottom row) in the CD16⁺ (shaded) and CD16⁻ (unshaded) subsets was plotted. <b>B.</b> Median fluorescence intensity (MFI) of CD44 staining in CD16⁺ and CD16⁻ DN T cells for all 7 mice is shown. Unpaired t tests p = 0.006 (CD16⁺ vs. CD16⁻ B6 DN T cells) and p = 0.0009 (CD16⁺ vs. CD16⁻ LPR DN T cells). <b>C.</b> MFI of CD62L staining in CD16⁺ and CD16⁻ DN T cells for all 7 mice is shown. Unpaired t tests p = 0.0036 (CD16⁺ vs. CD16⁻ B6 DN T cells) and p = 0.0165 (CD16⁺ vs. CD16⁻ LPR DN T cells).</p

CD8⁺ T cells proliferating in response to alloantigen are selectively killed by LPR DN T cells via the Fas pathway.

<p>CD8⁺ T cells (10⁵/well) from B6 (Fas^+/+) or B6.LPR (Fas^LPR/LPR) were labelled with CFSE and cocultured with irradiated CB6F1 splenocytes and IL-2 for 5 days without or with LPR DN T cells in the indicated ratios. After 5 days, the cultures were stained with anti-CD8-APC and 7-AAD and analyzed by flow cytometry. <b>A.</b> The percentage of undivided cells (CFSE^hi) was used to determine the percent suppression at each DN:CD8⁺ T cell ratio. Two-way ANOVA p<0.0001 for the effect of CD8⁺ T cell Fas expression. <b>B.</b> Representative histograms of 7-AAD staining gated on proliferated (CFSE-diluted) Fas^+/+ (top row) and Fas^LPR/LPR (bottom row) CD8⁺ T cells at the indicated DN:CD8⁺ ratios. Numbers inside histograms are the percentages of cells falling in the 7-AAD⁺ gate. <b>C.</b> The fold increase in cell death for proliferated Fas^+/+ (white bars), proliferated Fas^LPR/LPR (black bars), unproliferated Fas^+/+ (light grey bars), proliferated Fas^LPR/LPR (dark grey bars) CD8⁺ T cells is shown. Two-way ANOVA p<0.0001; Bonferrroni post-tests ***p<0.001 and *p<0.05. Data are derived from two independent experiments each with duplicate wells.</p

IFNγ secretion and signalling and FasL expression enable B6.lpr DN T cells to inhibit alloreactive CD4⁺ T cells in vitro and in vivo.

<p><b>A.</b> Preactivated ³H-thymidine labelled CD4⁺ T cells were cultured for 18h with preactivated B6.<i>lpr</i>, B6.<i>lpr</i>.IFNγ^−/−, or B6.<i>lpr</i>.IFNγR^−/− DN T cells at indicated ratios. Specific cytotoxicty was determined based on retention of ³H-thymidine in viable targets. Data are compiled from 4 experiments in which, respectively, B6.<i>lpr</i> (circles), B6.<i>lpr</i>. IFNγ^−/− (squares), or B6.<i>lpr</i>.IFNγR^−/− (triangles) DN T cells were used in 4, 3, and 2 experiments. Compared with B6.<i>lpr</i> DN, *Two-way ANOVA p = 0.0009 and **p<0.0001. <b>B.</b> In varying ratios, B6.<i>lpr</i> DN T cells were cultured with CFSE-labelled B6. Thy1.1 (Fas^+/+) or B6.<i>lpr</i> (Fas<i>^lpr/lpr</i>) CD4⁺ T cells, irradiated CB6F1 splenocytes and IL-2. CFSE dilution in live (7-AAD⁻) cells was determined after 5 days. Data from 2 independent experiments are shown; two-way ANOVA p<0.0001. <b>C.</b> Lethally irradiated CB6F1 mice received BM only (n = 5), BM+CD4⁺ (n = 8), BM+CD4⁺+2.5×10⁶ B6.<i>lpr</i> DN (n = 10, FasL⁺) or 2.5×10⁶ B6.<i>gld</i> DN (n = 12, FasL⁻). Data are from 2 independent experiments, each with 2–6 mice per group. *Log rank p = 0.006 vs. B6.<i>gld</i> DN.</p

DN T cells inhibit semi-allogeneic CD4⁺ T cell-induced GVHD.

<p><b>A.</b> Lethally irradiated CB6F1 mice were reconstituted with BALB/c TCD BM alone (BM only, n = 7) or with BALB/c CD4⁺ T cells, without (BM+CD4⁺, n = 6) or with BALB/c DN-enriched lymphocytes (BM+CD4⁺+DN, n = 7). A clinical score <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0047732#pone.0047732-Cooke1" target="_blank">[26]</a> incorporating posture, fur texture, activity level, skin integrity and weight loss was assigned 3 times weekly and survival was monitored daily. Data from 3 independent experiments (each with 2–3 mice per group) are shown; *log rank p = 0.0003 vs. BM+CD4⁺. <b>B.</b> CB6F1 recipients conditioned as in A were reconstituted with B6 TCD BM alone (BM only) or with B6.Thy1.1 CD4⁺ T cells, without (BM+CD4⁺) or with B6.<i>lpr</i> DN T cells (BM+CD4⁺+DN). Data are from 5 independent experiments, each with up to 6 mice per group; *log rank p = 0.0034 vs. BM+CD4⁺; **log rank p<0.0001 vs. BM+CD4⁺. <b>C.</b> Clinical score was determined 2 weeks after BMT in recipients of BM only (n = 17), BM+CD4⁺ (n = 24), BM+CD4⁺+DN (n = 14) and BM+DN (n = 8). Data are derived from 5 independent experiments, each with up to 6 mice per group. Kruskal-Wallis test p<0.0001; *Dunns post test p<0.05.</p