Suppression of gene expression in patients inoculated with E. coli 83972

Therapeutic urinary tract inoculation with the prototype ABU strain E. coli 83972 is a safe alternative approach for patients with therapy-resistant, recurrent UTIs (15–17). The strain establishes persistent bacteriuria, protecting patients against superinfection with more virulent strains (17). Using this protocol, we examined whether the establishment of asymptomatic bacterial carriage alters host gene expression. Patients were inoculated with E. coli 83972 (105 CFU/ml), and in 3 individuals, significant bacteriuria was established after 24 hours (Figure 1A). Prior to inoculation, the urine was sterile and neutrophil numbers and cytokine concentrations in serum and urine were low, contradicting a strong preexisting chronic inflammatory response in the urinary tract or a preceding systemic immune response (Figure 1B).

Figure 1 Inhibition of gene expression 24 hours after human inoculation with the ABU strain E. coli 83972. (A) E. coli 83972 CFUs in urine samples obtained 24 hours after inoculation. (B) Lack of systemic innate immune response after 24 hours. Neutrophil numbers and IL8 concentrations in urine 4 weeks after inoculation. Levels in patients with symptomatic UTIs are indicated for comparison (20). (C) Heatmap illustrating the suppression of gene expression in each patient (green represents suppressed; red represents activated). (D) Venn diagram indicating the number of individually or commonly regulated genes. (E) Main functional categories among the 160 shared regulated genes (individual genes and other functional categories are shown in Supplemental Table 1). (F) IPA-generated network illustrating the in vivo suppression of genes in a Pol II network (for specific genes, see Supplemental Tables 1 and 2). PMNs, polymorphonuclear neutrophils.

The urinary tract response to E. coli 83972 inoculation in a placebo-controlled study was previously examined (18) and low, but detectable, increases in urinary neutrophils and cytokines were observed. These responses were below the levels reported during symptomatic UTIs (19, 20). Here, systemic cytokine activation (IL6 or IL8) was not detected in the inoculated patients (Figure 1B). Urinary neutrophil numbers were transiently elevated in parallel with an IL8 response (Figure 1B).

We examined the effects of the ABU strain on host gene expression using peripheral blood leucocyte RNA isolated before and 24 hours after intravesical inoculation. Substantially altered genes identified by whole genome transcriptomic profiling were sorted according to their relative expression levels (unadjusted P values less than 0.05 and absolute log 2 fold change greater than 0.5).

Gene expression was markedly reduced in the 3 patients 24 hours after E. coli 83972 inoculation (63.3%, 5,278 of 8,337 probe sets) (Figure 1C). Shared genes, showing a similar change in expression in the 3 patients were mostly suppressed (87.5%, 140 of 160 probe sets, Venn diagram) (Figure 1D). Regulators of gene expression were most abundant in this group, including transcriptional repressors, transcriptional activators, and regulators of translation, chromatin, or DNA organization (Figure 1E and Supplemental Table 1; supplemental material available online with this article; doi: 10.1172/JCI66451DS1). We further analyzed the commonly regulated genes for network interactions. Based on established molecular pathways, we identified a network of genes directly interacting with Pol II, associating Pol II with transcription factors JUN and CREB1 (Figure 1F). In total, we found that 9 of 9 regulated genes in this network were suppressed. For other strongly regulated networks, see Supplemental Table 2.

Global changes in gene expression after in vitro infection of human uroepithelial cells

To address the mechanism of transcriptional inhibition, we infected human kidney epithelial A498 cells with the ABU strain or the virulent CFT073 (APN) strain. This cell line has been validated as a model of uropathogenic E. coli infection; the cells express functional receptors for bacterial virulence ligands, and the response to virulent strains reflects human UTI (21). RNA harvested after 4 hours was subjected to whole genome transcriptomic analysis. Raw data, normalized using cross-correlation (22), were of high quality with a replicate correlation greater than 0.98 and no systematic bias (MA plots, Supplemental Figure 1). Genes with empirical Bayes-adjusted P values less than 0.05 and log 2 fold changes greater than 0.585 were considered differentially expressed and were functionally characterized using the Database for Annotation, Visualization and Integrated Discovery (DAVID), Blast AceView, and Ingenuity Pathway Analysis (IPA) software.

In total, 1,957 cellular genes were substantially altered by the ABU and/or APN strains compared with uninfected cells (Figure 2A). After sorting according to relative expression levels, we identified 4 patterns; 96 genes were only regulated by the ABU strain and were designated as ABU specific (Figure 2D and Supplemental Tables 3 and 4). Six genes were inversely regulated between the ABU and APN strains (Supplemental Table 3 and Supplemental Figure 2), 1,186 genes were similarly regulated by both strains (Supplemental Table 5), and 669 genes were APN specific (Figure 2A). Overall, fewer genes were transcribed in response to the ABU strain compared with the APN strain (1,288 versus 1,861; P < 0.05) (Figure 2, B and C), suggesting either active suppression of gene expression or a lack of activation.

Figure 2 Gene expression in human uroepithelial cells infected with E. coli 83972 or CFT073. (A) Heatmap of genes sorted according to expression levels. (B) Scatter plot of log 2 normalized expression illustrating a higher number of regulated genes after CFT073 stimulation than after 83972 stimulation. (C) Venn diagram specifying the number of 83972-specific, CFT073-specific, or commonly or inversely regulated genes. (D) Main categories of deregulated 83972-specific genes. Transcriptional regulators comprised 42% of the 83972-specific genes, and 22.5% were Pol II related (for 83972-specific genes, see Supplemental Tables 3 and 4).

ABU-specific changes in gene expression

To identify the effects on host gene expression unique to the ABU strain, we sorted the 96 ABU-specific and 6 inversely regulated genes according to their known or suggested functions. Substantially regulated genes involved in transcriptional regulation are listed in Supplemental Table 3 according to fold change, and the most regulated gene categories are indicated in Figure 2D. These categories correspond to those found among the shared regulated genes in the patients (Figure 1F). The regulators of gene expression were most abundant, including transcriptional repressors, transcriptional activators, zinc finger proteins, regulators of translation, and transcriptional regulators of the innate immune response, confirming the in vivo observations (Figure 2D). Genes directly involved in Pol II transcription or in regulating Pol II–dependent pathways were strongly affected by the ABU strain (22.5% of the ABU-specific genes) (Figure 2D and Supplemental Table 3). Other regulated gene categories include innate immune receptors like NOD1, TLR5, and TNFRSF10D, as well as other innate immunity and inflammatory response genes, all of which were suppressed (Supplemental Table 4). IPA identified 1 network formed by the inversely regulated genes (Supplemental Figure 2), including FOSB, HSPA6, RN7SK, RGS4, and IFIT1. Three of these genes regulate Pol II through TATA box–binding protein (TBP) (23–25). We also identified networks involved in infection and immunity (Supplemental Figure 3). The transcriptomic profile and IPA did not identify oxidative stress or ER stress response pathways in infected cells.

Effects on RNA Pol II–dependent transcription

RNA Pol II controls eukaryotic gene expression through mRNA precursors, most small nuclear RNAs (snRNAs), and microRNAs (miRNAs) (26). The effects of the ABU strain on critical steps involved in the Pol II transcription cycle are shown in Figure 3A, and molecules whose expression was modified in an ABU-specific manner are listed in Supplemental Table 3 according to their role in Pol II–dependent gene expression (see legend, Figure 3A). In addition, genes inversely regulated by APN and ABU were included. A heatmap of genes modified by the ABU strain is shown in Figure 3B, and the corresponding effects of infection with the APN strain are shown for comparison.

Figure 3 Suppression of RNA Pol II–dependent transcription by E. coli 83972. (A) Overview of the Pol II–dependent transcription cycle, with genes modified by E. coli 83972 indicated in boxes adjacent to each step (modified from Fuda et al., ref. 38). For log 2 fold changes of individual genes, see Supplemental Table 3. Step 1: Chromatin opening and binding of the Pol II transcriptional complex to DNA is modified by specific activators or repressors. E. coli 83972 suppressed 6 transcriptional activators and activated 3 repressors. Step 2: Preinitiation complex formation. HSP70 interacting with GTFs was inhibited. Genes involved in steps 3 (initiation of transcription), 4 (promoter escape/clearance), and 5 (escape from pausing) were not affected. Step 6: Message elongation. Suppressed snRNA RN7SK, which binds to the transcription elongation factor P-TEFb. Step 7: Termination. Suppressed snRNA RNU6ATAC, involved in pre-mRNA splicing. See also Supplemental Figure 2 and Supplemental Table 3. (B) Heatmap of 83972-specific genes interacting with Pol II and regulating transcription. (C) Verification of the transcriptomic analysis. RT-PCR confirmed a reduction in transcript levels for genes in the Pol II cycle (FOS, LBH, HSPA6, RN7SK, and RNU6ATAC) in A498 cells infected with E. coli 83972. Data represent the means ± SEM. *P < 0.05; **P < 0.01. (D) Western blot analysis confirming suppression by E. coli 83972. Reduced levels of FOS, FOSB, LBH, and increased levels of DACH1 were detected.

Transcriptional activators and repressors (step 1). The ABU strain suppressed 7 activators of Pol II–dependent transcription (FOSB, LBH, OSR2, LHX1, CTNNB1, MN1, and TFAP2A) (Figure 3A and Supplemental Table 3). The FOS transcription factor family has pleiotropic cellular effects. LBH recruits Pol II to specific promoter sites and may regulate FOS through the serum-responsive element (SRE) in the FOS promoter (27). OSR2 regulates genes involved in proliferation and development (28). LHX1 is highly expressed in the urinary tract and is a potential transcription factor. CTNNB1 is a pleiotropic regulator that recruits Pol II to promoters for cell growth and adhesion, and recruits the transcriptional activators LBH (through WNT3A), MN1, SOX4, and BRIX1, which are necessary for ribosome biogenesis (29). MN1 is a transcriptional coregulator of genes induced by retinoic acid (30). TFAP2A belongs to the transcription factor AP2 superfamily, which is inhibited by viral infection (31).

Remarkably, we found that the ABU strain also activated several repressors of Pol II–dependent transcription, including DACH1, BANP, and DDX20, which prevent Pol II from accessing multiple promoters through local or global chromatin alterations. DACH1 regulates FOS by binding SRE in the FOS promoter (32), potentially providing a mechanism for suppression of the innate immune response by the ABU strain (Supplemental Table 3). BANP represses CCND1 transcription and promotes TP53 Ser15 phosphorylation, causing cell cycle arrest (33). DDX20 interacts directly with the RNA Pol II complex (34), associates with histone deacetylases, and enhances NFKB suppression by miRNAs (35).

Pol II complex formation and initiation of transcription (steps 2 and 3). The ABU strain suppressed 5 genes recognizing TBP (MDM2, CTNNB1, FOSB, RN7SK, and HSPA6). In contrast, the APN strain increased the expression of HSPA6, a member of the HSP70 family that has been suggested to facilitate transcription when TBP is inhibited (23). In addition, the APN strain upregulated HSPA9, another member of the HSP70 family that interacts with GTF2A1 (a TFIIA component) (24) and also upregulated 2 genes that interact with TFIIB (NR4A2 and GMNN, a repressor of transcription factor binding). The APN strain stimulated the expression of 3 mediator complex components (MED10, MED13, MED15) but the ABU strain had only a weak effect on MED13 (log fold change 0.65).

Genes regulating preinitiation complex release and productive elongation (steps 4–6). The ABU strain suppressed the snRNA RN7SK, a regulator of the Pol II transcription cycle that reversibly sequesters transcription elongation factor b (P-TEFb) into a large kinase-inactive complex (25). As RN7SK also directly interacts with TBP, this snRNA has great potential as a broad transcriptional regulator in ABU. RNU6ATAC, which regulates splicing of pre-mRNA (36), was the most strongly suppressed gene in ABU-stimulated cells (log fold change –4.14). In addition, the ABU strain inhibited the transcription of RNA28S5, which is Pol I dependent. The ABU strain increased the expression of PDCD4, which inhibits the helicase activity of translation initiation factors EIF4A and EIF4F and blocks their binding to RNA (37).

RT-PCR and Western blot validation

We confirmed the effects of the ABU strain on gene expression by RT-PCR of mRNA and snRNA obtained after stimulation of A498 cells (Figure 3C). FOS and LBH expression was reduced compared with uninfected cells, as was heat shock protein HSPA6 and snRNAs RN7SK and RNU6ATAC. Western blot analysis (Figure 3D) detected a reduction in FOS and LBH proteins, whereas the transcriptional repressor DACH1 was increased, consistent with the gene expression data. These genes were also suppressed in the patients (for a comparison with the in vitro data, see Supplemental Figure 4A). mRNA levels for genes involved in Pol II regulation and transcription were perturbed, including LBH and FOS, as well as transcription factors IFIT1, EGR1, and ID2, which were suppressed, and DACH1, which was increased (Supplemental Figure 4, A and B).

Inhibition of Pol II Ser2 phosphorylation by the ABU strain

Productive elongation of the mRNA requires Pol II phosphorylation (38). Carboxy-terminal domain (CTD) repeats on Ser5 are phosphorylated by the kinase subunit of the general transcription factor TFIIH, followed by the phosphorylation of Pol II CTD repeats on Ser2 by the kinase complex positive transcription elongation factor b (P-TEFb). Pol II hyperphosphorylation by TFIIH is necessary for breaking Pol II contacts with some promoter-bound factors and for its release from the core promoter region.

We observed changes in Pol II phosphorylation after infection with the ABU strain (for 4 hours) (Figure 4). We infected the A498 human kidney epithelial cell line or pediatric kidney cells in primary culture with the ABU strain, and detected phosphorylated Pol II by immuno-peroxidase staining using anti–phospho Ser2 antibodies. Cells infected with the APN strain were included as controls (Figure 4, A and B). For cell morphology, see Supplemental Figure 5A. A marked reduction in nuclear phosphorylated Pol II staining was detected in A498 cells infected with the ABU strain compared with uninfected cells (Figure 4A). The frequency of cells with negative staining (–) increased from 5% to 85% (P < 0.0001, χ2 test for independence). This effect was confirmed by Western blot analysis of phosphorylated Pol II in whole-cell extracts (Supplemental Figure 5B). Four additional ABU strains inhibited Pol II phosphorylation (Supplemental Figure 5C), but infection with the APN strain had no significant effect (P = 0.742) (Figure 4A).

Figure 4 Suppression of Pol II phosphorylation by E. coli 83972 (A) Active Ser2 phosphorylated Pol II was stained using monoclonal primary human phospho CTD Ser2 antibodies and peroxidase-labeled secondary antibodies (brown). E. coli 83972 markedly suppressed Pol II phosphorylation compared with uninfected or CFT073-infected human kidney cells (A498). E. coli CFT073 had a nonsuppressive effect. (B) Inhibition by E. coli 83972 of Pol II phosphorylation in primary human kidney cells (HRTEC) compared with uninfected or CFT073-infected cells. (C) The specific Pol II inhibitor DRB (60 μM) abrogated Pol II phosphorylation in response to E. coli CFT073 compared with A498 cells without an inhibitor. For dose-dependent inhibition of Pol II phosphorylation in uninfected A498 cells, see Supplemental Figure 7A. (D) Competition between E. coli CFT073 and 83972 in A498 cells. E. coli 83972 inhibited Pol II phosphorylation in the presence of E. coli CFT073. (A and C) Scale bars: 50 μm and 10 μm (insets). #P = compared with medium control; ##P = compared with CFT073-infected cells; χ2 test for independence.

We observed a parallel reduction in Pol II phosphorylation in primary human renal tubular cells. A dose-dependent reduction in Ser2 phosphorylation was detected in ABU-infected cells (P < 0.0001 compared with uninfected control cells) (Figure 4B). Furthermore, in the human bladder cell lines J82 and HTB-9, we observed a reduction in phosphorylation after exposure to the ABU strain (Supplemental Figure 6).

Effects of Pol II inhibition on the cellular response to infection

To specifically address whether Pol II inhibition alters the response to infection, we pretreated human kidney cells with 5,6-dichloro-1-b-D-ribofuranosylbenzimidazole (DRB). This adenosine analog has been proposed to specifically and reversibly inhibit Pol II transcription without directly affecting other cellular functions (39). We quantified the dose-dependent effect of DRB as the reduction in Ser2 staining in A498 cells (Supplemental Figure 7A). At these concentrations, no reduction in cell viability was observed (Supplemental Figure 8A). Pol II phosphorylation in APN-infected cells was specific (Supplemental Figure 8B) and was reduced by DRB (60 μM; P < 0.0001) (Figure 4C). We observed a similar reduction in Pol II phosphorylation in cells cochallenged with the ABU and APN strains compared with the APN strain alone, suggesting that the ABU strain inhibits the response to the APN strain (P < 0.0001) (Figure 4D).

We confirmed the broad suppression of gene expression by transcriptomic analysis in DRB-treated (60 μM) human kidney cells (Supplemental Figure 7, B and C) and found that the effect of the ABU strain was more restricted (1,577 versus 155 inhibited genes; absolute fold change greater than 2). Genes regulated by both DRB and the ABU strain included FOS, LBH, DACH1, and RNU6ATAC, as defined by RT-PCR. We found that HSPA6 was suppressed only by the ABU strain, and RN7SK was not regulated in this experiment (Supplemental Figure 7D). Several studies have proposed that DRB targets casein kinase II and that inhibition of casein kinase II may block Pol II carboxy-terminal domain phosphorylation (39–41). Furthermore, casein kinase II inhibits CREB1 and ATF1, which regulate the MAPK1 pathway (42, 43). These effects are consistent with the profile of suppression by the ABU strain, but the molecular basis for Pol II inhibition and the narrower spectrum of inhibited genes compared with DRB remain to be identified.

Inhibition of innate immune signaling defined by pathway analysis and phosphorylation arrays

Uropathogenic E. coli activate a pathogen-specific, TLR4/CREB/IRF3/7-dependent innate immune response, which is crucial for host resistance to acute pyelonephritis (9, 44). P fimbriae bind to glycosphingolipids, and ceramide release activates TLR4 signaling (44). Irf3–/– mice develop acute pyelonephritis with urosepsis, followed by tissue damage within 1 week of infection (9). Uropathogens also stimulate IFNβ expression and infected Ifnb mice develop kidney pathology, suggesting that type 1 IFNs are essential for a functional antibacterial defense in the kidneys (9).

These pathways were less strongly activated by the ABU strain than by the APN strain, as shown by transcriptomic analysis of infected A498 kidney cells (at 4 hours) (Figure 5A and Supplemental Figure 9) TLR4, MAPK14, FOS, JUN, and CCL5. In addition, IL1B expression was reduced in ABU-infected cells, and MAP2K3, MAP3K1, PRKCE, and IRF7 were not activated. IFNB was less strongly induced by the ABU strain, and IFIT1, downstream of IFNB, was suppressed. Importantly, the ABU strain still activated the NFKB-dependent pathway and TNF, IL6, and IL8 responses to the ABU and APN strains were similar in vitro (Supplemental Figure 10).

Figure 5 Pathogen-specific signaling in infected A498 cells. (A) Suppression by E. coli 83972 (right panels) of genes in the TLR4/CREB/IRF3/7 and type 1 IFN pathways. For log 2 fold changes of individual genes, see Supplemental Table 3. Activation by E. coli CFT073 is shown for comparison. Increased expression (red), reduced expression (green), and no change (gray). Color intensity reflects the fold change. (B) Phosphorylation of molecules in the TLR4/CREB/IRF3/7 signaling pathway examined by phosphoarray analysis of whole-cell extracts from E. coli 83972-infected A498 cells. (C) The ABU strain suppressed the phosphorylation of 7 targets compared with uninfected cells. Quantification based on pixel intensities of phosphorylated targets. Cutoff is set at a fold change less than –1.5 or greater than 1.5.

The ABU strain also reduced the phosphorylation of signaling molecules downstream of TLR4 (MAP2K1/2 and MAPK1, as well as MAPK14 and MAPK8), as determined by phosphoarray analysis of whole-cell extracts of ABU-infected cells (Figure 5, B and C). A corresponding increase in phosphorylation in response to virulent strains has been reported (9). MAPKs, and especially MAPK14, were previously shown to regulate the phosphorylation of CREB1 and the nuclear translocation of IRF3 (9), and MAPKs regulate AP1 formation from FOS and JUN.

Long-term suppression of pathogen-specific signaling in inoculated patients

We also detected the inhibition of TLR4/CREB/IRF3/7 and type 1 IFN signaling in the inoculated patients (Figure 6, Supplemental Figure 11, and Supplemental Table 1). After 24 hours, both pathways were suppressed compared with the preinoculation samples in each patient (reduced JUN, FOS, CREB, and PLCG1 expression) (Figure 6A). Type I IFN pathway genes were either suppressed or unchanged, including SOCS1,STAT1-, and IFN-stimulated genes (IFIT1, IFIT3, OAS1, MX1, CXCL10) (Figure 6A).

Figure 6 Suppression by E. coli 83972 of pathogen-specific signaling in inoculated patients. (A) Inhibition in inoculated patients of the TLR4/CREB/IRF3/7 pathway and suppression of type I IFN signaling. Each symbol is divided into 3 parts and colored according to individual gene expression levels in P1, P2, and P3. Log 2 fold change greater than 0.4 (red); log 2 fold change less than –0.4 (green); and no change (white). For log fold change of individual genes, see Supplemental Table 1. (B) Transcriptomic analysis of RNA samples obtained from each patient after 3, 24, 48 hours, and 7 days. Pol II cycle–related genes remained suppressed for at least 1 week after inoculation. DACH1 expression was increased, except after 1 week in P1 and P2.

To address whether the ABU strain continued to suppress host gene expression during in vivo carriage, we obtained repeated RNA samples from each patient (at 3, 24, 48 hours and 7 days) (Figure 6B). Suppression of Pol II cycle–related genes was detected up to 1 week after inoculation (FOS, HSPA5, functional analogs of HSPA6 and LBH). DACH1 expression was increased at all time points except after the first week in patient no. 1 (P1) and patient no. 2 (P2). The results show that the ABU strain actively suppresses essential regulators of gene expression and pathogen-specific innate immune signaling and that the suppressive effect is maintained in inoculated patients for at least 1 week.

Frequency of Pol II suppression among E. coli strains from APN, ABU, or the fecal flora

We examined Pol II suppression in an epidemiologically defined collection of ABU isolates (n = 75) from which E. coli 83972 was derived (10, 45, 46). APN strains (n = 88) were obtained from children with febrile UTIs and fecal strains (n = 46) from children in the same area who had no history of UTI and negative urine cultures at screening.

We infected human kidney cells with each strain, and the inhibition of Pol II phosphorylation was graded according to the overall staining intensity as strong, intermediate, or negative relative to uninfected control cells (Figure 7, A–C). Seventy-seven percent of the ABU strains inhibited phosphorylation and 37% were strongly inhibitory compared with 17% for the strongly inhibitory APN strains (P = 0.006, X2 test for independence) (Figure 7A). The fecal strains resembled the ABU group, but with the majority of the strains being intermediately inhibitory (P = 0.038 compared with the APN group).

Figure 7 Epidemiologic analysis of Pol II suppression. (A) Higher frequency of Pol II suppression in A498 cells infected with ABU or fecal strains than with APN strains. (B) Light microscopic images of Pol II phosphorylation at Ser2 in A498 cells, analyzed by ImageJ, using changed lookup tables and inverted colors. Scale bar: 50 μm (upper panel). Each strain was assigned a staining score based on the scale and is illustrated in the lower panel (scale bar: 10 μm). (C) Strongly inhibitory strains were more abundant in the ABU (44%) than in the APN group (9%, P < 0.001), and the staining score was lower (P = 0.001).

To further characterize the difference in Pol II phosphorylation, we quantified the staining in a subset of the ABU (n = 45) or APN (n = 43) strains (Figure 7B). The Pol II phosphorylation score was substantially lower in the ABU group than in the APN group (P < 0.001, χ2 test for independence) (Figure 7C). The results suggest that ABU strains inhibit Pol II activation more often than virulent strains and that this property is shared with certain E. coli strains from the intestinal flora.

To address whether the inhibitory effect might be relevant in the intestinal environment, we exposed 2 colon cancer cell lines (HT29 and DLD-1) to E. coli 83972 and stained for Pol II phosphorylation. The strain inhibited phosphorylation in both cell lines (Supplemental Figure 12, A and B). The cells were subsequently exposed to E. coli strains from the fecal flora, one defined as inhibitory and one as noninhibitory in the epidemiologic survey based on uroepithelial cells (Figure 7A and Supplemental Figure 12C). The pattern of inhibition was reproduced in the colonic epithelial cells. In addition, we exposed the 2 bladder epithelial cell lines, J82 and HTB-9, to two acute cystitis strains, one with inhibitory activity in A498 human kidney cells and one lacking such activity. The pattern of Pol II inhibition resembled that observed in A498 cells (Supplemental Figure 6, C and D).