Inhibition of TIR domain signaling by TcpC: MyD88-dependent and independent effects on Escherichia coli virulence

Abstract

Toll-like receptor signaling requires functional Toll/interleukin-1 (IL-1) receptor (TIR) domains to activate innate immunity. By producing TIR homologous proteins, microbes inhibit host response induction and improve their own survival. The TIR homologous protein TcpC was recently identified as a virulence factor in uropathogenic Escherichia coli (E. coli), suppressing innate immunity by binding to MyD88. This study examined how the host MyD88 genotype modifies the in vivo effects of TcpC and whether additional, TIR-domain containing proteins might be targeted by TcpC. In wild type mice (wt), TcpC enhanced bacterial virulence, increased acute mortality, bacterial persistence and tissue damage after infection with E. coli CFT073 (TcpC+), compared to a ΔTcpC deletion mutant. These effects were attenuated in Myd88(-/-) and Tlr4(-/-) mice. Transcriptomic analysis confirmed that TcpC inhibits MYD88 dependent gene expression in CFT073 infected human uroepithelial cells but in addition the inhibitory effect included targets in the TRIF and IL-6/IL-1 signaling pathways, where MYD88 dependent and independent signaling may converge. The effects of TcpC on bacterial persistence were attenuated in Trif (-/-) or Il-1β (-/-) mice and innate immune responses to ΔTcpC were increased, confirming that Trif and Il-1β dependent targets might be involved in vivo, in addition to Myd88. Furthermore, soluble TcpC inhibited Myd88 and Trif dependent TLR signaling in murine macrophages. Our results suggest that TcpC may promote UTI-associated pathology broadly, through inhibition of TIR domain signaling and downstream pathways. Dysregulation of the host response by microbial TcpC thus appears to impair the protective effects of innate immunity, while promoting inflammation and tissue damage.

Figure 1. TcpC acts as a virulence factor by promoting bacterial persistence in the urinary tract and abscess formation in the kidneys of wild type mice.

(A) Bacterial counts (CFUs) in kidneys of C57BL/6, Tlr4^−/− and Myd88^−/− on days 4 and 7 post-infection. (B) Bacterial counts (CFUs) in urine of C57BL/6, Tlr4^−/− and Myd88^−/− mice after infection with CFT073 or ΔTcpC. (C) MIP-2 response kinetics in urine samples of wt and mutant mice. (D) Neutrophil response kinetics in urine samples of wt and mutant mice. P values (*p<0.05, **p<0.01 and ***p<0.001 for CFT073 versus ΔTcpC mutant and for wt versus mutant mice; Fisher's exact test). Eight to ten mice were used per time point. (E) Abscess formation (arrows) in the kidneys of wt mice infected with CFT073 but not in Tlr4^−/− and Myd88^−/− mice infected with CFT073 or ΔTcpC (Scale bar, 2 mm).(F) Abscess formation after CFT073 or ΔTcpC infection, in percent of the total number of kidneys examined.

Figure 2. Deletion of TcpC abrogates abscess formation in kidney sections from wild type mice infected with CFT073.

(A) Overview of htx-eosin stained kidney section of wt mice infected with CFT073, showing abscesses (scale bar, 500 µm). Magnified areas of section A shown as A-I, A-II and A-III are stained with specific antibodies to neutrophils (green, NIMP-R14) and the PapG adhesin (red, antiserum to a synthetic PapG peptide) (scale bar, 100 µm). Abscesses in wt mice are shown by dotted lines. (B) Overview of kidney section of wt mice infected with ΔTcpC (scale bar, 300 µm). Magnified areas of section B shown as areas B-I and B-II are stained with specific antibodies as described above. (C) Kidney section of uninfected control mice with htx-eosin staining (scale bar, 200 µm) and inset picture showing negative control for anti-neutrophil and anti-PapG antibodies.

Figure 3. Morphology of intact kidney tissue in Tlr4 ^−/− and adaptor protein mutant mice infected with CFT073 and ΔTcpC.

(A) Overview of htx-eosin stained kidney section of Tlr4^−/−, Myd88^−/− and Trif ^−/− mice. Scale bar, 500 µm. (B) Histology of renal cortex stained with specific antibodies to neutrophils (green, NIMP-R14) or the PapG adhesion (red, antiserum to a synthetic PapG peptide). Scale bar, 100 µm. Neutrophils or bacteria were not detected in the tissues. The Tlr4^−/− and Myd88^−/− mice were compared to C57BL/6 wt mice and Trif ^−/− mice to C57BL6/129 wt mice.

Figure 4. Uroepithelial gene expression in response to in vitro infection with CFT073 or ΔTcpC.

(A) A two-dimensional hierarchical heat map illustrating differentially transcribed genes in human A498 cells infected with CFT073 or ΔTcpC versus the control group. Analysis was performed on 766 genes with a fold change of at least two over untreated cells, and classified based on whether they are CFT073 or ΔTcpC specific, or common to both infections. Induced genes are represented by brighter shades of red, while down regulated genes are represented by brighter shades of green (see color scale). (B) Venn diagram showing numerical distribution of differentially expressed genes in CFT073 (red) and ΔTcpC infected (blue) cells compared to mock infected cells. (C) A comparison of the 10 highest scoring biological pathways between infected cells and controls as analyzed by Ingenuity Pathway Analysis with default settings.

Figure 5. Identification of pathway-specific genes by Ingenuity Pathway Analysis in response to in vitro infection.

(A-I) Pattern recognition pathway. (A-II) IL-6/IL-1 pathway. Each node represents a protein whose functional class is represented by various shapes (ovals = transcription factors, spiral = kinases, dumbbell = transcription regulators, V shape = cytokine or growth factors, Y shape = transmembrane receptors, circles = others). Direct protein interactions are represented by solid lines, while dashed lines indicate indirect interaction. A red node indicates up regulation of a protein in response to CFT073, while a green node indicates down regulation by CFT073 relative to ΔTcpC.

Figure 6. Protein array analysis in response to in vitro infection with CFT073 or ΔTcpC.

(A) RT-PCR analysis of human kidney cell lines infected with CFT073 or ΔTcpC for 4 hrs. All values represent means ± SEMs of three experiments. (B) Uroepithelial cytokine responses to infection with CFT073, ΔTcpC or mock-infected controls. A498 cells were stimulated for 4 hours with the indicated bacterial strains. Supernatant cytokine levels are in pg/ml and represent means ± SEMs of three experiments. Significant differences are indicated by single * (p<0.05), double ** (p<0.01) or triple asterisks *** (p<0.001, Student's t-test).

Figure 7. Effects of TcpC virulence in Trif ^−/− and Trif ^Lps2/Lps2 mice infected with CFT073 or ΔTcpC.

(A) Bacterial burden (CFU) in kidneys of wt (C57BL6/129), Trif ^−/−, wt (C57BL/6) and Trif ^Lps2/Lps2 mice on day seven after infection with CFT073 or ΔTcpC. (B) Bacterial numbers in urine of wt and mutant mice after infection. (C) Kinetics of the MIP-2 response, measured in urine samples. (D) Neutrophil response kinetics in urine of wt and mutant mice. P values (*p<0.05, **p<0.01 and ***p<0.001 for CFT073 versus ΔTcpC mutant and for wt versus mutant mice; Fisher's exact test; ns = not significant). (E) Abscess formation in mouse kidneys, seven days post-infection (Scale bar, 2 mm). (F) Abscess formation after CFT073 or ΔTcpC infection, in percent of the total number of kidneys examined.

Figure 8. Inhibitory effects of the soluble TcpC TIR-domain on TLR signaling in murine macrophages.

(A) Wt BMDM were stimulated with Pam3Cys (1 µg/ml), poly(I:C) (2.5 µg/ml), ultrapure LPS from E. coli K12 (100 ng/ml), flagellin from S. typhimurium (1 µg/ml) and CpG 1826 (2 µM) in the presence of titrated amounts of the purified TIR domain of TcpC (TIR-TcpC) as indicated. TNF secretion was analyzed 3 hr after stimulation. (B) The experiment in (A) was repeated at the same time, side by side with Myd88^−/− BMDM. Error bars represent SD from three individual cultures. The experiment was repeated once with similar results.

Figure 9. Effects of TcpC virulence in Il-1β^−/− and Irf3^−/− mice infected with CFT073 or Δ TcpC.

(A) Bacterial burden (CFU) in kidneys of wt (CF7BL/6), Il-1β^−/− and Irf3^−/− mice on day seven after infection with CFT073 or ΔTcpC. (B) Bacterial numbers in urine after infection. (C) Kinetics of the MIP-2 response, measured in urine samples. (D) Neutrophil response kinetics in urine of wt and mutant mice. P values (**p<0.01 and ***p<0.001 for CFT073 versus ΔTcpC mutant and for wt versus mutant mice; Fisher's exact test; ns = not significant). (E) Abscess formation in mouse kidneys, seven days post-infection (Scale bar, 2 mm). (F) Abscess formation after CFT073 or ΔTcpC infection, in percent of the total number of kidneys examined.