Urinary tract infections are among the most common human bacterial infections and place a significant burden on healthcare systems due to associated morbidity, cost and antibiotic use. Despite being a facultative anaerobe, uropathogenic Escherichia coli, the primary cause of urinary tract infections, requires aerobic respiration to establish infection in the bladder. Here, by combining bacterial genetics with cell culture and murine models of infection, we demonstrate that the widely conserved respiratory quinol oxidase cytochrome bd is required for intracellular infection of urothelial cells. Through a series of genetic, biochemical and functional assays, we show that intracellular oxygen scavenging by cytochrome bd alters mitochondrial physiology by reducing the efficiency of mitochondrial respiration, stabilizing the hypoxia-inducible transcription factor HIF-1 and promoting a shift towards aerobic glycolysis. This bacterially induced rewiring of host metabolism antagonizes apoptosis, thereby protecting intracellular bacteria from urothelial cell exfoliation and preserving their replicative niche. These results reveal the metabolic basis for intracellular bacterial pathogenesis during urinary tract infection and identify subversion of mitochondrial metabolism as a bacterial strategy to facilitate persistence within the urinary tract.
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All data required for evaluation of these results are present in the text, figures or supplemental material. Transcriptional profiling data have been deposited in the Gene Expression Omnibus database (accession GSE188981) and are also contained in Supplementary Table 2a. Data from the following publicly available databases were used in this study: NCBI Protein Database (FASTA sequence: NP_415261.2) and the RCSB Protein Data Bank (PDB) (PDB IDs 6rko and 6rx4). Source data are provided with this paper.
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We thank K. Voss and H. Woods for technical assistance and helpful advice. This work was supported by National Institutes of Health (NIH) grants F30AI150077 (C.J.B.), T32GM007347 (C.J.B. and B.I.R.), F99NS125829 (G.L.R.), F30CA247202 (B.I.R.), F32CA250258 (A.M.B.), T32AI112541 (G.H.M.), T32GM007569 (J.R.B.), R01AI127793 (W.J.C.), R01AI101171 (W.J.C.), R01DK105550 (J.C.R.), R35GM128915 (V.G.), RF1MH123971 (V.G.), R01AI107052 (M.H.), P20DK123967 (M.H.) and the Howard Hughes Medical Institute Gilliam Fellowship (G.L.R.). Transmission electron microscopy was performed by W. Beatty at the Molecular Microbiology Imaging Facility (Washington University in St. Louis). Confocal laser scanning and structured illumination microscopy were performed at the Vanderbilt Cell Imaging Shared Resource, which is supported by NIH grant DK20593. NanoString analysis was performed at the Vanderbilt Technologies for Advanced Genomics core facility, which is supported by NIH grants UL1RR024975, P30CA68485, P30EY08126 and G20RR030956. Access to the Vanderbilt Advanced Computing Center for Research and Education was supported in part by NIH grants S10RR031634 and S10OD023680. Some images were created using BioRender.com.
The authors declare no conflicts of interest at the time of submission of this manuscript.
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Extended Data Fig. 1 Cytochrome bd does not influence type 1 pilus dependent adherence or invasion of urothelial cells.
a, Anti-FimA immunoblot performed on normalized samples boiled in acidified SDS to depolymerize type 1 pili. b, Anti-FimA immunoblot performed on normalized samples with polymerized type 1 pili. Each band represents FimA polymers of a different size. c, Bacterial hemagglutination titer with or without the FimH inhibitor mannose; mean ± SEM; one-way ANOVA with Dunnett’s test for multiple comparisons. d, Total, adherent, and intracellular bacterial titers in infected urothelial cells; geometric mean ± 95% CI; Kruskal-Wallis test with Dunn’s test for multiple comparisons. All experiments were performed with a minimum of three biological replicates. Each point represents a biological replicate. Exact p-values are provided in the figure, with bold values indicating statistical significance (p < 0.05).
Extended Data Fig. 2 Loss of cytochrome bd increases intracellular bacterial sensitivity to nitrofurantoin.
Percent survival of intracellular UPEC treated with ciprofloxacin or nitrofurantoin as compared to vehicle treated controls. Data is representative of six biological replicates per group. Each point represents a biological replicate; mean ± SEM; two-tailed unpaired t test. Exact p-values are provided in the figure, with bold values indicating statistical significance (p < 0.05).
Extended Data Fig. 3 Loss of cytochrome bd does not impair flagellar biosynthesis.
a, Representative images of wild-type and ∆cydAB transformed with a transcriptional reporter for FlhDC, the master regulator of flagellar expression. b, GFP intensity normalized to cell area; median ± 95% CI; two-tailed unpaired t test. c, Representative transmission electron microscopy images of bacterial cells. Arrows indicate flagella. Images are representative of three biological replicates. Exact p-values are provided in the figure, with bold values indicating statistical significance (p < 0.05).
Extended Data Fig. 4 Urothelial cells upregulated NOS2 in response to intracellular infection.
Normalized counts of NOS2 (inducible nitric oxide synthase, iNOS) transcript in intracellularly infected urothelial cells. Data is representative of at least three biological replicates per group; mean ± SEM; one-way ANOVA with Tukey’s test for multiple comparisons. Exact p-values are provided in the figure, with bold values indicating statistical significance (p < 0.05).
Extended Data Fig. 5 UPEC is not sensitive to rotenone or antimycin A.
OCR readings of wild-type UPEC treated with vehicle (open circles) or treated with oligomycin, FCCP, and rotenone/antimycin A (closed circles) presented as percent OCR of time = 0; mean ± SEM. Data was fit to a one phase decay model (R2 > 0.9 for both groups) and statistically analyzed by comparing k (p = 0.6402). Data is representative of five biological replicates, each with at least three technical replicates.
Extended Data Fig. 6 Expression of respiratory oxidases in intracellular bacterial populations.
a, Abundance of respiratory oxidase transcripts in the inoculum used for infections and intracellular bacterial populations compared to gyrB. Dotted line indicates gyrB abundance; mean ± SEM; two-tailed unpaired t test. b, Relative abundance of respiratory oxidase transcript in the inoculum and intracellular populations. c, Representative peptide nucleic acid in situ hybridization (PNA-FISH) image of IBCs. All experiments were performed with a minimum of three biological replicates. Each point represents a biological replicate. Exact p-values are provided in the figure, with bold values indicating statistical significance (p < 0.05).
Extended Data Fig. 7 Intracellular infection modulates urothelial cell transcriptional programs.
a, Volcano plot depicting changes in transcript abundance between wild-type and mock infected urothelial cells. Transcripts involved in glycolysis, glucose uptake, and hypoxia are denoted by color. b, Volcano plot depicting changes in the abundance of metabolic transcripts depicted in Fig. 5e between wild-type and mock infected urothelial cells. Data is representative of at least three biological replicates per group. Transcript normalization, differential expression calculations, and statistical comparisons were performed on the nSolver Advanced Analysis platform using the Benjamini-Yekutieli method.
Extended Data Fig. 8 Cytochrome bd has minimal impact on urothelial cell immune response to intracellular infection.
Directed pathway expression score for pathways not directly involved in central metabolism in wild-type and ∆cydAB infected compared to mock infected urothelial cells; mean ± SEM; two-tailed unpaired t test. Data is representative of at least three biological replicates per group. Exact p-values are provided in the figure, with bold values indicating statistical significance (p < 0.05).
Extended Data Fig. 9 Flow cytometry gating strategy.
Single cells were selected by gating on FSC-A and FSC-H. Debris and bacteria were subsequently excluded by gating out the FSC-Alow, SSC-Alow population. The remaining population was analyzed for UPEC (GFP) and annexin V (Pacific Blue) or caspase-3 activity (Pacific Blue). Because we are interested in quantifying cell death, dead cells were not specifically excluded from analyses. Each dot represents one cell. Relative population density is represented by a color spectrum (red indicates regions of high cell density, blue indicates regions of low cell density).
Extended Data Fig. 10 Proposed model.
Schematic depicting the proposed model of how intracellular infection modulates urothelial cell metabolism and survival. left, During bladder infection UPEC induces a strong inflammatory response that triggers urothelial cell apoptosis and exfoliation. Urothelial cell exfoliation exposes underlying tissue layers to infection and promotes bacterial persistence in the bladder. right, By consuming oxygen and activating HIF-1 signaling, intracellular bacterial aerobic respiration alters urothelial cell metabolism and antagonizes apoptosis, allowing UPEC to complete its intracellular infection cascade and evade exfoliation.
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Beebout, C.J., Robertson, G.L., Reinfeld, B.I. et al. Uropathogenic Escherichia coli subverts mitochondrial metabolism to enable intracellular bacterial pathogenesis in urinary tract infection. Nat Microbiol 7, 1348–1360 (2022). https://doi.org/10.1038/s41564-022-01205-w
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