Fig 1.
G. vaginalis bladder exposures induce E. coli emergence from latent bladder reservoirs.
(A) Schematic of mouse model. E. coli bladder reservoirs were established during a 4 week period following a ‘Primary E. coli UTI’; those with kidney infections (bacteriuria at 4 weeks) were excluded; cream shading indicates period of exposure to G. vaginalis, L. crispatus, or PBS; open circles, urine sample collections. (B) G. vaginalis and L. crispatus urine titers following first exposure in mice with E. coli reservoirs. (C) Percentage of mice with detectable E. coli urine titers following secondary exposures. N = 6 experiments totaling 155 mice. ** P < 0.001 by Fisher’s exact test. (D) Highest E. coli urine titer from each mouse after exposure 2 (N = 6 independent experiments). Dotted line = limit of detection. A Kruskal-Wallis test was performed (P < 0.0001), followed by post hoc pairwise comparisons. **** P < 0.0001; ** P < 0.002 by Mann-Whitney. (E) E. coli reservoir titers in bladder homogenates at 72 h after exposure 2 (N = 2). ** P < 0.01 by Mann-Whitney. (F) Example of Hema-3 stained urine sediments from a PBS-exposed mouse negative for E. coli bacteriuria (left) and from a G. vaginalis-exposed mouse positive for E. coli bacteriuria (right). Arrows—neutrophils.
Fig 2.
G. vaginalis causes urothelial exfoliation.
(A) Scanning electron micrographs of splayed bladders showing the superficial hexagonal urothelial cells that line the bladder lumen. N = 2 independent experiments. PBS, n = 3; G. vaginalis n = 7. ‘E’ denotes areas of exfoliation. (B) Immunofluorescence microscopy of bladder sections. Red—uroplakin IIIa (superficial epithelial cells); Blue—DAPI. White lines—epithelial basement membrane; L—bladder lumen. N = 3 independent experiments; n = 2–5 mice per group. (C) Blinded scoring of urothelial cells in urine sediments collected between 3 and 24 h after two PBS or G. vaginalis exposures. N = 3 independent experiments; PBS, n = 37; G. vaginalis, n = 59. * P = 0.0374 by Fisher’s exact test. (D) Schematic model of G. vaginalis-induced recurrent E. coli UTI.
Fig 3.
G. vaginalis induces apoptosis in the urothelium.
Data are from mice containing E. coli reservoirs that were exposed twice (12 h apart) to G. vaginalis or PBS. Bladders were collected 3 h (A) or 12 h (B-E) after the second exposure. (A) Scanning electron microscopy (SEM) images of bladders from mice exposed to G. vaginalis. Top: G. vaginalis association with membrane protrusions. Bottom: membrane blebbing consistent with apoptotic body formation. (B) TUNEL staining of bladder sections revealed TUNEL-positive cells (white arrows) within the lumen (L) and superficial urothelium of G. vaginalis-exposed bladders. White dotted line denotes the epithelial basement membrane. (C) Immunohistochemistry of bladder sections stained for cleaved caspase-3 (brown). (D) Percentage of bladders in each exposure group that stained positively for cleaved caspase-3. N = 2 independent experiments, 2–5 mice per group. (E) Level of pro-inflammatory cytokines in bladder homogenates. A D’Agostino & Pearson omnibus normality test was performed followed by appropriate pairwise analysis (either unpaired t-test or Mann-Whitney). * P < 0.05.
Fig 4.
G. vaginalis causes IL-1 receptor-mediated kidney injury and increases the incidence of severe E. coli kidney and systemic infection.
(A-C) Data are from mice that were exposed twice (12 h apart) to PBS or G. vaginalis as described in S3 Fig. (A) G. vaginalis titers in kidney tissue. (B) Cytokine/chemokine levels in kidney homogenates at 12 h after the second exposure. Kruskal-Wallis tests were performed followed by post hoc pairwise comparison. For comparison, data from an abscessed kidney collected at 72 h after exposure (not included in the statistical analysis) are denoted by symbols with a blue ‘X’. ** P < 0.005, * P < 0.05, Mann-Whitney (uncorrected P values). (C) Serum creatinine levels, a marker of acute kidney injury, at 12 h after the second bladder exposure. Mice received 2 intraperitoneal injections of PBS (vehicle) or anakinra at ~16h prior to and at the time of transurethral inoculation. N = 6 independent experiments. ** P < 0.005, * P < 0.05. (D) Incidence of E. coli kidney infection with abscessed kidney and splenomegaly at 72 h post exposure. Data are compiled from 12 experiments with two exposures (12 h and 1 wk apart, including mice injected with PBS as in C) and 1 experiment with 3 exposures, each 24 h apart. ** P = 0.0018, Fisher’s exact.
Fig 5.
Model of Gardnerella vaginalis “covert pathogenesis.”
In women, approximately half of recurrent urinary tract infection (rUTI) episodes are caused by an E. coli strain identical to the strain that caused the initial infection. Here we present a model of one mechanism of rUTI: activation of latent intracellular E. coli reservoirs in the bladder. G. vaginalis urinary tract exposure, likely a consequence of sexual activity, results in exfoliation of the bladder epithelium and damage to the kidney. Subsequent to exfoliation, E. coli emerges from intracellular reservoirs into the bladder lumen, where it can ascend into the kidney, sometimes causing severe inflammation and systemic infection. Often E. coli rUTI occurs after G. vaginalis clearance from the urinary tract. These findings have important implications for our understanding of rUTI etiology and point to G. vaginalis colonization as a potentially important marker of rUTI risk.