Mysorekar et al. 10.1073/pnas.0602136103. |
Supporting Figure 7
Supporting Figure 8
Supporting Figure 9
Supporting Figure 10
Supporting Text
Fig. 7
. Replicating bacteria within a superficial facet cell intracellular bacterial community (IBC). Multilabel immunofluorescence study of tissue sections from bladders of infected mice that received an intraperitoneal injection of BrdU 90 min before sacrifice show several BrdU+ (red; stained with Alexa Fluor 594-tagged goat polyclonal antibodies to BrdU) bacteria (yellow overlay) within a mature IBC (green stained with Alexa Fluor 488-tagged rabbit polyclonal antibodies to Escherichia coli) located in a superficial facet cell. Epithelial nuclei stained with bis-benzimide appear blue.Fig. 8
. Intracellular bacterial communities (IBCs) in PS pretreated bladders. Epiflourescence microscopy on whole-mounted bladders PS-treated for 12h and infected for 6h with UTI89 expressing green fluorescent protein (GFP) and showing multiple UPEC IBCs (green) exist in a single facet cell (red, stained with Alexa Fluor 594-tagged wheat germ agglutinin, r-WGA, to demarcate outlines of facet cell membranes). Epithelial nuclei stain blue with bis-benzimide. (Inset) High power view of a single facet cell with four distinct IBCs (green). (Scale bar, 50 m m.).Fig. 9
. Multilabel immunofluorescence study of bladder tissue sections show that Escherichia coli intracellular bacterial communities (IBCs) (green, stained with Alexa Fluor 488-tagged rabbit polyclonal Abs to E. coli) are intracellular within E-cadherin+ underlying transitional cells (red, with AlexaFluor594-tagged mouse mAb to E-cadherin). (Scale bar, 10 m m.)Fig. 10
. Model of recurrent urinary tract infection (UTI) depicting the sequence of events during the progression of establishment of a bacterial QIR and its subsequent re-emergence from the underlying transitional cells of the bladder epithelium to induce recurrence of infection. Bacteria (green) bind to and invade into superficial facet cells within 6h after infection and develop into intracellular bacterial communities (IBCs). IBC-containing facet cells exfoliate around 6-12h. Released bacteria can escape into the lumen or infect underlying transitional cells at 24-48h after infection and form IBCs. Subsequently, Escherichia coli organized as rosettes are enclosed within endosomal vesicles, which persist for at least 12 weeks. When the underlying transitional cells eventually differentiate into mature superficial facet cells, the E. coli QIRs can reemerge to cause a high-titer UTI.Protamine Sulfate (PS) Treatment.
To ensure that PS did not have bactericidal activity in vitro, 50 ml of 107 CFU UTI89 was grown overnight as a static culture at 37°C in LB containing 10 mg/ml solution of PS and plated on LB agar. No significant differences in CFUs were noted between PS treated versus untreated bacteria. To determine that PS does not affect bacterial growth or maturation in vivo, 50 ml of 10 mg/ml PS was co-injected with 107 CFU UPEC into mouse bladders (n = 5 mice per time point). Histopathological examination revealed evidence of IBCs at 3.5 and 6 h after inoculation, suggesting that PS did not have any affect on intracellular growth of bacteria and their progression through the IBC developmental cycle. Bladders pretreated for 12 h with 10 mg/ml PS and subsequently infected with 107 UPEC had 629 ± 45 IBC’s (n = 4 mice examined).At a concentration of 10 mg/ml PS in 50 ml of sterile PBS, 65% of facet cells exfoliated within 12 h, confirming the efficacy of PS in stripping host barriers. To determine whether higher doses of PS would impact bacterial colonization, 10 mg/ml PS was administered for 12 h followed by a booster dose of 50 mg/ml for 6 h to ensure maximal (>95%) facet cell exfoliation as described above. PS-treated mice were infected with 107 CFU of GFP-UTI89 and bladders isolated after 6 and 24 h. An occasional intact superficial facet cell could be seen containing loose individual bacteria with rod shaped morphology (n = 5 mice per time point examined). Twenty-four hours later, there was extensive epithelial cell division followed by facet cell regeneration. However, no IBCs were observed at any time, and underlying cells were devoid of bacteria, despite robust extracellular colonization.
Measurement of Extent of Epithelial Exfoliation.
Exfoliation calculated over an arbitrarily defined area of the bladder [150 mm (x axis) x 150 mm (y axis)]/whole mounted bladder/mouse. Analysis was performed on biz-benzimide- and tetramethylrhodamine-conjugated wheat germ agglutinin (r-WGA, Molecular Probes)-stained whole mounted bladders, three to four areas per mouse, n = 4 mice per experiment.