In normal uninfected bladder epithelial cells, Rho GTPases like Rac cycle between active GTP-bound and inactive GDP-bound states, regulated by GAPs and GEFs (guanine nucleotide exchange factors). CNF1 renders Rho GTPases constitutively active by catalyzing deamidation of glutamine 63 in RhoA (or glutamine 61 in Rac and Cdc42). CNF1-modified constitutively active Rac, in this example, is shown as a black oval. Activation of Rac and other Rho GTPases leads to cytoskeletal rearrangements resulting in increased cell spreading and membrane ruffling (1–4 h post-CNF1 intoxication). By 4 h postintoxication, constitutively activated Rho GTPases are ubiquitinated and targeted for proteasomal degradation. The resulting depletion of Rho GTPases promotes host cytoskeletal rearrangements, resulting in enhanced filopodia formation and stimulating bacterial uptake. Bacterial internalization continues to increase up to 24 h post-CNF1 intoxication. Rho depletion also stimulates bladder cell migration. Theoretically, this could result in redistribution of host cells within a stratified three-dimensional tissue such as the bladder epithelium, allowing UPEC to more efficiently disseminate and colonize underlying cells. The model shows the migration of host cells in the upper (yellow) and lower (blue) layers of an idealized tissue bilayer as a consequence of CNF1-stimulated Rac depletion.

FimH, localized at the distal tips of type 1 pili, bind and cluster unspecified host receptors, possibly concentrated within lipid raft domains (green regions). This triggers a signaling cascade leading to localized rearrangements of F-actin (red), which in turn results in the envelopment and uptake of adherent UPEC. Once internalized, UPEC is trafficked into membrane-bound compartments. From here, undefined signals derived from the host environment (and possibly associated with the host cell differentiation state) trigger UPEC replication, resulting in the formation of intracellular bacterial communities (IBCs) and the eventual efflux of UPEC. Alternatively, UPEC enters a nonreplicating or viable but nonculturable (VBNC) state that can persist for days or weeks, possibly serving as a reservoir for subsequent recurrent acute infections. PIPs phosphoinositides.

Transmission electron microscopy reveals that the human UPEC cystitis isolate UTI89 is trafficked into compartments with morphologic characteristics of MVBs (arrowheads). Images were taken 24 h postinfection of immortalized human bladder cells designated 5637. After an initial 2-h incubation, the host cell impermeable antibiotic gentamicin was added to the cell culture media to prevent extracellular bacterial growth. Scale bars, 5 μm. n, nucleus.

(A and B) Scanning electron microscopy shows filamentous UPEC (a cystitis isolate designated UTI89) emerging from and binding to mouse bladder superficial cells. Scale bars, (A) 5 μm and (B) 3 μm.