Ability of Proteus mirabilis to invade human urothelial cells is coupled to motility and swarming differentiation

Abstract

Proteus mirabilis causes serious kidney infections which can involve invasion of host urothelial cells. We present data showing that the ability to invade host urothelial cells is closely coupled to swarming, a form of cyclical multicellular behavior in which vegetative bacteria differentiate into hyperflagellated, filamentous swarm cells capable of coordinated and rapid population migration. Entry into the human urothelial cell line EJ/28 by P. mirabilis U6450 isolated at different stages throughout the swarming cycle was measured by the antibiotic protection assay method and confirmed by electron microscopy. Differentiated filaments entered urothelial cells within 30 min and were 15-fold more invasive (ca. 0.18% entry in 2 h) than an equivalent dry weight of vegetative cells isolated before differentiation, which attained only ca. 0.012% entry in the 2-h assay. The invasive ability of P. mirabilis was modulated in parallel with flagellin levels throughout two cycles of swarming. Septation and division of intracellular swarm cells produced between 50 and 300 vegetative bacteria per human cell, compared with 4 to 12 intracellular bacteria after incubation with vegetative cells. Transposon (Tn5) mutants of P. mirabilis with specific defects in motility and multicellular behavior were compared with the wild-type for the ability to invade. Mutants which lacked flagella (nonmotile nonswarming) were entirely noninvasive, and those which were motile but defective in swarm cell formation (motile nonswarming) were 25-fold less invasive than wild-type vegetative cells. Mutants with defects in the coordination of multicellular migration and the temporal control of consolidation (cyclical reversion of swarm cells to vegetative cells) were reduced ca. 3- to 12-fold in the ability to enter urothelial cells. In contrast, a nonhemolytic transposon mutant which swarmed normally retained over 80% of wild-type invasive ability. Swarm cells and early consolidation cells were at least 10-fold more cytolytic than vegetative cells as a result of their high-level production of hemolysin.