Date of Award
Doctor of Philosophy (PhD)
Chaperone-usher pathway (CUP) pili are a diverse set of extracellular organelles produced by Gram-negative bacteria that share a homologous assembly mechanism and mediate adhesion, colonization, and biofilm formation. Several CUP pili, including type 1, P and S pili, are involved in the pathogenesis of uropathogenic E. coli (UPEC), the primary cause of community acquired urinary tract infections (UTIs). As important virulence factors, it is vital to fully understand the function and regulation of these pili, in order to design and optimize anti-virulence therapeutics targeting them. With this work, we investigate several novel aspects of CUP pilus regulation, as well as the cross-regulation between CUP pili and other virulence factors. We also fully characterize the effects of small molecule inhibitors of CUP pili.
Although there are many known pathways coordinating the expression of CUP pili, here we document new regulatory pathways. We show that type 1 pilus regulation is sensitive to type 1 pilus assembly and function. A dedicated chaperone and usher pair assemble CUP pili by facilitating pilus subunit folding and subunit polymerization into the mature pilus. Type 1 pili, encoded by the fim operon, have phase variable expression, with transcription controlled by the fimS promoter, which is encoded on an invertible element that switched between phase ON (pilus producing) and phase OFF orientations. The CUP chaperone and usher are each necessary for pilus assembly, and we demonstrate that lack of the type 1 pilus chaperone, FimC, induces the fimS phase OFF state. Furthermore, type 1 pili use the FimH adhesin to mediate attachment to and invasion of the bladder epithelium by binding to mannosylated surface proteins. We demonstrate that genetic and chemical inactivation of FimH function also leads to the fimS phase OFF state. This is impressive given that FimH functions extracellularly, at a considerable distance from the bacterial outer membrane. Furthermore, we demonstrate that human urine impacts type 1 pilus regulation and expression, and therefore functions as an innate defense against UPEC UTIs. With this work, we provide insight into additional regulatory pathways controlling the expression of this important virulence factor.
We also characterize regulatory networks coordinating CUP pili with each other and with other virulence factors. Growth of the UTI89fim mutant in type 1 pili inducing conditions, leads to a compensatory response from other CUP pili, specifically P and S pili. We demonstrate the UTI89fim mutant produces increased P and S pilus transcript and protein levels. Furthermore, the UTI89fim mutant is hypermotile, indicating regulatory connections between type 1 pili and flagella, another virulence factor implicated in UPEC pathogenesis. Thus, there are complex regulatory networks coordinating the expression of different pili, which allow the bacteria to attach to different receptors and colonize diverse environments, and coordinating the expression of pili and flagella, which allow the bacteria to control when to migrate to new environments.
In order to decrease the virulence of UPEC, we developed small molecule inhibitors of CUP pili, termed pilicides, which were shown to inhibit type 1 and/or P piliation. A screen revealed that pilicide ec240 was the most potent type 1 pilus inhibitor. We show that ec240 induced the fimS phase OFF orientation of the type 1 pilus promoter. We investigated if there were downstream consequences of type 1 pilus inhibition, and if ec240 had other effects on UPEC with global transcriptional and proteomic analyses of UTI89 treated with ec240. We demonstrated that ec240 disregulated CUP pili, by decreasing type 1, but increasing S and P pilus transcript levels, and also altering transcription of motility and other virulence genes. Interestingly, although ec240 increased S and P pilus transcript levels, it reduced S and P piliation levels on the cell surface, indicating ec240 post-transcriptionally blocked S and P pilus assembly. The regulators SfaB, PapB, SfaX and PapX are likely involved in the ec240 effects on type 1 pilus phase state, and we show that these regulators are necessary for the ec240 effect on motility. Thus, ec240 disregulated several UPEC virulence factors at the transcriptional and/or post-transcriptional level, and has therapeutic potential to treat UPEC UTIs or other infections.
This dissertation has further elucidated the regulation of UPEC extracellular appendages and the genetic networks coordinating their expression and production. We have demonstrated how human urine manipulates this regulation to decrease the virulence of UPEC, and how small molecules can manipulate pilus regulation and assembly to decrease UPEC virulence.
Chair and Committee
Scott J. Hultgren
Michael G. Caparon, Daniel Goldberg, David A. Hunstad, Christina Stallings,
Greene, Sarah Elizabeth, "Regulation and Inhibition of Uropathogenic E. coli Extracellular Appendages" (2016). Arts & Sciences Electronic Theses and Dissertations. 737.
Available for download on Friday, May 15, 2116