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In the Hadjifrangiskou Lab (or in short, the H-team), we are interested in understanding regulatory mechanisms that underlie mutlicellular behavior and virulence in bacteria that cause urinary tract infections (UTI).

UTIs are among the most frequent bacterial infections, are highly prevalent among women and have a high degree of recurrence. Currently, antibiotics are the primary treatment option for UTI, however they oftentimes fail to eliminate infection, they perturb the host microbial flora and select for increased antibiotic resistance. This means that there is a pressing need for the development of alternative strategies for preventing and/or treating UTIs. Uropathogenic Escherichia coli (UPEC), which causes 85% of all UTIs, has evolved a remarkable mechanism to evade host immune defenses and establish infection, by forming biofilm-like intracellular bacterial communities (IBC) inside bladder cells, in addition to forming extracellular biofilms on host cell surfaces and on catheter implants. Type 1 pili are adhesive organelles that are vital for UPEC adherence and biofilm formation.

We are interested in identifying additional factors  and mechanisms that regulate UPEC biofilm formation and that could serve as new drug targets. A mutagenesis screen that has identified 41 such factors, including the QseBC signal transduction system, which we are currently characterizing in more detail. To do this, we use a wide variety of approaches, including molecular biology, cell-based and in vitro assays, chemical biology and 3 mouse models of UTI.

Current Projects include:

Characterization of the QseBC two-component system and its role in pathogenesis – Two-component system networks integrate signals, modulating gene expression changes to tailor adaptation to changing surrounding environments. In their simplest form, two-component systems consist of a sensor kinase that upon signal interception alters the phosphorylation state of a cognate response regulator, which then mediates transcription. Typically, two-component system interactions are restricted to cognate partners and non-cognate partner cross-talk while possible, is not strong. We have previously shown that in the case of QseBC, deletion of the QseC sensor in UPEC leads to gene dysregulation and virulence attenuation, due to over-activation of the QseB response regulator. This is because, in the absence of QseC, another sensor we have identified is able to activate QseB, but, unlike QseC it cannot deactivate QseB.

UPEC pathogenic cascade and QseC

We are currently exploring:

1) The possibility of targeting QseC activity as a means of attenuating UPEC virulence.

2) The identification of QseC residues responsible for QseB deactivation

3) The basis of interaction between QseB and the non-cognate sensor that activates it in the absence of QseC

4) The role of the non-cognate QseB activator in UPEC virulence

5) Characterization of QseBC in other uropathogens

Investigation of previously uncharacterized UPEC biofilm effectors, the disruption of which diminishes IBC formation and attenuates UPEC virulence- Using a transposon screen, several UPEC biofilm determinants were discovered, which also impact UPEC IBC formation and in vivo virulence. We are interested in further characterizing each category of mutants and understanding the mechanism by which they modulate IBC formation. One of these factors is VisC. Assessment of virulence factor production in this mutant demonstrated that, although type 1 pili and flagellar motility are inversely related, disruption of the visC gene impaired both motility and type 1 pili expression. We went on to show that non-polar deletion of visC, imparts resistance to energy-dependent aminoglycoside antibiotics and leads to higher persister cell formation, consistent with fluctuations in proton motive force (pmf). We verified a perturbation in
membrane potential using flow cytometry, providing evidence that VisC
contributes to the generation of pmf. Using qPCR, we discovered that in the absence of VisC, expression of the fimB recombinase is reduced, explaining the switch to the OFF orientation in the visC mutant. We are currently exploring:

1) The mechanism of fimB downregulation in the absence of VisC

2) The potential of targeting VisC to prevent UPEC biofilm formation

Understand the mechanism by which rationally designed small molecules act against UPEC pathogenesis- Our collaborator chemists have designed small molecular weight inhibitors to block UPEC pilus assembly, thereby impeding bacterial adherence to biotic and abiotic surfaces; we have come to discover that these molecules impact UPEC gene expression, causing pleiotropic effects on virulence gene regulation and inhibiting biofilm formation. In collaboration with labs at Washington University in Saint Louis and Umea University in Sweden, we are working towards understanding how these inhibitors function and how they can be optimized for use as treatments against UTIs.


April 2014