Biofilm formation on abiotic and biotic surfaces is a common survival strategy for many microorganisms. Biofilms allow pathogens to subvert immune defenses and antibiotic treatments. Therefore, strategies to interrupt bacterial biofilm formation are urgently required. Many Gram-negative bacteria including uropathogenic Escherichia coli produce functional amyloids called curli that serve as scaffolds for biofilm production. The ubiquitous existence of functional amyloids throughout cellular life is well established now. The precursor of curli fibers is the amyloidogenic protein CsgA. The potential intracellular amyloid toxicity of curli fibers in E. coli is prevented by specific chaperones and periplasmic proteins like CsgC. The human amyloidogenic protein transthyretin (TTR) has structural homology with CsgC and is known to inhibit amyloid-? (A?) aggregation in vitro and suppress the Alzheimer's-like phenotypes in a transgenic mouse model of A? deposition. It also appears to inhibit fibrillogenesis by other amyloid precursors (e.g. HypF-N, Ig L-chains, and Islet amyloid polypeptide) but has no effect on other forms of protein aggregation. New discoveries show that both human wild-type tetrameric TTR (WT-TTR) and its engineered non-tetramer forming monomer (M-TTR) inhibit CsgA amyloid formation in vitro with M-TTR being the more efficient inhibitor. Significantly TTR also inhibited amyloid-dependent biofilm formation in E. coli with no apparent bactericidal or bacteriostatic effects. However, the molecular details underlying the mechanism of TTR-mediated amyloid inhibition remain to be elucidated. The anti-amyloid and anti-biofilm activity of TTR against other bacterial amyloids and biofilms has not yet been studied.
In Aim 1, the mechanism of amyloid inhibition by TTR will be interrogated using a diverse array of biophysical and biochemical techniques that have been well established in the lab. The specific protein?protein interactions between TTR and its clients will be characterized to better understand amyloid inhibition.
In Aim 2, the ability of TTR to abolish amyloid assembly by various microbial amyloids like FapC, TasA, phenol soluble modulins (PSMs) will be determined. The efficacy of TTR-coated catheters to discourage amyloid and amyloid- dependent biofilm formation will be demonstrated. Work in Aim 3 will determine how bacterial colonization and biofilm formation in mice is influenced by genetic overexpression or deletion of TTR. The consequence of stabilizing the TTR tetramer on bacterial infections will be tested using a well-established murine urinary tract infection model. Collectively, the discoveries from this proposal will determine if TTR can be utilized as an effective and versatile anti-amyloid and anti-biofilm agent that will potentiate antibiotic efficacy in infections associated with significant biofilm formation. Finally, the work will demonstrate how a protein fold shared by CsgC and TTR, which is largely independent of primary sequence conservation, can direct protein-protein interactions that discourage amyloid formation and is functional across biological kingdoms.
Escherichia coli and related enteric bacteria form communities called biofilms that are resistant to host immune defenses and antibiotic treatment. Extracellular assembly of protein amyloids is the critical determinant of biofilm formation. We propose to investigate how a human protein (transthyretin) can block amyloid assembly and biofilm formation in diverse bacteria, so that new avenues to combat biofilm-associated infections can be developed.
| Deshmukh, Maya; Evans, Margery L; Chapman, Matthew R (2018) Amyloid by Design: Intrinsic Regulation of Microbial Amyloid Assembly. J Mol Biol 430:3631-3641 |
