Sequence-targeted Peptides Divert Functional Bacterial Amyloid Towards Destabilized Aggregates and Reduce Biofilm Formation.

Functional bacterial amyloid provides structural stability in biofilm, making it a promising target for anti-biofilm therapeutics. Fibrils formed by CsgA, the major amyloid component in E. coli are extremely robust and can withstand very harsh conditions. Like other functional amyloids, CsgA contains relatively short aggregation-prone regions (APR) which drive amyloid formation. Here, we demonstrate the use of aggregation-modulating peptides to knock down CsgA protein into aggregates with low stability and altered morphology. Remarkably, these CsgA-peptides also modulate fibrillation of the unrelated functional amyloid protein FapC from Pseudomonas, possibly through recognition of FapC segments with structural and sequence similarity with CsgA. The peptides also reduce the level of biofilm formation in E. coli and P. aeruginosa, demonstrating the potential for selective amyloid targeting to combat bacterial biofilm.

Mapping the sequence specificity of heterotypic amyloid interactions enables the identification of aggregation modifiers.

Heterotypic amyloid interactions between related protein sequences have been observed in functional and disease amyloids. While sequence homology seems to favour heterotypic amyloid interactions, we have no systematic understanding of the structural rules determining such interactions nor whether they inhibit or facilitate amyloid assembly. Using structure-based thermodynamic calculations and extensive experimental validation, we performed a comprehensive exploration of the defining role of sequence promiscuity in amyloid interactions. Using tau as a model system we demonstrate that proteins with local sequence homology to tau amyloid nucleating regions can modify fibril nucleation, morphology, assembly and spreading of aggregates in cultured cells. Depending on the type of mutation such interactions inhibit or promote aggregation in a manner that can be predicted from structure. We find that these heterotypic amyloid interactions can result in the subcellular mis-localisation of these proteins. Moreover, equilibrium studies indicate that the critical concentration of aggregation is altered by heterotypic interactions. Our findings suggest a structural mechanism by which the proteomic background can modulate the aggregation propensity of amyloidogenic proteins and we discuss how such sequence-specific proteostatic perturbations could contribute to the selective cellular susceptibility of amyloid disease progression.

Mast cell activation test: A new asset in the investigation of the chlorhexidine cross-sensitization profile.

BACKGROUND: Insights into the IgE cross-sensitization and possible cross-reactivity patterns of sera reactive to chlorhexidine (CHX) are still incomplete and are likely to benefit from a functional exploration using a passive mast cell activation test (pMAT). Therefore, we want to study whether the pMAT with CHX-specific IgE (sIgE) enables to depict effector cell degranulation in response to alexidine (ALX), octenidine (OCT) and/or polyhexamethylene biguanide (PHMB) indicative of cross-reactivity between these compounds and CHX. METHODS: Serum of 10 CHX-allergic patients, nine individuals with an isolated sIgE CHX and five healthy controls were included. Human cultured mast cells (MCs) were, before and after sensitization, challenged with CHX, ALX, OCT or PHMB. Degranulation was measured via quantification of upregulation of CD63. RESULTS: Mast cell responsiveness to ALX and OCT was demonstrable with 4/10 and 3/10 of the sera of CHX-allergic patients respectively. Percentage of degranulation varied between 12 and 34% for ALX-reactive MCs and between 4 and 22% for OCT-reactive MCs. No reactivity to ALX or OCT was demonstrable when using sera obtained from individuals with an isolated sIgE CHX or from healthy controls. Unlike CHX, ALX and OCT, PHMB turned out to be a direct MC activator via occupation of MRGPRX2. PHMB-reactive sIgEs were demonstrable in some patients with an isolated sIgE CHX but were unable to trigger PHMB-induced degranulation in MRGPRX2 knockdown MCs. CONCLUSION: Mast cells constitute an attractive tool to explore cross-reactivity between structurally similar compounds. Along with the identification of safe alternatives for the individual patient, the pMAT can advance our insights into sIgE cross-reactivity patterns including assessment of molecules not yet approved for human use.