FapA is an Intrinsically Disordered Chaperone for Pseudomonas Functional Amyloid FapC

Rasmussen, H.H.; Kumar, Amit; Shin, Ben; Stylianou, Fisentzos; Sewell, Lee; Xu, Yingqi; Otzen, Daniel E.; Pedersen, Jan Skov; Matthews, Steve

doi:10.1016/j.jmb.2022.167878

Cited by 14 publications

(30 citation statements)

References 57 publications

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“…56 FapA is an IDP similar to FapC based on the 2D HSQC fingerprint spectrum from the unlabeled FapA by our work here, Figure SI3, as well as recent work by others. 17 Recently it was shown that FapA dramatically affects FapC fibrilization monitored by ThT assay, but speculated that this effect was through an "NMR invisible oligomeric species" which did not affect the NMR spectra of FapC. Even though it is not straightforward to think of an IDP acting as a chaperone, the data we present here clearly shows that FapA acts as a chaperone for monomeric FapC in vitro though specific interactions during functional amyloid formation and hence suppresses FapC fibrillation.…”

Section: Introductionmentioning

confidence: 55%

“…Examples from both pathologic and functional amyloid perspectives cover a wide range of chaperone effect on amyloid fibrils, ranging from slowing down to the prevention or even to their reversal. 17,35,37,[46][47][48] CsgE was shown to slow down the fibril formation of CsgA functional amyloid. Similarly, pathologic amyloid fibril formation of aSynuclein was slowed down by HSP90 family chaperones.…”

Section: Introductionmentioning

confidence: 97%

“…6,14 These unstructured monomers common to almost all of the functional and pathologic amyloids can be studied by only a few techniques at atomic resolution, with high-resolution solution state NMR (solution NMR) spectroscopy being a powerful choice. [15][16][17] For small fragments of amyloid proteins that can be crystallized, X-Ray crystallography established the first atomic models of different systems, 3,18,19 however, longer fragments of amyloids are not crystalline. For the full-length insoluble filamentous amyloid fibrils, solid-state magicangle spinning (MAS) NMR (ssNMR) had been the only available technique to study these non-crystalline insoluble proteins until the resolution revolution by cryo electron microscopy (Cryo-EM) in the last decade.…”

Section: Introductionmentioning

confidence: 99%

“…Compared to the HSP family or other folded chaperones, FapA belongs to a unique class of proteins that are intrinsically unfolded, but still can act as a chaperone. 17 Understanding details of the molecular mechanism of FapA chaperone is crucial to develop strategies to prevent amyloid formation in biofilm context and to prevent related chronic infections.…”

Section: Introductionmentioning

confidence: 99%

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Identifying interaction interface betweenPseudomonasmajor biofilm forming functional amyloid FapC and disordered chaperone FapA during fibrillation deceleration

Byeon

Hansen

Jeffrey

et al. 2023

Preprint

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Functional bacterial amyloids (FuBA) are involved in biofilm formation in Pseudomonas aeruginosa. Biofilm forming bacterial colonies are difficult to treat, cause most of the chronic bacterial infections and are relevant in the context of antimicrobial resistance. Here in this work by focusing on the major biofilm forming FapC protein we demonstrate the initial steps of the chaperone aided amyloid formation. Solution NMR spectroscopy gave atomic resolution information on the prefibrillar soluble monomeric FapC that is an intrinsically disordered protein (IPD). Moreover, we show that the chaperone FapA within the same biofilm forming protein operon interacts with FapC. FapA is itself an IDP and remarkably slows down the FapC fibril formation without modifying the final fibril morphology. Monitoring of the small but significant chemical shift perturbations allowed the determination of the interaction interface which was not observed previously. To the best of our knowledge, our work provides the first atomic-level identification of a rather specific interaction of an IDP chaperone, FapA, to modulate and slow-down the functional amyloid protein FapC towards its path to mature fibril formation. Our results establish a better understanding of the functional amyloid formation in bacterial biofilms and its molecular level tuning or prevention by chaperones. We foresee that future therapeutic treatments of biofilms and relevant infections could emerged from these structural and mechanistic insights.

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Section: Introductionmentioning

confidence: 55%

Section: Introductionmentioning

confidence: 97%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 2 more Smart Citations

Identifying interaction interface betweenPseudomonasmajor biofilm forming functional amyloid FapC and disordered chaperone FapA during fibrillation deceleration

Byeon

Hansen

Jeffrey

et al. 2023

Preprint

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“…It came to our attention at the time of our submission that another preprint shows the solution-state NMR signal assignment of FL FapC with different sample conditions with additives such as a chaperone. 11 No BMRB data was available at the time of our submission, and we present results solely based on our own manual NMR signal assignment work performed last year and the data is deposited to the BMRB database.…”

mentioning

confidence: 99%

Solution-state NMR Assignment and Secondary Structural Propensities of the Full-Length and Minimalistic-Truncated Prefibrillar Monomeric Form of Biofilm-Forming Functional-Amyloid FapC fromPseudomonas aeruginosa

Akbey

Byeon

et al. 2023

Preprint

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Functional bacterial amyloids provide structural scaffolding to bacterial biofilms. In contrast to the pathological amyloids, they have a role in vivo and are tightly regulated. Their presence is essential to the integrity of the bacterial communities surviving in biofilms and may cause serious health complications. Targeting amyloids in biofilms could be a novel approach to prevent chronic infections. However, structural information is very scarce on them in both soluble monomeric and insoluble fibrillar forms, hindering our molecular understanding and strategies to fight biofilm related diseases. Here, we present solution-state NMR assignment of 250 amino acid long biofilm-forming functional-amyloid FapC from Pseudomonas aeruginosa. We studied the full-length and shorter minimalistic-truncated FapC constructs without signal-sequence that is required for secretion. 91% and 100% backbone NH resonance assignment for FL and short constructs, respectively, indicates that soluble monomeric FapC is predominantly disordered, with sizeable secondary structural propensities mostly as PP2 helices, but also as alpha-helices and beta-sheets highlighting hotspots for fibrillation initiation interface. Shorter construct showing almost identical NMR chemical shifts highlights the promise of utilizing it for more demanding solid-state NMR studies that requires methods to alleviate signal redundancy due to almost identical repeat units. This study provides key NMR resonance assignment for future structural studies of soluble, pre-fibrillar and fibrillar forms of FapC.

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In vitro and in silico evaluation of the serrapeptase effect on biofilm and amyloids of Pseudomonas aeruginosa

Katsipis,

Avgoulas,

Geromichalos

et al. 2023

Appl Microbiol Biotechnol

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Pseudomonas aeruginosa is an emerging threat for hospitalized and cystic fibrosis patients. Biofilm, a microbial community embedded in extracellular polymeric substance, fortifies bacteria against the immune system. In biofilms, the expression of functional amyloids is linked with highly aggregative, multi-resistant strains, and chronic infections. Serrapeptase (SPT), a protease possessing similar or superior anti-microbial properties with many antibiotics, presents anti-amyloid potential. However, studies on the employment of SPT against Pseudomonas biofilms and Fap amyloid, or the possible mechanisms of action are scarce. Here, SPT inhibited biofilm formation of P. aeruginosa ATCC 27853 on both plastic and glass surfaces, with an IC50 of 11.26 µg/mL and 0.27 µg/mL, respectively. The inhibitory effect of SPT on biofilm was also verified with optical microscopy of crystal violet-stained biofilms and with confocal microscopy. Additionally, SPT caused a dose-dependent decrease of bacterial viability (IC50 of 3.07 µg/mL) as demonstrated by MTT assay. Reduction of bacterial functional amyloids was also demonstrated, employing both fluorescence microscopy with thioflavin T and photometrical determination of Congo-red-positive compounds. Both viability and functional amyloids correlated significantly with biofilm inhibition. Finally, in silico molecular docking studies provided a mechanistic insight into the interaction of SPT with FapC or FapD, proving that both peptides are possible targets of SPT. These results offer new insights into the biofilm formation of P. aeruginosa and potentiate the involvement of SPT in the prevention and eradication of Pseudomonas biofilms. Graphical abstract Key points • Serrapeptase inhibits biofilm formation of P. aeruginosa on plastic and glass. • Biofilm inhibition correlated with reduced viability and functional amyloid levels. • In silico studies indicated that serrapeptase may target FapC and FapD peptides.

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FapA is an Intrinsically Disordered Chaperone for Pseudomonas Functional Amyloid FapC

Cited by 14 publications

References 57 publications

Identifying interaction interface betweenPseudomonasmajor biofilm forming functional amyloid FapC and disordered chaperone FapA during fibrillation deceleration

Identifying interaction interface betweenPseudomonasmajor biofilm forming functional amyloid FapC and disordered chaperone FapA during fibrillation deceleration

Solution-state NMR Assignment and Secondary Structural Propensities of the Full-Length and Minimalistic-Truncated Prefibrillar Monomeric Form of Biofilm-Forming Functional-Amyloid FapC fromPseudomonas aeruginosa

In vitro and in silico evaluation of the serrapeptase effect on biofilm and amyloids of Pseudomonas aeruginosa

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