Journal of Molecular Biology
Volume 430, Issue 20, 12 October 2018, Pages 3751-3763
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Protein Engineering Reveals Mechanisms of Functional Amyloid Formation in Pseudomonas aeruginosa Biofilms

https://doi.org/10.1016/j.jmb.2018.06.043Get rights and content

Highlights

  • FapC protein from P. aeruginosa makes amyloid fibrils which strengthen biofilm.

  • We identify hexapeptide repeat sequences implicated in FapC amyloid formation.

  • Their removal slows aggregation and destabilizes the end-state fibrils.

  • They also increase sensitivity to EGCG and restrict the fibrillation pH range.

  • Removal of C-terminal Cys eliminates oligomers without affecting fibrillation.

Abstract

Amyloids are typically associated with neurodegenerative diseases, but recent research demonstrates that several bacteria utilize functional amyloid fibrils to fortify the biofilm extracellular matrix and thereby resist antibiotic treatments. In Pseudomonas aeruginosa, these fibrils are composed predominantly of FapC, a protein with high-sequence conservation among the genera. Previous studies established FapC as the major amyloid subunit, but its mechanism of fibril formation in P. aeruginosa remained largely unexplored. Here, we examine the FapC sequence in greater detail through a combination of bioinformatics and protein engineering, and we identify specific motifs that are implicated in amyloid formation. Sequence regions of high evolutionary conservation tend to coincide with regions of high amyloid propensity, and mutation of amyloidogenic motifs to a designed, non-amyloidogenic motif suppresses fibril formation in a pH-dependent manner. We establish the particular significance of the third repeat motif in promoting fibril formation and also demonstrate emergence of soluble oligomer species early in the aggregation pathway. The insights reported here expand our understanding of the mechanism of amyloid polymerization in P. aeruginosa, laying the foundation for development of new amyloid inhibitors to combat recalcitrant biofilm infections.

Introduction

Pseudomonas aeruginosa is one of the most prominent causes of health care-associated infections due to its unique arsenal of virulence factors, resistance to a range of antibiotics, and its ability to form biofilms [1], [2], [3]. Infections with P. aeruginosa often manifest in the form of ventilator-associated pneumonia, which demonstrates mortality rates as high as 30% in patients with comorbidities [4]. The plastic endotracheal tube readily provides a colonization site for P. aeruginosa, and aerosolization of the mature biofilm by mechanical ventilation or tracheal suctioning can lead to ventilator-associated pneumonia [5]. Mucoid, biofilm-associated P. aeruginosa, is also found in chronic lung infections of patients with cystic fibrosis [6]. When bacteria dwell in biofilms, cells co-associate using a self-produced extracellular matrix (EM). The EM comprises a protective network of polysaccharides, DNA, lipids, and proteins that encases the cells, mediates attachment, and promotes metabolite transport [7]. Proteins in the EM take on a variety of roles, but a growing body of research seeks to understand the role of amyloid fibrils in this complex biological material [8].

Amyloid fibrils are non-branching, rigid protein structures characterized by high β-sheet content and alignment of β-strands perpendicular to the fibril axis [9] (Fig. 1a). Insoluble amyloid deposits form as a result of protein folding dysfunction in diseases like Alzheimer's and Parkinson's, but recent evidence suggests that several microbes intentionally produce amyloid fibrils to serve functional roles [10]. Biofilm-associated bacteria, in particular, utilize amyloids as a building material to reinforce the EM and resist dispersion by chemical or mechanical agents [8], [11]. These fibrils may also promote antibiotic resistance and act as a reservoir for small molecules involved in quorum sensing [12], [13]. Amyloid fibrils polymerize in the absence of an energy source, so they serve as a metabolically advantageous molecular scaffold despite the limited resources of the EM environment [14].

In P. aeruginosa, the production of functional amyloids is controlled by a single six-gene operon known as (functional amyloid in Pseudomonas) fap[15]. The mature form of the major amyloid subunit, FapC, consists of 316-amino-acid residues without its 24-residue signal peptide, and it includes three imperfect sequence repeats (R1, R2, R3) separated by two “linker” regions (L1, L2) [16] (Fig. 1b, c). The three repeats are highly conserved among pseudomonads and related genera, with 56% ± 25% average residue conservation observed among 65 strains [17]. As such, the current model of FapC fibril formation designates the repeat regions as drivers of amyloid polymerization that ultimately constitute the core of the mature fibril. Analogous to the curli amyloids found in Escherichia coli [18], the fap system also includes a nucleator protein FapB, which demonstrates 38% sequence identity to FapC and is believed to serve as a template for rapid fibril polymerization on the exterior of the cell. Small amounts of FapB are also found in mature fibrils, so it may play an additional role in modulating properties of the fibrils [17]. The remaining proteins encoded by the fap operon serve as outer membrane pores for translocation of amyloid precursors (FapF) [19], chaperones to guide monomers through the periplasm (FapA), or auxiliary regulators and proteases (FapE, FapD) [16]. Wild-type P. aeruginosa PAO1 expresses fap constitutively, with peak promoter activity occurring during the exponential growth phase, but laboratory growth conditions limit the strain's ability to produce large quantities of functional amyloid. Conversely, overexpression of the fap operon in P. aeruginosa PAO1 leads to highly aggregative phenotypes with five to six times more biofilm than the wild-type strain, and similar effects are observed for overexpression of fap in E. coli [15], [16], [20].

Despite extensive characterization of Fap proteins under sessile growth conditions, their mechanisms of fibril formation remain largely unexplored. We studied the FapC sequence in greater detail through a combination of bioinformatics and protein engineering. Sequence analysis of the repeat regions predicts that a specific, conserved hexapeptide motif—GVNXAA—is responsible for a significant amyloidogenic effect in each of the three repeats. Mutation of the amyloidogenic motif to a highly soluble, non-amyloidogenic hexapeptide changes aggregation kinetics compared to wild-type FapC in a direction consistent with our predictions. These effects are pH dependent, and we demonstrate the particular significance of the third sequence repeat, R3, in promoting fibril formation. Finally, we highlight a minor role for the FapC disulfide bond in forming small, pre-amyloid oligomers. The insights reported here reveal important mechanistic details of FapC polymerization, paving the way for new strategies to inhibit functional amyloid formation and ultimately provide new therapeutic avenues for biofilm-associated infections.

Section snippets

Amyloid-prone segments of FapC coincide with conserved regions

The rapid polymerization of FapC in vitro precludes its structural characterization by traditional means, such as NMR or X-ray crystallography, but a combination of evolutionary sequence analysis and bioinformatics tools helped us to identify potential “hot spots” for aggregation. The three repeat regions of the FapC sequence are highly conserved among pseudomonads, and therefore, we hypothesized that these regions are critical for the macromolecular assembly of amyloid fibrils. A variety of

Discussion

Through detailed sequence examination, we identified specific motifs that are implicated in amyloid formation of the P. aeruginosa FapC protein. Our analysis revealed sequence regions with high levels of both evolutionary conservation and amyloid propensity, which facilitated biophysical study of these regions with protein engineering. Mutation of highly conserved segments to corresponding segments with much lower aggregation propensity confirmed the role of evolution in maintaining FapC

Sequence analysis

The FASTA sequence for PAO1 FapC (without the 24-residue signal peptide) was imported from the UniProtKB database. The sequence was then analyzed for amyloid “hot spots” in ZipperDB (http://services.mbi.ucla.edu/zipperdb/) with threshold − 23 kcal/mol and FISH Amyloid (http://www.comprec.pwr.wroc.pl/fish/) with threshold 0.19. Predicted hot spots were compared to conserved regions among 31 FapC homologs using sequence alignment in Clustal Omega [25].

Peptide microarrays

Printed peptide libraries were designed to

Acknowledgments

We extend thanks to Dr. Morten Simonsen Dueholm (Aalborg University) and Line F. B. Christiansen (Aarhus University) for helpful discussions, and to Ivan Vulovic and Shijie Cao (University of Washington) for assistance with equipment. This research was supported by the Danish Council for Independent Research (6111-00241B to D.E.O. and 7093-00001B to D.E.O. and A.B.) and the National Science Foundation (GROW Fellowship, ID 2015176941 to A.B.).

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    Present address: Rudolf Virchow Center for Experimental Biomedicine and Biocenter, Department of Biotechnology and Biophysics, University of Wuerzburg, 97,080 Wuerzburg, Germany.

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