Fusobacterium nucleatum secretes amyloid‐like FadA to enhance pathogenicity
2021; Springer Nature; Volume: 22; Issue: 7 Linguagem: Inglês
10.15252/embr.202152891
ISSN1469-3178
AutoresQing H. Meng, Qiuqiang Gao, Shebli Mehrazarin, Kamonchanok Tangwanichgapong, Yu Wang, Yiming Huang, Yutong Pan, Samuel T. Robinson, Ziwen Liu, Amirali Zangiabadi, Renate Lux, Panos N. Papapanou, Xiao Guo, Harris H. Wang, Luke E. Berchowitz, Yiping W. Han,
Tópico(s)Oral microbiology and periodontitis research
ResumoArticle29 June 2021free access Source DataTransparent process Fusobacterium nucleatum secretes amyloid-like FadA to enhance pathogenicity Qing Meng Qing Meng Section of Oral, Diagnostic and Rehabilitation Sciences, Division of Periodontics, College of Dental Medicine, Columbia University Irving Medical Center, New York, NY, USAThese authors contributed equally to this work as first authors Search for more papers by this author Qiuqiang Gao Qiuqiang Gao Section of Oral, Diagnostic and Rehabilitation Sciences, Division of Periodontics, College of Dental Medicine, Columbia University Irving Medical Center, New York, NY, USAThese authors contributed equally to this work as first authors Search for more papers by this author Shebli Mehrazarin Shebli Mehrazarin Section of Oral, Diagnostic and Rehabilitation Sciences, Division of Periodontics, College of Dental Medicine, Columbia University Irving Medical Center, New York, NY, USAThese authors contributed equally to this work as second authors Search for more papers by this author Kamonchanok Tangwanichgapong Kamonchanok Tangwanichgapong orcid.org/0000-0001-6425-3312 Section of Oral, Diagnostic and Rehabilitation Sciences, Division of Periodontics, College of Dental Medicine, Columbia University Irving Medical Center, New York, NY, USAThese authors contributed equally to this work as second authors Search for more papers by this author Yu Wang Yu Wang Section of Oral, Diagnostic and Rehabilitation Sciences, Division of Periodontics, College of Dental Medicine, Columbia University Irving Medical Center, New York, NY, USAThese authors contributed equally to this work as second authors Search for more papers by this author Yiming Huang Yiming Huang Department of Systems Biology, Vagelos College of physicians and Surgeons, Columbia University Irving Medical Center, New York, NY, USA Search for more papers by this author Yutong Pan Yutong Pan Section of Oral, Diagnostic and Rehabilitation Sciences, Division of Periodontics, College of Dental Medicine, Columbia University Irving Medical Center, New York, NY, USA Search for more papers by this author Samuel Robinson Samuel Robinson Department of Biomedical Engineering, Fu Foundation School of Engineering and Applied Sciences, Columbia University, New York, NY, USA Search for more papers by this author Ziwen Liu Ziwen Liu Section of Oral, Diagnostic and Rehabilitation Sciences, Division of Periodontics, College of Dental Medicine, Columbia University Irving Medical Center, New York, NY, USA Search for more papers by this author Amirali Zangiabadi Amirali Zangiabadi Electron Microscopy Labs, Columbia Nano Initiative, Columbia University, New York, NY, USA Search for more papers by this author Renate Lux Renate Lux Department of Oral Biology, UCLA School of Dentistry, Los Angeles, CA, USA Search for more papers by this author Panos N Papapanou Panos N Papapanou Section of Oral, Diagnostic and Rehabilitation Sciences, Division of Periodontics, College of Dental Medicine, Columbia University Irving Medical Center, New York, NY, USA Search for more papers by this author X Edward Guo X Edward Guo Department of Biomedical Engineering, Fu Foundation School of Engineering and Applied Sciences, Columbia University, New York, NY, USA Search for more papers by this author Harris Wang Harris Wang Department of Systems Biology, Vagelos College of physicians and Surgeons, Columbia University Irving Medical Center, New York, NY, USA Search for more papers by this author Luke E Berchowitz Corresponding Author Luke E Berchowitz [email protected] orcid.org/0000-0002-3388-7723 Department of Genetics and Development, Vagelos College of Physicians and Surgeons, Columbia University Irving Medical Center, New York, NY, USA Taub Institute for Research on Alzheimer's and the Aging Brain, New York, NY, USA Search for more papers by this author Yiping W Han Corresponding Author Yiping W Han [email protected] orcid.org/0000-0002-8444-499X Section of Oral, Diagnostic and Rehabilitation Sciences, Division of Periodontics, College of Dental Medicine, Columbia University Irving Medical Center, New York, NY, USA Department of Microbiology and Immunology, Vagelos College of Physicians and Surgeons, Columbia University Irving Medical Center, New York, NY, USA Herbert Irving Comprehensive Cancer Center, Columbia University Irving Medical Center, New York, NY, USA Search for more papers by this author Qing Meng Qing Meng Section of Oral, Diagnostic and Rehabilitation Sciences, Division of Periodontics, College of Dental Medicine, Columbia University Irving Medical Center, New York, NY, USAThese authors contributed equally to this work as first authors Search for more papers by this author Qiuqiang Gao Qiuqiang Gao Section of Oral, Diagnostic and Rehabilitation Sciences, Division of Periodontics, College of Dental Medicine, Columbia University Irving Medical Center, New York, NY, USAThese authors contributed equally to this work as first authors Search for more papers by this author Shebli Mehrazarin Shebli Mehrazarin Section of Oral, Diagnostic and Rehabilitation Sciences, Division of Periodontics, College of Dental Medicine, Columbia University Irving Medical Center, New York, NY, USAThese authors contributed equally to this work as second authors Search for more papers by this author Kamonchanok Tangwanichgapong Kamonchanok Tangwanichgapong orcid.org/0000-0001-6425-3312 Section of Oral, Diagnostic and Rehabilitation Sciences, Division of Periodontics, College of Dental Medicine, Columbia University Irving Medical Center, New York, NY, USAThese authors contributed equally to this work as second authors Search for more papers by this author Yu Wang Yu Wang Section of Oral, Diagnostic and Rehabilitation Sciences, Division of Periodontics, College of Dental Medicine, Columbia University Irving Medical Center, New York, NY, USAThese authors contributed equally to this work as second authors Search for more papers by this author Yiming Huang Yiming Huang Department of Systems Biology, Vagelos College of physicians and Surgeons, Columbia University Irving Medical Center, New York, NY, USA Search for more papers by this author Yutong Pan Yutong Pan Section of Oral, Diagnostic and Rehabilitation Sciences, Division of Periodontics, College of Dental Medicine, Columbia University Irving Medical Center, New York, NY, USA Search for more papers by this author Samuel Robinson Samuel Robinson Department of Biomedical Engineering, Fu Foundation School of Engineering and Applied Sciences, Columbia University, New York, NY, USA Search for more papers by this author Ziwen Liu Ziwen Liu Section of Oral, Diagnostic and Rehabilitation Sciences, Division of Periodontics, College of Dental Medicine, Columbia University Irving Medical Center, New York, NY, USA Search for more papers by this author Amirali Zangiabadi Amirali Zangiabadi Electron Microscopy Labs, Columbia Nano Initiative, Columbia University, New York, NY, USA Search for more papers by this author Renate Lux Renate Lux Department of Oral Biology, UCLA School of Dentistry, Los Angeles, CA, USA Search for more papers by this author Panos N Papapanou Panos N Papapanou Section of Oral, Diagnostic and Rehabilitation Sciences, Division of Periodontics, College of Dental Medicine, Columbia University Irving Medical Center, New York, NY, USA Search for more papers by this author X Edward Guo X Edward Guo Department of Biomedical Engineering, Fu Foundation School of Engineering and Applied Sciences, Columbia University, New York, NY, USA Search for more papers by this author Harris Wang Harris Wang Department of Systems Biology, Vagelos College of physicians and Surgeons, Columbia University Irving Medical Center, New York, NY, USA Search for more papers by this author Luke E Berchowitz Corresponding Author Luke E Berchowitz [email protected] orcid.org/0000-0002-3388-7723 Department of Genetics and Development, Vagelos College of Physicians and Surgeons, Columbia University Irving Medical Center, New York, NY, USA Taub Institute for Research on Alzheimer's and the Aging Brain, New York, NY, USA Search for more papers by this author Yiping W Han Corresponding Author Yiping W Han [email protected] orcid.org/0000-0002-8444-499X Section of Oral, Diagnostic and Rehabilitation Sciences, Division of Periodontics, College of Dental Medicine, Columbia University Irving Medical Center, New York, NY, USA Department of Microbiology and Immunology, Vagelos College of Physicians and Surgeons, Columbia University Irving Medical Center, New York, NY, USA Herbert Irving Comprehensive Cancer Center, Columbia University Irving Medical Center, New York, NY, USA Search for more papers by this author Author Information Qing Meng1, Qiuqiang Gao1, Shebli Mehrazarin1, Kamonchanok Tangwanichgapong1,10, Yu Wang1,11, Yiming Huang2, Yutong Pan1, Samuel Robinson3, Ziwen Liu1, Amirali Zangiabadi4, Renate Lux5, Panos N Papapanou1, X Edward Guo3, Harris Wang2, Luke E Berchowitz *,6,7 and Yiping W Han *,1,8,9 1Section of Oral, Diagnostic and Rehabilitation Sciences, Division of Periodontics, College of Dental Medicine, Columbia University Irving Medical Center, New York, NY, USA 2Department of Systems Biology, Vagelos College of physicians and Surgeons, Columbia University Irving Medical Center, New York, NY, USA 3Department of Biomedical Engineering, Fu Foundation School of Engineering and Applied Sciences, Columbia University, New York, NY, USA 4Electron Microscopy Labs, Columbia Nano Initiative, Columbia University, New York, NY, USA 5Department of Oral Biology, UCLA School of Dentistry, Los Angeles, CA, USA 6Department of Genetics and Development, Vagelos College of Physicians and Surgeons, Columbia University Irving Medical Center, New York, NY, USA 7Taub Institute for Research on Alzheimer's and the Aging Brain, New York, NY, USA 8Department of Microbiology and Immunology, Vagelos College of Physicians and Surgeons, Columbia University Irving Medical Center, New York, NY, USA 9Herbert Irving Comprehensive Cancer Center, Columbia University Irving Medical Center, New York, NY, USA 10Present address: Division of Periodontology, Department of Biomedical Sciences, Faculty of Dentistry, Khon Kaen University, Khon Kaen, Thailand 11Present address: Department of Periodontics, School of Dental Medicine, University of Pennsylvania, Philadelphia, PA, USA **Corresponding author: Tel: +1 212 305 7003; E-mail: [email protected] ***Corresponding author: Tel: +1 212 342 1790; E-mail: [email protected] EMBO Reports (2021)22:e52891https://doi.org/10.15252/embr.202152891 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions Figures & Info Abstract Fusobacterium nucleatum (Fn) is a Gram-negative oral commensal, prevalent in various human diseases. It is unknown how this common commensal converts to a rampant pathogen. We report that Fn secretes an adhesin (FadA) with amyloid properties via a Fap2-like autotransporter to enhance its virulence. The extracellular FadA binds Congo Red, Thioflavin-T, and antibodies raised against human amyloid β42. Fn produces amyloid-like FadA under stress and disease conditions, but not in healthy sites or tissues. It functions as a scaffold for biofilm formation, confers acid tolerance, and mediates Fn binding to host cells. Furthermore, amyloid-like FadA induces periodontal bone loss and promotes CRC progression in mice, with virulence attenuated by amyloid-binding compounds. The uncleaved signal peptide of FadA is required for the formation and stability of mature amyloid FadA fibrils. We propose a model in which hydrophobic signal peptides serve as “hooks” to crosslink neighboring FadA filaments to form a stable amyloid-like structure. Our study provides a potential mechanistic link between periodontal disease and CRC and suggests anti-amyloid therapies as possible interventions for Fn-mediated disease processes. Synopsis Fusobacterium nucleatum, an oral commensal anaerobe, secretes amyloid-like FadA to enhance its pathogenicity. Intact pre-FadA is a critical component of amyloid-like FadA, which is secreted via the autotransporter Fap2. Fusobacterium nucleatum (Fn) secretes amyloid-like FadA, promoting periodontal bone loss and colorectal cancer. Amyloid-binding compounds disrupt Fn pathogenicity in a FadA-dependent manner. Amyloid-like FadA facilitates Fn biofilm formation, acid tolerance and binding to host cells. Introduction Fusobacterium nucleatum (Fn) is a filamentous Gram-negative anaerobe ubiquitous in the oral cavity. As an opportunistic commensal, it is the most predominant core component in the subgingival microbiome in both health and disease. Outside the oral cavity, Fn is absent or infrequently detected under healthy conditions (Segata et al, 2012). Under disease conditions, however, Fn is one of the most prevalent species involved in organ abscesses, atherosclerosis, pregnancy complications, rheumatoid arthritis, respiratory tract infections, and GI disorders, e.g., appendicitis, inflammatory bowel disease, esophageal, gastric, pancreatic, and colorectal cancers (CRC) (Han et al, 2009; Ortiz et al, 2009; Han et al, 2010; Strauss et al, 2011; Swidsinski et al, 2011; Castellarin et al, 2012; Kostic et al, 2012; Wang et al, 2013; Mitsuhashi et al, 2015; Hsieh et al, 2018; Liu et al, 2019; Thomas et al, 2019). It is not known how Fn acts both as a common commensal and a rampant pathogen. Understanding the molecular signal and mechanism is critical for controlling Fn pathogenesis. Several lines of evidence implicate that the FadA adhesin (for Fusobacterium adhesin A) plays a critical role in the pathogenicity of Fn. FadA is conserved among Fn, Fusobacterium periodonticum and Fusobacterium necrophorum, but absent in most other Fusobacteria species (Han et al, 2005; Umana et al, 2019). In human colonic tissues, the fadA gene levels increase stepwise from normal to adenoma and from adenoma to carcinoma (Rubinstein et al, 2013). Meta-analysis of diverse populations showed that fadA is also consistently enriched in the fecal microbiome of CRC patients (Wirbel et al, 2019). The pathogenesis mechanisms of FadA in CRC and pregnancy complications have been reported previously (Ikegami et al, 2009; Rubinstein et al, 2013; Rubinstein et al, 2019). FadA mediates Fn binding and invasion of epithelial and endothelial cells and colonization in the murine placenta (Han et al, 2005; Xu et al, 2007; Ikegami et al, 2009; Fardini et al, 2011). It binds VE-cadherin on endothelial cells, loosening of the cell–cell junctions enabling systemic bacterial dissemination (Ikegami et al, 2009; Fardini et al, 2011). It also enables Fn to preferentially bind CRC cells expressing Annexin A1, a β-catenin modulator required for CRC cell growth. Upon binding to CRC cells, FadA further elevates Annexin A1 expression. This positive feedback loop between FadA and Annexin A1 exacerbates CRC progression (Rubinstein et al, 2019). While the implication of FadA in various pathologies is well documented, how this virulence factor functions in health and disease is not known. In this study, we report that FadA undergoes dramatic biochemical changes to become “amyloid-like” to enhance Fn virulence. Amyloids are fibrous protein aggregates that are implicated in numerous human diseases such as Alzheimer’s, Parkinson’s, and prion diseases. A related, but biochemically distinct, group of protein aggregates is known as ‘amyloid-like’. This class shares a subset of biochemical properties of the disease-related amyloids including fiber formation, stable cross-beta sheets, and/or resistance to ionic detergents (Berchowitz et al, 2015; Boke et al, 2016). FadA readily forms fibers in vitro with a diameter similar to well-studied amyloids (Nithianantham et al, 2009). These preliminary data led us to speculate that FadA forms functional amyloid-like assemblies and that the formation of these structures is critical for Fn pathogenesis. Results Fn produces amyloid-like FadA regulated by growth phase A distinctive property of amyloid and amyloid-like assemblies is their ability to bind amyloidophilic compounds such as Congo Red and Thioflavin-T (Tukel et al, 2009; Evans et al, 2018). To determine whether Fn expresses amyloid-like FadA, we assessed whether Fn and its mutants bind Congo Red. Wild-type Fn 12230 clearly bound Congo Red, depleting it from the solution in a density-dependent manner, while the fadA-deletion mutant US1 (ΔfadA) was defective (Fig 1A). In order to compare with the well-characterized bacterial amyloid-like adhesin curli (Evans et al, 2018), wild-type curli-producing E. coli and curli mutants were tested in the same assay (Fig 1A). The similarities observed between Fn and E. coli support that FadA is an amyloid-like adhesin. Figure 1. Fn produces amyloid-like FadA in stationary phase Congo Red depletion assay. Fn 12230 (black), the fadA-deletion mutant US1 (ΔfadA) (orange), and the spontaneous lam mutant (blue) (top panel) were grown to OD600 > 0.8. E. coli MC4100, Δcurli, and ΔcsgA (bottom panel) were grown on TSA-blood agar plate at 26°C for 48 h. The bacteria were suspended in PBS to OD600 of 0.5, 1.0, 1.5, and 2.0, followed by incubation in 10 μg/ml Congo Red (CR) for 10 min. After centrifugation, the supernatants were measured at OD500. The results shown are the average of five independent experiments, each performed in duplicate. The error bars indicate SD. *P < 0.05, **P < 0.01, ***P < 0.001 (compared to wild type, t-test). Kinetics of FadA production in Fn. Fn 12230 and US1 were inoculated to OD600 = 0.1. 10 OD600 units of bacteria were harvested by centrifugation at 12-h intervals. Bacteria were lysed with 2 mg/ml lysozyme, followed by incubation in 1% sarkosyl at 4°C for 20 min, which became the ‘total’ sample. Lysates were then centrifuged at 100,000 g for 20 min, and the supernatants and pellets were collected. An aliquot of 5 μl is loaded onto each lane, followed by immunoblot using anti-FadA monoclonal antibody (mAb) 7H7 at 1:4,000 dilution to detect FadA protein in total, supernatant, and pellet fractions. Ponceau staining of lysozyme is shown as a loading control. Pre-FadA and mFadA are pointed by arrows. Note pre-FadA migrates faster than mFadA on SDS–PAGE as previously reported (Xu et al, 2007). Analysis of detergent-resistant FadA polymers in wild-type Fn and its mutants by semi-denaturing detergent agarose gel electrophoresis (SDD–AGE) and Western blot analysis using mAb 7H7. Fn12230 (wt), fadA-deletion mutant US1 (ΔfadA) and spontaneous mutant lam (lam) were grown to log (OD600 0.3–0.4) or stationary phase (OD600 > 0.8), E. coli MC4100, Δcurli, and ΔcsgA were grown to stationary phase. The bacteria were harvested by centrifugation. Following sequential incubation in lysis buffer containing 2 mg/ml lysozyme and 1% sarkosyl, the insoluble pellets were obtained by centrifugation. An aliquot of 100 μg of each pellet was loaded onto 1.7% agarose gel followed by overnight electrophoresis in 0.5xTAE and 0.1% SDS. Following transfer to nitrocellulose membrane, Western blot was performed using anti-FadA mAb 7H7 at 1:4,000 dilution or anti-CsgA antibody at 1:15,000 dilution. The large heterogeneous detergent-resistant FadA polymers were detected in wild-type Fn in the stationary phase, defective in US1 and lam, absent in log phase. Immunohistochemical analysis of FadA in log and stationary phases. Fn12230 (Fn), fadA-deletion mutant US1 (ΔfadA), and spontaneous mutant lam in log and stationary phase were fixed and incubated with mAb 7H7 at 1:800 dilution, followed by incubation of HRP-conjugated anti-mouse IgG and developed by DAB. The large FadA aggregates were detected specifically in wild-type Fn 12230 in the stationary but not in log phase. FadA was detected on lam, which was defective in secreted aggregates. No FadA was detected in US1. The images were taken using a 40× objective. Scale bar equals 20 μm. Double immunofluorescent staining of Fn12230, fadA-deletion mutant US1 (ΔfadA) and spontaneous mutant lam in log and stationary phases using mAb 7H7 at 1:800 dilution and polyclonal anti-human β-amyloid antibody A11 at 1:500 dilution, followed by incubation with Alexa Fluor 680-conjugated donkey anti-rabbit and Alexa Fluor 555-conjugated goat anti-mouse, both at 1:1,000 dilution. Co-staining of FadA and A11 was observed in Fn in stationary phase, not in log phase, or in US1 or lam. The images were taken using a 60X objective. Scale bar equals 5 μm. Scanning electron microscopy of Fn 12230, US1 (ΔfadA) and lam in log and stationary phase at 10,000× magnification. Note the fibrous structure coating Fn in stationary phase, pointed by the clear arrows, but not in log phase. Scale bar equals 1 μm. Source data are available online for this figure. Source Data for Figure 1 [embr202152891-sup-0007-SDataFig1.pdf] Download figure Download PowerPoint Because Congo Red was depleted in a bacterial density-dependent manner, we conducted a kinetic analysis to examine production of amyloid-like FadA over the course of growth. Fn cultures were harvested and lysed at increasing time points following subculture and incubated with 1% sarkosyl. Resistance to ionic detergent is a characteristic of amyloid-like proteins (Sondheimer & Lindquist, 2000). Sarkosyl-resistant (i.e., amyloid-like) FadA (pellet) increased over time, and by 48 h (late stationary phase), FadA was only detected in the pellet but not in supernatant, indicating the existence of FadA as predominantly insoluble aggregates (Fig 1B). [Note that on SDS–PAGE, pre-FadA migrates faster than mFadA (Xu et al, 2007).] This result indicates that although FadA is constitutively expressed in Fn, its molecular characteristics change during growth. To examine the production of amyloid-like FadA aggregates, we employed semi-denaturing detergent agarose gel electrophoresis (SDD–AGE), followed by Western blot analysis using anti-FadA monoclonal antibody (mAb) 7H7. Amyloid-like (as opposed to amorphous or globular) assemblies are SDS-resistant, and SDD-AGE allows for the resolution of these structures based on their size and resistance to the ionic detergent SDS. Fn produced massive and heterogeneously-sized amyloid-like FadA assemblies in stationary phase, similar as the curli-producing E. coli, but not in log phase (Fig 1C). This observation suggests FadA assembly could be regulated by nutrient depletion, a feature observed with other amyloid-like proteins (Berchowitz et al, 2015). Image analyses were then conducted to visually examine the FadA aggregates. Increased secretion of FadA aggregates by wild-type Fn was detected by immunohistochemistry (IHC) in stationary phase compared to log phase (Fig 1D). To further examine the amyloid-like properties of extracellular FadA aggregates, double-immunofluorescent staining was performed using mAb 7H7 and polyclonal anti-human β-amyloid antibody A11, which specifically recognizes amyloid oligomers independent of amino-acid (aa) sequence. Co-staining between A11 and FadA was observed in stationary phase but not in log phase of wild-type Fn (Fig 1E). Scanning electron microscopy (SEM) confirmed the existence of extracellular “plaque-like” fibrils coating wild-type Fn in stationary phase, but not in log phase, which were significantly reduced in US1 (ΔfadA) (Fig 1F). Together, these data confirm that Fn produces amyloid-like FadA as the bacterium enters stationary phase, possibly in response to nutrient deprivation. Pre-FadA is a key component of amyloid-like FadA secreted by Fap2-like Type V autotransporter FadA is an unusual adhesin in that it exists in two forms: a full-length peptide consisting of 129 aa residues (15.5 kDa), termed pre-FadA, and a cleaved “mature” form consisting of 111 aa residues (13.6 kDa) without the signal peptide, termed mFadA (Fig 2A). Crystallographic analysis of mFadA reveals a filamentous structure, with predominantly α-helical monomers linked in a head-to-tail pattern through a “leucine chain” motif (Nithianantham et al, 2009). The mFadA alone exhibits little virulence, and pre-FadA by itself is insoluble under neutral pH. Together, these two forms constitute a heterogeneous complex termed FadAc (Xu et al, 2007). Previous studies have demonstrated that recombinant FadAc, but not mFadA, is the active form for binding and invading host cells and stimulating CRC growth (Xu et al, 2007; Fardini et al, 2011; Rubinstein et al, 2013; Rubinstein et al, 2019). Pre-FadA plays a critical role in the size, heterogeneity and function of FadAc. With increasing pre-FadA, the multimeric complex becomes more heterogeneous, and the complex size and cell-binding activities also increase (Xu et al, 2007; Temoin et al, 2012a). Figure 2. Pre-FadA is a key component of amyloid-like FadA Single-letter amino-acid (aa) sequence of FadA. The intact pre-FadA consists of 129 aa, with the first 18-aa (top row) constitute the signal peptide. The remaining 111 aa constitute mFadA. The residues that are replaced in the mutant proteins used in this study are shown in red. Analysis of detergent-resistant recombinant FadA polymers expressed in E. coli. An aliquot of 50 μg detergent-resistant pellets prepared from E. coli BL21(DE3) carrying the cloning vector pET21(b), pYWH417-6 (p_FadAc), or pYWH418 (p_mFadA) were analyzed by SDD-AGE, followed by Western blot analysis. FadA polymers were only detected in E. coli expressing FadAc, but not in mFadA or the vector control. Reactivity of recombinant FadA and its variants with anti-amyloid fibril antibody OC. A total of 10 μg each of recombinant protein mFadA, FadAc, FadA-L-9A, FadA-L14A, FadA-S71A, and FadA-L76A purified from E. coli was slot-blotted onto nitrocellulose membrane and incubated with polyclonal OC antibody at 1:5,000 dilution. Amyloid-like recombinant Rim4 (Berchowitz et al, 2015) was used as a positive control and histone H1 was used as a negative control. Ponceau stain (pink color) is shown as a loading control. Thioflavin-T binding assay. FadA proteins at indicated concentrations were incubated with 10 μM Thioflavin-T at room temperature for 10 min. The fluorescent intensity was measured at excitation wavelength of 440 nm and emission wavelength of 500 nm. The experiment was performed in duplicate and repeated three times. The error bars indicate SD. *P < 0.05, **P < 0.01, ***P < 0.001 (compared to BSA, t-test). Thioflavin-T binding assay in the presence of 0.1% SDS. FadA proteins (0.8 mg/ml) were incubated with 10 μM Thioflavin-T as described above. The experiment was performed in duplicate and repeated three times. The error bars indicate SD. *P < 0.05, **P < 0.01 (compared to BSA, t-test). Western blot analysis of Fn12230 (Fn), fadA-deletion mutant US1 (ΔfadA), and spontaneous mutant lam following SDS–PAGE. A total of 400 μl of each culture grown to OD600 of 1.0 was pelleted and loaded onto 12% SDS–PAGE. FadA protein was detected using anti-FadA mAb 7H7 at 1:4,000 dilution. Compared to the wild type, lam produced significantly less pre-FadA, but not mFadA. Analysis of amyloid-like FadA produced by Fn ATCC 23726 and its Δfap2 mutant. An aliquot of 50 μg detergent-resistant pellets was loaded onto each lane, followed by SDD-AGE and Western blot analysis as described above. The mutant produced significantly less amyloid-like polymers than the wild type. Source data are available online for this figure. Source Data for Figure 2 [embr202152891-sup-0008-SDataFig2.pdf] Download figure Download PowerPoint Using SDD-AGE and Western blot analysis, we found that FadAc expressed in E. coli also formed detergent-resistant polymers, although the sizes were smaller than those produced by Fn. In contrast, mFadA did not form polymers (Fig 2B). These results indicate that while the full-size amyloid-like FadA polymers likely involve additional factors and processes that are specific to Fn, FadAc has intrinsic capability to assemble into amyloid-like structure and that pre-FadA is a key component for amyloid formation. To evaluate whether, and to what degree, FadA and its variants self-assemble into amyloid-like aggregates, we assessed binding of recombinant FadA proteins to two different antibodies that recognize amyloid structures: polyclonal anti-human amyloid-β antibodies OC and A11. The difference between these two antibodies is that A11 recognizes pre-fibril oligomers, while OC recognizes mature amyloid fibrils (Kayed et al, 2007). Only A11 is suitable for immunofluorescence staining, but both can be used for the analysis of purified proteins by slot blot. Among the recombinant FadA mutants tested, L14A and L76A did not produce pre-FadA (Fig EV1A) and exhibited no filamentous structure or cell-binding function (Xu et al, 2007). These mutant proteins did not react with either OC or A11, similar as the negative control protein histone H1 (Figs 2C and EV1B). In contrast, FadAc, L-9A (carrying Leu-Ala mutation at position −9 in the signal peptide) and S71A reacted with both OC and A11, similar as the positive control protein Rim4, which readily forms β-sheet-rich amyloid-like aggregates (Figs 2C and EV1B) (Berchowitz et al, 2015). Previous study showed these latter group all retained filamentous structure and were functional (Temoin et al, 2012a). Interestingly, they all express both forms of FadA (Fig EV1A). Notably, mFadA reacted weakly with the
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