A protein called Aβ is thought to cause neuronal death in Alzheimer’s disease (AD). Aβ forms insoluble aggregates in the brains of patients with AD, which are a hallmark of the disease. Aβ and its propensity for aggregation are widely viewed as intrinsically abnormal. However, in new work, Kumar et al. show that Aβ is a natural antibiotic that protects the brain from infection. Most surprisingly, Aβ aggregates trap and imprison bacterial pathogens. It remains unclear whether Aβ is fighting a real or falsely perceived infection in AD. However, in any case, these findings identify inflammatory pathways as potential new drug targets for treating AD.

The amyloid-β peptide (Aβ) is a key protein in Alzheimer’s disease (AD) pathology. We previously reported in vitro evidence suggesting that Aβ is an antimicrobial peptide. We present in vivo data showing that Aβ expression protects against fungal and bacterial infections in mouse, nematode, and cell culture models of AD. We show that Aβ oligomerization, a behavior traditionally viewed as intrinsically pathological, may be necessary for the antimicrobial activities of the peptide. Collectively, our data are consistent with a model in which soluble Aβ oligomers first bind to microbial cell wall carbohydrates via a heparin-binding domain. Developing protofibrils inhibited pathogen adhesion to host cells. Propagating β-amyloid fibrils mediate agglutination and eventual entrapment of unatttached microbes. Consistent with our model, Salmonella Typhimurium bacterial infection of the brains of transgenic 5XFAD mice resulted in rapid seeding and accelerated β-amyloid deposition, which closely colocalized with the invading bacteria. Our findings raise the intriguing possibility that β-amyloid may play a protective role in innate immunity and infectious or sterile inflammatory stimuli may drive amyloidosis. These data suggest a dual protective/damaging role for Aβ, as has been described for other antimicrobial peptides.

Members of the evolutionarily ancient family of proteins, collectively known as antimicrobial peptides (AMPs), share many of Aβ’s purportedly abnormal activities, including oligomerization and fibrillization ( 3 , 4 ). For AMPs, these activities mediate key protective roles in innate immunity. AMPs are the first line of defense against pathogens and act as potent broad-spectrum antibiotics and immunomodulators that target bacteria, mycobacteria, enveloped viruses, fungi, protozoans, and, in some cases, transformed or cancerous host cells ( 5 ). AMPs are widely expressed and are abundant in brain and other immunoprivileged tissues where actions of the adaptive immune system are constrained. Although AMPs are normally protective, AMP dysregulation can lead to host cell toxicity, chronic inflammation, and degenerative pathologies ( 6 – 8 ). Particularly germane to Aβ’s role in AD, AMPs are deposited as amyloid in several disorders ( 3 , 4 , 9 ), including senile seminal vesicle amyloid and isolated atrial amyloidosis, two of the most common human amyloidopathies. Consistent with identity as an AMP, we recently reported that synthetic Aβ exhibits potent in vitro antimicrobial activity toward eight common and clinically relevant microbial pathogens ( 3 ). Furthermore, whole-brain homogenates from AD patients show Aβ-mediated activity against Candida albicans. More recently, synthetic Aβ has been shown to protect cultured cells from influenza A virus ( 10 ) and herpes simplex virus ( 11 ). However, the biological relevance of protective in vitro Aβ activities requires validation in vivo. Here, we extend our original findings and show that Aβ expression inhibits infection in a transgenic mouse model of AD (5XFAD), in the nematode Caenorhabditis elegans, and in cultured mammalian cell models. Mice lacking the amyloid precursor protein (APP) that have low Aβ expression also show a trend toward attenuated survival after bacterial infection. Most surprisingly, oligomerization and fibrillization appear to mediate Aβ’s protective activity, and cerebral infection with microbial cells seeds and markedly accelerates β-amyloid deposition in 5XFAD mice and transgenic C. elegans.

Neurodegeneration in Alzheimer’s disease (AD) is mediated by soluble oligomeric intermediates generated during fibrillization of the amyloid-β protein (Aβ) ( 1 ). Overwhelming evidence supports Aβ’s pivotal role in AD. However, despite remarkably high sequence conservation across diverse species (humans share Aβ42 sequences with coelacanths, a 400 million–year–old fish taxon) ( 2 ) and extensive data showing broad activity spectra for Aβ, the peptide has traditionally been characterized as a functionless catabolic byproduct. Activities identified for Aβ in vivo are most often described as stochastic pathological behaviors. Oligomerization, in particular, is viewed as a pathogenic pathway, and Aβ oligomers are assumed to be intrinsically abnormal. Scant consideration has been given to possible physiological roles for Aβ.

Four-week-old WT mice or transgenic 5XFAD animals expressing high levels of human Aβ were injected intracerebrally with viable S. Typhimurium bacteria. Mice were also injected with heat-treated S. Typhimurium cell debris as a negative control for the injection procedure. ( A and B ) Mouse brain sections were prepared 24 (A) or 48 hours (B) after infection. Signals shown include visible (VIS), anti-Salmonella immunoreactivity (α-Salmonella), enhanced Thioflavin S fluorescence (ThS) or anti-Aβ immunoreactivity (α-Aβ), and superimposed (Overlay) signals. Panels are representative images of multiple images captured as Z-sections using CFM. Yellow denotes signal colocalization (Z-series projections showing β-amyloid surrounding and entrapping bacterial colonies in a rotating three-dimensional section of 5XFAD mouse brain are also included in video S1). Micrographs are representative of data from three replicate experiments and multiple discrete image fields (table S1C).

Four-week-old 5XFAD mouse brain is normally negative for β-amyloid deposits ( 12 ). However, Thioflavin S and anti-Aβ staining of 5XFAD mouse brain revealed widespread β-amyloid deposition 48 hours after infection with S. Typhimurium ( Fig. 7 , A and B). Moreover, anti-Salmonella and β-amyloid signal colocalized in the 5XFAD mouse brain, suggesting that bacterial cells may have induced Aβ fibrillization. TEM analysis also revealed that bacterial cells were embedded in fibrous material labeled by anti–Aβ-Au nanoparticles in 5XFAD but not wild-type mouse brain sections (fig. S8). A video of Z-section projections rotating through 360° shows that bacteria are not confined to the surface of Aβ accretions but are embedded within the β-amyloid deposits (video S1). Consistent with fibrillization driven by proliferation of S. Typhimurium cells, β-amyloid deposits were absent from sham-infected 1-month-old 5XFAD control mice injected with heat-killed bacteria. Thioflavin S staining and anti–β-amyloid antibodies did not label mouse brain from negative control nontransgenic littermates ( Fig. 7A ).

Aβ42-expressing GMC101 C. elegans were infected with C. albicans (Candida) and probed for anti-Aβ immunoreactivity and β-amyloid markers using TEM and confocal fluorescence microscopy (CFM). ( A ) Micrograph shows positive labeling of yeast cell surface in GMC101 worm gut by immunogold nanoparticles coated with anti-Aβ antibodies (α–Aβ-Au) after Candida ingestion. ( B to D ) Visible (VIS) and fluorescence signals from freeze-fracture nematode sections with advanced Candida infections. (B) Comparison of uninfected and infected worms. (C and D) Thioflavin S and anti-Aβ staining for gut yeast aggregates. Signals include anti-Candida immunoreactivity (α-Candida), Thioflavin S–enhanced fluorescence (ThS), anti-Aβ immunoreactivity (α-Aβ), and superimposed (Overlay) signals. Yellow denotes signal colocalization. Uninfected and infected CL2122 nematode controls were negative for anti-Aβ immunoreactivity and enhanced Thioflavin S fluorescence (figs. S2 and S8). Micrographs are representative of data from three or more replicate experiments and multiple discrete image fields (table S1B).

We also investigated infection-associated Aβ fibrillization in our nematode and mouse infection models. Consistent with Aβ targeting and binding to yeast cells in our cell culture model, Candida in the gut of recently infected (2 hours after ingestion) GMC101 nematodes were labeled by anti–Aβ-Au nanoparticles ( Fig. 6A ). Yeast cells in the gut of the control CL2122 nematode were not labeled by anti–Aβ-Au (fig. S8A). Aβ fibrillization in GMC101 worms is normally confined to the body wall muscle. However, compared to infection-free nematodes, GMC101 worms with late-stage Candida infection showed enhanced Thioflavin S fluorescence in nonmuscle tissue, including the gastrointestinal tract ( Fig. 6B ). High-resolution micrographs of yeast cells in the gastrointestinal tract of GMC101 nematodes revealed clumped Candida embedded in the material that showed enhanced fluorescence after Thioflavin S staining ( Fig. 6C ) and was labeled by anti-Aβ antibodies ( Fig. 6D ). Consistent with Aβ-specific labeling, anti-Aβ signal (fig. S2B) and enhanced Thioflavin S fluorescence (fig. S8B) were absent from uninfected or Candida-infected negative control CL2122 nematodes that did not express Aβ. Findings for C. albicans–infected GMC101 nematodes were consistent with the agglutinating and entrapment roles of Aβ fibrils observed in our cell culture infection models. Thus, Aβ fibrillization on the surface of yeast cells infecting the gut of GMC101 nematodes may mediate the resistance to infection observed for these worms.

After overnight incubation with H4-Aβ42 medium, yeast (C. albicans) aggregates were harvested and probed for β-amyloid markers. ( A and B ) Visible yeast aggregates (VIS), yeast aggregates stained with green fluorescent Thioflavin S (ThS FLU), yeast aggregates probed with red fluorescent anti-Aβ (α-Aβ FLU) antibodies, and superimposed images (VIS/FLU overlay). Yeast aggregates generated with the control synthetic LL-37 peptide (A) are negative for Thioflavin S–enhanced fluorescence. (B) Yellow denotes colocalization of anti-Aβ and Thioflavin S signals. Colocalization of these signals is the hallmark of Aβ. ( C ) SEM analysis revealed fibrous material in H4-Aβ42 yeast aggregates that is absent from control C. albicans pellets prepared by centrifugation in H4-N medium. ( D ) H4-Aβ42 yeast aggregates incubated with immunogold nanoparticles coated with anti-Aβ antibodies (α–Aβ-Au) and analyzed by TEM. The first and second panels show labeling of fibrous material by α–Aβ-Au. The third panel shows inhibition of α–Aβ-Au nanoparticle binding by soluble synthetic Aβ peptide (α–Aβ-Au + Aβ peptide), consistent with specific labeling of β-amyloid. Micrographs are representative of data from two or more replicate experiments and multiple discrete image fields (table S1A).

Epifluorescence micrographs of Thioflavin S–stained late-stage (>12 hours after infection) H4-Aβ42 yeast aggregates displayed the enhanced fluorescence and red shifts that mark the presence of amyloid fibrils ( Fig. 5A ). Enhanced fluorescence was not observed for negative control yeast agglutinates ( Fig. 5A ). Thioflavin S fluorescence within H4-Aβ42 yeast aggregates colocalized with the signal for anti-Aβ immunoreactivity ( Fig. 5B ). Congo red–stained H4-Aβ42 yeast aggregates also showed birefringence under polarized light, another marker for β-amyloid (fig. S7). Scanning electron microscopy (SEM) micrographs of yeast aggregates from H4-Aβ42 medium revealed an irregular material adhering to cell surfaces not present in Candida pellets prepared by centrifugation in Aβ-free medium ( Fig. 5C ). Analysis of the Candida cell surface by TEM revealed the adhering material to be filamentous and immunoreactive to anti–Aβ-Au ( Fig. 5D ). Co-incubation of soluble synthetic Aβ40 peptide abolished anti–Aβ-Au binding. Collectively, the data are consistent with microbial agglutination and entrapment mediated by Aβ fibrillization in our cell culture infection model.

Early-stage C. albicans aggregates harvested from H4-Aβ42 conditioned medium were probed with α–Aβ-Au nanoparticles and analyzed by TEM. ( A ) Yeast agglutination is mediated by fibrillar structures. The micrograph shows fibrils binding cells within yeast aggregates and linking C. albicans clusters. ( B ) Fibrillar structures extending from yeast cell surfaces. ( C and D ) α–Aβ-Au nanoparticle labeling of short fibrillar structures extending from C. albicans surfaces and long fibrils running between yeast clumps. ( E ) Absorption experiment showing ablated α–Aβ-Au binding of fibrils extending from yeast in the presence of soluble synthetic Aβ peptide. Data are consistent with specific α–Aβ-Au labeling of β-amyloid fibrils. Micrographs are representative of data from three replicate experiments and multiple discrete image fields (table S1A).

Binding by Aβ of glycosaminoglycans found in brain tissue induces peptide fibrillization ( 34 ). Aβ’s binding of cell wall and glycocalyx carbohydrates at microbial surfaces seemed likely to also generate Aβ fibrils. Although viewed solely as a part of Aβ’s pathophysiology, fibrillization among AMPs is a normal protective behavior that mediates antimicrobial activities, including microbial cell and viral agglutination ( 35 ) and bacterial membrane perturbation ( 3 , 4 ). Most recently, studies have shown that the human AMP α-defensin-6 (HD6) forms fibrils that entangle and trap microbial cells ( 36 ). Thus, we next investigated a possible role for Aβ fibrillization in the peptide’s protective AMP activities. Analysis of early-stage (<3 hours after infection) Candida agglutination in H4-Aβ42 medium using transmission electron microscopy (TEM) revealed clumped microbial cells entwined and linked by fibrils propagating from cell surfaces ( Fig. 4 , A to D). C. albicans lack flagella and are not reported to produce extended fibrillar structures. Moreover, the fibrillar structures on the Candida cell surface were labeled by anti–Aβ immunogold nanoparticles (anti–Aβ-Au). Anti–Aβ-Au binding to fibrils was ablated by co-incubation with synthetic Aβ peptide, consistent with Aβ-specific labeling ( Fig. 4D ). TEM analysis of early-stage S. Typhimurium agglutinates in H4-Aβ42 conditioned medium confirmed that bacterial cells were also bound and linked by fibrils (fig. S3F).

We further characterized Aβ’s binding to C. albicans and S. Typhimurium using a new binding immunoassay. For this assay, samples were incubated in wells containing immobilized intact hyphal Candida or S. Typhimurium cells, and bound Aβ was detected immunochemically with an Aβ42-specific antibody. Aβ binding to Candida and S. Typhimurium was concentration-dependent ( Fig. 3I and fig. S3E). Consistent with binding mediated by Aβ’s VHHQKL domain, the anti-Aβ signal from H4-Aβ42 medium was significantly attenuated in the presence of glucan (P = 0.008) or mannan (P = 0.004) ( Fig. 3J ). The anti-Aβ signal in wells was also significantly reduced (P = 0.006) for anti-Aβ–immunodepleted H4-Aβ42 medium (negative control), which was consistent with assay specificity for Aβ42 binding. Consistent with findings for antimicrobial activities, cell-generated Aβ oligomers showed increased binding to immobilized yeast compared to synthetic monomeric peptide ( Fig. 3I ). Previous studies have shown that Aβ oligomerization greatly increases carbohydrate-binding activity ( 31 ). Heparin-binding AMP oligomers also show potentiated carbohydrate binding compared to monomeric species ( 33 ). Overall, our findings are consistent with soluble Aβ oligomers having an enhanced propensity to bind to cell walls, engendering greater adhesion inhibition and agglutination activities compared to monomeric synthetic peptide.

Binding of AMP peptides to microbial surfaces is a prerequisite step for adhesion inhibition and agglutination activities. LL-37 contains an XBBXBX heparin-binding motif (where X is a hydrophobic or uncharged residue and B is a basic residue) that mediates inhibition of host cell adhesion and agglutination activities by facilitating attachment of oligomeric species ( 26 , 30 ) to microbial cell wall carbohydrates ( 22 ). Aβ also contains an XBBXBX heparin-binding motif between residues 12 to 17 (VHHQKL) ( 31 ). Competitive inhibition by soluble microbial sugars is a hallmark for AMPs with activities mediated by lectin-like carbohydrate binding ( 22 ). Indeed, fungal and bacterial pathogens secrete specialized scavenging exopolysaccharides that target the heparin-binding domains of AMPs as a countermeasure to defenses mounted by hosts. Soluble forms of mannan and glucan, the two most abundant carbohydrates in the yeast cell wall, have been shown to inhibit XBBXBX-mediated binding of LL-37 to Candida ( 22 , 32 ). We investigated whether the adhesion inhibition and agglutination activities of Aβ were similarly inhibited by soluble mannan and glucan. Live yeast cells were incubated in H4-Aβ40, H4-Aβ42, and CHO-CAB conditioned medium in the presence or absence of mannan or glucan. Consistent with anti-Candida activity mediated by Aβ’s heparin-binding domain, mannan and glucan significantly attenuated adhesion inhibition (P < 0.008) and agglutination (P < 0.003) activities of conditioned medium from Aβ-expressing transformed cells ( Fig. 3 , G and H).

To test whether oligomerization modulates Aβ’s AMP activity, we generated synthetic Aβ oligomers and compared the antimicrobial activities of Aβ42 monomer, soluble oligomeric ADDLs (amyloid-β–derived diffusible oligomeric ligands) ( 24 ), and high-order protofibril (>600 kD) preparations. Compared to monomeric peptide, ADDLs exhibited potentiated, and protofibrils attenuated, adhesion inhibition ( Fig. 3E ) and agglutination ( Fig. 3F ) activities. Our data are consistent with a central role for soluble Aβ low-order (2 to 30 monomer units) oligomers in mediating the peptide’s AMP activities. Consistent with such a role, soluble Aβ is overwhelmingly oligomeric in vivo ( 25 ), and oligomers are key for the protective activities of a wide range of AMPs ( 26 – 29 ) including LL-37 ( 26 , 30 ).

Serial dilution experiments showed that adhesion inhibition and agglutination activities were dose-dependent for both synthetic and cell-derived Aβ ( Fig. 3 , C and D). However, synthetic Aβ peptide preparations had lower specific activities compared to cell-derived material. Cofactors secreted by cultured cells were unlikely to account for the increased potency of cell-derived Aβ because synthetic peptide incubations were performed in Aβ42-depleted conditioned medium (H4-Aβ42-ID) from H4-Aβ42 cell cultures. Anti-Aβ antibodies used to clear Aβ42 from H4-Aβ42 culture medium before addition of synthetic peptides were specific for Aβ and not likely to deplete species acting as cofactors. Oligomerization has been shown to modulate a range of Aβ activities. Moreover, conditioned medium from experimental cell lines has been reported to contain oligomeric Aβ ( 23 ), whereas our synthetic peptide preparations were pretreated to remove oligomer species. Synthetic peptide pretreatments included fractionation by preparative size exclusion chromatography to remove species >6 kD. Characterization experiments using analytical size exclusion chromatography confirmed that immediately before experimental inoculation with yeast, cell-derived material contained a polydisperse population of soluble Aβ oligomers of between 8 and 50 kD, whereas synthetic peptides remained overwhelmingly monomeric (fig. S5C).

C. albicans adhesion to abiotic surfaces and agglutination in the bulk phase were characterized in the presence of cell-derived or synthetic Aβ. After 36 hours of conditioning, host cell–free culture medium was collected from control nontransformed (H4-N or CHO-N) or transformed Aβ-overexpressing (H4-Aβ40, H4-Aβ42, or CHO-CAB) cultured cells. Aβ-immunodepleted (ID α-Aβ) and control immunodepleted [ID IgG (immunoglobulin G)] media were prepared by incubation with immobilized anti-Aβ or nonspecific antibodies. Experimental synthetic peptides included Aβ (Aβ40 and Aβ42), AMP positive control (LL-37), and negative control scrambled Aβ42 (scAβ42). ( A and B ) Comparison of ID α-Aβ and ID IgG medium’s adhesion inhibition (*P = 0.009, **P = 0.001, and ***P = 0.004) and agglutination (*P = 0.001, **P = 0.0005, and ***P = 0.004) activities. ( C and D ) Comparison of anti-Candida activities of serially diluted conditioned medium and synthetic peptides. ( E and F ) Activities of synthetic Aβ42 monomer, soluble oligomeric ADDLs, and protofibril preparations. ( G and H ) Conditioned culture medium adhesion inhibition (*P = 0.003 and **P < 0.0003) and agglutinating (*P < 0.02 and **P < 0.003) source activities alone (Neat) or in the presence of soluble yeast wall carbohydrates (+Glucan or +Mannan). ( I ) Synthetic monomeric Aβ42 and cell-generated peptide from H4-Aβ42 cells were compared for Candida binding using an Aβ/Candida binding ELISA. ( J ) Untreated, immunodepleted, or glucan (Glu)- or mannan (Man)–spiked H4-Aβ42 conditioned media were incubated with intact immobilized yeast cells in an Aβ/Candida binding ELISA assay (*P = 0.006, **P = 0.008, and ***P < 0.004). Synthetic peptide incubations (C to F and I) were performed in H4-Aβ42 conditioned culture medium pretreated to remove cell-derived Aβ by α-Aβ immunodepletion. Symbols and bars for (A) to (J) are statistical means of six replicate wells ± SEM. Statistical significance was by t test.

We next characterized cell-free conditioned culture medium for Aβ-mediated adhesion inhibition and agglutinating activities. Yeast adhesion and agglutination were assayed in 96-well plates using the methods of Tsai et al. ( 22 ). Briefly, synchronized hyphal C. albicans were incubated (2 hours, 37°C) with conditioned medium samples in the absence of host cells. After washing, yeast adhering to well surfaces were stained with Calcofluor white, and fluorescence was measured. Well images were analyzed for yeast aggregation after overnight incubation. Immunodepletion with anti-Aβ antibodies significantly attenuated H4-Aβ42, H4-Aβ40, and CHO-CAB medium adhesion inhibition (P = 0.009, P = 0.001, and P = 0.004, respectively) and agglutination (P = 0.001, P = 0.0005, and P = 0.004, respectively) activities against C. albicans ( Fig. 3 , A and B). Analysis confirmed that anti-Aβ immunodepletion removed >95% of the Aβ from samples used in experiments to confirm that the anti-Candida activities of transformed cell culture medium were specific for Aβ (fig. S5, A and B).

Whereas the amount of Aβ in conditioned cell culture medium (fig. S5, A and B) fell within the physiological ranges reported for human cerebrospinal fluid (CSF) (2 to 20 ng/ml) ( 21 ), concentrations were two orders of magnitude (log 10 ) lower than the minimal inhibitory concentration (MIC) for fungicidal activities in microdilution MIC assays ( 3 ). We have previously reported that Aβ’s antimicrobial activities show close parallels with those of LL-37 ( 3 ), an archetypal human AMP that remains protective at subfungicidal concentrations ( 22 ). Two linked, yet distinct activities mediate LL-37’s protective anti-Candida actions at low peptide concentrations ( 22 ). The first is disruption of C. albicans adhesion to host cells. Host cell attachment is a prerequisite step for infection by many pathogens, including C. albicans. The second is agglutination of the resulting unattached yeast cells. Agglutination limits microbial access to host cells and also generates high local AMP concentrations within peptide/microbe aggregates. Accordingly, we next tested Aβ for adhesion inhibition and agglutination activities using the cell culture infection model. Hyphal C. albicans was incubated (2 hours, 37°C) in preconditioned medium with transformed or nontransformed cell cultures prepared in slide chambers. Microscopic examination revealed fewer C. albicans attached to transformed Aβ-expressing cells compared to nontransformed monolayers ( Fig. 2C and fig. S6A). To confirm these data, we repeated C. albicans–cell culture incubation experiments in 96-well microtiter plates, and we assayed the Candida load in wells immunochemically using anti-Candida antibodies. Data confirmed visual observations with statistically significant attenuation of C. albicans adhesion to transformed H4-Aβ42 (P = 0.001), H4-Aβ40 (P = 0.001), and CHO-CAB (P = 0.004) cells compared to naive control lines ( Fig. 2D ). Additionally, after overnight incubation, marked microbial agglutination was observed in wells containing transformed, but not nontransformed, host cells ( Fig. 2E and fig. S6B). Images of wells were analyzed for yeast aggregation. Candida aggregation was significantly elevated in transformed H4-Aβ42 (P = 0.00004), H4-Aβ40 (P = 0.0003), and CHO-CAB (P = 0.002) samples compared to naive controls ( Fig. 2F ). For H4 cell lines, adhesion inhibition and agglutination activities were consistent with host viability data, with rank orders H4-Aβ42 > H4-Aβ40 > H4-N.

We first compared nontransformed and transformed host cells for survival after infection with C. albicans. Host cells were prelabeled with bromodeoxyuridine (BrdU). After infection, host cell viability was determined by assaying for anti-BrdU immunofluorescence. Consistent with findings for 5XFAD mice and GMC101 nematodes, survival 28 hours after infection was significantly increased for Aβ-overexpressing H4-Aβ40 (P = 0.002) and H4-Aβ42 (P = 0.001) transformed cell lines compared to control H4-N cells, with rank order H4-Aβ42 > H4-Aβ40 > H4-N ( Fig. 2B ). Survival of transformed CHO-CAB cells was also significantly higher (P = 0.004) than that of control CHO-N cell lines. Additional independent assays of host cell viability (fig. S4, A and B) were performed to confirm increased resistance of transformed H4-Aβ42 cells to C. albicans infection. Attenuated C. albicans load for H4-Aβ42 cells was also independently confirmed by comparing wells for yeast CFU (fig. S4C).

To address the mechanism of protection, we next tested the ability of Aβ to protect cell monolayers from infection using transformed cultured human brain neuroglioma (H4) and Chinese hamster ovary (CHO) cells. H4 lines included stably transformed H4-Aβ40 and H4-Aβ42 cells that selectively secrete the 1–40 residue Aβ isoform (Aβ40) or Aβ42 isoform, respectively ( 19 ). Processing of a BRI-Aβ fusion protein expressed by transformed H4 cells led to constitutive high-level expression and secretion of the encoded Aβ protein. For double transfected CHO cells (CHO-CAB), overexpression of APP and the APP-processing protease β-secretase leads to APP cleavage and the generation of multiple Aβ isoforms ( 20 ). Nontransformed H4 (H4-N) and CHO (CHO-N) cells were used as control cell lines. C. albicans has been extensively characterized in cell culture infection models and was used in our experiments as an infectious agent.

Aβ-mediated protection against C. albicans (Candida) was characterized in C. elegans and cultured host cell monolayer mycosis models. Experimental nematodes included control (Cont.) non-Aβ expressing (CL2122) and transgenic (Tg) human Aβ-expressing (GMC101) strains. Host cell lines included control nontransformed (H4-N and CHO-N) and transformed Aβ-overexpressing (H4-Aβ40, H4-Aβ42, and CHO-CAB) cells. ( A ) Survival curves for CL2122 (n = 61) and GMC101 (n = 57) nematodes after infection with Candida (P < 0.00001). ( B ) Viability of nontransformed and transformed host cell monolayers after 28-hour incubation with Candida. Host cell viability was followed by prelabeling host cell monolayers with BrdU and then comparing wells for an anti-BrdU signal. Signal of infected wells shown as percentage of uninfected control wells (*P = 0.002, **P = 0.001, and ***P = 0.004). ( C ) Candida adherence to host cells. Fluorescence micrograph of Calcofluor white–stained Candida adhering to control H4-N or transformed H4-Aβ42 host cell monolayers after 2 hours of co-incubation in preconditioned culture medium. ( D ) Quantitative analysis of Candida host cell colonization. Adhering Candida were detected using an immunochemical luminescence assay with anti-Candida antibodies (*P = 0.003, **P = 0.001, and ***P = 0.004). Well comparisons use arbitrary luminescence units (AU). ( E ) Phase-contrast micrographs of agglutinated Candida after overnight incubation with H4-N or H4-Aβ42 host cells. ( F ) Quantitative analysis of Candida agglutination. Wells were compared for yeast aggregate surface area using image analysis software (*P = 0.007, **P = 0.002, and ***P = 0.009). Bars in (B), (D), and (F) are means of six replicate wells ± SEM. Statistical significance was calculated by log-rank (Mantel-Cox) test for nematode survival (A) and statistical mean comparisons by t test (B, D, and F). Micrographs (C and E) are representative of data from three replicate experiments and multiple discrete image fields (table S1A).

C. albicans [American Type Culture Collection (ATCC) 90028] is an Aβ-sensitive dimorphic fungus ( 3 ) and a well-characterized C. elegans intestinal pathogen that causes distention, penetrative filamentation, and death among wild-type nematodes 2 days after ingestion. Links between fungal brain infections and AD pathology have also recently emerged, including for C. albicans ( 17 ) and closely related Candida glabrata ( 18 ). We compared survival of control CL2122 (n = 56) and GMC101 (n = 59) nematodes after incubation (2 hours, 25°C) on C. albicans lawns. Consistent with Aβ-mediated protection, GMC101 nematodes infected with C. albicans showed significantly (P < 0.00001) reduced mortality as compared to control CL2122 worms that did not express Aβ ( Fig. 2A ). Consistent with mouse data, Aβ-expressing nematodes were also protected from the C. elegans intestinal pathogen S. Typhimurium with GMC101 worms showing statistically significant (P = 0.0005) increased survival compared to CL2122 controls after infection with the bacterium (fig. S3A).

To further explore the ability of Aβ to afford protection against infection, we next tested transgenic C. elegans for resistance to Candida. Our nematode infection model uses two previously described C. elegans transgenic strains: GMC101 that expresses the 1–42 residue human Aβ isoform (Aβ42) ( 14 ) and CL2122, a control strain that expresses intestinal green fluorescent protein (GFP) (mtl-2:gfp) marker (as does GMC101) but does not express Aβ. Adult GMC101 nematodes ultimately develop age-progressive paralysis and β-amyloid deposition in the body wall muscle. For our experiments, developmentally synchronized L4 larvae were infected 5 days before the onset of paralysis. Aβ expression is driven by the unc-54 promoter (which encodes a myosin heavy chain), active in body wall muscle ( 14 ) as well as in other tissues, including muscle cells of the gastrointestinal tract ( 15 ). Amyloidogenic peptides under the unc-54 promoter have also been shown to translocate via vesicular transport to the gut of transgenic worms, and Aβ has been proposed as a likely candidate for translocation via this mechanism ( 16 ). Immunohistological analysis of adult GMC101 using three different anti-Aβ antibodies confirmed Aβ localization in the body wall muscle and the gut lumen (fig. S2, A and B). Anti-Aβ antibodies did not label negative control strain CL2122 intestine or body wall cells. In addition, excreta from healthy GMC101 but not CL2122 worms were positive for anti-Aβ signal by immunoblot (fig. S2C). Although an origin for gut Aβ remains unclear, strong empirical evidence supports the localization of Aβ peptides in the intestinal lumen of GMC101 nematodes. Thus, transgenic GMC101 nematodes appear to be suitable models for testing Aβ-mediated protective activities against intestinal pathogens.

Transgenic (5XFAD) mice expressing human Aβ and mice lacking murine APP (APP-KO) were compared to genetically unmodified littermates [wild type (WT)] for resistance to S. Typhimurium meningitis. One-month-old mice received single ipsilateral intracranial injections of S. Typhimurium, and clinical progression was followed to moribundity. ( A to C ) Performance of 5XFAD (n = 12) mice compared to WT (n = 11) are shown after infection for survival (P = 0.009) (A), clinical score (P < 0.0001) (B), and percent weight loss (P = 0.0008) (C). ( D ) S. Typhimurium load 24 hours after infection in 5XFAD (n = 4) and WT (n = 4) mouse brain hemisphere homogenates shown as mean CFU ± SEM (*P = 0.03 and **P = 0.04). ( E ) APP-KO mice (n = 15) show a trend (P = 0.104) toward reduced survival compared to WT (n = 15) littermates after infection. ( F ) No mortality was observed among control sham-infected WT (n = 6) or 5XFAD (n = 6) mice injected with heat-killed S. Typhimurium. Statistical significance was calculated by log-rank (Mantel-Cox) test for survival (A, E, and F), linear regression for clinical score and weight (B and C), and statistical means compared by t test for brain bacterial loads (D). For survival and clinical analysis (A to C), data were pooled from three independent experiments.

We first used genetically modified mice to test for protective effects of elevated Aβ expression and attenuated resistance with decreased peptide. Four-week-old 5XFAD transgenic mice constitutively express human Aβ in the brain at high levels but lack the β-amyloid deposits and features of neuroinflammation found in older animals ( 12 ). APP knockout (APP-KO) mice lack the precursor protein required for murine Aβ generation ( 13 ). One-month-old 5XFAD mice (n = 12), APP-KO mice (n = 15), and wild-type littermates (n = 11 and 15, respectively) received a single intracerebral injection of 65,000 colony-forming units (CFU) of S. Typhimurium. Clinical progression to the moribund state was followed according to established grading criteria for mouse encephalomyelitis (fig. S1A). Survival of Aβ-expressing 5XFAD mice was significantly increased compared to that of nontransgenic littermates (P = 0.009) ( Fig. 1A ). Consistent with increased resistance to infection, 5XFAD mice also ranked significantly higher in clinical tests grading mouse encephalomyelitis progression (P < 0.0001). 5XFAD mice also showed reduced weight loss (P = 0.0008) and lower cerebral S. Typhimurium loads (P = 0.03) compared to wild-type controls ( Fig. 1 , B to D). Consistent with immunodeficiency associated with low Aβ, APP-KO mice showed a trend (P = 0.10) toward increased mortality after infection ( Fig. 1E ). Control injections using heat-killed bacteria did not lead to clinical decline or death in 5XFAD and wild-type mice ( Fig. 1F ), consistent with mouse mortality being mediated by S. Typhimurium infection. Next, we confirmed high amounts of soluble Aβ and low amounts of insoluble Aβ in 4-week-old 5XFAD mouse brain using formic acid extraction and anti–β-amyloid enzyme-linked immunosorbent assays (ELISAs) (fig. S1B). To confirm that inflammation did not immunologically prime and protect 5XFAD mice against infection, we compared the immune profiles in 1-month-old transgenic and wild-type mouse brains. Consistent with previous reports showing an absence of immune activation ( 12 ), there was no significant increase in glial fibrillary acidic protein–positive (GFAP + ) astrocytes, lba1 + microglia, and the amounts of 10 cytokines in 4-week-old 5XFAD mice compared to wild-type littermates (fig. S1, C to E).

DISCUSSION

Our findings are consistent with a potential protective role for Aβ in vivo as an AMP. Expression of Aβ was associated with increased host survival in cell culture, nematode, and mouse infection models (Figs. 1 and 2). Low Aβ expression was associated with higher mortality after infection of APP-KO mice. Our data are consistent with a protective role for Aβ in innate immunity that uses a classic AMP mechanism characterized by reduced microbial adhesion to host cells and agglutination and entrapment of microbes by Aβ fibrils. Moreover, well-characterized Aβ activities mediate the peptide’s antimicrobial actions. However, these same properties, oligomerization, fibrillization, and carbohydrate binding, are also linked to Aβ’s pathophysiology. Whereas a protective/damaging duality is a new proposition for Aβ’s activities, this is not the case for classical AMPs. For example, LL-37 offers a germane model for the potential pathological consequences of normally protective AMP actions. LL-37 is essential for normal immune function, and low expression leads to lethal infections (37). However, at elevated concentrations, LL-37 is cytotoxic to host cells, particularly smooth muscle cells (38). The cytotoxic and proinflammatory activities of LL-37 are implicated in the pathogenesis of several major late-life diseases, including rheumatoid arthritis, lupus erythematosus, and atherosclerosis (39). Thus, a normally protective Aβ activity spectrum that, when dysregulated, also leads to AD pathology is consistent with the actions of classical human AMPs.

Adhesion blocking and agglutination activities are distinct from AMP microbicidal activities, which typically require micromolar concentrations of peptide and involve different mechanisms (22). The adhesion inhibition and agglutination activities that we observed in vitro for cell-derived Aβ (Fig. 3) fall within physiological concentration ranges reported for normal human CSF (1 to 5 ng/ml). Consistent with a normal in vivo protective role, the highest cerebral concentrations of Aβ are in the leptomeninges (10 to 50 ng/ml) (40), the brain’s first line of defense against infection and a tissue enriched for LL-37 and other innate immune proteins (41). The high specific activity observed for cell-derived material is consistent with our previous finding that Aβ in human brain extracts is a potent anti-Candida agent (3). Classical AMP expression can be either constitutive or inducible (5). In our transgenic mouse, nematode, and cell culture models, constitutive expression of Aβ is maintained artificially. Hence, our models are not suitable for testing whether infection normally results in Aβ up-regulation. However, data from other investigators suggest that Aβ may be an inducible AMP. Host cell exposure to herpes simplex virus–1 (42), HIV-1 (42), spirochetes (43), or Chlamydia (44, 45) increases Aβ expression.

In in vitro assays, cell-derived and synthetic Aβ oligomers were more potent against Candida than were monomeric forms (Fig. 3, C to F, and fig. S5C). The specific activities of synthetic ADDLs, although higher than nonoligomerized peptide, remain lower than cell-derived Aβ species. Peptide posttranslational modifications may enhance the AMP activity of cell-derived Aβ oligomers. However, oligomer conformation is also likely to play a key role. Neurotoxicity has been shown to be highly dependent on the arrangement of Aβ peptides within oligomeric assemblies. Oligomer morphology may also modulate Aβ’s protective antimicrobial activities. Protocols for preparing ADDLs and other synthetic Aβ assemblies are optimized for oligomer populations with neurotoxic, not antimicrobial, activities. Future protocols optimized for enhanced AMP activities may generate soluble synthetic Aβ oligomers with potencies that approach that of cell-derived material.

Aβ pathophysiology is thought to arise from an abnormal propensity to generate soluble oligomers. However, oligomerization is not a pathogenic behavior for AMPs, and it plays a key role in normal protective activities across this diverse group of proteins, including microbe agglutination and entrapment (35), the targeting (26, 30) and disruption of microbial cell membranes (4, 46), resistance to bacterial proteases (26, 27, 46), and expanding of the molecular diversity and protective functions of AMP families without commensurate genome expansion (28, 29). Our data and the widespread involvement of oligomerization in the protective actions of AMPs suggest that the brain’s pool of soluble Aβ may normally include physiologically functional oligomeric species that mediate protective antimicrobial activities. The intrinsic polymorphic stoichiometry of Aβ oligomers may also play a protective physiological role. As has been shown with classical AMPs, diverse polymorphic oligomer pools target a broader spectrum of pathogens and are more resistant to AMP-targeting microbial proteases than are homogeneous peptide populations.

The lectin activity of Aβ oligomers is thought to promote brain amyloidosis (34). Studies to date have focused on accelerated Aβ fibrillization induced by binding of endogenous brain proteoglycans and glycosaminoglycans. However, our findings suggest that Aβ oligomers also bind to microbial carbohydrates with high affinity (Fig. 3, G to J). Carbohydrate-binding activity among AMPs is widespread and normally protective, playing a key role in helping peptides to recognize and bind to microbial pathogens (22). Heparin-binding AMPs have high affinities for the unique microbial carbohydrates found in cell walls but also bind to host glycosaminoglycans (47). Consistent with our findings for Aβ, binding of classical AMPs to microbial carbohydrates can lead to rapid peptide fibrillization and amyloid-mediated antimicrobial activities (48). Dysregulated carbohydrate binding by Aβ may play a role in AD amyloidogenesis. However, a normal role as an AMP would suggest that polymeric microbial cell surface carbohydrates may be the normal in vivo target for the heparin-binding activity of oligomeric Aβ species.

Long recognized as a key defensive strategy among lower organisms, AMP-mediated microbial agglutination is also emerging as an important part of human immunity (49). AMP fibrillization appears to play a central role in this important protective activity (35). Most recently, in vivo fibrillization of HD6 has been shown to mediate not only agglutination but also microbial entrapment within an amyloid fibril network (36). Our findings suggest that fibrillization is also involved in Aβ-mediated agglutination and leads to the entrapment of microbial cells by Aβ fibrils. On the basis of our findings, we propose a three-stage model for the protective activity of Aβ in vivo. Our model parallels the agglutination and entrapment actions of amyloidogenic HD6 (36). First, the VHHQKL heparin-binding domain of Aβ mediates targeting and binding of soluble oligomeric species to cell wall carbohydrates (fig. S10A). Bound oligomers then provide a nidus and anchor for Aβ fibril propagation. Second, growing protofibrils interfere with microbial adhesion to host cells (fig. S10B). Third, Aβ fibrils link, agglutinate, and then entrap the unattached microbial cells in a protease-resistant network of β-amyloid (fig. S10C). Consistent with our model for the antimicrobial activities of Aβ, classical human AMPs have also been shown to generate amyloid fibrils on microbial surfaces that agglutinate pathogens and inhibit infection (35).

Consistent with our AMP model for Aβ, APP-KO mice show a trend for reduced pathogen resistance (Fig. 1E). However, the increase in infection-driven mortality among APP-KO mice was less marked than the increase in survival observed in the 5XFAD mouse model (Fig. 1A). For AMP-deficient models, immune impairment is often moderate because redundant activities among related members of AMP families can partially offset the loss of protection associated with low expression of individual AMP species (50). The well-studied human AMP LL-37 that serves as our model for Aβ’s AMP activity (3) is a member of the cathelicidin protein family. In humans, serious immunodeficiency associated with low LL-37 expression typically leads to fatal infections in childhood if untreated (37). However, mice lacking the murine LL-37 precursor protein (mCRAMP) show only a modest increase in mortality (≈10%) due to bacterial meningitis (51). Conversely, survival with infection among transgenic mice overexpressing human LL-37 is increased several-fold (52). APP-KO mice generate at least two Aβ homologs from amyloid precursor-like protein 1 (APLP1) and 2 (APLP2), which may help to mitigate loss of Aβ-mediated protection (53). Consistent with this model, APP, APLP1, and APLP2 and their nonamyloidogenic processing products show extensive functional redundancy (54), likely because of the gene duplication origin for this protein family. APP-KO mice also have an important additional limitation as models for the loss of Aβ-mediated protection. APP itself may be involved in central nervous system (CNS) immunity (55). It remains unclear how loss of activities normally mediated by full-length APP can be excluded as the source of attenuated infection resistance in APP-KO mice.

Genetically modified mice that lack proteases [BACE1 (β-site APP cleaving enzyme 1) and BACE2] for generating the Aβ family of peptides provide an alternative Aβ-null model. Consistent with our data, knockout BACE-KO mice that lack BACE have been reported to have marked immunodeficiency. Whereas neonatal mortality is below 2% under sterile conditions, in less stringently antiseptic environments, up to half of pups born to BACE-KO mice die from infections within the first 2 weeks of life (56). Benchmark tests for adaptive immunity have failed to identify defects in the response of BACE-KO mice to immune challenges. Findings for BACE-KO mice appear consistent with an innate immune deficiency and a possible normal protective role for Aβ. However, as with APP-KO mice, it is unclear how to demonstrate that the immunodeficiency in BACE-KO mice is specific for a loss of members of the Aβ family of peptides. Additional data are required to conclusively link the etiology of BACE-KO mouse immunodeficiency to low Aβ.

Our findings for Aβ and β-amyloid may have corollaries for amyloidopathies beyond AD. Protein fibrillization may be important not only for Aβ’s AMP activities but also for the normal actions of other amyloidosis-causing proteins. An association between amyloidosis and chronic bacterial infections has been recognized for almost a century (57), but the potential protective activities of host-generated amyloid have only recently emerged (4, 35, 58). At least six amyloidosis-associated peptides show antimicrobial activities, including amylin (59), atrial natriuretic factor (9), prion protein (60), cystatin C (61), lysozyme (5), and superoxide dismutase (62). Conversely, host AMPs have been identified that generate protective amyloids localized to infection sites (4). AA-type amyloidosis involves both systemic deposition of the acute-phase opsonin AMP serum amyloid A and has an infection-driven etiology (63). It remains to be determined whether serum amyloid A or other amyloidosis-causing AMPs also engage in nonpathogenic fibrillization pathways that help to protect against infection. However, should this prove to be the case, Aβ may be the first member of a new class of AMPs in which amyloid-generating activities protect against local infections but can also lead to widespread pathological amyloidosis.

If confirmed, our model carries important implications for understanding the pathogenesis of amyloidosis in AD. Excessive β-amyloid deposition may arise not from an intrinsically abnormal propensity of Aβ to aggregate but instead may be mediated by dysregulation of the brain’s innate immune system, for example, the consequence of an immune response mounted to microbial or sterile inflammatory stimuli. Our new model is congruent with the amyloid hypothesis and the importance of Aβ and β-amyloid in the neurodegenerative cascade of AD. However, our model would shift the modality of Aβ’s pathophysiology from abnormal stochastic behavior toward dysregulated antimicrobial activities.

Our study used genetically modified cell and animal models to generate data consistent with a normal physiological role for Aβ as an AMP. However, it remains unclear from these data how important a role Aβ plays in normal infection resistance. To address this question, additional data will be needed from wild-type animals modeling common physiological routes of infection. Further investigation will also be needed to clarify the extent to which the normal antimicrobial activities of Aβ identified in our study affect AD pathology.

It is important to emphasize that although infection of 5XFAD mice with S. Typhimurium seeded and accelerated β-amyloid deposition, the presence of a CNS infection is not implicit in our proposed AD amyloidosis model. Our work has identified what we believe is the normal role of Aβ. What drives widespread β-amyloid deposition in AD remains unclear. Among sterile inflammatory diseases, dysregulated innate immune responses rather than infections are emerging as drivers of pathology. Notably, two of the three confirmed AMP amyloidopathies are not linked to obvious infections (4, 9, 64). However, a large body of data accrued over nearly a century suggests that genuine infection may also play a role in AD etiology (65). Moreover, although a causal link to amyloidosis remains to be conclusively demonstrated, recent epidemiological findings have given increased prominence to the “infection hypothesis,” including studies linking brain fungal infection to AD (17, 18) and data showing that risk for the disease increases with infectious burden (66). Our findings do not constitute direct evidence of a role for infection in AD etiology. However, they do suggest a possible mechanism for pathogen-driven β-amyloid amyloidosis. Our data also suggest the possibility that a range of microbial organisms may be able to induce β-amyloid deposition, a possible reason for why a single pathogen species has not yet been identified that is overwhelmingly associated with AD. Future studies systematically characterizing microbial pathogens (viral, bacterial, and fungal) in the brains of AD patients, for example, by RNASeq, will be necessary to further interrogate whether specific clinical pathogens seed β-amyloid as part of the brain’s innate immune system. In any case, whether infectious or sterile inflammatory stimuli drive AD pathology, the pathways that regulate innate immunity in the brain may offer significant new targets for therapeutic intervention.