Human α-defensin 6 promotes mucosal innate immunity through self-assembled peptide nanonets

Paneth cells are specialized small intestinal epithelial cells that maintain intestinal homeostasis, in part by expressing and secreting antimicrobial peptides and proteins (1-3). Deficiencies of two human Paneth cell peptides, α-defensins HD5 and HD6, are associated with Crohn's disease, a chronic inflammatory bowel disease (3-5). HD5 is broadly antimicrobial (6-8) and can shape the gut microbiota in vivo (9). In contrast, HD6 exerts little antibacterial activity (Fig. S1, (10) and (8)), and its function is unknown.

To investigate HD6, we developed a transgenic mouse model wherein HD6 gene (DEFA6) expression was controlled by its endogenous promoter and restricted to Paneth cells (Fig. S2A,B). Transgenic HD6 expression occurred at levels commensurate with endogenous human and murine Paneth cell α-defensin production, and did not alter expression of other murine Paneth cell-derived antimicrobial (poly)peptides (Fig. S2C,D).

When HD6 transgenic mice and littermate controls were challenged intragastrically with 2 × 10⁸ colony forming units (CFU) of S. Typhimurium (STM), 50 % of wild-type animals but no transgenic mice had died six days post infection (P < 0.05) (Fig. 1A). Consistent with HD6's lack of direct antibacterial activity, the intestinal lumen of transgenic and wild-type mice contained similar bacterial numbers four days post infection. However, STM counts were 100-fold lower in Peyer’s patches (P < 0.001) and 10-fold lower in spleens (P < 0.001) of HD6 transgenic mice, than that in their wild-type littermates (Fig. 1B). The decreased bacterial burden in these tissues resembled findings in wild-type mice infected with an invasion-deficient STM invA mutant (11).

To determine whether HD6 inhibited invasion, intestinal epithelial cells were challenged with α-defensin-treated or untreated STM. The ability of HD6-treated bacteria to invade T84 epithelial cells was impaired in a dose-dependent manner (Fig. 1C,D). HD6 could reduce invasion by wild-type STM to levels of the minimally invasive invA mutant, which lacks a functional invasion-associated type III secretion system (T3SS) (Fig. 1C). Another human intestinal cell line, INT-407, and a mouse small intestinal cell line, MODE-K, exhibited the same pattern of HD6-mediated inhibition of STM invasion (Fig. S3). The same concentrations of HD5, the other human Paneth cell α-defensin (Fig. 1C), or the mouse Paneth cell α-defensins, cryptdins 2 and 3 (Fig. S4A), did not inhibit STM invasion. Immunofluorescence microscopy confirmed that HD6 treatment blocked invasion into intestinal epithelial cells (Fig. S5).

Similar STM invasion occurred in untreated and HD6-treated intestinal epithelial cells, suggesting that HD6 does not protect by altering host cells (Fig. S4B). The hypothesis that HD6 blocked invasion by interfering with synthesis of T3SS proteins was rejected by findings that STM treated with protective concentrations of HD6 showed normal expression of flhD and fliC, and even induced expression of hilA (a key SPI-1 activator) and invG, a structural protein of the T3SS-1 system (Fig. S6). Also excluded were the possibilities that HD6 reduced invasion by inhibiting bacterial adhesion (Fig. S7), or by altering the intestinal microbiota of the transgenic mice (Fig. S8).

To investigate the specificity of HD6, we performed invasion assays with Yersinia enterocolitica. This enteropathogenic bacterium invades cells by a different mechanism, involving attachment of its invasin protein (Inv) to host β-integrins (12). Since HD6 reduced Yersinia invasion to levels similar to the invasion-deficient Δinv mutant (Fig. S9), its ability to prevent bacterial invasion was not pathogen-specific.

Recent studies identified the Trp residue of human neutrophil α-defensin peptide (HNP)1 as a key functional amino acid (13). Whereas most Hominid α-defensins, including HD5, have a nonpolar aromatic residue, Trp, Tyr or Phe, at the corresponding position, HD6 and its Hominid orthologs contain His (Fig. S10). When we replaced His27 of HD6 with Trp, the resulting H27W-HD6 failed to block invasion (Fig. 1C, S4, S9), indicating that His27 was critical for this activity.

Seeking insight into the protective properties of HD6 and the role of His27, we used surface plasmon resonance (SPR) to examine binding to bacterial proteins. HD5 bound rapidly to immobilized Y. enterocolitica invasin and reached an equilibrium plateau by 3 min (Fig. 2A). In marked contrast, HD6 binding was less extensive and much slower. Binding of H27W-HD6 was enhanced relative to that of HD6, and like HD5, the H27W-HD6 binding curve rapidly reached a plateau denoting equal rates of association and dissociation. Because binding of HD6 to invasin was continuing to increase when the conventional 3 min binding period ended, we tested longer binding intervals, eventually adopting a multi-cycle approach to monitor binding over much longer periods (Fig. 2B,C). Because other α-defensin molecules self-associate (13-16), we measured the ability of solution-phase HD6 to bind HD6 immobilized on a biosensor. HD6 self-association rose progressively during a six-cycle experiment (Fig. 2B). In contrast, HD5 binding to HD5 reached a molar ratio close to 1 in each cycle, signifying dimer formation, and then declined to baseline during each intervening "washout" period. Unlike HD6, H27W-HD6 showed minimal self-association, indicating that His27 was critical to this property. Similarly, H27A-HD6, containing a less bulky Ala substitution, showed kinetics like H27W-HD6 (Fig. S11). Therefore, HD6 forms higher order assemblages with slower kinetics of association and dissociation, while HD5 and other α-defensins rapidly (and reversibly) form dimeric complexes.

In a six-cycle binding experiment using immobilized invasin as the target, HD5 oscillated in each binding cycle, rapidly reaching a plateau and then falling by about half during each wash period (Fig. 2C). The oscillations likely represented reversible association and dissociation of solution-phase HD5 to molecules of HD5 that bound the immobilized invasin. Whereas H27W-HD6 binding to invasin showed similar cyclic oscillations, , HD6 binding to invasin rose progressively, with a 100-fold increase from cycle one to cycle six (Fig. 2C). Similar results were observed when immobilized Salmonella flagellin anchored progressive HD6 self-assembly (Fig. 2D). These data suggested that HD6 molecules bound directly to immobilized proteins served as points of origin for progressive assembly of an extended structure composed of HD6 molecules. Importantly, even a low-level binding of HD6 to a target protein created anchoring sites for progressive HD6 self-assembly.

The SPR experiments indicated that HD6 binding to target proteins lacks the specificity typical of high affinity interactions. To explore this further, we tested HD6 binding to HIV glycoproteins gp41 and gp120. HD6 bound both proteins poorly until they were enzymatically deglycosylated (Fig. 2E,F). Thus, whereas primary binding of HD6 appears nonspecific with respect to the target protein's sequence, glycosylation can greatly reduce its binding.

Previous crystallographic studies suggested that HD6 can assemble into an elongated, highly ordered helical structure (15), significantly different from the dimeric structures formed by HD5 and HNPs (13-16). HD6 monomers associate into dimers (Fig 3A), but the respective monomers slide apart and form fewer backbone H-bonds between β-strands than that for HD5 or the HNPs. This atypical dimer is critically stabilized by dimer-dimer association (Fig. 3A), resulting in a stable tetramer – the repeating unit of the elongated HD6 structure (Fig. S12). HD6 tetramerization is mediated by extensive reciprocal inter-dimer interactions that include electrostatic interactions of the His27 imidazole side chain with the C-terminal carboxyl group of Leu32 (17). The molecular surface buried within the HD6 tetramer (an average value of 760 Ų/monomer) is quite large given the peptide's small size. Comparative structural analyses of HD6 with H27W-HD6 reveal that His27-mediated electrostatic interactions are critical for HD6 tetramer formation (Fig. 3) and higher-order oligomerization (Fig. S12-S14). The H27W substitution eliminates His27-Leu32 interactions important for HD6 dimer-dimer association (Fig. 3B, S14), as further discussed (18).

By scanning electron microscopy, HD6-treated STM were aggregated (Fig. 4A-C), and higher magnification revealed a net-like meshwork of fibrils that arose from the bacterial surface to entangle the bacteria and their flagella. Such nanonets were again observed in experiments using human small intestinal aspirates (Fig. S15). Latex beads coated with a bacterial protein supported formation of similar nanofibrils with wild-type HD6, but not with H27W-HD6, indicating that the structures seen in electron microscopy were not of bacterial origin (Fig. 4D, S16).

HD6 nanonets formed in vivo in a murine ileal loop model (19) wherein HD6 transgenic and littermate wild-type controls received pilocarpine to induce Paneth cell secretion, and ileal loops were inoculated with STM. Abundant nanonet structures that entangled and aggregated STM were observed (Fig. 4G,H, S17). In contrast, no nanonets were observed in wild-type mouse ileal loops (Fig. 4E,F, S17). Thus, HD6 can form small intestinal nanonets that entangled STM in vivo.

Because HD6 lacks lectin-like activity (20), it seemed unlikely to target carbohydrates on the bacterial surface. We therefore directed our attention to the predominant proteinaceous surface appendages of STM, flagellae and type I fimbriae (21). Compared to wild-type bacteria, we observed a decreased aggregation by HD6 of STM mutants lacking type I fimbriae or flagella (Fig. S18). Neither nanofibrils nor nanonets nor aggregation was evident in an HD6-treated Salmonella mutant lacking both fimbriae and flagella (Fig. 4I-L), suggesting that appendages function as HD6 anchoring points that trigger HD6 nanonet formation.

Together these data support a model wherein secreted HD6 molecules bind stochastically (i.e., randomly and at low levels) to any of the various proteinaceous bacterial surface molecules they encounter. These initially bound HD6 molecules provide a nidus that triggers a dynamic and deterministic process of self-assembly, building nanonets that can aggregate bacteria and/or impede the close physical contact with epithelial cells required for attachment or invasion. Highly expressed in Paneth cells at the base of small intestinal crypts, secreted HD6 will accumulate in the crypt and pericellular space beneath the mucus layer. Host proteins in this location are heavily glycosylated, leaving microbial intruders as the prime targets for HD6 binding.

Any microbial intruder that enters the small intestinal crypt, be it bacterial, fungal or protozoan, is a potential threat – especially since the stem cells that continually renew the intestinal epithelium reside at the crypt base (3). An intruder in this location would encounter HD6 and provide anchorage for the initial, low-level binding of this peptide. As this binding is not target sequence specific, HD6 can be effective against diverse threats. Because HD6 concentrations in the crypt are orders of magnitude higher than the highest concentrations (10 μg/ml) used in our experiments (22, 23), fibril and nanonet formation around such intruding microbes should be faster and in greater abundance than those described above. Since mouse Paneth cells lack an ortholog of HD6 (24), the transgenic mice benefit from expression of this human peptide. On the other hand, it is noteworthy that individuals with Crohn’s disease characteristically have an accumulation of invasive bacteria at the epithelial surface of their small intestine (5). A predisposition to this inflammatory bowel disease may, in part, be a result of a deficiency of HD6, as supported by the decreased expression of both HD5 and HD6 observed in Crohn’s disease of the ileum (4). Although the present experiments dealt primarily with two model Gram-negative bacteria, the general mechanism that emerged could apply to other bacteria, as well as fungi and protozoans.