(A-B) In vivo challenge of mice with an intragastric inoculum of S. Typhimurium (2 × 10⁸ CFU). (A) Kaplan-Meyer survival curve of 6 week female wild-type (n = 15) and HD6 +/− transgenic littermates (n = 9) with a scheduled experimental endpoint of 6 days. Log-rank test, * P < 0.05. The data are representative of three independent experiments. (B) S. Typhimurium bacterial load from colon fecal pellets, Peyer’s patches (3/mouse), and spleen tissue of 6 week female wild-type (n = 4) and HD6 +/− transgenic female littermate mice (n = 6) 4 days post-infection. Error bars represent SEM. ** P < 0.001, t-test. The data are representative of three independent experiments. (C-E) In vitro invasion assays. (C) S. Typhimurium were pre-treated for 1 h with either 10 μg/ml of HD5, HD6 or H27W-HD6, or with vehicle alone (WT untreated). Invasion-deficient S. Typhimurium (invA mutant) were treated with vehicle alone. Bacteria were then allowed to invade the epithelial cells (MOI of 10) for 1 h. Cells were then washed, treated with gentamicin, and then lysed to quantitate intracellular bacteria (expressed as % of initial inoculum). Data represent the average of three experiments, and are representative of more than 9 independent experiments. (D) Concentration-dependence of the HD6 activity in assays as described in (C). Error bars represent SEM. *P < 0.01, ** P < 0.001, *** P < 0.0001 compared to WT vehicle, t-test. Data represent the average of three experiments, and are representative of more than 9 independent experiments.

The SPR biosensors presented the following amounts of immobilized ligands (molecule covalently ligated to the biosensor surface), in resonance units (RU): invasin, 2338 RU; HD5, 1158 RU; HD6, 1100 RU, H27W-HD6, 2204 RU; flagellin, 2206 RU; gp120, 8200 RU; gp41, 5600 RU. The defensin peptides were prepared in HBS-EP buffer and traversed the biosensors at either 50 μl/min (A) or 25 μl/min (B - F). Each 15 min cycle in panels B - F included a binding period of 12.5 min, followed by a wash period of approximately 2.5 min. Molar ratio (MR) calculations, which take into account the relative masses of the analytes and ligands and the number of RU presented by each biosensor, are described in the methods. Shown are representative data from at least three separate experiments. (E) Binding of HD6 to gp120-LAV before (triangles) and after enzymatic deglycosylation (closed circles) of the same biosensor chip. Shown are averaged data from six experiments, with error bars representing SEM. P < 0.002, Mann-Whitney Rank-sum test. Also shown are data from a second set of biosensor chips (n=6) presenting the same quantity of deglycosylated gp120 (open circles). (F) Binding of HD6 to gp41-LAV before and after enzymatic deglycosylation of the same biosensor chip. Six experiments were done before enzymatic deglycosylation and six more were done afterwards. Error bars represent SEM. The differences in each cycle were statistically significant with a P < 0.01, Mann-Whitney Rank-sum test.

(A) The previously described HD6 tetramer formed by four crystallographically independent monomers in the asymmetric unit of crystal (PDB code 1ZMQ) (). ‘Canonical’ dimers are colored yellow (monomers a and b) and green (monomers c and d), hydrogen bonds shown as blue dashes. HD6 dimerization is mediated primarily through the hydrogen bonds formed by the backbone atoms of the β2 strands, involving two reciprocal hydrogen bonds between a pair of Thr21 and a single hydrogen bond between Met23 N and Thr19 O. Dimer-dimer association is mediated by the backbone atoms of the β1 strands, forming four H-bonds between Phe2 and Cys4 of monomers a and c and two between Ala1 N of monomers a and c and Cys6 O of monomers d and b. In addition, the HD6 tetramer is stabilized by electrostatic interactions involving the side chains of two His27 residues (red arrow) and the C-terminal carboxyl groups of two Leu32 residues, among others (). The orientation of the imidazole ring of His27 is supported by side-chain stacking interactions with Phe2. (B) Equivalent putative tetrameric assembly of four H27W-HD6 monomers from two symmetry-related ‘canonical’ dimers in orange and cyan. The H27W mutation has little impact on the tertiary structure of the mutant defensin (), and does not change the inter-chain backbone H-bonding pattern seen in wild-type HD6. However, what does change is the quaternary structure (), where the mutation significantly debilitates HD6 molecules to assemble into high-order oligomers.

(A-D) Scanning electron microscopy (SEM) of in vitro nanonet formation. Wild-type S. Typhimurium incubated with vehicle (A) or HD6 (10 μg/ml, B,C) in Tris-maleate buffer, as described in the methods. Bar = 5 μm. Magnification 5000X (B) and 10,000X (C). The white rectangle (B) and the asterisks (B,C) highlight a prominent nanonet. Note that the longer and wider structures are flagellae (white arrowheads), many of which are entangled with other cobweb-like nanonets, but are not evident with ΔfliC fljB (). SEM of protein A-coated polystyrene beads (D) incubated with HD6 (1 μg/ml) for 5 min at room temperature in 50 mM Tris-maleate buffer. The beads were then washed, fixed, and processed for SEM. Data are representative of six independent experiments. (E-H) SEM of WT (E,F) and HD6 transgenic (G,H) mouse ileal loop directly inoculated with S. Typhimurium. Bar = 2 μm. In a double-blinded data acquisition and scoring evaluation, three individuals scored 12 of 12 such images correctly for mouse genotype. P < 0.001, Fisher’s exact test. See methods for details. Data are representative of two independent experiments. (I-L) SEM of wild-type S. Typhimurium (I,J) and Δfim ΔfliC fljB (K,L) treated with vehicle alone (I,K) or 10 μg/ml HD6 (J,L). Bar = 10 μm. See methods for details. Data are representative of three independent experiments.