Figure 3. Staphylococcus aureus PSMα Is Increased on Netherton syndrome Skin and Promotes Epidermal Protease Activity.

(A) Normalized counts of the gene psmα detected from metagenomic samples.

(B) Relative abundance of S. aureus psmα mRNA isolated from skin swabs of healthy and NS non-lesional and lesional skin (n, number of swabs assessed per condition). Results represent mean ± SEM, and a non-parametric unpaired Kruskal-Wallis test was used to determine statistical significance: *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001.

(C) Spearman correlation between S. aureus (SA) CFU/cm² and the relative abundance of S. aureus psmα mRNA isolated from skin swabs. Each dot represents an individual swab.

(D) Pearson correlation between the trypsin activity induced in neonatal human epidermal keratinocytes (NHEKs) after culture for 24 h with 5% supernatant of clinical S. aureus (SA) isolates from NS skin and the relative abundance of psmα mRNA level in the same SA isolates. Each dot represents an individual SA isolate.

(E–G) Epicutaneous application of 1e⁷ CFU/cm² of S. aureus clinical isolates on murine back skin for 48 h (n = 3 per group). For each NS subject, one lesional S. aureus isolate with a high psmα expression was selected for mouse skin application.

(E) Visual representation of murine back skin after 48 h colonization with 1e⁷ CFU/cm² SA isolates. (F and G) Analysis of (F) epidermal barrier damage (TEWL), trypsin activity, and (G) qPCR analysis of inflammatory cytokines stimulated in murine skin by clinical SA NS isolates 1–10. qPCR cytokine levels (Ifng, Il4, Il17a, Il17f, Il6, and Il1b) are normalized to the housekeeping gene Gapdh.

(H) Relative abundance of SPINK5 mRNA in NHEKs that were treated with scrambled control or SPINK5 siRNA (iSPINK5) (n = 3 per condition). Each dot represents an individual sample. Results represent mean ± SEM, and Student’s t test was used to determine statistical significance: *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001.

(I) Trypsin activity from conditioned medium of NHEKs that were pretreated with scrambled control or SPINK5 siRNA (iSPINK5) and then cultured for 24 h with S. aureus synthetic PSMα3 peptide (0, 1, 2.5, 5, and 10 μg/mL) (n = 4 per condition). Results represent mean ± SEM, and two-way ANOVA was used to determine statistical significance: *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001.

In (E)–(I), experiments are representative of two independent experiments. See also Figure S5.