Adaptively evolved human oral actinomyces-sourced defensins show therapeutic potential (2021)
Figure 5

Figure 5. The therapeutic potential of AMSIN.

A An inhibition zone-based assay for evaluating the stability of AMSIN in H2O or normal mouse serum. The bacterium used was BM and the peptide dose was 0.2 nmol/well. Mean ± SD of three biological replicates is displayed. P values were obtained by Mann-Whitney U test (*P<0.05, ns: no significance; exact P values are listed in Table EV7).

B Hemolysis by AMSIN. Meucin-18 was used as a positive control. Mean ± SD of three technical replicates is displayed.

C Comparison of cytotoxic effects of AMSIN and LL-37 on HL-60 cells. Mean ± SD of three biological replicates is displayed. P values were obtained by Student's t-test or Mann-Whitney U test (the latter indicated by a red asterisk; *P<0.05, ***P<0.001, ns: no significance; exact P values are listed in Table EV7).

D An inhibition-zone assay showing the inability of AMSIN to bind DNA. The bacterium used was MRSA P1374 and the peptide dose was 1.0 nmol/well. Mean ± SD of three biological replicates is displayed. P values were obtained by Mann-Whitney U (ns: no significance; exact P values are listed in Table EV7).

E The effect of AMSIN on human ion channels expressed in the CNS and heart. The asterisks mark steady state current traces after administering 100 μM AMSIN.

F Peptide-induced IL-8 release. A549 cells were treated with AMSIN or LL-37 for 24 h. IL-8 levels in culture supernatants from the FBS-containing medium (left) or the serum-free medium (right) were measured with an ELISA, as described in Materials and Methods. Mean ± SD of three biological replicates is displayed. P values were obtained by Student's t-test or Mann-Whitney U test (the latter indicated by a red asterisk or ns; *P<0.05, **P<0.01, ***P<0.001, ns: no significance; exact P values are listed in Table EV7).

G Peptide-induced MCP-1 release. HL-60 cells were treated with AMSIN or LL-37 for 24 h. MCP-1 levels in culture supernatants from the FBS-containing medium (left) or the serum-free medium (right) were measured with an ELISA. Mean ± SD of three biological replicates is displayed. P values were obtained by Student's t-test or Mann-Whitney U test (the latter indicated by a red asterisk or ns; *P<0.05, **P<0.01, ns: no significance; exact P values are listed in Table EV7).

H Surface plasmon resonance (SPR)-based assay detecting anti-AMSIN antibodies in mouse serum using Biacore T100. Sera were taken from three AMSIN-treated mice (Samples 1-3) and one control mouse only injected with 0.9% NaCl. The sensorgrams present data of subtracting the background SPR signals from the control. Note: the abnormal peaks at 30 and 90 s yielded by the equipment due to sample injections were artificially removed. Insect, schematic diagram of SPR experiment in which 1:100 diluted serum was used as analyte to flow over the AMSIN-immobilized CM5 chip.

I Treatment of pneumococcal pneumonia. Five mice per sampling point were inoculated with SP D39 through the nasopharynx and 24 h later the animals received AMSIN or penicillin (as a positive control) at different doses (AMSIN, at the doses of 8.35, 16.7 and 33 mg per kg; penicillin at 8.35, 16.7 and 30 mg per kg) via intramuscular injection (i.m.). One day after treatment, the animals were necropsied and their lungs were removed and homogenized for the determination of the level of SP, as described in the Materials and Methods section. Each point represents the determination from a single animal and the line shows the mean log value. Mean ± SD of five to ten biological replicates is displayed. P values were obtained by Student's t-test or Mann-Whitney U test (the latter indicated by red asterisks; **P<0.01, ***P<0.001 denotes significant reduction in CFU compared with the saline group; exact P values are listed in Table EV7).

J Survival after peritoneal infection with MRSA P1374. The survival fraction of the vehicle-treated control group was zero out of 11 mice and the treatment group contained six mice and all survived.

Source data are available online for this figure.