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. 2022 Jun 7;61(11):1029-1040.
doi: 10.1021/acs.biochem.1c00762. Epub 2022 May 24.

Temporin B Forms Hetero-Oligomers with Temporin L, Modifies Its Membrane Activity, and Increases the Cooperativity of Its Antibacterial Pharmacodynamic Profile

Philip M Ferguson  1 , Maria Clarke  1 , Giorgia Manzo  1 , Charlotte K Hind  2 , Melanie Clifford  2 , J Mark Sutton  1   2 , Christian D Lorenz  3 , David A Phoenix  4 , A James Mason  1
Affiliations

Temporin B Forms Hetero-Oligomers with Temporin L, Modifies Its Membrane Activity, and Increases the Cooperativity of Its Antibacterial Pharmacodynamic Profile

Philip M Ferguson et al. Biochemistry. .

Abstract

The pharmacodynamic profile of antimicrobial peptides (AMPs) and their in vivo synergy are two factors that are thought to restrict resistance evolution and ensure their conservation. The frog Rana temporaria secretes a family of closely related AMPs, temporins A-L, as an effective chemical dermal defense. The antibacterial potency of temporin L has been shown to increase synergistically in combination with both temporins B and A, but this is modest. Here we show that the less potent temporin B enhances the cooperativity of the in vitro antibacterial activity of the more potent temporin L against EMRSA-15 and that this may be associated with an altered interaction with the bacterial plasma membrane, a feature critical for the antibacterial activity of most AMPs. Addition of buforin II, a histone H2A fragment, can further increase the cooperativity. Molecular dynamics simulations indicate temporins B and L readily form hetero-oligomers in models of Gram-positive bacterial plasma membranes. Patch-clamp studies show transmembrane ion conductance is triggered with lower amounts of both peptides and more quickly when used in combination, but conductance is of a lower amplitude and pores are smaller. Temporin B may therefore act by forming temporin L/B hetero-oligomers that are more effective than temporin L homo-oligomers at bacterial killing and/or by reducing the probability of the latter forming until a threshold concentration is reached. Exploration of the mechanism of synergy between AMPs isolated from the same organism may therefore yield antibiotic combinations with advantageous pharmacodynamic properties.

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Conflict of interest statement

The authors declare no competing financial interest.

Figures

Figure 1
Figure 1
Pharmacodynamic response of EMRSA-15 to the antibiotic challenge in MHB. EMRSA-15 was challenged with increasing concentrations of temporin L (TL), a 1:1 mol/mol ratio combination of temporin B and temporin L (TB/TL) or clinically relevant antibiotics. Curves shown are fits of averages of three independent repeated experiments (A). The cooperativity (kappa), pharmacodynamic MIC (zMIC), and maximum (ψmax) and minimum (ψmin) growth rates are provided in Table 2, while one-way ANOVA with Tukey posthoc test multiple comparisons for kappa, highlighting the differences in cooperativity between the AMPs and antibiotics (B). ns p > 0.05; *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.
Figure 2
Figure 2
Temporin L and temporin B form hetero-oligomers in MD simulations of POPG bilayer challenge. Top zoom views of snapshots (A–C) and analysis of the average number of contacts for each residue involved in any homo- or hetero-oligomerization (D–F) in simulations of eight temporin B (A/D), four temporin L (blue) and four temporin B (green) (B/E), or eight temporin L (C/F) peptides inserting into a 512 POPG lipid bilayer. Time-resolved analysis of the maximum number of peptides in any assembly (G) and the number of any such assemblies (H).
Figure 3
Figure 3
Hetero-oligomerization reduces local membrane disordering by temporin L in MD simulations of POPG bilayer challenge. Order parameter profiles, averaged over the duration of the 200 ns simulations, are shown for lipids within 4 Å of each inserting peptide (A,B) or for the whole bilayer (C,D). Comparisons are provided for temporin B (A,C) or temporin L (B,D). Data is an average of two independently repeated simulations for each condition.
Figure 4
Figure 4
Patch-clamp analysis of the challenge of a DPhPG bilayer with a combination of temporin L and temporin B. The concentrations of temporin L (0.84 μM) and temporin B (2.92 μM) used correspond to the minimum amount of the combination needed to induce conductance and are equal to 1/12 of the concentrations needed to induce conductance when each peptide is applied alone. A representative of six traces (A) together with a frequency plot of events of varying amplitude across all six traces (B). The average time taken for conductance to begin after peptide addition (latency) shows conductance begins more rapidly for the combination than temporin L alone (C). One-way ANOVA with a Tukey posthoc test, *p < 0.05, ***p < 0.001.
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