BioSAXS Measurements Reveal That Two Antimicrobial Peptides Induce Similar Molecular Changes in Gram-Negative and Gram-Positive Bacteria.

Two highly active short broad-spectrum AMPs (14D and 69D) with unknown mode of action have been investigated in regards to their effect against the Gram-negative bacteria Escherichia and the Gram-positive bacteria methicillin-resistant (MRSA). Minimal inhibitory concentration (MIC) measurements using a cell density of 10 cfu/ml resulted in values between 16 and 32 µg/ml. Time-kill experiments using 10 cfu/ml revealed complete killing, except for 69D in combination with MRSA, where bacterial load was reduced a million times. Small-angle X-ray scattering of biological samples (BioSAXS) at 10 cfu/ml was applied to investigate the ultrastructural changes in and MRSA in response to these two broad-spectrum AMPs. In addition, electron microscopy (EM) was performed to visualize the treated and non-treated bacteria. As expected, the scattering curves generated using BioSAXS show the ultrastructure of the Gram-positive and Gram-negative bacteria to be very different (BioSAXS is not susceptible to the outer shape). After treatment with either peptide, the scattering curves of and MRSA cells are much more alike. Whereas in EM, it is notoriously difficult to observe changes for spherical Gram-positives; the BioSAXS results are superior and reveal strongly similar effects for both peptides induced in Gram-positive as well as Gram-negative bacteria. Given the high-throughput possibility and robust statistics, BioSAXS can support and speed up mode of action research in AMPs and other antimicrobial compounds, making a contribution toward the development of urgently needed drugs against resistant bacteria.