The increasing development of microbial resistance to classical antimicrobial agents has led to the search for novel antimicrobials. Antimicrobial peptides (AMPs) derived from scorpion and snake venoms offer an attractive source for the development of novel therapeutics. Smp24 (24 amino acids [aa]) and Smp43 (43 aa) are broad-spectrum AMPs that have been identified from the venom gland of the Egyptian scorpion Scorpio maurus palmatus and subsequently characterized. Using a DNA microarray approach, we examined the transcriptomic responses of Escherichia coli to subinhibitory concentrations of Smp24 and Smp43 peptides following 5 h of incubation. Seventy-two genes were downregulated by Smp24, and 79 genes were downregulated by Smp43. Of these genes, 14 genes were downregulated in common and were associated with bacterial respiration. Fifty-two genes were specifically upregulated by Smp24. These genes were predominantly related to cation transport, particularly iron transport. Three diverse genes were independently upregulated by Smp43. Strains with knockouts of differentially regulated genes were screened to assess the effect on susceptibility to Smp peptides. Ten mutants in the knockout library had increased levels of resistance to Smp24. These genes were predominantly associated with cation transport and binding. Two mutants increased resistance to Smp43. There was no cross-resistance in mutants resistant to Smp24 or Smp43. Five mutants showed increased susceptibility to Smp24, and seven mutants showed increased susceptibility to Smp43. Of these mutants, formate dehydrogenase knockout (fdnG) resulted in increased susceptibility to both peptides. While the electrostatic association between pore-forming AMPs and bacterial membranes followed by integration of the peptide into the membrane is the initial starting point, it is clear that there are numerous subsequent additional intracellular mechanisms that contribute to their overall antimicrobial effect. IMPORTANCE The development of life-threatening resistance of pathogenic bacteria to the antibiotics typically in use in hospitals and the community today has led to an urgent need to discover novel antimicrobial agents with different mechanisms of action. As an ancient host defense mechanism of the innate immune system, antimicrobial peptides (AMPs) are attractive candidates to fill that role. Scorpion venoms have proven to be a rich source of AMPs. Smp24 and Smp43 are new AMPs that have been identified from the venom gland of the Egyptian scorpion Scorpio maurus palmatus, and these peptides can kill a wide range of bacterial pathogens. By better understanding how these AMPs affect bacterial cells, we can modify their structure to make better drugs in the future.

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The global increase in antimicrobial resistance development has created a critical need for the development of novel antimicrobial agents. Antimicrobial peptides (AMPs) are one such class of novel antimicrobials, but they have several disadvantages such as high manufacturing cost and low selectivity that must be addressed before they can achieve clinical use. In this study the relatively large size and high cytotoxic activity of the natural, venom derived AMP Smp24 were addressed using several different approaches. A formulation approach was investigated by incorporation of the peptide into sol-gel coatings in order to inhibit or prevent the growth of bacterial biofilms, showing that the unique physiochemical properties of the peptide can be utilized to improve and control the properties of the coating. Furthermore, a drug design approach was also used, based on several stages. Firstly, an investigation of the peptide structure was done creating a regional breakdown of the 3D structure and mechanism of action of the early stages of the pore formation using planar patch clamp electrophysiology. A bridge was built between the structure and mechanism via the use of molecular dynamics simulations of the peptide-bilayer interactions in order to establish a structure mechanism relationship. Based on this relationship several truncated variants of the parent peptide were designed an evaluated, first in silico showing that the simulations can give direct feedback during the design process and in vitro showing that two of the truncated analogs had both smaller size, improved antimicrobial properties and reduced cytotoxic properties compared to the parent peptide. The best analogs were further developed via amino acids substitutions in two iterations, with a total of 19 analogs evaluated in silico with 11 of those also being evaluated in vitro. Again, multiple analogs were produced with improved antimicrobial activity and selectivity. Overall, additional insight into the structure and biophysical behaviour of Smp24 was gained and utilized to guide the design of new analogs with significantly smaller size and improved selectivity without loss of antimicrobial activity relative to the parent peptide.