Antimicrobial Peptide Induced-Stress Renders Staphylococcus aureus Susceptible to Toxic Nucleoside Analogs.

Cationic antimicrobial peptides (AMPs) are active immune effectors of multicellular organisms and are also considered as new antimicrobial drug candidates. One of the problems encountered when developing AMPs as drugs is the difficulty of reaching sufficient killing concentrations under physiological conditions. Here, using pexiganan, a cationic peptide derived from a host defense peptide of the African clawed frog and the first AMP developed into an antibacterial drug, we studied whether sub-lethal effects of AMPs can be harnessed to devise treatment combinations. We studied the pexiganan stress response of Staphylococcus aureus at sub-lethal concentrations using quantitative proteomics. Several proteins involved in nucleotide metabolism were elevated, suggesting a metabolic demand. We then show that Staphylococcus aureus is highly susceptible to antimetabolite nucleoside analogs when exposed to pexiganan, even at sub-inhibitory concentrations. These findings could be used to enhance pexiganan potency while decreasing the risk of resistance emergence, and our findings can likely be extended to other antimicrobial peptides.

Resistance Evolution Against Antimicrobial Peptides in Staphylococcus aureus Alters Pharmacodynamics Beyond the MIC.

Antimicrobial peptides (AMPs) have been proposed as a promising class of new antimicrobials partly because they are less susceptible to bacterial resistance evolution. This is possibly caused by their mode of action but also by their pharmacodynamic characteristics, which differ significantly from conventional antibiotics. Although pharmacodynamics of antibiotic resistant strains have been studied, such data are lacking for AMP resistant strains. Here, we investigated if the pharmacodynamics of the Gram-positive human pathogen Staphylococcous aureus evolve under antimicrobial peptide selection. Interestingly, the Hill coefficient (kappa κ) evolves together with the minimum inhibition concentration (MIC). Except for one genotype, strains harboring mutations in menF and atl, all mutants had higher kappa than the non-selected sensitive controls. Higher κ results in steeper pharmacodynamic curve and, importantly, in a narrower mutant selection window. S. aureus selected for resistance to melittin displayed cross resistant against pexiganan and had as steep pharmacodynamic curves (high κ) as pexiganan-selected lines. By contrast, the pexiganan-sensitive tenecin-selected lines displayed lower κ. Taken together, our data demonstrate that pharmacodynamic parameters are not fixed traits of particular drug/strain interactions but actually evolve under drug treatment. The contribution of factors such as κ and the maximum and minimum growth rates on the dynamics and probability of resistance evolution are open questions that require urgent attention.

In vivo exposure of insect AMP resistant Staphylococcus aureus to an insect immune system.

Antimicrobial peptides (AMPs) are important immune effectors in insects. Bacteria have a limited number of ways to resist AMPs, and AMP-resistance is often costly. Recently, it has become clear that AMP activities in vitro and in vivo differ. Although some studies have followed the in vivo survival of AMP resistant pathogens, studying a pathogen resistant to the AMPs of that particular host has never been reported. Here, we infected the mealworm beetle Tenebrio molitor with Staphylococcus aureus strains that were evolved in vitro in the presence of one or two antimicrobial peptides from T. molitor. We found that the Tenebrio immune system could clear mutant Tenecin resistant strains at least as efficiently as sensitive controls. The bacterial load of Tenecin resistant S. aureus segregated by mutation. Strains with mutations in both the pmt and rpo operons showed the highest in vivo survival and therefore showed the lowest fitness cost amongst the evolved resistance mutations. In contrast, Tenecin resistant strains with mutations in the nsa and rpo operons showed much lower survival within the hosts. Our study shows that Tenecin resistant strains are phagocytosed at a lower rate. The nsa/rpo mutants were phagocytosed at a higher rate than other Tenecin resistant S. aureus strains. The differences in resistance against AMPs and phagocytosis did not translate into changes in virulence. AMP resistance, while a prerequisite for an infection in vertebrates, does not provide a survival advantage to S. aureus in a host environment that is dominated by AMPs.

Genomics of experimental adaptation of Staphylococcus aureus to a natural combination of insect antimicrobial peptides.

Antimicrobial peptides (AMP) are highly conserved immune effectors across the tree of life and are employed as combinations. In the beetle Tenebrio molitor, a defensin and a coleoptericin are highly expressed in vivo after inoculation with S. aureus. The defensin displays strong in vitro activity but no survival benefit in vivo. The coleoptericin provides a survival benefit in vivo, but no activity in vitro. This suggests a potentiating effect in vivo, and here we wanted to investigate the effects of this combination on resistance evolution using a bottom-approach in vitro starting with a combination of two abundant AMPs only. We experimentally evolved S. aureus in the presence of the defensin and a combination of the defensin and coleoptericin. Genome re-sequencing showed that resistance was associated with mutations in either the pmt or nsa operons. Strains with these mutations show longer lag phases, slower Vmax, and nsa mutants reach lower final population sizes. Mutations in the rpo operon showed a further increase in the lag phase in nsa mutants but not in pmt mutants. In contrast, final MICs (minimum inhibitory concentrations) do not differ according to mutation. All resistant lines display AMP but not antibiotic cross-resistance. Costly resistance against AMPs readily evolves for an individual AMP as well as a naturally occurring combination in vitro and provides broad protection against AMPs. Such non-specific resistance could result in strong selection on host immune systems that rely on cocktails of AMPs.

Lucifensin II, a defensin of medicinal maggots of the blowfly Lucilia cuprina (Diptera: Calliphoridae).

A novel homolog of insect defensin, designated lucifensin II (Lucilia cuprina Wiedemann [Diptera: Calliphoridae] defensin), was purified from hemolymph extract from larvae of the blowfly L. cuprina. The full-length primary sequence of this peptide of 40 amino acid residues and three intramolecular disulfide bridges was determined by electrospray ionization-orbitrap mass spectrometry and Edman degradation and is almost identical to the previously identified sequence of lucifensin (Lucilia sericata Meigen defensin). Lucifensin II sequence differs from that of lucifensin by only one amino acid residue, that is, by isoleucine instead of valine at position 11. The presence of lucifensin II also was detected in the extracts of other larval tissues, such as gut, salivary glands, fat body, and whole body extract.