Investigating the mechanism of action of aggregation-inducing antimicrobial Pept-ins

Abstract

Aggregation can be selectively induced by aggregation-prone regions (APRs) contained in the target proteins.Aggregation inducing antimicrobial peptides (Pept-ins) contain homologous sequences to APRs of target proteins and exert their bactericidal effect by causing aggregation of a large number of proteins. To better understand the mechanism of action of Pept-ins and the resistance mechanisms, we analysed the phenotypic, lipidomic, transcriptomic as well as genotypic changes of laboratory derived Pept-in-resistant E. coli mutator cells. The analysis showed that Pept-in resistance mechanism is dominated by a decreased Pept-in uptake, both in laboratory derived mutator cells and clinical isolates. Our data indicates that Pept-in uptake involves the electrostatic attraction between Pept-in and bacterial membrane and follows a complex mechanism potentially involving many transporters. Furthermore, it seems more challenging for bacteria to become resistant towards Pept-ins which are less dependent on electrostatic attraction for uptake, suggesting future Pept-ins should be selected for this property.

Is It Possible to Create Antimicrobial Peptides Based on the Amyloidogenic Sequence of Ribosomal S1 Protein of P. aeruginosa?

“…The special attention should be paid to the use of the amyloidogenic properties of bacterial proteins for targeted protein aggregation [35]. The development of synthetic amyloidogenic peptides has been shown to be a promising approach to combat pathogenic bacteria [36,37]. At the same time, it should be borne in mind that bacteria have different mechanisms of counteracting protein aggregation, for example, by directing amyloids into inclusion bodies [35].…”

Bacterial Protein Homeostasis Disruption as a Therapeutic Intervention

“…Comparing the transcriptomic profiles of P2-resistant strains to their ancestors showed that translation was the most affected gene ontology category and translation-related genes were predominantly down-regulated in P2-resistant strains ( Figure 3H ). This seems to confirm that Pept-ins act co-translationally: reducing translation rates and thereby decreasing the exposure of APRs could rendered bacteria somewhat resistant to Pept-in treatment, but the extent of this potential mechanism is limited since bacteria of course depend on translation for continued survival ( Wu et al, 2020 ). We expected a high translation rate to render bacterial proteostasis more susceptible to perturbation, but confusingly P2 induced a significantly higher amount of aggregation events in the CH184 mutant strain that has a slower translation elongation rate compared to wild-type E. coli .…”

“…No resistance development to Pept-ins was observed in our studies of wild-type bacteria ( Bednarska et al, 2016 ; Khodaparast et al, 2018 ) therefore we used a mutator strain to develop strains resistant to Pept-ins. Resistance development was slow and low-grade even in the mutator strain after serial-passaging the bacteria in the presence of sub-MIC concentration of P2 for 27 days ( Wu et al, 2020 ). Comparing the transcriptomic profiles of P2-resistant strains to their ancestors showed that translation was the most affected gene ontology category and translation-related genes were predominantly down-regulated in P2-resistant strains ( Figure 3H ).…”

“…This was a surprising result and needs to be further investigated, but seems to confirm that it is the high volume of protein turnover (the high number of polypeptide chains that are in the process of translation at any given time) that makes the proteostasis of bacteria vulnerable and not the high speed of translation itself. Currently, we think that the slower elongation rate in CH184 strain gives P2 a longer time window to act on the unfolded proteins during translation, rendering these proteins more prone to aggregation in the presence of Pept-ins and thus making CH184 more susceptible towards Pept-ins ( Wu et al, 2020 ).…”

“…However, this resistance mechanism was not observed in the resistant strains, indicating a) the clear benefits of designing antibiotics targeting a large number of targets and b) the difficulty of changing the targeted regions (the APRs that form part of the hydrophobic core of the protein) because this usually requires multiple mutations ( Langenberg et al, 2020 ). Phenotypic, lipidomic, transcriptomic, as well as genotypic changes of laboratory-derived Pept-in-resistant E. coli mutator cells revealed that preventing uptake was the main resistance mechanism to Pept-ins ( Wu et al, 2020 ) ( Figure 3I ).…”