An inhibitory mechanism of action of coiled‐coil peptides against type three secretion system from enteropathogenic Escherichia coli

Abstract

Human pathogenic gram‐negative bacteria, such as enteropathogenic Escherichia coli (EPEC), rely on type III secretion systems (T3SS) to translocate virulence factors directly into host cells. The coiled‐coil domains present in the structural proteins of T3SS are conformed by amphipathic alpha‐helical structures that play an important role in the protein‐protein interaction and are essential for the assembly of the translocation complex. To investigate the inhibitory capacity of these domains on the T3SS of EPEC, we synthesized peptides between 7 and 34 amino acids based on the coiled‐coil domains of proteins that make up this secretion system. This analysis was performed through in vitro hemolysis assays by assessing the reduction of T3SS‐dependent red blood cell lysis in the presence of the synthesized peptides. After confirming its inhibitory capacity, we performed molecular modeling assays using combined techniques, docking‐molecular dynamic simulations, and quantum‐mechanic calculations of the various peptide‐protein complexes, to improve the affinity of the peptides to the target proteins selected from T3SS. These techniques allowed us to demonstrate that the peptides with greater inhibitory activity, directed against the coiled‐coil domain of the C‐terminal region of EspA, present favorable hydrophobic and hydrogen bond molecular interactions. Particularly, the hydrogen bond component is responsible for the stabilization of the peptide‐protein complex. This study demonstrates that compounds targeting T3SS from pathogenic bacteria can indeed inhibit bacterial infection by presenting a higher specificity than broad‐spectrum antibiotics. In turn, these peptides could be taken as initial structures to design and synthesize new compounds that mimic their inhibitory pharmacophoric pattern.

Peptidomimetics as Potential Anti-Virulence Drugs Against Resistant Bacterial Pathogens

“…Synthetic coiled-coil peptides also interfered with the assembly and functionality of NF-T3SS in enteropathogenic Escherichia coli (EPEC), enterohemorrhagic E. coli O157:H7 (EHEC), and the murine pathogen Citrobacter rodentium ( Larzabal et al, 2010 , 2013 ). These peptides appear to act by disrupting the oligomerization of the NF-T3SS system EspA protein by interacting with the C-terminal coiled-coil domain of EspA ( Larzabal et al, 2019 ). Coiled-coil peptides protected mice from C. rodentium infection, avoiding intestinal damage ( Larzabal et al, 2013 ).…”

“…On the other hand, as an emergent strategy, there is an urgent demand for discovery, design, and development of anti-virulence drugs. Natural peptide-based drugs have been identified as promising anti-virulence agents; however, these types of drugs tend to be associated with proteolytic instability, immunogenicity, toxicity, and low bioavailability, which could limit their applicability as anti-virulence agents ( Overhage et al, 2008 ; Kudryashova et al, 2017 ; Larzabal et al, 2019 ; Lenci and Trabocchi, 2020 ; Rezende et al, 2021 ). In this context, peptidomimetic compounds could be a valuable source of anti-virulence drugs, since they can overcome some of the shortcomings associated with natural peptide-based drugs ( De La Fuente-Núñez et al, 2015 ; Duprez et al, 2015 ).…”

On the road to structure-based development of anti-virulence therapeutics targeting the type III secretion system injectisome

“…28,59,62 The mediating PxxP turn is highly conserved in all SctF homologues, 28,61 with mutational analysis showing elevated effector secretion, suggesting that this structural motif may play a role in regulation of secretion. 61 Notably, this common feature of the T3SS has been exploited for drug design of coiled-coil peptide inhibitors, 68,69 as will be discussed below.…”

“…Building off of previous data, 68 Larzàbal and colleagues (2019) sought to understand the mechanism of inhibition of these inhibitory peptides using molecular modelling techniques. 69 Using the truncated EspA structure from the chaperone (CesA)-substrate complex as their model, the authors used molecular dynamic simulations to design and evaluate peptides that may inhibit the oligomerization of the EspA filament (tip -SctA) structure. In their previous work, two promising peptides were developed, termed CoilA and CoilB, both exhibiting approximately 95% inhibition of EPEC-induced RBC haemolysis and protected murine models from colonic injury during C. rodentium (possesses the same T3SS-encoded pathogenicity island -LEEas EPEC/EHEC) infection.…”

Antimicrobial peptides properties beyond growth inhibition and bacterial killing

“…Beyond the anti-T3SS of natural peptides, the strategy of using polypeptides that mimic some components of the systems and that compete with the binding of the natural bacterial components was effective to inhibit the system in Chlamydia, Salmonella, and Shigella, blocking their entrance to eukaryotic cells in cultures ( McShan & De Guzman, 2015 ). Similarly, in enteropathogenic E. coli (EPEC), coiled-coil peptide mimetics, analogs of the EspA, EscF, and CesA proteins (CoilA, Coil B and CesA2) of its T3SS, inhibit the T3SS mediated hemolysis ( Larzábal et al, 2019 ).…”