In light of an increasing number of antibiotic-resistant bacterial strains, it is essential to understand an action imposed by various antimicrobial agents on bacteria at the molecular level. One of the leading mechanisms of killing bacteria is related to the alteration of their plasmatic membrane. We study bio-inspired peptides originating from natural antimicrobial proteins colicins, which can disrupt membranes of bacterial cells. Namely, we focus on the α-helix H1 of colicin U, produced by bacterium Shigella boydii, and compare it with analogous peptides derived from two different colicins. To address the behavior of the peptides in biological membranes, we employ a combination of molecular simulations and experiments. We use molecular dynamics simulations to show that all three peptides are stable in model zwitterionic and negatively charged phospholipid membranes. At the molecular level, their embedment leads to the formation of membrane defects, membrane permeation for water, and, for negatively charged lipids, membrane poration. These effects are caused by the presence of polar moieties in the considered peptides. Importantly, simulations demonstrate that even monomeric H1 peptides can form toroidal pores. At the macroscopic level, we employ experimental co-sedimentation and fluorescence leakage assays. We show that the H1 peptide of colicin U incorporates into phospholipid vesicles and disrupts their membranes, causing leakage, in agreement with the molecular simulations. These insights obtained for model systems seem important for understanding the mechanisms of antimicrobial action of natural bacteriocins and for future exploration of small bio-inspired peptides able to disrupt bacterial membranes.

• Klíčová slova
• Colicins, Leakage, Lipid membranes, Molecular dynamics, Permeation, Poration,
• MeSH
• fosfatidylcholiny chemie   MeSH
• fosfatidylethanolaminy chemie   MeSH
• fosfolipidy chemie   MeSH
• koliciny chemie   metabolismus   farmakologie   MeSH
• konformace proteinů, alfa-helix MeSH
• permeabilita účinky léků   MeSH
• sekvence aminokyselin MeSH
• Shigella boydii metabolismus   MeSH
• simulace molekulární dynamiky MeSH
• unilamelární lipozómy chemie   metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• 1-palmitoyl-2-oleoylphosphatidylcholine MeSH Prohlížeč
• 1-palmitoyl-2-oleoylphosphatidylethanolamine MeSH Prohlížeč
• fosfatidylcholiny MeSH
• fosfatidylethanolaminy MeSH
• fosfolipidy MeSH
• koliciny MeSH
• unilamelární lipozómy MeSH

Colicin U is a protein produced by the bacterium Shigella boydii (serovars 1 and 8). It exerts antibacterial activity against strains of the enterobacterial genera Shigella and Escherichia Here, we report that colicin U forms voltage-dependent pores in planar lipid membranes; its single-pore conductance was found to be about 22 pS in 1 M KCl at pH 6 under 80 mV in asolectin bilayers. In agreement with the high degree of homology between their C-terminal domains, colicin U shares some pore characteristics with the related colicins A and B. Colicin U pores are strongly pH dependent, and as we deduced from the activity of colicin U in planar membranes at different protein concentrations, they have a monomeric pore structure. However, in contrast to related colicins, we observed a very low cationic selectivity of colicin U pores (1.5/1 of K+/Cl- at pH 6) along with their atypical voltage gating. Finally, using nonelectrolytes, we determined the inner diameter of the pores to be in the range of 0.7 to 1 nm, which is similar to colicin Ia, but with a considerably different inner profile.IMPORTANCE Currently, a dramatic increase in antibiotic resistance is driving researchers to find new antimicrobial agents. The large group of toxins called bacteriocins appears to be very promising from this point of view, especially because their narrow killing spectrum allows specific targeting against selected bacterial strains. Colicins are a subgroup of bacteriocins that act on Gram-negative bacteria. To date, some colicins are commercially used for the treatment of animals (1) and tested as a component of engineered species-specific antimicrobial peptides, which are studied for the potential treatment of humans (2). Here, we present a thorough single-molecule study of colicin U which leads to a better understanding of its mode of action. It extends the range of characterized colicins available for possible future medical applications.

• Klíčová slova
• Shigella boydii, black lipid membrane, colicin U, ion-selectivity, membrane pores,
• MeSH
• buněčná membrána metabolismus   MeSH
• chlorid draselný farmakologie   MeSH
• gating iontového kanálu MeSH
• koliciny metabolismus   MeSH
• koncentrace vodíkových iontů MeSH
• lipidové dvojvrstvy metabolismus   MeSH
• permeabilita MeSH
• Shigella boydii metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• chlorid draselný MeSH
• koliciny MeSH
• lipidové dvojvrstvy MeSH

A novel colicin, designated colicin U, was found in two Shigella boydii strains of serovars 1 and 8. Colicin U was active against bacterial strains of the genera Escherichia and Shigella. Plasmid pColU (7.3 kb) of the colicinogenic strain S. boydii M592 (serovar 8) was sequenced, and three colicin genes were identified. The colicin U activity gene, cua, encodes a protein of 619 amino acids (Mr, 66,289); the immunity gene, cui, encodes a protein of 174 amino acids (Mr, 20,688); and the lytic protein gene, cul, encodes a polypeptide of 45 amino acids (Mr, 4,672). Colicin U displays sequence similarities to various colicins. The N-terminal sequence of 130 amino acids has 54% identity to the N-terminal sequence of bacteriocin 28b produced by Serratia marcescens. Furthermore, the N-terminal 36 amino acids have striking sequence identity (83%) to colicin A. Although the C-terminal pore-forming sequence of colicin U shows the highest degree of identity (73%) to the pore-forming C-terminal sequence of colicin B, the immunity protein, which interacts with the same region, displays a higher degree of sequence similarity to the immunity protein of colicin A (45%) than to the immunity protein of colicin B (30.5%). Immunity specificity is probably conferred by a short sequence from residues 571 to residue 599 of colicin U; this sequence is not similar to that of colicin B. We showed that binding of colicin U to sensitive cells is mediated by the OmpA protein, the OmpF porin, and core lipopolysaccharide. Uptake of colicin U was dependent on the TolA, -B, -Q, and -R proteins. pColU is homologous to plasmid pSB41 (4.1 kb) except for the colicin genes on pColU. pSB41 and pColU coexist in S. boydii strains and can be cotransformed into Escherichia coli, and both plasmids are homologous to pColE1.

• MeSH
• buněčná membrána metabolismus   MeSH
• DNA bakterií MeSH
• koliciny biosyntéza   chemie   genetika   metabolismus   MeSH
• molekulární sekvence - údaje MeSH
• plazmidy MeSH
• proteiny vnější bakteriální membrány genetika   metabolismus   MeSH
• sekvence aminokyselin MeSH
• sekvence nukleotidů MeSH
• sekvenční delece MeSH
• sekvenční homologie aminokyselin MeSH
• Shigella boydii genetika   metabolismus   MeSH
• vazebná místa MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• DNA bakterií MeSH
• koliciny MeSH
• proteiny vnější bakteriální membrány MeSH

• MeSH
• bacilární dyzentérie imunologie   MeSH
• bakteriální toxiny imunologie   MeSH
• dítě MeSH
• dospělí MeSH
• imunoglobulin A sekreční metabolismus   MeSH
• léčebná irigace MeSH
• lidé MeSH
• lipopolysacharidy imunologie   MeSH
• protilátky bakteriální krev   MeSH
• shiga toxiny MeSH
• Shigella boydii imunologie   MeSH
• Shigella dysenteriae imunologie   MeSH
• Shigella flexneri imunologie   MeSH
• sliny imunologie   MeSH
• slizniční imunita MeSH
• střevní sliznice imunologie   MeSH
• Check Tag
• dítě MeSH
• dospělí MeSH
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, U.S. Gov't, Non-P.H.S. MeSH
• Názvy látek
• bakteriální toxiny MeSH
• imunoglobulin A sekreční MeSH
• lipopolysacharidy MeSH
• protilátky bakteriální MeSH
• shiga toxiny MeSH

• MeSH
• Enterobacteriaceae izolace a purifikace   MeSH
• lidé středního věku MeSH
• lidé MeSH
• Shigella boydii izolace a purifikace   MeSH
• Shigella dysenteriae izolace a purifikace   MeSH
• Check Tag
• lidé středního věku MeSH
• lidé MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• Geografické názvy
• Československo MeSH

• MeSH
• bakteriofágy * MeSH
• bakteriologické techniky MeSH
• bakteriolýza MeSH
• dítě v ústavní péči MeSH
• dítě MeSH
• dyzenterie mikrobiologie   MeSH
• fagotypizace * MeSH
• kolifágy MeSH
• kultivace virů MeSH
• lidé MeSH
• Shigella boydii klasifikace   MeSH
• Shigella klasifikace   MeSH
• Check Tag
• dítě MeSH
• lidé MeSH
• Publikační typ
• časopisecké články MeSH