R-Type Fonticins Produced by Pragia fontium Form Large Pores with High Conductance.

Fonticins are phage tail-like bacteriocins produced by the Gram-negative bacterium Pragia fontium from the family Budviciaceae. This bacterium produces contractile-type particles that adsorb on the surface of sensitive bacteria and penetrate the cell wall, probably during contraction, in a way similar to the type VI secretion system. We characterized the pore-forming activity of fonticins using both living cells and in vitro model membranes. Using a potassium leakage assay, we show that fonticins are able to permeabilize sensitive cells. On black lipid membranes, single-pore conductance is about 0.78 nS in 1 M NaCl and appears to be linearly dependent on the increasing molar strength of NaCl solution, which is a property of considerably large pores. In agreement with these findings, fonticins are not ion selective for Na+, K+, and Cl-. Polyethylene glycol 3350 (PEG 3350) molecules of about 3.5 nm in diameter can enter the fonticin pore lumen, whereas the larger molecules cannot pass the pore. The size of fonticin pores was confirmed by transmission electron microscopy. The terminal membrane-piercing complex of the fonticin tube probably creates a selective barrier restricting passage of macromolecules. IMPORTANCE Phage tail-like bacteriocins are now the subject of research as potent antibacterial agents due to their narrow host specificity and single-hit mode of action. In this work, we focused on the structure and mode of action of fonticins. According to some theories, related particles were initially adapted for passage of double-stranded DNA (dsDNA) molecules, but fonticins changed their function during the evolution; they are able to form large pores through the bacterial envelope of Gram-negative bacteria. As various pore-forming proteins are extensively used for nanopore sequencing and stochastic sensing, we decided to investigate the pore-forming properties of fonticin protein complexes on artificial lipid membranes. Our research revealed remarkable structural properties of these particles that may have a potential application as a nanodevice.

Colicin U from Shigella boydii Forms Voltage-Dependent Pores.

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.

LEGO-lipophosphonoxins: length of hydrophobic module affects permeabilizing activity in target membranes of different phospholipid composition.

In the past few decades, society has faced rapid development and spreading of antimicrobial resistance due to antibiotic misuse and overuse and the immense adaptability of bacteria. Difficulties in obtaining effective antimicrobial molecules from natural sources challenged scientists to develop synthetic molecules with antimicrobial effect. We developed modular molecules named LEGO-Lipophosphonoxins (LEGO-LPPO) capable of inducing cytoplasmic membrane perforation. In this structure-activity relationship study we focused on the role of the LEGO-LPPO hydrophobic module directing the molecule insertion into the cytoplasmic membrane. We selected three LEGO-LPPO molecules named C9, C8 and C7 differing in the length of their hydrophobic chain and consisting of an alkenyl group containing one double bond. The molecule with the long hydrophobic chain (C9) was shown to be the most effective with the lowest MIC and highest perforation rate both in vivo and in vitro. We observed high antimicrobial activity against both G+ and G- bacteria with significant differences in LEGO-LPPOs mechanism of action on these two cell types. We observed a highly cooperative mechanism of LEGO-LPPO action on G- bacteria as well as on liposomes resembling G- bacteria. LEGO-LPPO action on G- bacteria was significantly slower compared to G+ bacteria suggesting the role of the outer membrane in affecting the LEGO-LPPOs perforation rate. This notion was supported by the higher sensitivity of the E. coli strain with a compromised outer membrane. Finally, we noted that the composition of the cytoplasmic membrane affects the activity of LEGO-LPPOs since the presence of phosphatidylethanolamine increases their membrane disrupting activity.

LEGO-Lipophosphonoxins: A Novel Approach in Designing Membrane Targeting Antimicrobials.

The alarming rise of bacterial antibiotic resistance requires the development of new compounds. Such compounds, lipophosphonoxins (LPPOs), were previously reported to be active against numerous bacterial species, but serum albumins abolished their activity. Here we describe the synthesis and evaluation of novel antibacterial compounds termed LEGO-LPPOs, loosely based on LPPOs, consisting of a central linker module with two attached connector modules on either side. The connector modules are then decorated with polar and hydrophobic modules. We performed an extensive structure-activity relationship study by varying the length of the linker and hydrophobic modules. The best compounds were active against both Gram-negative and Gram-positive species including multiresistant strains and persisters. LEGO-LPPOs act by first depleting the membrane potential and then creating pores in the cytoplasmic membrane. Importantly, their efficacy is not affected by the presence of serum albumins. Low cytotoxicity and low propensity for resistance development demonstrate their potential for therapeutic use.

Semi-synthetic puwainaphycin/minutissamide cyclic lipopeptides with improved antifungal activity and limited cytotoxicity.

Microbial cyclic lipopeptides are an important class of antifungal compounds with applications in pharmacology and biotechnology. However, the cytotoxicity of many cyclic lipopeptides limits their potential as antifungal drugs. Here we present a structure-activity relationship study on the puwainaphycin/minutissamide (PUW/MIN) family of cyclic lipopeptides isolated from cyanobacteria. PUWs/MINs with variable fatty acid chain lengths differed in the dynamic of their cytotoxic effect despite their similar IC50 after 48 hours (2.8 µM for MIN A and 3.2 µM for PUW F). Furthermore, they exhibited different antifungal potency with the lowest MIC values obtained for MIN A and PUW F against the facultative human pathogen Aspergillus fumigatus (37 µM) and the plant pathogen Alternaria alternata (0.6 µM), respectively. We used a Grignard-reaction with alkylmagnesium halides to lengthen the lipopeptide FA moiety as well as the Steglich esterification on the free hydroxyl substituents to prepare semi-synthetic lipopeptide variants possessing multiple fatty acid tails. Cyclic lipopeptides with extended and branched FA tails showed improved strain-specific antifungal activity against A. fumigatus (MIC = 0.5-3.8 µM) and A. alternata (MIC = 0.1-0.5 µM), but with partial retention of the cytotoxic effect (∼10-20 µM). However, lipopeptides with esterified free hydroxyl groups possessed substantially higher antifungal potencies, especially against A. alternata (MIC = 0.2-0.6 µM), and greatly reduced or abolished cytotoxic activity (>20 µM). Our findings pave the way for a generation of semi-synthetic variants of lipopeptides with improved and selective antifungal activities.

Outer membrane and phospholipid composition of the target membrane affect the antimicrobial potential of first- and second-generation lipophosphonoxins.

Lipophosphonoxins (LPPOs) are small modular synthetic antibacterial compounds that target the cytoplasmic membrane. First-generation LPPOs (LPPO I) exhibit an antimicrobial activity against Gram-positive bacteria; however they do not exhibit any activity against Gram-negatives. Second-generation LPPOs (LPPO II) also exhibit broadened activity against Gram-negatives. We investigated the reasons behind this different susceptibility of bacteria to the two generations of LPPOs using model membranes and the living model bacteria Bacillus subtilis and Escherichia coli. We show that both generations of LPPOs form oligomeric conductive pores and permeabilize the bacterial membrane of sensitive cells. LPPO activity is not affected by the value of the target membrane potential, and thus they are also active against persister cells. The insensitivity of Gram-negative bacteria to LPPO I is probably caused by the barrier function of the outer membrane with LPS. LPPO I is almost incapable of overcoming the outer membrane in living cells, and the presence of LPS in liposomes substantially reduces their activity. Further, the antimicrobial activity of LPPO is also influenced by the phospholipid composition of the target membrane. A higher proportion of phospholipids with neutral charge such as phosphatidylethanolamine or phosphatidylcholine reduces the LPPO permeabilizing potential.

Bacillus subtilis cardiolipin protects its own membrane against surfactin-induced permeabilization.

Surfactin, a cyclic lipoheptapeptide produced by Bacillus subtilis, is a surface-active antimicrobial that targets the barrier function of lipid membranes. It inserts itself into the membrane, where it forms conductive pores. Depending on its concentration, it eventually disintegrates the membrane in a detergent-like manner. The molecular details of this activity are not yet sufficiently understood, nor are the mechanisms that the surfactin producer employs to resist its own toxic product. We have previously shown that B. subtilis modifies its membrane lipid composition upon the onset of surfactin production, mainly increasing the cardiolipin content. Here we show that the increased cardiolipin content leads to a decreased surfactin-induced leakage of liposomes reconstituted from lipids isolated from the surfactin producer. This stabilizing effect of cardiolipin is concentration-dependent. Using a propidium iodide-based cell permeabilization assay, we further confirmed that the cytoplasmic membrane of the mutant B. subtilis strain lacking cardiolipin was substantially more susceptible to the action of surfactin, even though the amount of bound surfactin was the same as in the wild-type strain. We propose that membrane remodelling; due to the increase in cardiolipin content, contributes to the surfactin tolerance of B. subtilis.

Residues 529 to 549 participate in membrane penetration and pore-forming activity of the Bordetella adenylate cyclase toxin.

The adenylate cyclase toxin-hemolysin (CyaA, ACT or AC-Hly) of pathogenic Bordetellae delivers its adenylyl cyclase (AC) enzyme domain into the cytosol of host cells and catalyzes uncontrolled conversion of cellular ATP to cAMP. In parallel, the toxin forms small cation-selective pores that permeabilize target cell membrane and account for the hemolytic activity of CyaA on erythrocytes. The pore-forming domain of CyaA is predicted to consist of five transmembrane α-helices, of which the helices I, III, IV and V have previously been characterized. We examined here the α-helix II that is predicted to form between residues 529 to 549. Substitution of the glycine 531 residue by a proline selectively reduced the hemolytic capacity but did not affect the AC translocating activity of the CyaA-G531P toxin. In contrast, CyaA toxins with alanine 538 or 546 replaced by diverse residues were selectively impaired in the capacity to translocate the AC domain across cell membrane but remained fully hemolytic. Such toxins, however, formed pores in planar asolectin bilayer membranes with a very low frequency and with at least two different conducting states. The helix-breaking substitution of alanine 538 by a proline residue abolished the voltage-activated increase of membrane activity of CyaA in asolectin bilayers. These results reveal that the predicted α-helix comprising the residues 529 to 549 plays a key role in CyaA penetration into the target plasma membrane and pore-forming activity of the toxin.