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.
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.
- MeSH
- antibakteriální látky chemická syntéza farmakologie MeSH
- Bacillus subtilis chemie cytologie účinky léků MeSH
- Escherichia coli chemie cytologie účinky léků MeSH
- fosfatidylcholiny analýza metabolismus MeSH
- fosfatidylethanolaminy analýza metabolismus MeSH
- kationické antimikrobiální peptidy chemická syntéza farmakologie MeSH
- lipidové dvojvrstvy MeSH
- membránové potenciály účinky léků MeSH
- mikrobiální testy citlivosti MeSH
- permeabilita buněčné membrány MeSH
- vnější bakteriální membrána chemie účinky léků metabolismus MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
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.
Daptomycin is a calcium-dependent lipodepsipeptide antibiotic clinically used to treat serious infections caused by Gram-positive pathogens. Its precise mode of action is somewhat controversial; the biggest issue is daptomycin pore formation, which we directly investigated here. We first performed a screening experiment using propidium iodide (PI) entry to Bacillus subtilis cells and chose the optimum and therapeutically relevant conditions (10 µg/ml daptomycin and 1.25 mM CaCl2) for the subsequent analyses. Using conductance measurements on planar lipid bilayers, we show that daptomycin forms nonuniform oligomeric pores with conductance ranging from 120 pS to 14 nS. The smallest conductance unit is probably a dimer; however, tetramers and pentamers occur in the membrane most frequently. Moreover, daptomycin pore-forming activity is exponentially dependent on the applied membrane voltage. We further analyzed the membrane-permeabilizing activity in B. subtilis cells using fluorescence methods [PI and DiSC3(5)]. Daptomycin most rapidly permeabilizes cells with high initial membrane potential and dissipates it within a few minutes. Low initial membrane potential hinders daptomycin pore formation.
- MeSH
- antibakteriální látky farmakologie MeSH
- Bacillus subtilis účinky léků metabolismus MeSH
- biologický transport fyziologie MeSH
- cytotoxické proteiny tvořící póry farmakologie MeSH
- daptomycin farmakologie MeSH
- membránové potenciály účinky léků MeSH
- mikrobiální testy citlivosti MeSH
- permeabilita buněčné membrány účinky léků MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem 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.
- 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
After cold shock, the Bacillus subtilis desaturase Des introduces double bonds into the fatty acids of existing membrane phospholipids. The synthesis of Des is regulated exclusively by the two-component system DesK/DesR; DesK serves as a sensor of the state of the membrane and triggers Des synthesis after a decrease in membrane fluidity. The aim of our work is to investigate the biophysical changes in the membrane that are able to affect the DesK signalling state. Using linear alcohols (ethanol, propanol, butanol, hexanol, octanol) and benzyl alcohol, we were able to suppress Des synthesis after a temperature downshift. The changes in the biophysical properties of the membrane caused by alcohol addition were followed using membrane fluorescent probes and differential scanning calorimetry. We found that the membrane fluidization induced by alcohols was reflected in an increased hydration at the lipid-water interface. This is associated with a decrease in DesK activity. The addition of alcohol mimics a temperature increase, which can be measured isothermically by fluorescence anisotropy. The effect of alcohols on the membrane periphery is in line with the concept of the mechanism by which two hydrophilic motifs located at opposite ends of the transmembrane region of DesK, which work as a molecular caliper, sense temperature-dependent variations in membrane properties.
- MeSH
- alkoholy farmakologie MeSH
- aminokyselinové motivy MeSH
- Bacillus subtilis metabolismus MeSH
- bakteriální proteiny metabolismus MeSH
- buněčná membrána účinky léků fyziologie MeSH
- desaturasy mastných kyselin biosyntéza genetika MeSH
- diferenciální skenovací kalorimetrie MeSH
- enzymová indukce účinky léků MeSH
- fluidita membrány účinky léků MeSH
- fluorescenční polarizace MeSH
- fosforylace MeSH
- hydrofobní a hydrofilní interakce MeSH
- mastné kyseliny metabolismus MeSH
- nízká teplota MeSH
- posttranslační úpravy proteinů * MeSH
- proteinkinasy metabolismus MeSH
- regulace genové exprese u bakterií účinky léků MeSH
- rekombinantní fúzní proteiny metabolismus MeSH
- reportérové geny MeSH
- signální transdukce účinky léků MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- srovnávací studie MeSH
Diamond nanoparticles (DNPs) of various types have been recently reported to possess antibacterial properties. Studies have shown a decrease of the colony forming ability on agar plates of the bacteria that had been previously co-incubated with DNPs in the suspension. Before plating, bacteria with DNPs were adequately diluted in order to obtain a suitable number of colony forming units. However, residual DNPs were still present on an agar plate, concentrated on the surface during the plating process; this introduces a potential artifact which might affect colony growth. The effect of DNPs remaining on the surface, alongside growing bacteria, has not been previously investigated. In this work, we present the experiments designed to investigate the effect of DNPs on bacterial survival and on the growth of the bacterial colony on a solid media. We employed Escherichia coli and Bacillus subtilis as models of Gram-negative and Gram-positive bacteria, respectively, and Proteus mirabilis as a model of bacterium exhibiting swarming motility on the surfaces. We analyzed the number, area, and weight of bacterial colonies grown on the agar surface covered with DNPs. We did not observe any bactericidal effect of such applied DNPs. However, in all bacterial species used in this work, we observed the appreciable reduction of colony area, which suggests that DNPs obstruct either bacterial growth or motility. The most obvious effect on colony growth was observed in the case of motile P. mirabilis. We show that DNPs act as the mechanical barrier blocking the lateral colony growth.
- MeSH
- antibakteriální látky chemie farmakologie MeSH
- Bacillus subtilis účinky léků růst a vývoj MeSH
- Bacteria cytologie účinky léků růst a vývoj MeSH
- diamant chemie farmakologie MeSH
- Escherichia coli účinky léků růst a vývoj MeSH
- mikrobiální testy citlivosti MeSH
- nanočástice chemie MeSH
- povrchové vlastnosti MeSH
- Proteus mirabilis účinky léků růst a vývoj MeSH
- velikost částic MeSH
- vztah mezi dávkou a účinkem léčiva MeSH
- vztahy mezi strukturou a aktivitou MeSH
- Publikační typ
- časopisecké články MeSH
Changes in environmental temperature represent one of the major stresses faced by microorganisms as they affect the function of the cytoplasmic membrane. In this study, we have analyzed the thermal adaptation in two closely related respiratory pathogens Bordetella pertussis and Bordetella bronchiseptica Although B. pertussis represents a pathogen strictly adapted to the human body temperature, B. bronchiseptica causes infection in a broad range of animals and survives also outside of the host. We applied GC-MS to determine the fatty acids of both Bordetella species grown at different temperatures and analyzed the membrane fluidity by fluorescence anisotropy measurement. In parallel, we also monitored the effect of growth temperature changes on the expression and production of several virulence factors. In response to low temperatures, B. pertussis adapted its fatty acid composition and membrane fluidity to a considerably lesser extent when compared with B. bronchiseptica Remarkably, B. pertussis maintained the production of virulence factors at 24 °C, whereas B. bronchiseptica cells resumed the production only upon temperature upshift to 37 °C. This growth temperature-associated differential modulation of virulence factor production was linked to the phosphorylation state of transcriptional regulator BvgA. The observed differences in low-temperature adaptation between B. pertussis and B. bronchiseptica may result from selective adaptation of B. pertussis to the human host. We propose that the reduced plasticity of the B. pertussis membranes ensures sustained production of virulence factors at suboptimal temperatures and may play an important role in the transmission of the disease.
- MeSH
- aklimatizace * MeSH
- anizotropie MeSH
- bakteriální proteiny metabolismus MeSH
- Bordetella bronchiseptica cytologie fyziologie MeSH
- Bordetella pertussis cytologie fyziologie MeSH
- buněčná membrána metabolismus MeSH
- cytoplazma metabolismus MeSH
- druhová specificita MeSH
- faktory virulence metabolismus MeSH
- fluorescenční spektrometrie MeSH
- fosforylace MeSH
- lidé MeSH
- mastné kyseliny chemie MeSH
- plynová chromatografie s hmotnostně spektrometrickou detekcí MeSH
- signální transdukce MeSH
- tělesná teplota MeSH
- teplota * MeSH
- transkripční faktory metabolismus MeSH
- virulence MeSH
- životní prostředí MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- MeSH
- dějiny lékařství MeSH
- krysa rodu rattus MeSH
- lidé MeSH
- mor * dějiny prevence a kontrola přenos MeSH
- pandemie dějiny prevence a kontrola MeSH
- přenos infekční nemoci dějiny prevence a kontrola MeSH
- Xenopsylla MeSH
- Yersinia pestis * patogenita MeSH
- zvířata MeSH
- Check Tag
- krysa rodu rattus MeSH
- lidé MeSH
- zvířata MeSH
