A real-time analysis of protein transport via the twin arginine translocation pathway in response to different components of the protonmotive force.

The twin arginine translocation (Tat) pathway transports folded protein across the cytoplasmic membrane in bacteria, archaea, and across the thylakoid membrane in plants as well as the inner membrane in some mitochondria. In plant chloroplasts, the Tat pathway utilizes the protonmotive force (PMF) to drive protein translocation. However, in bacteria, it has been shown that Tat transport depends only on the transmembrane electrical potential (Δψ) component of PMF in vitro. To investigate the comprehensive PMF requirement in Escherichia coli, we have developed the first real-time assay to monitor Tat transport utilizing the NanoLuc Binary Technology in E. coli spheroplasts. This luminescence assay allows for continuous monitoring of Tat transport with high-resolution, making it possible to observe subtle changes in transport in response to different treatments. By applying the NanoLuc assay, we report that, under acidic conditions (pH = 6.3), ΔpH, in addition to Δψ, contributes energetically to Tat transport in vivo in E. coli spheroplasts. These results provide novel insight into the mechanism of energy utilization by the Tat pathway.

An experiment-informed signal transduction model for the role of the Staphylococcus aureus MecR1 protein in ß-lactam resistance.

The treatment of hospital- and community-associated infections by methicillin-resistant Staphylococcus aureus (MRSA) is a perpetual challenge. This Gram-positive bacterium is resistant specifically to ß-lactam antibiotics, and generally to many other antibacterial agents. Its resistance mechanisms to ß-lactam antibiotics are activated only when the bacterium encounters a ß-lactam. This activation is regulated by the transmembrane sensor/signal transducer proteins BlaR1 and MecR1. Neither the transmembrane/metalloprotease domain, nor the complete MecR1 and BlaR1 proteins, are isolatable for mechanistic study. Here we propose a model for full-length MecR1 based on homology modeling, residue coevolution data, a new extensive experimental mapping of transmembrane topology, partial structures, molecular simulations, and available NMR data. Our model defines the metalloprotease domain as a hydrophilic transmembrane chamber effectively sealed by the apo-sensor domain. It proposes that the amphipathic helices inserted into the gluzincin domain constitute the route for transmission of the ß-lactam-binding event in the extracellular sensor domain, to the intracellular and membrane-embedded zinc-containing active site. From here, we discuss possible routes for subsequent activation of proteolytic action. This study provides the first coherent model of the structure of MecR1, opening routes for future functional investigations on how ß-lactam binding culminates in the proteolytic degradation of MecI.

Spheroplast-Mediated Carbapenem Tolerance in Gram-Negative Pathogens.

Antibiotic tolerance, the ability to temporarily sustain viability in the presence of bactericidal antibiotics, constitutes an understudied and yet potentially widespread cause of antibiotic treatment failure. We have previously shown that the Gram-negative pathogen Vibrio cholerae can tolerate exposure to the typically bactericidal ß-lactam antibiotics by assuming a spherical morphotype devoid of detectable cell wall material. However, it is unclear how widespread ß-lactam tolerance is. Here, we tested a panel of clinically significant Gram-negative pathogens for their response to the potent, broad-spectrum carbapenem antibiotic meropenem. We show that clinical isolates of Enterobacter cloacae, Klebsiella aerogenes, and Klebsiella pneumoniae, but not Escherichia coli, exhibited moderate to high levels of tolerance of meropenem, both in laboratory growth medium and in human serum. Importantly, tolerance was mediated by cell wall-deficient spheroplasts, which readily recovered wild-type morphology and growth upon removal of antibiotic. Our results suggest that carbapenem tolerance is prevalent in clinically significant bacterial species, and we suggest that this could contribute to treatment failure associated with these organisms.

Membrane potential is vital for rapid permeabilization of plasma membranes and lipid bilayers by the antimicrobial peptide lactoferricin B.

Lactoferricin B (LfcinB) is a cationic antimicrobial peptide, and its capacity to damage the bacterial plasma membrane is suggested to be a main factor in LfcinB's antimicrobial activity. However, the specific processes and mechanisms in LfcinB-induced membrane damage are unclear. In this report, using confocal laser-scanning microscopy, we examined the interaction of LfcinB with single Escherichia coli cells and spheroplasts containing the water-soluble fluorescent probe calcein in the cytoplasm. LfcinB induced rapid calcein leakage from single E. coli cells and from single spheroplasts, indicating that LfcinB interacts directly with the plasma membrane and induces its rapid permeabilization. The proton ionophore carbonyl cyanide m-chlorophenylhydrazone suppressed this leakage. Next, we used the single giant unilamellar vesicle (GUV) method to examine LfcinB's interaction with GUVs comprising polar lipid extracts of E. coli containing a water-soluble fluorescent probe, Alexa Fluor 647 hydrazide (AF647). We observed that LfcinB stochastically induces local rupture in single GUVs, causing rapid AF647 leakage; however, higher LfcinB concentrations were required for AF647 leakage from GUVs than from E. coli cells and spheroplasts. To identify the reason for this difference, we examined the effect of membrane potential on LfcinB-induced pore formation, finding that the rate of LfcinB-induced local rupture in GUVs increases greatly with increasing negative membrane potential. These results indicate that membrane potential plays an important role in LfcinB-induced local rupture of lipid bilayers and rapid permeabilization of E. coli plasma membranes. On the basis of these results, we discuss the mode of action of LfcinB's antimicrobial activity.

Calcium ion induces outer membrane fusion of Deinococcus grandis spheroplasts to generate giant spheroplasts with multiple cytoplasms.

Generally, enlarged spheroplasts of the Gram-negative bacterium Deinococcus grandis contain a single cytoplasm and a large periplasmic space. Enlargement of D. grandis spheroplasts requires the presence of divalent cation Ca2+ or Mg2+. In this study, we elucidated the effects of concentrations of these divalent cations on the enlargement of spheroplasts. We compared the cell sizes of the spheroplasts at five different concentrations (16.2, 62, 100, 200 and 333 mM) of CaCl2 or MgCl2. At the lowest concentration (16.2 mM) of CaCl2 or MgCl2, the inner membrane of D. grandis spheroplasts collapsed and the spheroplasts did not enlarge. At the highest concentration (333 mM) of CaCl2 or MgCl2, enlargement was inhibited. At 200 mM of CaCl2, the outer membranes of D. grandis spheroplasts were fused repeatedly, but the inner membranes were not fused. Thus, at 200 mM of CaCl2, giant cells that have multiple cytoplasms were observed and were ≥ 500 µm in diameter. However, cell fusions were not observed in any concentrations of MgCl2. This indicates that Ca2+ induces lipopolysaccharide dehydration more strongly than Mg2+ and outer membranes may be fused by hydrophobic bonding. Our findings show the different functions of Ca2+ and Mg2+ on the outer membrane stability.

Endonucleolytic cleavage in the expansion segment 7 of 25S rRNA is an early marker of low-level oxidative stress in yeast.

The ability to detect and respond to oxidative stress is crucial to the survival of living organisms. In cells, sensing of increased levels of reactive oxygen species (ROS) activates many defensive mechanisms that limit or repair damage to cell components. The ROS-signaling responses necessary for cell survival under oxidative stress conditions remain incompletely understood, especially for the translational machinery. Here, we found that drug treatments or a genetic deficiency in the thioredoxin system that increase levels of endogenous hydrogen peroxide in the yeast Saccharomyces cerevisiae promote site-specific endonucleolytic cleavage in 25S ribosomal RNA (rRNA) adjacent to the c loop of the expansion segment 7 (ES7), a putative regulatory region located on the surface of the 60S ribosomal subunit. Our data also show that ES7c is cleaved at early stages of the gene expression program that enables cells to successfully counteract oxidative stress and is not a prerequisite or consequence of apoptosis. Moreover, the 60S subunits containing ES7c-cleaved rRNA cofractionate with intact subunits in sucrose gradients and repopulate polysomes after a short starvation-induced translational block, indicating their active role in translation. These results demonstrate that ES7c cleavage in rRNA is an early and sensitive marker of increased ROS levels in yeast cells and suggest that changes in ribosomes may be involved in the adaptive response to oxidative stress.

Membrane perturbing activities and structural properties of the frog-skin derived peptide Esculentin-1a(1-21)NH2 and its Diastereomer Esc(1-21)-1c: Correlation with their antipseudomonal and cytotoxic activity.

Antimicrobial peptides (AMPs) represent new alternatives to cope with the increasing number of multi-drug resistant microbial infections. Recently, a derivative of the frog-skin AMP esculentin-1a, Esc(1-21), was found to rapidly kill both the planktonic and biofilm forms of the Gram-negative bacterium Pseudomonas aeruginosa with a membrane-perturbing activity as a plausible mode of action. Lately, its diastereomer Esc(1-21)-1c containing two d-amino acids i.e. DLeu14 and DSer17 revealed to be less cytotoxic, more stable to proteolytic degradation and more efficient in eradicating Pseudomonas biofilm. When tested in vitro against the free-living form of this pathogen, it displayed potent bactericidal activity, but this was weaker than that of the all-l peptide. To investigate the reason accounting for this difference, mechanistic studies were performed on Pseudomonas spheroplasts and anionic or zwitterionic membranes, mimicking the composition of microbial and mammalian membranes, respectively. Furthermore, structural studies by means of optical and nuclear magnetic resonance spectroscopies were carried out. Our results suggest that the different extent in the bactericidal activity between the two isomers is principally due to differences in their interaction with the bacterial cell wall components. Indeed, the lower ability in binding and perturbing anionic phospholipid bilayers for Esc(1-21)-1c contributes only in a small part to this difference, while the final effect of membrane thinning once the peptide is inserted into the membrane is identical to that provoked by Esc(1-21). In addition, the presence of two d-amino acids is sufficient to reduce the α-helical content of the peptide, in parallel with its lower cytotoxicity.

The Lysis of Pathogenic Escherichia coli by Bacteriophages Releases Less Endotoxin Than by ß-Lactams.

BACKGROUND.: Other than numerous experimental data assessing phage therapy efficacy, questions regarding safety of this approach are not sufficiently addressed. In particular, as phages can kill bacterial cells within <10 minutes, the associated endotoxin release (ER) in severe infections caused by gram-negative bacteria could be a matter of concern. METHODS.: Two therapeutic virulent phages and 4 reference antibiotics were studied in vitro for their ability to kill 2 pathogenic strains of Escherichia coli and generate an ER. The early interaction (first 3 hours) between these actors was assessed over time by studying the instantaneous cell viability, the colony-forming unit count, the concentration of free endotoxin released, and the cell morphology under light microscope. RESULTS.: While ß-lactams have a relatively slow effect, both tested phages, as well as amikacin, were able to rapidly abolish the bacterial growth. Even when considering the fastest phage (cell lysis in 9 minutes), the concentrations of phage-induced ER never reached the highest values, which were recorded with antibiotic treatments. Cumulative concentrations of endotoxin over time in phage-treated conditions were lower than those observed with ß-lactams and close to those observed with amikacin. Whereas ß-lactams were responsible for strong cell morphology changes (spheroplast with imipenem, filamentous cells with cefoxitin and ceftriaxone), amikacin and phages did not modify cell shape but produced intracellular inclusion bodies. CONCLUSIONS.: This work provides important and comforting data regarding the safety of phage therapy. Therapeutically relevant phages, with their low endotoxin release profile and fast bactericidal effect, are not inferior to ß-lactams.

Straightforward approach to produce recombinant scorpion toxins-Pore blockers of potassium channels.

Scorpion venom peptide blockers (KTx) of potassium channels are a valuable tool for structure-functional studies and prospective candidates for medical applications. Low yields of recombinant KTx hamper their wide application. We developed convenient and efficient bioengineering approach to a large-scale KTx production that meets increasing demands for such peptides. Maltose-binding protein was used as a carrier for cytoplasmic expression of folded disulfide-rich KTx in E. coli. TEV protease was applied for in vitro cleavage of the target peptide from the carrier. To produce KTx with retained native N-terminal sequence, the last residue of TEV protease cleavage site (CSTEV) was occupied by the native N-terminal residue of a target peptide. It was shown that decreased efficiency of hydrolysis of fusion proteins with non-canonical CSTEV can be overcome without by-product formation. Disulfide formation and folding of a target peptide occurred in cytoplasm eliminating the need for renaturation procedure in vitro. Advantages of this approach were demonstrated by producing six peptides with three disulfide bonds related to four KTx sub-families and achieving peptide yields of 12-22mg per liter of culture. The developed approach can be of general use for low-cost production of various KTx, as well as other disulfide-rich peptides and proteins.

Bacterial Spheroplasts as a Model for Visualizing Membrane Translocation of Antimicrobial Peptides.

Studies attempting to characterize the membrane translocation of antimicrobial and cell-penetrating peptides are frequently limited by the resolution of conventional light microscopy. This study shows that spheroplasts provide a valuable approach to overcome these limits. Spheroplasts produce less ambiguous images and allow for more systematic analyses of localization. Data collected with spheroplasts are consistent with studies using normal bacterial cells and imply that a particular peptide may not always follow the same mechanism of action.

Mode of Action of Antimicrobial Peptides on E. coli Spheroplasts.

We investigated the phenomena of antimicrobial peptides (AMPs) directly attacking the cytoplasmic membranes of Escherichia coli spheroplasts. We developed a procedure for fluorescence recovery after photobleaching to examine dye leakage through bacterial membranes as AMPs in solution bound to the membranes. We found that the AMP binding did not increase the apparent membrane area of a spheroplast, contrary to the response of a lipid-bilayer vesicle, which always showed a membrane area expansion by AMP binding. The permeability through the bacterial membrane increased in a sigmoidal fashion as the AMP binding increased in time, exhibiting a cooperative behavior of AMPs. The analysis of fluorescence recovery after photobleaching showed that the fluxes of dye molecules into and out of the cell were consistent with diffusion of molecules through a number of pores that increased with binding of AMPs and then saturated to a steady level. We discovered a new, to our knowledge, experimental parameter called the flux rate that characterizes the AMP-induced permeability of dye molecules through bacterial membranes. The phenomena observed in bacterial membranes are consistent with the pore-forming activities of AMPs previously observed in lipid bilayers. The experimental value of the flux rate per pore is much smaller than a theoretical value that assumes no friction for the dye molecule's permeation through the pore. We believe that experimental studies of the flux rate will be useful for further analysis of AMPs' permeabilization mechanisms.

Requirement of dark culture condition for enlargement of spheroplasts of the aerobic anoxygenic photosynthetic marine bacterium Erythrobacter litoralis.

In the present study, spheroplasts from the aerobic anoxygenic photosynthetic marine bacterium Erythrobacter litoralis were generated and cultivated. In the presence of penicillin, the spheroplasts grew and enlarged in marine broth without undergoing cell division. However, continuous light inhibited their enlargement, and they were therefore cultivated in the dark. Cellular DNA was quantified at various time points (0, 24, and 48 h) and temperatures (20°C, 25°C, and 30°C) using real-time quantitative PCR. The DNA content was highest at 30°C in the absence of penicillin, whereas there was no observable change with exposure to penicillin at all evaluated temperatures. During growth, larger spheroplasts were more frequently observed at 25°C in the presence of penicillin. These results demonstrate that the optimal culture conditions for the enlargement of spheroplasts in E. litoralis differ from those required for cell division.

The effects of LPS on the activity of Trp-containing antimicrobial peptides against Gram-negative bacteria and endotoxin neutralization.

A series of synthesized Trp-containing antimicrobial peptides showed significantly different antimicrobial activity against Gram-negative bacteria despite having similar components and amino acid sequences and the same net positive charge and hydrophobicity. Lipopolysaccharide (LPS) in the outer membrane is a permeability barrier to prevent antimicrobial peptides from crossing into Gram-negative bacteria. We investigated the interaction of five Trp-containing peptides, I1W, I4W, L5W, L11W and L12W, with LPS using circular dichroism (CD), IR spectroscopy, isothermal titration calorimetry (ITC), dynamic light scattering (DLS), zeta-potential measurements and confocal laser scanning microscopy, to address whether bacterial LPS is responsible for the different susceptibilities of Gram-negative bacteria to Trp-containing peptides. Our data indicate that I1W and I4W penetrated the LPS layer and killed Gram-negative bacteria by a "self-promoted uptake" pathway in which the peptides first approach LPS by electrostatic forces and then dissociate LPS micelle. This process results in disorganization of the LPS leaflet and promotes the ability of the peptide to cross the outer membrane into the inner membrane and disrupt the cytoplasmic membrane. Although L5W, L11W and L12W strongly bind to LPS bilayers and depolarize bacterial cytoplasmic membranes, similar to I1W and I4W, they are unable to destabilize LPS aggregates and traverse through the tightly packed LPS molecules. This study increases our understanding of the mechanism of action of these peptides in the LPS outer membrane and will help in the development of a potent broad-spectrum antibiotic for future therapeutic purposes. STATEMENT OF SIGNIFICANCE: Tryptophan (Trp) residues show a strong preference for the interfacial region of biological membranes, and this property endows Trp-containing peptides with the unique ability to interact with the surface of bacterial cell membranes. In this manuscript, we report the membrane interaction of Trp-containing peptide to address whether bacterial LPS is responsible for the different susceptibilities of Gram-negative bacteria to Trp-containing peptides. Based on the data collected, we propose a molecular mechanism for the peptide-LPS interactions that allows the peptides to traverse or prevents them from transversing the LPS layer and the target inner membrane. The data should help in the development of a potent broad-spectrum antibiotic for future therapeutic purposes.

NP24 induces apoptosis dependent on caspase-like activity in Saccharomyces cerevisiae.

Tomato NP24 is a homolog of osmotin, a PR-5 protein from tobacco that can initiate apoptosis in yeast via PHO36 in the plasma membrane. We cloned and sequenced NP24 from tomato cv. Momotaro. Based on phylogenetic analysis, NP24 from Momotaro belonged to the Solanaceae clade. The amino acid sequence was identical to that of cv. Ailsa Craig including signal peptide, but the residues predicted to interact with the adiponectin receptor, ADIPOR, were slightly different from osmotin. Recombinant NP24 (rNP24) was expressed in a reductase-deficient mutant of Escherichia coli as host cell, and purified from cell extract by affinity chromatography. Purified rNP24 significantly inhibited growth of Saccharomyces cerevisiae wild-type spheroplasts. In contrast, growth of PHO36 deletion mutant (ΔIzh2) spheroplasts was not inhibited. Moreover, rNP24 induced significant activity of reactive oxygen species, caspase-like activity, and also nuclear fragmentation in wild-type spheroplast cells. These results demonstrated that rNP24 from Momotaro greatly influenced cell viability due to triggering apoptosis through PHO36. Notably, apoptosis induced by NP24 was caspase-like protease dependent.

Connectivity of the intracytoplasmic membrane of Rhodobacter sphaeroides: a functional approach.

The photosynthetic apparatus in the bacterium Rhodobacter sphaeroides is mostly present in intracytoplasmic membrane invaginations. It has long been debated whether these invaginations remain in topological continuity with the cytoplasmic membrane, or form isolated chromatophore vesicles. This issue is revisited here by functional approaches. The ionophore gramicidin was used as a probe of the relative size of the electro-osmotic units in isolated chromatophores, spheroplasts, or intact cells. The decay of the membrane potential was monitored from the electrochromic shift of carotenoids. The half-time of the decay induced by a single channel in intact cells was about 6 ms, thus three orders of magnitude slower than in isolated chromatophores. In spheroplasts obtained by lysis of the cell wall, the single channel decay was still slower (~23 ms) and the sensitivity toward the gramicidin concentration was enhanced 1,000-fold with respect to isolated chromatophores. These results indicate that the area of the functional membrane in cells or spheroplasts is about three orders of magnitude larger than that of isolated chromatophores. Intracytoplasmic vesicles, if present, could contribute to at most 10% of the photosynthetic apparatus in intact cells of Rba. sphaeroides. Similar conclusions were obtained from the effect of a ∆pH-induced diffusion potential in intact cells. This caused a large electrochromic response of carotenoids, of similar amplitude as the light-induced change, indicating that most of the system is sensitive to a pH change of the external medium. A single internal membrane and periplasmic space may offer significant advantages concerning renewal of the photosynthetic apparatus and reallocation of the components shared with other bioenergetic pathways.

Unidirectional incorporation of a bacterial mechanosensitive channel into liposomal membranes.

The bacterial mechanosensitive channel of small conductance (MscS) plays a crucial role in the protection of bacterial cells against hypo-osmotic shock. The functional characteristics of MscS have been extensively studied using liposomal reconstitution. This is a widely used experimental paradigm and is particularly important for mechanosensitive channels as channel activity can be probed free from cytoskeletal influence. A perpetual issue encountered using this paradigm is unknown channel orientation. Here we examine the orientation of MscS in liposomes formed using 2 ion channel reconstitution methods employing the powerful combination of patch clamp electrophysiology, confocal microscopy, and continuum mechanics simulation. Using the previously determined electrophysiological and pharmacological properties of MscS, we were able to determine that in liposomes, independent of lipid composition, MscS adopts the same orientation seen in native membranes. These results strongly support the idea that these specific methods result in uniform incorporation of membrane ion channels and caution against making assumptions about mechanosensitive channel orientation using the stimulus type alone.

Microbial Uptake, Toxicity, and Fate of Biofabricated ZnS:Mn Nanocrystals.

Despite their importance in nano-environmental health and safety, interactions between engineered nanomaterials and microbial life remain poorly characterized. Here, we used the model organism E. coli to study the penetration requirements, subcellular localization, induction of stress responses, and long-term fate of luminescent Mn-doped ZnS nanocrystals fabricated under "green" processing conditions with a minimized ZnS-binding protein. We find that such protein-coated quantum dots (QDs) are unable to penetrate the envelope of unmodified E. coli but readily translocate to the cytoplasm of cells that have been made competent by chemical treatment. The process is dose-dependent and reminiscent of bacterial transformation. Cells that have internalized up to 0.5 µg/mL of nanocrystals do not experience a significant activation of the unfolded protein or SOS responses but undergo oxidative stress when exposed to high QD doses (2.5 µg/mL). Finally, although they are stable in quiescent cells over temperatures ranging from 4 to 42°C, internalized QDs are rapidly diluted by cell division in a process that does not involve TolC-dependent efflux. Taken together, our results suggest that biomimetic QDs based on low toxicity inorganic cores capped by a protein shell are unlikely to cause significant damage to the microbial ecosystem.

Alteration of ROS homeostasis and decreased lifespan in S. cerevisiae elicited by deletion of the mitochondrial translocator FLX1.

This paper deals with the control exerted by the mitochondrial translocator FLX1, which catalyzes the movement of the redox cofactor FAD across the mitochondrial membrane, on the efficiency of ATP production, ROS homeostasis, and lifespan of S. cerevisiae. The deletion of the FLX1 gene resulted in respiration-deficient and small-colony phenotype accompanied by a significant ATP shortage and ROS unbalance in glycerol-grown cells. Moreover, the flx1Δ strain showed H2O2 hypersensitivity and decreased lifespan. The impaired biochemical phenotype found in the flx1Δ strain might be justified by an altered expression of the flavoprotein subunit of succinate dehydrogenase, a key enzyme in bioenergetics and cell regulation. A search for possible cis-acting consensus motifs in the regulatory region upstream SDH1-ORF revealed a dozen of upstream motifs that might respond to induced metabolic changes by altering the expression of Flx1p. Among these motifs, two are present in the regulatory region of genes encoding proteins involved in flavin homeostasis. This is the first evidence that the mitochondrial flavin cofactor status is involved in controlling the lifespan of yeasts, maybe by changing the cellular succinate level. This is not the only case in which the homeostasis of redox cofactors underlies complex phenotypical behaviours, as lifespan in yeasts.

Energetics of gating MscS by membrane tension in azolectin liposomes and giant spheroplasts.

Mechanosensitive (MS) ion channels are molecular sensors that detect and transduce signals across prokaryotic and eukaryotic cell membranes arising from external mechanical stimuli or osmotic gradients. They play an integral role in mechanosensory responses including touch, hearing, and proprioception by opening or closing in order to facilitate or prevent the flow of ions and organic osmolytes. In this study we use a linear force model of MS channel gating to determine the gating membrane tension (γ) and the gating area change (ΔA) associated with the energetics of MscS channel gating in giant spheroplasts and azolectin liposomes. Analysis of Boltzmann distribution functions describing the dependence of MscS channel gating on membrane tension indicated that the gating area change (ΔA) was the same for MscS channels recorded in both preparations. The comparison of the membrane tension (γ) gating the channel, however, showed a significant difference between the MscS channel activities in these two preparations.