Lucienne Letellier

Quantitative time-resolved measurement of membrane protein- ligand interactions using microcantilever array sensors

Membrane proteins are central to many biological processes, and the interactions between transmembrane protein receptors and their ligands are of fundamental importance in medical research. However, measuring and characterizing these interactions is challenging. Here we report that sensors based on arrays of resonating microcantilevers can measure such interactions under physiological conditions. A protein receptor--the FhuA receptor of Escherichia coli--is crystallized in liposomes, and the proteoliposomes then immobilized on the chemically activated gold-coated surface of the sensor by ink-jet spotting in a humid environment, thus keeping the receptors functional. Quantitative mass-binding measurements of the bacterial virus T5 at subpicomolar concentrations are performed. These experiments demonstrate the potential of resonating microcantilevers for the specific, label-free and time-resolved detection of membrane protein-ligand interactions in a micro-array format.

Colicin A unfolds during its translocation in Escherichia coli cells and spans the whole cell envelope when its pore has formed

The addition of the pore forming colicin A to Escherichia coli cells results in an efflux of cytoplasmic potassium. This efflux is preceded by a lag time which is related to the time needed for the translocation of the toxin through the envelope. Denaturing the colicin A with urea, before adding it to the cells, did not affect the properties of the pore but decreased the lag time. After renaturation, the lag time was similar to that of the native colicin. This suggests that the unfolding of colicin A accelerates its translocation. The addition of trypsin, which has access neither to the periplasmic space nor to the cytoplasmic membrane, resulted in an immediate arrest of the potassium efflux induced by colicins A and B. The possibility that trypsin may act on a bacterial component required for colicin reception and/or translocation was ruled out. It is thus likely that the arrest of the efflux corresponds to a closing of the pores. This long distance effect of trypsin suggests that part of the polypeptide chain of the colicins may still be in contact with the external medium even when the pore has formed in the inner membrane.

FhuA, a transporter of the Escherichia coli outer membrane, is converted into a channel upon binding of bacteriophage T5.

The Escherichia coli outer membrane protein FhuA catalyzes the transport of Fe3+(‐)ferrichrome and is the receptor of phage T5 and phi 80. The purified protein inserted into planar lipid bilayers showed no channel activity. Binding of phage T5 and FhuA resulted in the appearance of high conductance ion channels. The electrophysiological characteristics of the channels (conductance, kinetic behavior, substates, ion selectivity including the effect of ferrichrome) showed similarities with those of the channel formed by a FhuA derivative from which the ‘gating loop’ (delta 322–355) had been removed. binding of phage T5 to FhuA in E.coli cells conferred SDS sensitivity to the bacteria, suggesting that such channels also exist in vivo. These data suggest that binding of T5 to loop 322–355 of FhuA, which constitutes the T5 binding site, unmasks an inner channel in FhuA. Both T5 and ferrichrome bind to the closed state of the channel but only T5 can trigger its opening.

Real-Time Imaging of DNA Ejection from Single Phage Particles

Infection by tailed dsDNA phages is initiated by release of the viral DNA from the capsid and its polarized injection into the host. The driving force for the genome transport remains poorly defined. Among many hypothesis [1], it has been proposed that the internal pressure built up during packaging of the DNA in the capsid is responsible for its injection [2-4]. Whether the energy stored during packaging is sufficient to cause full DNA ejection or only to initiate the process was tested on phage T5 whose DNA (121,400 bp) can be released in vitro by mere interaction of the phage with its E. coli membrane receptor FhuA [5-7]. We present a fluorescence microscopy study investigating in real time the dynamics of DNA ejection from single T5 phages adsorbed onto a microfluidic cell. The ejected DNA was fluorescently stained, and its length was measured at different stages of the ejection after being stretched in a hydrodynamic flow. We conclude that DNA release is not an all-or-none process but occurs in a stepwise fashion and at a rate reaching 75,000 bp/sec. The relevance of this stepwise ejection to the in vivo DNA transfer is discussed.

Phage T5 Straight Tail Fiber Is a Multifunctional Protein Acting as a Tape Measure and Carrying Fusogenic and Muralytic Activities

We report a bioinformatic and functional characterization of Pb2, a 121-kDa multimeric protein that forms phage T5 straight fiber and is implicated in DNA transfer into the host. Pb2 was predicted to consist of three domains. Region I (residues 1–1030) was mainly organized in coiled coil and shared features of tape measure proteins. Region II (residues 1030–1076) contained two α-helical transmembrane segments. Region III (residues 1135–1148) included a metallopeptidase motif. A truncated version of Pb2 (Pb2-Cterm, residues 964–1148) was expressed and purified. Pb2-Cterm shared common features with fusogenic membrane polypeptides. It formed oligomeric structures and inserted into liposomes triggering their fusion. Pb2-Cterm caused β-galactosidase release from Escherichia coli cells and in vitro peptidoglycan hydrolysis. Based on these multifunctional properties, we propose that binding of phage T5 to its receptor triggers large conformational changes in Pb2. The coiled coil region would serve as a sensor for triggering the opening of the head-tail connector. The C-terminal region would gain access to the host envelope, permitting the local degradation of the peptidoglycan and the formation of the DNA pore by fusion of the two membranes.

PHAGE DNA TRANSPORT ACROSS MEMBRANES

Phage nucleic acid transport is atypical in bacterial membrane transport: it is unidirectional and concerns a unique molecule the size of which may represent 50 times that of the bacterium. The rate of DNA transport, although it varies from one phage to another, can reach values as high as 3000 bp s(-1). This raises the following questions which will be discussed in this review. Is there a single mechanism of transport for all types of phages? Does the phage genome cross the outer and inner membranes by a unique mechanism? Is it transported as a free molecule or in association with proteins? How does it avoid periplasmic nucleases? Is such transport dependent on phage and/or host cell components? What is the driving force for transport? Recent cryoelectron microscopy experiments will be presented which show that it is possible to encapsulate a phage genome (121000 bp) into unilamellar liposomes. The interest of such a model system in gene delivery and in the study of the mechanisms of DNA compaction will be discussed.

Microcin E492 antibacterial activity: evidence for a TonB‐dependent inner membrane permeabilization on Escherichia coli

The mechanism of action of microcin E492 (MccE492) was investigated for the first time in live bacteria. MccE492 was expressed and purified to homogeneity through an optimized large‐scale procedure. Highly purified MccE492 showed potent antibacterial activity at minimal inhibitory concentrations in the range of 0.02–1.2 µM. The microcin bactericidal spectrum of activity was found to be restricted to Enterobacteriaceae and specifically directed against Escherichia and Salmonella species. Isogenic bacteria that possessed mutations in membrane proteins, particularly of the TonB–ExbB–ExbD complex, were assayed. The microcin bactericidal activity was shown to be TonB‐ and energy‐dependent, supporting the hypothesis that the mechanism of action is receptor mediated. In addition, MccE492 depolarized and permeabilized the E. coli cytoplasmic membrane. The membrane depolarization was TonB dependent. From this study, we propose that MccE492 is recognized by iron‐siderophore receptors, including FepA, which promote its import across the outer membrane via a TonB‐ and energy‐dependent pathway. MccE492 then inserts into the inner membrane, whereupon the potential becomes destabilized by pore formation. Because cytoplasmic membrane permeabilization of MccE492 occurs beneath the threshold of the bactericidal concentration and does not result in cell lysis, the cytoplasmic membrane is not hypothesized to be the sole target of MccE492.

Androctonin, a hydrophilic disulphide-bridged non-haemolytic anti-microbial peptide: a plausible mode of action

Androctonin is a 25-residue non-haemolytic anti-microbial peptide isolated from the scorpion Androctonus australis and contains two disulphide bridges. Androctonin is different from known native anti-microbial peptides, being a relatively hydrophilic and non-amphipathic molecule. This raises the possibility that the target of androctonin might not be the bacterial membrane, shown to be a target for most amphipathic lytic peptides. To shed light on its mode of action on bacteria and its non-haemolytic activity, we synthesized androctonin, its fluorescent derivatives and its all-D-amino acid enantiomer. The enantiomer preserved high activity, suggesting a lipid-peptide interaction between androctonin and bacterial membranes. In Gram-positive and (at higher concentrations) Gram-negative bacteria, androctonin induced an immediate perturbation of the permeability properties of the cytoplasmic membrane of the bacterial energetic state, concomitant with perturbation of the morphology of the cell envelope as revealed by electron microscopy. Androctonin binds only to negatively charged lipid vesicles and induces the leakage of markers at high concentrations and with a slow kinetics, in contrast with amphipathic alpha-helical anti-microbial peptides that bind and permeate negatively charged vesicles, and to a smaller extent also zwitterionic ones. This might explain the selective lytic activity of androctonin towards bacteria but not red blood cells. Polarized attenuated total reflection-Fourier transform infrared spectroscopy revealed that androctonin adopts a beta-sheet structure in membranes and did not affect the lipid acyl chain order, which supports a detergent-like effect. The small size of androctonin, its hydrophilic character and its physicochemical properties are favourable features for its potential application as a replacement for commercially available antibiotics to which bacteria have developed resistance.

Reconstitution of FhuA, an Escherichia coli Outer Membrane Protein, into Liposomes BINDING OF PHAGE T5 TO FhuA TRIGGERS THE TRANSFER OF DNA INTO THE PROTEOLIPOSOMES

The Escherichia coli outer membrane protein FhuA catalyzes the transport of ferrichrome and is the receptor of bacteriophage T5. Purified FhuA was reconstituted into liposomes. The size of the proteoliposomes and the distribution of the proteins in the vesicles were determined by freeze fracture electron microscopy. Unilamellar vesicles with a diameter larger than 200 nm were observed frequently. FhuA was symetrically oriented in the proteoliposomes. Reconstituted FhuA was functional as binding of phage T5 induced the release of phage DNA and its transfer inside the vesicles.

Studies on the Energetics of Proaerolysin Secretion across the Outer Membrane of Aeromonas Species EVIDENCE FOR A REQUIREMENT FOR BOTH THE PROTONMOTIVE FORCE AND ATP

Aeromonas spp. secrete the channel-forming protein proaerolysin across their inner and outer membranes in separate steps using the general secretion pathway. Here we show that treating A. hydrophila or A. salmonicida with the protonophore carbonyl cyanide m-chorophenyl hydrazone blocks the second step in transport, secretion across the outer membrane from the periplasm, under conditions where the ATP levels in the cell are no different than the levels in control, secreting cells. A threshold for ΔΨ was observed in the region of 120 mV, below which secretion by both species was inhibited. Treatment of cells with arsenate, which lowered ATP levels but did not affect ΔΨ, also reduced secretion from the periplasm, an indication that there is an ATP requirement for this step independent of the requirement for ΔΨ. Secretion across the outer membrane was also arrested by increasing the osmotic pressure of the medium, even though cellular ATP levels and ΔΨ were not affected. This may be due to disruption of some necessary association between the inner and outer membranes.