Manuela Zanetti

Plasticity of Listeriolysin O Pores and its Regulation by pH and Unique Histidine

Pore formation of cellular membranes is an ancient mechanism of bacterial pathogenesis that allows efficient damaging of target cells. Several mechanisms have been described, however, relatively little is known about the assembly and properties of pores. Listeriolysin O (LLO) is a pH-regulated cholesterol-dependent cytolysin from the intracellular pathogen Listeria monocytogenes, which forms transmembrane β-barrel pores. Here we report that the assembly of LLO pores is rapid and efficient irrespective of pH. While pore diameters at the membrane surface are comparable at either pH 5.5 or 7.4, the distribution of pore conductances is significantly pH-dependent. This is directed by the unique residue H311, which is also important for the conformational stability of the LLO monomer and the rate of pore formation. The functional pores exhibit variations in height profiles and can reconfigure significantly by merging to other full pores or arcs. Our results indicate significant plasticity of large β-barrel pores, controlled by environmental cues like pH.

Asymmetric Mach–Zehnder Interferometer Based Biosensors for Aflatoxin M1 Detection

In this work, we present a study of Aflatoxin M1 detection by photonic biosensors based on Si3N4 Asymmetric Mach–Zehnder Interferometer (aMZI) functionalized with antibodies fragments (Fab′). We measured a best volumetric sensitivity of 104 rad/RIU, leading to a Limit of Detection below 5 × 10−7 RIU. On sensors functionalized with Fab′, we performed specific and non-specific sensing measurements at various toxin concentrations. Reproducibility of the measurements and re-usability of the sensor were also investigated.

DOPAL derived alpha-synuclein oligomers impair synaptic vesicles physiological function.

Parkinson’s disease is a neurodegenerative disorder characterized by the death of dopaminergic neurons and by accumulation of alpha-synuclein (aS) aggregates in the surviving neurons. The dopamine catabolite 3,4-dihydroxyphenylacetaldehyde (DOPAL) is a highly reactive and toxic molecule that leads to aS oligomerization by covalent modifications to lysine residues. Here we show that DOPAL-induced aS oligomer formation in neurons is associated with damage of synaptic vesicles, and with alterations in the synaptic vesicles pools. To investigate the molecular mechanism that leads to synaptic impairment, we first aimed to characterize the biochemical and biophysical properties of the aS-DOPAL oligomers; heterogeneous ensembles of macromolecules able to permeabilise cholesterol-containing lipid membranes. aS-DOPAL oligomers can induce dopamine leak in an in vitro model of synaptic vesicles and in cellular models. The dopamine released, after conversion to DOPAL in the cytoplasm, could trigger a noxious cycle that further fuels the formation of aS-DOPAL oligomers, inducing neurodegeneration.

Biosensors based on Si3N4 asymmetric Mach-Zehnder interferometers

In this work, we present a study on photonic biosensors based on Si3N4 asymmetric Mach-Zehnder Interferometers (aMZI) for Aflatoxin M1 (AFM1) detection. AFM1 is an hepatotoxic and a carcinogenic toxin present in milk. The biosensor is based on an array of four Si3N4 aMZI that are optimized for 850nm wavelength. We measure the bulk Sensitivity (S) and the Limit of Detection (LOD) of our devices. In the array, three devices are exposed and have very similar sensitivities. The fourth aMZI, which is covered by SiO2, is used as an internal reference for laser (a VCSEL) and temperature fluctuations. We measured a phase sensitivity of 14300±400 rad/RIU. To characterize the LOD of the sensors, we measure the uncertainty of the experimental readout system. From the measurements on three aMZI, we observe the same value of LOD, which is ≈ 4.5×10−7 RIU. After the sensor characterization on homogeneous sensing, we test the surface sensing performances by flowing specific Aflatoxin M1 and non-specific Ochratoxin in 50 mM MES pH 6.6 buffer on the top of the sensors functionalized with Antigen-Recognising Fragments (Fab’). The difference between specific and non-specific signals shows the specificity of our sensors. A moderate regeneration of the sensors is obtained by using glycine solution.

Corrigendum: Plasticity of Listeriolysin O Pores and its Regulation by pH and Unique Histidine

Scientific Reports 5: Article number: 9623; 10.1038/srep09623published online: April082015; updated: November062015 The original version of this Article contained a typographical error in the title of the paper, where the word “Listeriolysin” was incorrectly given as “Lysteriolysin”. In addition, there was a typographical error in the legend of Figure 4: “(a) Time course of pore formation by the wild type LLO on SLB at pH 5.5. Numbers at the upper left side of each image show the time in seconds”. now reads: “(a) Time course of pore formation by the wild type LLO on SLB at pH 5.5. Numbers at the upper left side of each image show the time in minutes”. These errors have now been corrected in the PDF and HTML versions of the Article.

Properties of Pores Formed by Cholesterol-Dependent Cytolysins and Actinoporins

Planar lipid membrane (PLM) measurements allow direct observation of pores in model lipid membranes. This biophysical approach was very important for our understanding of how transmembrane pores are formed by cholesterol-dependent cytolysins (CDCs), a toxin family from pathogenic bacteria, and actinoporins, cytolysins from sea anemones. In this review we discuss current knowledge of pore formation by these two protein families and how the PLM approach revealed mechanisms by which these two unrelated protein families porate membranes. Interestingly, for both toxins, the protein portion constituting the pore walls has an alpha helical configuration in the secreted water soluble form. This structure is maintained for actinoporins in the membrane inserted configuration, while the pore of CDCs necessitates a drastic change in secondary structure, which transforms to beta hairpins in the membrane. Both proteins are able to form toroidal proteo-lipid pores.