Infrared and 2-Dimensional Correlation Spectroscopy Study of the Effect of CH3NH3PbI3 and CH3NH3SnI3 Photovoltaic Perovskites on Eukaryotic Cells

We studied the effect of the exposure of human A549 and SH-SY5Y cell lines to aqueous solutions of organic/inorganic halide perovskites CH3NH3PbI3 (MAPbI3) and CH3NH3SnI3 (MASnI3) at the molecular level by using Fourier transform infrared microspectroscopy. We monitored the infrared spectra of some cells over a few days following exposure to the metals and observed the spectroscopic changes dominated by the appearance of a strong band at 1627 cm−1. We used Infrared (IR) mapping to show that this change was associated with the cell itself or the cellular membrane. It is unclear whether the appearance of the 1627 cm−1 band and heavy metal exposure are related by a direct causal relationship. The spectroscopic response of exposure to MAPbI3 and MASnI3 was similar, indicating that it may arise from a general cellular response to stressful environmental conditions. We used 2D correlation spectroscopy (2DCOS) analysis to interpret spectroscopic changes. In a novel application of the method, we demonstrated the viability of 2DCOS for band assignment in spatially resolved spectra. We assigned the 1627 cm−1 band to the accumulation of an abundant amide or amine containing compound, while ruling out other hypotheses. We propose a few tentative assignments to specific biomolecules or classes of biomolecules, although additional biochemical characterization will be necessary to confirm such assignments

Structure of viral membrane-penetrating machines by electron cryo-microscopy and tomography

Sergey Nazarov

My PhD thesis aims to obtain the structure of several bacteriophages, including so-called “jumbo” phages, using the electron cryo-microscopy (cryo-EM) and to study the structural transformation of these viruses as they attach and infect the host cell. We aim to understand the process of host cell membrane penetration at the molecular level and to achieve the level of knowledge necessary to understand how the genome and proteins, which are originally packaged into the capsid, are delivered into the host cell. This structural information can be of great importance since bacteriophage therapy is a potentially promising alternative to antibiotics, which are urgently needed. Bacteriophages, or phages are the most abundant and diverse form of life on Earth, and have developed various strategies through which to infect a susceptible bacterial host. A vast majority of phages have evolved to use a special organelle, called a “tail”, for host recognition, attachment and genome delivery into the cell. The host cell envelope is penetrated with the help of the tail. Unlike most eukaryotic viruses, infection of a host by tailed bacteriophages usually requires only one virion per bacterium, indicating that tailed bacteriophages have evolved an extremely efficient infection mechanism. Bacteriophages with contractile tails are complex viruses that infect Bacteria and Archaea. They share a common evolutionary origin and some functional aspect with other large macromolecular machines, such as the Serratia entomophila antifeeding prophage, the Photorhabdus virulence cassette, and the type VI secretion system (T6SS), which function to attack other cells by translocating toxic effector proteins into the target cell’s cytoplasm. The T6SS and the so-called “jumbo” phages are the most complex of these contractile injection systems. Jumbo phages have genomes exceeding 250kb and virus particle sizes of 300 nm and greater. Hundreds of different proteins are involved in the morphogenesis and assembly of a jumbo phage particle. A number of bacteriophages infecting important pathogens were isolated and characterized by means of cryo-EM and bioinformatics tools. Bacteriophage ɸ92 is a virus infecting a wide variety of acapsular Escherichia coli laboratory strains, Salmonella strains, and pathogenic E.Coli strains carrying a polysialic acid, which cause severe invasive infections including meningitis, pneumonia, septicaemia, osteomyelitis, septic arthritis and pyelonephritis. A cryo-EM reconstructions of the ɸ92 capsid, baseplate and long contractile tail were calculated, and complete three-dimensional model of the phage virion was built. Two bacteriophages, ɸEco32 infecting mastitis-causing strains of E.Coli and 7-11 infecting Salmonella strains, were isolated. They are a members of the Podoviridae family with the rare C3 morphotype, which occurs in

EPFL2015

Association of Helicobacter pylori vacuolating toxin (VacA) with lipid rafts

Yiming Li, Françoise Gisou van der Goot Grunberg

A variety of extracellular ligands and pathogens interact with raft domains in the plasma membrane of eukaryotic cells. In this study, we examined the role of lipid rafts and raft-associated glycosylphosphatidylinositol (GPI)-anchored proteins in the process by which Helicobacter pylori vacuolating toxin (VacA) intoxicates cells. We first investigated whether GPI-anchored proteins are required for VacA toxicity by analyzing wild-type Chinese hamster ovary (CHO) cells and CHO-LA1 mutant cells that are defective in production of GPI-anchored proteins. Whereas wild-type and mutant cells differed markedly in susceptibility to aerolysin (a bacterial toxin that binds to GPI-anchored proteins), they were equally susceptible to VacA. We next determined whether VacA physically associates with lipid rafts. CHO or HeLa cells were incubated with VacA, and Triton-insoluble membranes then were separated by sucrose density gradient centrifugation. Immunoblot analysis revealed that a substantial proportion of cell-associated toxin was associated with detergent-resistant membranes (DRMs). DRM association required acid activation of the purified toxin prior to contact with cells, and acid activation also was required for VacA cytotoxicity. Treatment of cells with methyl-beta-cyclodextrin (a cholesterol-depleting agent) did not inhibit VacA-induced depolarization of the plasma membrane, but interfered with the internalization or intracellular localization of VacA and inhibited the capacity of the toxin to induce cell vacuolation. Treatment of cells with nystatin also inhibited VacA-induced cell vacuolation. These data indicate that VacA associates with lipid raft microdomains in the absence of GPI-anchored proteins and suggest that association of the toxin with lipid rafts is important for VacA cytotoxicity.

2002

Shigella phagocytic vacuolar membrane remnants participate in the cellular response to pathogen invasion and are regulated by autophagy

Nicolas Dupont, Françoise Gisou van der Goot Grunberg

Intracellular pathogens like Shigella flexneri enter host cells by phagocytosis. Once inside, the pathogen breaks the vacuolar membrane for cytosolic access. The fate and function of the vacuolar membrane remnants are not clear. Examining Shigella-infected nonmyeloid cells, we observed that proteins associated with vacuolar membrane remnants are polyubiquinated, recruit the autophagy marker LC3 and adaptor p62, and are targeted to autophagic degradation. Further, inflammasome components and caspase-1 were localized to these membranes and correlated with dampened inflammatory response and necrotic cell death. In Atg4B mutant cells in which autophagosome maturation is blocked, polyubiquitinated proteins and P62 accumulated on membrane remnants, and as in autophagy-deficient Atg5(-/-) cells, the early inflammatory and cytokine response was exacerbated. Our results suggest that host membranes, after rupture by an invading cytoplasm-targeted bacterium, contribute to the cellular responses to infection by acting as a signaling node, with autophagy playing a central role in regulating these responses.

2009

Structure of viral membrane-penetrating machines by electron cryo-microscopy and tomography

Sergey Nazarov

EPFL2015