CryoEM structures of membrane pore and prepore complex reveal cytolytic mechanism of Pneumolysin

Many pathogenic bacteria produce pore-forming toxins to attack and kill human cells. We have determined the 4.5 Å structure of the ~2.2 MDa pore complex of pneumolysin, the main virulence factor of Streptococcus pneumoniae, by cryoEM. The pneumolysin pore is a 400 Å ring of 42 membrane-inserted monomers. Domain 3 of the soluble toxin refolds into two ~85 Å β-hairpins that traverse the lipid bilayer and assemble into a 168-strand β-barrel. The pore complex is stabilized by salt bridges between β-hairpins of adjacent subunits and an internal α-barrel. The apolar outer barrel surface with large sidechains is immersed in the lipid bilayer, while the inner barrel surface is highly charged. Comparison of the cryoEM pore complex to the prepore structure obtained by electron cryo-tomography and the x-ray structure of the soluble form reveals the detailed mechanisms by which the toxin monomers insert into the lipid bilayer to perforate the target membrane.

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The Glycosyltransferase Domain of Penicillin-Binding Protein 2a from Streptococcus pneumoniae Catalyzes the Polymerization of Murein Glycan Chains

Journal of Bacteriology ◽

10.1128/jb.185.15.4418-4423.2003 ◽

2003 ◽

Vol 185 (15) ◽

pp. 4418-4423 ◽

Cited By ~ 31

Author(s):

Anne Marie Di Guilmi ◽

Andréa Dessen ◽

Otto Dideberg ◽

Thierry Vernet

Keyword(s):

Streptococcus Pneumoniae ◽

Pathogenic Bacteria ◽

Catalytic Domain ◽

Soluble Form ◽

Lipid Ii ◽

Extracellular Region ◽

Catalytically Active ◽

Bacterial Peptidoglycan

ABSTRACT The bacterial peptidoglycan consists of glycan chains of repeating β-1,4-linked N-acetylglucosaminyl-N-acetylmuramyl units cross-linked through short peptide chains. The polymerization of the glycans, or glycosyltransfer (GT), and transpeptidation (TP) are catalyzed by bifunctional penicillin-binding proteins (PBPs). The β-lactam antibiotics inhibit the TP reaction, but their widespread use led to the development of drug resistance in pathogenic bacteria. In this context, the GT catalytic domain represents a potential target in the antibacterial fight. In this work, the in vitro polymerization of glycan chains by the extracellular region of recombinant Streptococcus pneumoniae PBP2a, namely, PBP2a* (the asterisk indicates the soluble form of the protein) is presented. Dansylated lipid II was used as the substrate, and the kinetic parameters K m and k cat/K m were measured at 40.6 μM (± 15.5) and 1 × 10−3 M−1 s−1, respectively. The GT reaction catalyzed by PBP2a* was inhibited by moenomycin and vancomycin. Furthermore, the sequence between Lys 78 and Ser 156 is required for enzymatic activity, whereas it is dispensable for lipid II binding. In addition, we confirmed that this region of the protein is also involved in membrane interaction, independently of the transmembrane anchor. The characterization of the catalytically active GT domain of S. pneumoniae PBP2a may contribute to the development of new inhibitors, which are urgently needed to renew the antibiotic arsenal.

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Visualization of lipid bilayer in the crystals by solvent contrast modulation

Acta Crystallographica Section A Foundations and Advances ◽

10.1107/s2053273314085015 ◽

2014 ◽

Vol 70 (a1) ◽

pp. C1498-C1498

Author(s):

Yoshiyuki Norimatsu ◽

Junko Tsueda ◽

Ayami Hirata ◽

Shiho Iwasawa ◽

Chikashi Toyoshima

Keyword(s):

Lipid Bilayer ◽

Electron Density ◽

Lipid Bilayers ◽

Transmembrane Helix ◽

Real Space ◽

New Method ◽

Imaging Plate ◽

Salt Bridges ◽

Transmembrane Helices ◽

X Ray

A new method of X-ray solvent contrast modulation was developed to visualize lipid bilayers in crystals of membrane proteins at a high enough resolution to resolve individual phospholipids molecules (~3.5 Å ). Visualization of lipid bilayer has been escaping from conventional crystallographic methods due to its extreme flexibility, and our knowledge on the behavior of lipid bilayer is still very much limited. Here we applied the new method of X-ray solvent contrast modulation to crystals of Ca2+-ATPase in 4 different physiological states. As phospholipids have to be added to make crystals of Ca2+-ATPase, it is expected that lipid bilayers are present in the crystals. Moreover, transmembrane helices of Ca2+-ATPase rearrange drastically during the reaction cycle and some of them show substantial movements perpendicular to the bilayer plane. Thus these crystals provide a rare opportunity to directly visualize phospholipids interacting with a membrane protein in different conformations. Complete diffraction data covering from 200 to 3.2 Å resolution were collected at BL41XU, Spring-8, using an R-Axis V imaging plate detector for crystals soaked in solvent of different electron density. A new concept "solvent exchange probability", which should be 1 in the bulk solvent, 0 inside the protein and an intermediate at interface, was introduced and used as a restraint for real space phase improvement. The electron density maps thus obtained clearly show that: (i) Phospholipid molecules surrounding the protein are fixed apparently by Arg/Lys-phosphate salt bridges or Trp-carbonyl hydrogen bonds and follow the movements of transmembrane helices. Movements of as large as 12 Å are allowed. (ii) If the movement of a transmembrane helix exceeds this limit, associated phospholipids change the partners for fixation or change the orientation of the entire protein molecule.

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Evaluation of Antibacterial and Antidiabetic Potential of Silver Nanoparticles using Eugenia Uniflora L. Seed Extract

International Journal of Pharmaceutical Sciences and Nanotechnology ◽

10.37285/ijpsn.2020.13.6.6 ◽

2020 ◽

Vol 13 (6) ◽

pp. 5217-5225

Author(s):

Guru Kumar Dugganaboyana ◽

Chethankumar Mukunda ◽

Suresh Darshini Inakanally

Keyword(s):

Biomedical Applications ◽

Pathogenic Bacteria ◽

Seed Extract ◽

Drug Formulation ◽

Visible Spectroscopy ◽

Plant Materials ◽

X Ray ◽

Eugenia Uniflora ◽

Uv Visible

In recent years, green nanotechnology-based approaches using plant materials have been accepted as an environmentally friendly and cost-effective approach with various biomedical applications. In the current study, AgNPs were synthesized using the seed extract of the Eugenia uniflora L. (E.uniflora). Characterization was done using UV-Visible spectroscopy, X-ray diffraction (XRD), scanning electronic microscopy (SEM) and energy-dispersive X-ray spectroscopy (EDX) analyses. The formation of AgNPs has confirmed through UV-Visible spectroscopy (at 466 nm) by the change of color owing to surface Plasmon resonance. Based on the XRD pattern, the crystalline property of AgNPs was established. The functional group existing in seed of E.uniflora extract accountable for the reduction of Ag+ ion and the stabilization of AgNPs was investigated. The morphological structures and elemental composition was determined by SEM and EDX analysis. With the growing application of AgNPs in biomedical perspectives, the biosynthesized AgNPs were evaluated for their antibacterial and along with their antidiabetic potential. The results showed that AgNPs are extremely effective with potent antidiabetic potential at a very low concentration. It also exhibited potential antibacterial activity against the three tested human pathogenic bacteria. Overall, the results highlight the effectiveness and potential applications of AgNPs in biomedical fields such as in the treatment of acute illnesses as well as in drug formulation for treating various diseases such as cancer and diabetes. It could be concluded that E. uniflora seed extract AgNPs can be used efficiently for in vitro evaluation of their antibacterial and antidiabetic effects with potent biomedical applications.

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The Relationship between Colonization by Moraxella catarrhalis and Tonsillar Hypertrophy

Canadian Journal of Infectious Diseases and Medical Microbiology ◽

10.1155/2018/5406467 ◽

2018 ◽

Vol 2018 ◽

pp. 1-9

Author(s):

Mirela C. M. Prates ◽

Edwin Tamashiro ◽

José L. Proenca-Modena ◽

Miriã F. Criado ◽

Tamara H. Saturno ◽

...

Keyword(s):

Staphylococcus Aureus ◽

Pseudomonas Aeruginosa ◽

Streptococcus Pneumoniae ◽

Haemophilus Influenzae ◽

Moraxella Catarrhalis ◽

Pathogenic Bacteria ◽

Simultaneous Detection ◽

Tonsillar Hypertrophy ◽

Palatine Tonsils ◽

Adenotonsillar Hypertrophy

We sought to investigate the prevalence of potentially pathogenic bacteria in secretions and tonsillar tissues of children with chronic adenotonsillitis hypertrophy compared to controls. Prospective case-control study comparing patients between 2 and 12 years old who underwent adenotonsillectomy due to chronic adenotonsillar hypertrophy to children without disease. We compared detection of Streptococcus pneumoniae, Haemophilus influenzae, Staphylococcus aureus, Pseudomonas aeruginosa, and Moraxella catarrhalis by real-time PCR in palatine tonsils, adenoids, and nasopharyngeal washes obtained from 37 children with and 14 without adenotonsillar hypertrophy. We found high frequency (>50%) of Haemophilus influenzae, Streptococcus pneumoniae, Moraxella catarrhalis, and Pseudomonas aeruginosa in both groups of patients. Although different sampling sites can be infected with more than one bacterium and some bacteria can be detected in different tissues in the same patient, adenoids, palatine tonsils, and nasopharyngeal washes were not uniformly infected by the same bacteria. Adenoids and palatine tonsils of patients with severe adenotonsillar hypertrophy had higher rates of bacterial coinfection. There was good correlation of detection of Moraxella catarrhalis in different sampling sites in patients with more severe tonsillar hypertrophy, suggesting that Moraxella catarrhalis may be associated with the development of more severe hypertrophy, that inflammatory conditions favor colonization by this agent. Streptococcus pneumoniae, Staphylococcus aureus, Haemophilus influenzae, and Moraxella catarrhalis are frequently detected in palatine tonsils, adenoids, and nasopharyngeal washes in children. Simultaneous detection of Moraxella catarrhalis in adenoids, palatine tonsils, and nasopharyngeal washes was correlated with more severe tonsillar hypertrophy.

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The Lipid Bilayer Structure of the Abnormal Human Plasma Lipoprotein X. An X-Ray Small-Angle-Scattering Study

European Journal of Biochemistry ◽

10.1111/j.1432-1033.1977.tb11654.x ◽

1977 ◽

Vol 77 (1) ◽

pp. 165-171 ◽

Cited By ~ 34

Author(s):

Peter LAGGNER ◽

Otto GLATTER ◽

Karl MULLER ◽

Otto KRATKY ◽

Gerhard KOSTNER ◽

...

Keyword(s):

Human Plasma ◽

Lipid Bilayer ◽

Small Angle ◽

Small Angle Scattering ◽

Plasma Lipoprotein ◽

Bilayer Structure ◽

X Ray ◽

Angle Scattering ◽

Scattering Study

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The effect of polydispersity, shape fluctuations and curvature on small unilamellar vesicle small-angle X-ray scattering curves

Journal of Applied Crystallography ◽

10.1107/s1600576721001461 ◽

2021 ◽

Vol 54 (2) ◽

pp. 557-568

Author(s):

Veronica Chappa ◽

Yuliya Smirnova ◽

Karlo Komorowski ◽

Marcus Müller ◽

Tim Salditt

Keyword(s):

Lipid Bilayer ◽

Density Profile ◽

Small Angle ◽

Thermal Fluctuations ◽

Electron Density Profile ◽

Membrane Asymmetry ◽

X Ray ◽

X Ray Scattering ◽

Vesicle Shape ◽

Small Unilamellar Vesicles

Small unilamellar vesicles (20–100 nm diameter) are model systems for strongly curved lipid membranes, in particular for cell organelles. Routinely, small-angle X-ray scattering (SAXS) is employed to study their size and electron-density profile (EDP). Current SAXS analysis of small unilamellar vesicles (SUVs) often employs a factorization into the structure factor (vesicle shape) and the form factor (lipid bilayer electron-density profile) and invokes additional idealizations: (i) an effective polydispersity distribution of vesicle radii, (ii) a spherical vesicle shape and (iii) an approximate account of membrane asymmetry, a feature particularly relevant for strongly curved membranes. These idealizations do not account for thermal shape fluctuations and also break down for strong salt- or protein-induced deformations, as well as vesicle adhesion and fusion, which complicate the analysis of the lipid bilayer structure. Presented here are simulations of SAXS curves of SUVs with experimentally relevant size, shape and EDPs of the curved bilayer, inferred from coarse-grained simulations and elasticity considerations, to quantify the effects of size polydispersity, thermal fluctuations of the SUV shape and membrane asymmetry. It is observed that the factorization approximation of the scattering intensity holds even for small vesicle radii (∼30 nm). However, the simulations show that, for very small vesicles, a curvature-induced asymmetry arises in the EDP, with sizeable effects on the SAXS curve. It is also demonstrated that thermal fluctuations in shape and the size polydispersity have distinguishable signatures in the SAXS intensity. Polydispersity gives rise to low-q features, whereas thermal fluctuations predominantly affect the scattering at larger q, related to membrane bending rigidity. Finally, it is shown that simulation of fluctuating vesicle ensembles can be used for analysis of experimental SAXS curves.

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Intermedilysin cytolytic activity depends on heparan sulfates and membrane composition

PLoS Genetics ◽

10.1371/journal.pgen.1009387 ◽

2021 ◽

Vol 17 (2) ◽

pp. e1009387

Author(s):

Gediminas Drabavicius ◽

Dirk Daelemans

Keyword(s):

Pathogenic Bacteria ◽

Biological Membrane ◽

Cell Lysis ◽

Cytolytic Activity ◽

Asymmetric Distribution ◽

Membrane Composition ◽

Membrane Phospholipids ◽

Knock Out ◽

Target Membrane ◽

Heparan Sulfates

Cholesterol-dependent cytolysins (CDCs), of which intermedilysin (ILY) is an archetypal member, are a group of pore-forming toxins secreted by a large variety of pathogenic bacteria. These toxins, secreted as soluble monomers, oligomerize upon interaction with cholesterol in the target membrane and transect it as pores of diameters of up to 100 to 300 Å. These pores disrupt cell membranes and result in cell lysis. The immune receptor CD59 is a well-established cellular factor required for intermedilysin pore formation. In this study, we applied genome-wide CRISPR-Cas9 knock-out screening to reveal additional cellular co-factors essential for ILY-mediated cell lysis. We discovered a plethora of genes previously not associated with ILY, many of which are important for membrane constitution. We show that heparan sulfates facilitate ILY activity, which can be inhibited by heparin. Furthermore, we identified hits in both protein and lipid glycosylation pathways and show a role for glucosylceramide, demonstrating that membrane organization is important for ILY activity. We also cross-validated identified genes with vaginolysin and pneumolysin and found that pneumolysin’s cytolytic activity strongly depends on the asymmetric distribution of membrane phospholipids. This study shows that membrane-targeting toxins combined with genetic screening can identify genes involved in biological membrane composition and metabolism.

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