| 1. |
- De Samber, Björn, et al.
(författare)
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Effect of sample preparation techniques upon single cell chemical imaging : A practical comparison between synchrotron radiation based X-ray fluorescence (SR-XRF) and Nanoscopic Secondary Ion Mass Spectrometry (nano-SIMS)
- 2020
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Ingår i: Analytica Chimica Acta. - : Elsevier. - 0003-2670 .- 1873-4324. ; 1106, s. 22-32
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Tidskriftsartikel (refereegranskat)abstract
- Analytical capabilities of Nanoscopic Secondary Ion Mass Spectrometry (nano-SIMS) and Synchrotron Radiation based X-ray Fluorescence (SR nano-XRF) techniques were compared for nanochemical imaging of polymorphonuclear human neutrophils (PMNs). PMNs were high pressure frozen (HPF), cryosubstituted, embedded in Spurr's resin and cut in thin sections (500 nm and 2 mu m for both techniques resp.) Nano-SIMS enabled nanoscale mapping of isotopes of C, N, O, P and S, while SR based nano-XRF enabled trace level imaging of metals like Ca, Mn, Fe, Ni, Cu and Zn at a resolution of approx. 50 nm. The obtained elemental distributions were compared with those of whole, cryofrozen PMNs measured at the newly developed ID16A nano-imaging beamline at the European Synchrotron Radiation Facility (ESRF) in Grenoble, France. Similarities were observed for elements more tightly bound to the cell structure such as phosphorus and sulphur, while differences for mobile ions such as chlorine and potassium were more pronounced. Due to the observed elemental redistribution of mobile ions such as potassium and chlorine, elemental analysis of high pressure frozen (HPF), cryo-substituted and imbedded cells should be interpreted critically. Although decreasing analytical sensitivity occurs due to the presence of ice, analysis of cryofrozen cells - close to their native state - remains the golden standard. In general, we found nanoscale secondary ion mass spectrometry (nano-SIMS) and synchrotron radiation based nanoscopic X-ray fluorescence (SR nano-XRF) to be two supplementary alternatives for nanochemical imaging of single cells at the nanoscale.
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| 2. |
- Freire, Rafael V.M., et al.
(författare)
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Antimicrobial peptide induced colloidal transformations in bacteria-mimetic vesicles : combining in silico tools and experimental methods
- 2021
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Ingår i: Journal of Colloid and Interface Science. - : Elsevier. - 0021-9797 .- 1095-7103. ; 596, s. 352-363
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Tidskriftsartikel (refereegranskat)abstract
- With the growing challenges of bacteria becoming resistant to conventional antibiotics, antimicrobial peptides (AMPs) may offer a potential alternative. One of the most studied AMPs, the human cathelicidin derived AMP LL-37 is notable for its antimicrobial activity even though its mechanism of action is not fully understood yet. This work investigates the interaction of LL-37 with 1-Palmitoyl-2-oleoyl-sn-glycero-3-phospho-rac-(1-glycerol) (POPG) vesicles, which were employed as a bacterial membrane model given the common presence of this phospholipid in the bacterial membrane. Experimental techniques including small angle X-ray scattering, transmission electron microscopy and dynamic light scattering were used to characterize the interactions among LL-37 and POPG. Molecular dynamics simulations complement the experimental studies with molecular-level insights into the process. LL-37 was discovered to actively and critically interact with the POPG vesicles, modifying the membrane curvature that eventually leads to structural transformations from vesicles to mixed micelles. The results shed light on the mechanisms underlying the interactions among LL-37 and bacteria mimetic vesicles and can guide the further development of AMP based antimicrobial materials and therapies.
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| 3. |
- Gineste, Charlotte, et al.
(författare)
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Enzymatically dissociated muscle fibers display rapid dedifferentiation and impaired mitochondrial calcium control
- 2022
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Ingår i: iScience. - : Elsevier. - 2589-0042. ; 25:12
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Tidskriftsartikel (refereegranskat)abstract
- Cells rapidly lose their physiological phenotype upon disruption of their extracellular matrix (ECM)-intracellular cytoskeleton interactions. By comparing adult mouse skeletal muscle fibers, isolated either by mechanical dissection or by collagenase-induced ECM digestion, we investigated acute effects of ECM disruption on cellular and mitochondrial morphology, transcriptomic signatures, and Ca2+ handling. RNA-sequencing showed striking differences in gene expression patterns between the two isolation methods with enzymatically dissociated fibers resembling myopathic phenotypes. Mitochondrial appearance was grossly similar in the two groups, but 3D electron microscopy revealed shorter and less branched mitochondria following enzymatic dissociation. Repeated contractions resulted in a prolonged mitochondrial Ca2+ accumulation in enzymatically dissociated fibers, which was partially prevented by cyclophilin inhibitors. Of importance, muscle fibers of mice with severe mitochondrial myopathy show pathognomonic mitochondrial Ca2+ accumulation during repeated contractions and this accumulation was concealed with enzymatic dissociation, making this an ambiguous method in studies of native intracellular Ca2+ fluxes.
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| 4. |
- Iakovleva, Irina, et al.
(författare)
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Structural basis for transthyretin amyloid formation in vitreous body of the eye
- 2021
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Ingår i: Nature Communications. - : Nature Publishing Group. - 2041-1723. ; 12:1
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Tidskriftsartikel (refereegranskat)abstract
- Amyloid transthyretin (ATTR) amyloidosis is characterized by the abnormal accumulation of ATTR fibrils in multiple organs. However, the structure of ATTR fibrils from the eye is poorly understood. Here, we used cryo-EM to structurally characterize vitreous body ATTR fibrils. These structures were distinct from previously characterized heart fibrils, even though both have the same mutation and type A pathology. Differences were observed at several structural levels: in both the number and arrangement of protofilaments, and the conformation of the protein fibril in each layer of protofilaments. Thus, our results show that ATTR protein structure and its assembly into protofilaments in the type A fibrils can vary between patients carrying the same mutation. By analyzing and matching the interfaces between the amino acids in the ATTR fibril with those in the natively folded TTR, we are able to propose a mechanism for the structural conversion of TTR into a fibrillar form.
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| 5. |
- Jalalvand, Farshid, et al.
(författare)
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Protein domain-dependent vesiculation of Lipoprotein A, a protein that is important in cell wall synthesis and fitness of the human respiratory pathogen Haemophilus influenzae
- 2022
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Ingår i: Frontiers in cellular and infection microbiology. - : Frontiers Media SA. - 2235-2988. ; 12
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Tidskriftsartikel (refereegranskat)abstract
- The human pathogen Haemophilus influenzae causes respiratory tract infections and is commonly associated with prolonged carriage in patients with chronic obstructive pulmonary disease. Production of outer membrane vesicles (OMVs) is a ubiquitous phenomenon observed in Gram-negative bacteria including H. influenzae. OMVs play an important role in various interactions with the human host; from neutralization of antibodies and complement activation to spread of antimicrobial resistance. Upon vesiculation certain proteins are found in OMVs and some proteins are retained at the cell membrane. The mechanism for this phenomenon is not fully elucidated. We employed mass spectrometry to study vesiculation and the fate of proteins in the outer membrane. Functional groups of proteins were differentially distributed on the cell surface and in OMVs. Despite its supposedly periplasmic and outer membrane location, we found that the peptidoglycan synthase-activator Lipoprotein A (LpoA) was accumulated in OMVs relative to membrane fractions. A mutant devoid of LpoA lost its fitness as revealed by growth and electron microscopy. Furthermore, high-pressure liquid chromatography disclosed a lower concentration (55%) of peptidoglycan in the LpoA-deficient H. influenzae compared to the parent wild type bacterium. Using an LpoA-mNeonGreen fusion protein and fluorescence microscopy, we observed that LpoA was enriched in “foci” in the cell envelope, and further located in the septum during cell division. To define the fate of LpoA, C-terminally truncated LpoA-variants were constructed, and we found that the LpoA C-terminal domain promoted optimal transportation to the OMVs as revealed by flow cytometry. Taken together, our study highlights the importance of LpoA for H. influenzae peptidoglycan biogenesis and provides novel insights into cell wall integrity and OMV production.
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| 6. |
- Paulsson, Magnus, et al.
(författare)
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Peptidoglycan-Binding Anchor Is a Pseudomonas aeruginosa OmpA Family Lipoprotein With Importance for Outer Membrane Vesicles, Biofilms, and the Periplasmic Shape
- 2021
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Ingår i: Frontiers in Microbiology. - : Frontiers Media S.A.. - 1664-302X. ; 12
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Tidskriftsartikel (refereegranskat)abstract
- The outer membrane protein A (OmpA) family contains an evolutionary conserved domain that links the outer membrane in Gram-negative bacteria to the semi-rigid peptidoglycan (PG) layer. The clinically significant pathogen Pseudomonas aeruginosa carries several OmpA family proteins (OprF, OprL, PA0833, and PA1048) that share the PG-binding domain. These proteins are important for cell morphology, membrane stability, and biofilm and outer membrane vesicle (OMV) formation. In addition to other OmpAs, in silico analysis revealed that the putative outer membrane protein (OMP) with gene locus PA1041 is a lipoprotein with an OmpA domain and, hence, is a potential virulence factor. This study aimed to evaluate PA1041 as a PG-binding protein and describe its effect on the phenotype. Clinical strains were confirmed to contain the lipoprotein resulting from PA1041 expression with Western blot, and PG binding was verified in enzyme-linked immunosorbent assay (ELISA). By using a Sepharose bead-based ELISA, we found that the lipoprotein binds to meso-diaminopimelic acid (mDAP), an amino acid in the pentapeptide portion of PGs. The reference strain PAO1 and the corresponding transposon mutant PW2884 devoid of the lipoprotein were examined for phenotypic changes. Transmission electron microscopy revealed enlarged periplasm spaces near the cellular poles in the mutant. In addition, we observed an increased release of OMV, which could be confirmed by nanoparticle tracking analysis. Importantly, mutants without the lipoprotein produced a thick, but loose and unorganized, biofilm in flow cells. In conclusion, the lipoprotein from gene locus PA1041 tethers the outer membrane to the PG layer, and mutants are viable, but display severe phenotypic changes including disordered biofilm formation. Based upon the phenotype of the P. aeruginosa PW2884 mutant and the function of the protein, we designate the lipoprotein with locus tag PA1041 as “peptidoglycan-binding anchor” (Pba).
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| 7. |
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| 8. |
- Su, Yu Ching, et al.
(författare)
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Non-typeable Haemophilus influenzae major outer membrane protein P5 contributes to bacterial membrane stability, and affects the membrane protein composition crucial for interactions with the human host
- 2023
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Ingår i: Frontiers in Cellular and Infection Microbiology. - : Frontiers Media S.A.. - 2235-2988. ; 13
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Tidskriftsartikel (refereegranskat)abstract
- Non-typeable Haemophilus influenzae (NTHi) is a Gram-negative human pathogen that causes a wide range of airway diseases. NTHi has a plethora of mechanisms to colonize while evading the host immune system for the establishment of infection. We previously showed that the outer membrane protein P5 contributes to bacterial serum resistance by the recruitment of complement regulators. Here, we report a novel role of P5 in maintaining bacterial outer membrane (OM) integrity and protein composition important for NTHi-host interactions. In silico analysis revealed a peptidoglycan-binding motif at the periplasmic C-terminal domain (CTD) of P5. In a peptidoglycan-binding assay, the CTD of P5 (P5CTD) formed a complex with peptidoglycan. Protein profiling analysis revealed that deletion of CTD or the entire P5 changed the membrane protein composition of the strains NTHi 3655Δp5CTD and NTHi 3655Δp5, respectively. Relative abundance of several membrane-associated virulence factors that are crucial for adherence to the airway mucosa, and serum resistance were altered. This was also supported by similar attenuated pathogenic phenotypes observed in both NTHi 3655Δp5CTD and NTHi 3655Δp5. We found (i) a decreased adherence to airway epithelial cells and fibronectin, (ii) increased complement-mediated killing, and (iii) increased sensitivity to the β-lactam antibiotics in both mutants compared to NTHi 3655 wild-type. These mutants were also more sensitive to lysis at hyperosmotic conditions and hypervesiculated compared to the parent wild-type bacteria. In conclusion, our results suggest that P5 is important for bacterial OM stability, which ultimately affects the membrane proteome and NTHi pathogenesis.
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| 9. |
- Söderholm, Niklas, et al.
(författare)
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Exploring the bacterial nano-universe
- 2020
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Ingår i: Current opinion in structural biology. - : Elsevier. - 0959-440X .- 1879-033X. ; 64, s. 166-173
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Forskningsöversikt (refereegranskat)abstract
- Since the days of the first acknowledged microscopist, Antonie van Leeuwenhoek, the 'animalcules', that is, bacteria and other microbes have been subject to increasingly detailed visualization. With the currently most sophisticated molecular imaging method; cryo electron tomography (Cryo-ET), we are reaching the milestone of being able to image an entire organism in a single dataset at nanometer resolution. Cryo-ET will enable the next revolution in our understanding of bacterial cells, their ultra-structure and intricate molecular nanomachines. Here, we highlight recent research discoveries based on constantly progressing technology developments. We discuss advantages and challenges of using Cryo-ET to visualize spatial structure of microorganisms and macromolecular complexes in their native environment.
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| 10. |
- Thofte, Oskar, et al.
(författare)
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Nontypeable haemophilus influenzae P5 binds human C4b-binding protein, promoting serum resistance
- 2021
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Ingår i: Journal of Immunology. - : American Association of Immunologists. - 0022-1767 .- 1550-6606. ; 207:6, s. 1566-1577
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Tidskriftsartikel (refereegranskat)abstract
- Nontypeable Haemophilus influenzae (NTHi) is a Gram-negative human pathogen that causes infections mainly in the upper and lower respiratory tract. The bacterium is associated with bronchitis and exacerbations in patients suffering from chronic obstructive pulmonary disease and frequently causes acute otitis media in preschool children. We have previously demonstrated that the binding of C4b binding protein (C4BP) is important for NTHi complement evasion. In this study, we identified outer membrane protein 5 (P5) of NTHi as a novel ligand of C4BP. Importantly, we observed significantly lower C4BP binding and decreased serum resistance in P5-deficient NTHi mutants. Surface expression of recombinant P5 on Escherichia coli conferred C4BP binding and consequently increased serum resistance. Moreover, P5 expression was positively correlated with C4BP binding in a series of clinical isolates. We revealed higher levels of P5 surface expression and consequently more C4BP binding in isolates from the lower respiratory tract of chronic obstructive pulmonary disease patients and tonsil specimens compared with isolates from the upper respiratory tract and the bloodstream (invasive strains). Our results highlight P5 as an important protein for protecting NTHi against complement-mediated killing.
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