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Zheng, W., Andersson, M., Mortezaei, N., Bullitt, E. & Egelman, E. (2019). Cryo-EM structure of the CFA/I pilus rod. IUCrJ, 6(5), 815-821
Open this publication in new window or tab >>Cryo-EM structure of the CFA/I pilus rod
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2019 (English)In: IUCrJ, ISSN 0972-6918, E-ISSN 2052-2525, Vol. 6, no 5, p. 815-821Article in journal (Refereed) Published
Abstract [en]

Enterotoxigenic Escherichia coli (ETEC) are common agents of diarrhea for travelers and a major cause of mortality in children in developing countries. To attach to intestinal cells ETEC express colonization factors, among them CFA/I, which are the most prevalent factors and are the archetypical representative of class 5 pili. The helical quaternary structure of CFA/I can be unwound under tensile force and it has been shown that this mechanical property helps bacteria to withstand shear forces from fluid motion. We report in this work the CFA/I pilus structure at 4.3 Å resolution from electron cryomicroscopy (cryo-EM) data, and report details of the donor strand complementation. The CfaB pilins modeled into the cryo-EM map allow us to identify the buried surface area between subunits, and these regions are correlated to quaternary structural stability in class 5 and chaperone–usher pili. In addition, from the model built using the EM structure we also predicted that residue 13 (proline) of the N-terminal β-strand could have a major impact on the filament's structural stability. Therefore, we used optical tweezers to measure and compare the stability of the quaternary structure of wild type CFA/I and a point-mutated CFA/I with a propensity for unwinding. We found that pili with this mutated CFA/I require a lower force to unwind, supporting our hypothesis that Pro13 is important for structural stability. The high-resolution CFA/I pilus structure presented in this work and the analysis of structural stability will be useful for the development of novel antimicrobial drugs that target adhesion pili needed for initial attachment and sustained adhesion of ETEC.

Keywords
fimbriae, bacterial adhesion, helical reconstruction, force spectroscopy, electron cryomicroscopy, 3D reconstruction, 3D image processing, integrative structural biology
National Category
Structural Biology
Identifiers
urn:nbn:se:umu:diva-163321 (URN)10.1107/S2052252519007966 (DOI)000484171300007 ()2-s2.0-85071914577 (Scopus ID)
Available from: 2019-09-16 Created: 2019-09-16 Last updated: 2019-11-22Bibliographically approved
Zhang, H., Söderholm, N., Sandblad, L., Wiklund, K. & Andersson, M. (2019). DSeg: a dynamic image segmentation program to extract backbone patterns for filamentous bacteria and hyphae structures. Microscopy and Microanalysis, 25(3), 711-719
Open this publication in new window or tab >>DSeg: a dynamic image segmentation program to extract backbone patterns for filamentous bacteria and hyphae structures
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2019 (English)In: Microscopy and Microanalysis, ISSN 1431-9276, E-ISSN 1435-8115, Vol. 25, no 3, p. 711-719Article in journal (Refereed) Published
Abstract [en]

Analysis of numerous filamentous structures in an image is often limited by the ability of algorithms to accurately segment complex structures or structures within a dense population. It is even more problematic if these structures continuously grow when recording a time-series of images. To overcome these issues we present DSeg; an image analysis program designed to process time-series image data, as well as single images, to segment filamentous structures. The program includes a robust binary level-set algorithm modified to use size constraints, edge intensity, and past information. We verify our algorithms using synthetic data, differential interference contrast images of filamentous prokaryotes, and transmission electron microscopy images of bacterial adhesion fimbriae. DSeg includes automatic segmentation, tools for analysis, and drift correction, and outputs statistical data such as persistence length, growth rate, and growth direction. The program is available at Sourceforge.

Place, publisher, year, edition, pages
Cambridge University Press, 2019
Keywords
filamentous, hyphae, image segmentation, MATLAB, software, quantitative measurement
National Category
Biophysics
Research subject
Computerized Image Analysis; cell research
Identifiers
urn:nbn:se:umu:diva-150686 (URN)10.1017/S1431927619000308 (DOI)000474798800016 ()30894244 (PubMedID)
Note

Originally included in thesis in manuscript form.

The program is available at https://sourceforge.net/projects/dseg-software

Available from: 2018-08-15 Created: 2018-08-15 Last updated: 2019-08-07Bibliographically approved
Jonsson, M., Andersson, M., Fick, J., Brodin, T., Klaminder, J. & Piovano, S. (2019). High-speed imaging reveals how antihistamine exposure affects escape behaviours in aquatic insect prey. Science of the Total Environment, 648, 1257-1262
Open this publication in new window or tab >>High-speed imaging reveals how antihistamine exposure affects escape behaviours in aquatic insect prey
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2019 (English)In: Science of the Total Environment, ISSN 0048-9697, E-ISSN 1879-1026, Vol. 648, p. 1257-1262Article in journal (Refereed) Published
Abstract [en]

Aquatic systems receive a wide range of pharmaceuticals that may have adverse impacts on aquatic wildlife. Among these pharmaceuticals, antihistamines are commonly found, and these substances have the potential to influence the physiology of aquatic invertebrates. Previous studies have focused on how antihistamines may affect behaviours of aquatic invertebrates, but these studies probably do not capture the full consequences of antihistamine exposure, as traditional recording techniques do not capture important animal movements occurring at the scale of milliseconds, such as prey escape responses. In this study, we investigated if antihistamine exposure can impact escape responses in aquatic insect, by exposing damselfly (Coenagrion hastulatum) larvae to two environmentally relevant concentrations (0.1 and 1 μg L−1) of diphenhydramine. Importantly, we used a high-speed imaging approach that with high-time resolution captures details of escape responses and, thus, potential impacts of diphenhydramine on these behaviours. Our results show overall weak effects of antihistamine exposure on the escape behaviours of damselfly larvae. However, at stage 2 of the C-escape response, we found a significant increase in turning angle, which corresponds to a reduced swimming velocity, indicating a reduced success at evading a predator attack. Thus, we show that low concentrations of an antihistamine may affect behaviours strongly related to fitness of aquatic insect prey – effects would have been overlooked using traditional recording techniques. Hence, to understand the full consequences of pharmaceutical contamination on aquatic wildlife, high-speed imaging should be incorporated into future environmental risk assessments.

Place, publisher, year, edition, pages
Elsevier, 2019
Keywords
Damselfly larvae, Diphenhydramine, Escape response, Pharmaceutical pollution
National Category
Ecology
Identifiers
urn:nbn:se:umu:diva-150912 (URN)10.1016/j.scitotenv.2018.08.226 (DOI)2-s2.0-85052146409 (Scopus ID)
Available from: 2018-08-20 Created: 2018-08-20 Last updated: 2018-11-12Bibliographically approved
Dahlberg, T., Stangner, T., Hanqing, Z., Wiklund, K., Lundberg, P., Edman, L. & Andersson, M. (2018). 3D printed water-soluble scaffolds for rapid production of PDMS micro-fluidic flow chambers. Scientific Reports, 8(1), Article ID 3372.
Open this publication in new window or tab >>3D printed water-soluble scaffolds for rapid production of PDMS micro-fluidic flow chambers
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2018 (English)In: Scientific Reports, ISSN 2045-2322, E-ISSN 2045-2322, Vol. 8, no 1, article id 3372Article in journal (Refereed) Published
Abstract [en]

We report a novel method for fabrication of three-dimensional (3D) biocompatible micro-fluidic flow chambers in polydimethylsiloxane (PDMS) by 3D-printing water-soluble polyvinyl alcohol (PVA) filaments as master scaffolds. The scaffolds are first embedded in the PDMS and later residue-free dissolved in water leaving an inscription of the scaffolds in the hardened PDMS. We demonstrate the strength of our method using a regular, cheap 3D printer, and evaluate the inscription process and the channels micro-fluidic properties using image analysis and digital holographic microscopy. Furthermore, we provide a protocol that allows for direct printing on coverslips and we show that flow chambers with a channel cross section down to 40 x 300 μm can be realized within 60 min. These flow channels are perfectly transparent, biocompatible and can be used for microscopic applications without further treatment. Our proposed protocols facilitate an easy, fast and adaptable production of micro-fluidic channel designs that are cost-effective, do not require specialized training and can be used for a variety of cell and bacterial assays. To help readers reproduce our micro-fluidic devices, we provide: full preparation protocols, 3D-printing CAD files for channel scaffolds and our custom-made molding device, 3D printer build-plate leveling instructions, and G-code.

Place, publisher, year, edition, pages
Nature Publishing Group, 2018
National Category
Other Materials Engineering Other Engineering and Technologies not elsewhere specified Other Physics Topics
Identifiers
urn:nbn:se:umu:diva-144631 (URN)10.1038/s41598-018-21638-w (DOI)000425500300044 ()
Funder
Swedish Research Council, 2013-5379The Kempe Foundations, JCK-1622
Available from: 2018-02-08 Created: 2018-02-08 Last updated: 2018-08-16Bibliographically approved
Wiklund, K., Zhang, H., Stangner, T., Singh, B., Bullitt, E. & Andersson, M. (2018). A drag force interpolation model for capsule-shaped cells in fluid flows near a surface. Microbiology, 164(4), 483-494
Open this publication in new window or tab >>A drag force interpolation model for capsule-shaped cells in fluid flows near a surface
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2018 (English)In: Microbiology, ISSN 1350-0872, E-ISSN 1465-2080, Vol. 164, no 4, p. 483-494Article in journal (Refereed) Published
Abstract [en]

We report an interpolation model to calculate the hydrodynamic force on tethered capsule-shaped cells in micro-fluidic flows near a surface. Our model is based on numerical solutions of the full Navier–Stokes equations for capsule-shaped objects considering their geometry, aspect ratio and orientation with respect to fluid flow. The model reproduced the results from computational fluid dynamic simulations, with an average error of <0.15 % for objects with an aspect ratio up to 5, and the model exactly reproduced the Goldman approximation of spherical objects close to a surface. We estimated the hydrodynamic force imposed on tethered Escherichia coli cells using the interpolation model and approximate models found in the literature, for example, one that assumes that E. coli is ellipsoid shaped. We fitted the 2D-projected area of a capsule and ellipsoid to segmented E. coli cells. We found that even though an ellipsoidal shape is a reasonable approximation of the cell shape, the capsule gives 4.4 % better agreement, a small difference that corresponds to 15 % difference in hydrodynamic force. In addition, we showed that the new interpolation model provides a significantly better agreement compared to estimates from commonly used models and that it can be used as a fast and accurate substitute for complex and computationally heavy fluid dynamic simulations. This is useful when performing bacterial adhesion experiments in parallel-plate flow channels. We include a MATLAB script that can track cells in a video time-series and estimate the hydrodynamic force using our interpolation formula.

Place, publisher, year, edition, pages
Microbiology Society, 2018
Keywords
E. coli, adhesion, Goldman’s approximation, tethered cells, micro-fluidics
National Category
Other Physics Topics Other Biological Topics
Research subject
Physics
Identifiers
urn:nbn:se:umu:diva-144499 (URN)10.1099/mic.0.000624 (DOI)29509130 (PubMedID)2-s2.0-85045149561 (Scopus ID)
Available from: 2018-02-05 Created: 2018-02-05 Last updated: 2018-08-16Bibliographically approved
Barbercheck Epler, C. R., Bullitt, E. & Andersson, M. (2018). Bacterial adhesion pili. In: J. Robin Harris, Egbert Boekema (Ed.), Membrane protein complexes: structure and function (pp. 1-18). Springer Publishing Company
Open this publication in new window or tab >>Bacterial adhesion pili
2018 (English)In: Membrane protein complexes: structure and function / [ed] J. Robin Harris, Egbert Boekema, Springer Publishing Company, 2018, , p. 18p. 1-18Chapter in book (Refereed)
Abstract [en]

Escherichia coli bacterial cells produce multiple types of adhesion pili that mediate cell-cell and cell-host attachments. These pili (also called 'fimbriae') are large biopolymers that are comprised of subunits assembled via a sophisticated micro-machinery into helix-like structures that are anchored in the bacterial outer membrane. They are commonly essential for initiation of disease and thus provide a potential target for antibacterial prevention and treatment. To develop new therapeutics for disease prevention and treatment we need to understand the molecular mechanisms and the direct role of adhesion pili during pathogenesis. These helix-like pilus structures possess fascinating and unique biomechanical properties that have been thoroughly investigated using high-resolution imaging techniques, force spectroscopy and fluid flow chambers. In this chapter, we first discuss the structure of pili and the micro-machinery responsible for the assembly process. Thereafter, we present methods for measurement of the biomechanics of adhesion pili, including optical tweezers. Data demonstrate unique biomechanical properties of pili that allow bacteria to sustain binding during in vivo fluid shear forces. We thereafter summarize the current biomechanical findings related to adhesion pili and show that pili biomechanical properties are niche-specific. That is, the data suggest that there is an organ-specific adaptation of pili that facilitates infection of the bacteria's target tissue. Thus, pilus biophysical properties are an important part of Escherichia coli pathogenesis, allowing bacteria to overcome hydrodynamic challenges in diverse environments.

Place, publisher, year, edition, pages
Springer Publishing Company, 2018. p. 18
Series
Subcellular Biochemistry, ISSN 0306-0225 ; 87
Keywords
Fimbriae, Pilins, Pathogenesis, Virulence factors, Optical tweezers
National Category
Structural Biology Other Physics Topics
Identifiers
urn:nbn:se:umu:diva-145352 (URN)10.1007/978-981-10-7757-9_1 (DOI)978-981-10-7757-9 (ISBN)978-981-10-7756-2 (ISBN)
Available from: 2018-03-01 Created: 2018-03-01 Last updated: 2018-06-09Bibliographically approved
Stangner, T., Dahlberg, T., Svenmarker, P., Zakrisson, J., Wiklund, K., Oddershede, L. B. & Andersson, M. (2018). Cooke-Triplet-Tweezers: More compact, robust and efficient optical tweezers. Optics Letters, 43(9), 1990-1993
Open this publication in new window or tab >>Cooke-Triplet-Tweezers: More compact, robust and efficient optical tweezers
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2018 (English)In: Optics Letters, ISSN 0146-9592, E-ISSN 1539-4794, Vol. 43, no 9, p. 1990-1993Article in journal (Refereed) Published
Abstract [en]

We present a versatile three-lens optical design to improve the overall compactness, efficiency, and robustness for optical tweezers based applications. The design, inspired by the Cooke–Triplet configuration, allows for continuous beam magnifications of 2–10× , and axial as well as lateral focal shifts can be realized without switching lenses or introducing optical aberrations. We quantify the beam quality and trapping stiffness and compare the Cooke–Triplet design with the commonly used double Kepler design through simulations and direct experiments. Optical trapping of 1 and 2 μm beads shows that the Cooke–Triplet possesses an equally strong optical trap stiffness compared to the double Kepler lens design but reduces its lens system length by a factor of 2.6. Finally, we demonstrate how a Twyman–Green interferometer integrated in the Cooke–Triplet optical tweezers setup provides a fast and simple method to characterize the wavefront aberrations in the lens system and how it can help in aligning the optical components perfectly.

National Category
Atom and Molecular Physics and Optics
Identifiers
urn:nbn:se:umu:diva-145899 (URN)10.1364/OL.43.001990 (DOI)000431179400013 ()29714728 (PubMedID)
Available from: 2018-03-21 Created: 2018-03-21 Last updated: 2018-06-09Bibliographically approved
Zheng, W., Spaulding, C., Schreiber, H., Dodson, K., Conover, M., Wang, F., . . . Egelman, E. H. (2018). Cryo-Em Structure of Type 1 Pilus. Biophysical Journal, 114(3)
Open this publication in new window or tab >>Cryo-Em Structure of Type 1 Pilus
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2018 (English)In: Biophysical Journal, ISSN 0006-3495, E-ISSN 1542-0086, Vol. 114, no 3Article in journal, Meeting abstract (Other academic) Published
National Category
Biophysics
Identifiers
urn:nbn:se:umu:diva-144910 (URN)10.1016/j.bpj.2017.11.2052 (DOI)
Available from: 2018-02-15 Created: 2018-02-15 Last updated: 2018-06-09Bibliographically approved
Zheng, W., Spaulding, C. N., Schreiber, H. L., Dodson, K. W., Conover, M. S., Wang, F., . . . Egelman, E. H. (2018). Cryo-Em Structure of Type 1 Pilus. Paper presented at 62nd Annual Meeting of the Biophysical-Society, FEB 17-21, 2018, San Francisco, CA. Biophysical Journal, 114(3), 370a-370a
Open this publication in new window or tab >>Cryo-Em Structure of Type 1 Pilus
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2018 (English)In: Biophysical Journal, ISSN 0006-3495, E-ISSN 1542-0086, Vol. 114, no 3, p. 370a-370aArticle in journal, Meeting abstract (Other academic) Published
Abstract [en]

Urinary tract infections (UTIs) are caused by a wide range of pathogens, but the most common causative agent of UTIs is uropathogenic Escherichia coli (UPEC). Virtually all uropathogenic strains of E. coli encode filamentous surface adhesive organelles called type 1 pili, which are a subset of Chaperone-usher pathway (CUP) pili. CUP pili are also ubiquitously expressed on the surface of many other Gram-negative bacterial pathogens. They are important virulence factors facilitating host-pathogen interactions that are crucial for the establishment and persistence of an infection, and involved in regulating other key processes such as biofilm formation. We have solved the 4.2 Å resolution cryo-EM structure of the type 1 pilus, which was present as a background contaminant in a prep of type 4 pili. We have taken advantage of the strength of cryo-EM to separate different molecules and conformations present in solution to show that filament images which might otherwise have been discarded as a contaminant can actually be used to build an atomic model. The model reveals the residues that allow a long chain of FimA subunits, linked by the insertion of a β-strand of one subunit into the β-sheet of an adjacent subunit, to coil into a rigid rod. We show that site-specific mutation of these residues reduces the force needed to unwind the rod. Strikingly, one mutation (A22R) which showed the greatest reduction in unwinding force, eliminated bladder infections in a mouse model. This is presumably due to the fact that the altered mechanics of the A22R pilus rod cannot withstand the shear forces due to urinary flow in the bladder and bacteria harboring this mutation are cleared from the bladder. This provides new insights into the important role of pili mechanics in bacterial pathogenesis.

Place, publisher, year, edition, pages
Biophysical Society, 2018
National Category
Biophysics
Identifiers
urn:nbn:se:umu:diva-148031 (URN)10.1016/j.bpj.2017.11.2052 (DOI)000430450000342 ()
Conference
62nd Annual Meeting of the Biophysical-Society, FEB 17-21, 2018, San Francisco, CA
Available from: 2018-05-30 Created: 2018-05-30 Last updated: 2018-06-09Bibliographically approved
Spaulding, C. N., Schreiber, H. L., Zheng, W., Dodson, K. W., Hazen, J. E., Conover, M. S., . . . Egelman, E. H. (2018). Functional role of the type 1 pilus rod structure in mediating host-pathogen interactions. eLIFE, 7, Article ID e31662.
Open this publication in new window or tab >>Functional role of the type 1 pilus rod structure in mediating host-pathogen interactions
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2018 (English)In: eLIFE, E-ISSN 2050-084X, Vol. 7, article id e31662Article in journal (Refereed) Published
Abstract [en]

Uropathogenic E. coli (UPEC), which cause urinary tract infections (UTI), utilize type 1 pili, a chaperone usher pathway (CUP) pilus, to cause UTI and colonize the gut. The pilus rod, comprised of repeating FimA subunits, provides a structural scaffold for displaying the tip adhesin, FimH. We solved the 4.2 Å resolution structure of the type 1 pilus rod using cryo-electron microscopy. Residues forming the interactive surfaces that determine the mechanical properties of the rod were maintained by selection based on a global alignment of fimA sequences. We identified mutations that did not alter pilus production in vitro but reduced the force required to unwind the rod. UPEC expressing these mutant pili were significantly attenuated in bladder infection and intestinal colonization in mice. This study elucidates an unappreciated functional role for the molecular spring-like property of type 1 pilus rods in host-pathogen interactions and carries important implications for other pilus-mediated diseases.

Place, publisher, year, edition, pages
eLife Sciences Publications, 2018
National Category
Structural Biology Other Physics Topics Microbiology in the medical area
Identifiers
urn:nbn:se:umu:diva-144046 (URN)10.7554/eLife.31662 (DOI)000435201600001 ()29345620 (PubMedID)2-s2.0-85042100644 (Scopus ID)
Available from: 2018-01-19 Created: 2018-01-19 Last updated: 2018-09-14Bibliographically approved
Projects
Anmälan om utnyttjande av återvändarbidrag för beviljade postdoktorsstipendier [2010-02495_VR]; Umeå UniversityIdentification of key adhesion mechanisms in pathogenic Gram-negative bacteria - Characterization and analysis of bacterial adhesion [2013-05379_VR]; Umeå University
Organisations
Identifiers
ORCID iD: ORCID iD iconorcid.org/0000-0002-9835-3263

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