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Exploring the impact of antibodies on the mechanics of bacterial fimbriae
Umeå University, Faculty of Science and Technology, Department of Physics.
2016 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

The discovery of antibiotics in 1928 seemed like a win in the battle against infectious diseases. But, the ability of bacterial pathogens to adapt to these life-saving medicines was underestimated. The bacterial evolution, indeed, led to the emergence of antibiotic resistance as soon as the clinical consumption of antibiotics started. Today, certain bacteria including some strains of the gram-negative Escherichia coli are resistant to all major antibiotics. To overcome this problem, identifying new therapeutic targets in bacteria is essential, which necessitates scrutinizing the bacterial infection mechanism. An initial step in the bacterial infection mechanism is identification of and adherence to host tissue. Thus, blocking bacterial adhesion is considered as a potential target in the battle against infectious diseases. Gram-negative bacteria generally establish their adhesion by variety of proteinaceous structures known as fimbriae. The strains of Escherichia coli associated with gastrointestinal and urinary tract infections, for instance, colonize their host via a variety of adhesion fimbriae. These adhesion organelles are comprised of subunits assembled into a helix-like structure with remarkable biomechanical properties. For example, fimbriae can be significantly extended under force and are therefore very flexible. Fimbrial flexibility is considered to be beneficial for attachment and adhesion of bacteria in fluidic regions.

The aims of this thesis are: to provide insight into the structural and biomechanical differences of fimbriae expressed by enterotoxigenic and uropathogenic Escherichia coli, and to investigate how fimbrial mechanics are affected in the presence of anti-fimbrial antibodies. To achieve these aims we put together data acquired using different technical approaches. We used force measuring optical tweezers to characterize the force-extension responses of fimbriae in the absence and presence of antibodies. High-resolution imaging was employed to explore the structural features of fimbriae as well as monitoring the antibody-fimbriae interactions. Our results demonstrate that each type of fimbria explored shows unique force spectroscopy responses. For example, the fimbriae expressed by uropathogenic Escherichia coli require a higher unwinding force in comparison to enterotoxigenic Escherichia coli fimbriae. These observations suggest that bacteria adapt to the environment wherein they establish colonization by expressing fimbriae with different biophysical features. Such evolutionary adaptation can thereby help in the bacterial adhesion process. Furthermore, we found that antibodies significantly alter the biophysical features of fimbriae, implying that antibodies significantly interfere with the mechanics of fimbriae. We suggest further elucidation of how antibodies disrupt fimbrial mechanics, providing insights for the development of antibody-based therapeutics.

Place, publisher, year, edition, pages
Umeå: Umeå universitet , 2016. , 70 p.
National Category
Biophysics
Identifiers
URN: urn:nbn:se:umu:diva-119694ISBN: 978-91-7601-464-6 (print)OAI: oai:DiVA.org:umu-119694DiVA: diva2:922844
Public defence
2016-05-20, Naturvetarhuset N420, Umeå, 13:00 (English)
Opponent
Supervisors
Available from: 2016-04-29 Created: 2016-04-25 Last updated: 2016-04-29Bibliographically approved
List of papers
1. P-fimbriae in the presence of anti-PapA antibodies: new insight of antibodies action against pathogens
Open this publication in new window or tab >>P-fimbriae in the presence of anti-PapA antibodies: new insight of antibodies action against pathogens
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2013 (English)In: Scientific Reports, ISSN 2045-2322, E-ISSN 2045-2322, Vol. 3, Article Number: 3393- p.Article in journal (Refereed) Published
Abstract [en]

Uropathogenic strains of Escherichia coli establish urinary tract infections by attaching to host epithelial cells using adhesive organelles called fimbriae. Fimbriae are helix-like structures with a remarkable adaptability, offering safeguarding for bacteria exposed to changing fluid forces in the urinary tract. We challenged this property of P-fimbriae by cross-linking their subunits with shaft-specific antibodies and measuring the corresponding force response at a single organelle level. Our data show compromised extension and rewinding of P-fimbriae in the presence of antibodies and reduced fimbrial elasticity, which are important properties of fimbriae contributing to the ability of bacteria to cause urinary tract infections. The reduced elasticity found by cross-linking fimbrial subunits could thus be another assignment for antibodies; in addition to marking bacteria as foreign, antibodies physically compromise fimbrial function. We suggest that our assay and results will be a starting point for further investigations aimed at inhibiting sustained bacterial adhesion by antibodies.

Place, publisher, year, edition, pages
Nature Publishing Group, 2013
Keyword
optical tweezers, E coli, antibodies, IgG, adhesion
National Category
Other Physics Topics Immunology Microbiology Biophysics
Research subject
Physics
Identifiers
urn:nbn:se:umu:diva-82964 (URN)10.1038/srep03393 (DOI)
Funder
Swedish Research Council, VR-MH 2010-3031, VR-NT 2012-4638, 349-2007-8673
Available from: 2013-11-14 Created: 2013-11-14 Last updated: 2017-12-06Bibliographically approved
2. Structure and function of enterotoxigenic Escherichia coli fimbriae from differing assembly pathways
Open this publication in new window or tab >>Structure and function of enterotoxigenic Escherichia coli fimbriae from differing assembly pathways
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2015 (English)In: Molecular Microbiology, ISSN 0950-382X, E-ISSN 1365-2958, Vol. 95, no 1, 116-126 p.Article in journal (Refereed) Published
Abstract [en]

Pathogenic enterotoxigenic Escherichia coli (ETEC) are the major bacterial cause of diarrhea in young children in developing countries and in travelers, causing significant mortality in children. Adhesive fimbriae are a prime virulence factor for ETEC, initiating colonization of the small intestinal epithelium. Similar to other Gram-negative bacteria, ETEC express one or more diverse fimbriae, some assembled by the chaperone-usher pathway and others by the alternate chaperone pathway. Here, we elucidate structural and biophysical aspects and adaptations of each fimbrial type to its respective host niche. CS20 fimbriae are compared with colonization factor antigen I (CFA/I) fimbriae, which are two ETEC fimbriae assembled via different pathways, and with P-fimbriae from uropathogenic E.coli. Many fimbriae unwind from their native helical filament to an extended linear conformation under force, thereby sustaining adhesion by reducing load at the point of contact between the bacterium and the target cell. CFA/I fimbriae require the least force to unwind, followed by CS20 fimbriae and then P-fimbriae, which require the highest unwinding force. We conclude from our electron microscopy reconstructions, modeling and force spectroscopy data that the target niche plays a central role in the biophysical properties of fimbriae that are critical for bacterial pathophysiology.

Place, publisher, year, edition, pages
John Wiley & Sons, 2015
Keyword
fimbriae, pili, macromolecules, optical tweezers
National Category
Other Physics Topics Medical and Health Sciences
Identifiers
urn:nbn:se:umu:diva-95561 (URN)10.1111/mmi.12847 (DOI)000346656200008 ()25355550 (PubMedID)
Funder
Swedish Research Council, 621-2013-5379
Available from: 2014-10-31 Created: 2014-10-31 Last updated: 2017-12-05Bibliographically approved
3. Biomechanical and Structural features of CS2 fimbriae of Enterotoxigenic Escherichia coli 
Open this publication in new window or tab >>Biomechanical and Structural features of CS2 fimbriae of Enterotoxigenic Escherichia coli 
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2015 (English)In: Biophysical Journal, ISSN 0006-3495, E-ISSN 1542-0086, Vol. 109, no 1, 49-56 p.Article in journal (Refereed) Published
Abstract [en]

Enterotoxigenic Escherichia coli (ETEC) are a major cause of diarrhea worldwide, and infection of children in underdeveloped countries often leads to high mortality rates. Isolated ETEC express a plethora of colonization factors (fimbriae/pili), of which CFA/I and CFA/II that are assembled via the alternate chaperone pathway (ACP), are amongst the most common. Fimbriae are filamentous structures, whose shafts are primarily composed of helically arranged single pilin-protein subunits, with a unique biomechanical capability allowing them to unwind and rewind. A sustained ETEC infection, under adverse conditions of dynamic shear forces, is primarily attributed to this biomechanical feature of ETEC fimbriae. Recent understandings about the role of fimbriae as virulence factors are pointing to an evolutionary adaptation of their structural and biomechanical features. In this work, we investigated the biophysical properties of CS2 fimbriae from the CFA/II group. Homology modelling its major structural subunit CotA reveals structural clues and these are related to the niche in which they are expressed. Using optical tweezers force spectroscopy we found that CS2 fimbriae unwind at a constant force of 10 pN and have a corner velocity of 1300 nm/s, i.e., the velocity at which the force required for unwinding rises exponentially with increased speed. The biophysical properties of CS2 fimbriae assessed in this work classify them into a low-force unwinding group of fimbriae together with the CFA/I and CS20 fimbriae expressed by ETEC strains. The three fimbriae are expressed by ETEC, colonize in similar gut environments, and exhibit similar biophysical features, but differ in their biogenesis. Our observation suggests that the environment has a strong impact on the biophysical characteristics of fimbriae expressed by ETEC.

Place, publisher, year, edition, pages
Elsevier, 2015
Keyword
pili, optical tweezers, bacteria, pathogenesis, virulence factors
National Category
Other Physics Topics
Research subject
Physics
Identifiers
urn:nbn:se:umu:diva-103247 (URN)10.1016/j.bpj.2015.05.022 (DOI)
Funder
Swedish Research Council, 621-2013-5379
Note

This work was supported by NIH (GM05722 and RR025434 to E.B.), the Swedish Research Council (621-2013-5379 to M.A. and the Carl Trygger foundation to M.A.

Available from: 2015-05-19 Created: 2015-05-19 Last updated: 2017-12-04Bibliographically approved
4. Antibody-mediated disruption of the mechanics of CS20 fimbriae of enterotoxigenic Escherichia coli
Open this publication in new window or tab >>Antibody-mediated disruption of the mechanics of CS20 fimbriae of enterotoxigenic Escherichia coli
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2015 (English)In: Scientific Reports, ISSN 2045-2322, E-ISSN 2045-2322, Vol. 5, 13678Article in journal (Refereed) Published
Abstract [en]

Preventive vaccines against enterotoxigenic Escherichia coli (ETEC) are being developed, many of which target common fimbrial colonization factors as the major constituent, based on empirical evidence that these function as protective antigens. Particularly, passive oral administration of ETEC anti-fimbrial antibodies prevent ETEC diarrhea. Little is, however, known regarding the specific mechanisms by which intestinal antibodies against ETEC fimbriae function to prevent disease. Using coli surface antigen 20 (CS20) fimbriae as a model ETEC colonization factor, we show using force spectroscopy that anti-fimbrial antibodies diminish fimbrial elasticity by inhibiting their natural capacity to unwind and rewind. In the presence of anti-CS20 antibodies the force required to unwind a single fimbria was increased several-fold and the extension length was shortened several-fold. Similar measurements in the presence of anti-CS20 Fab fragments did not show any effect, indicating that bivalent antibody binding is required to reduce fimbrial elasticity. Based on these findings, we propose a model for an in-vivo mechanism whereby antibody-mediated disruption of the biomechanical properties of CS20 fimbriae impedes sustained adhesion of ETEC to the intestinal mucosal surface. Further elucidation of the role played by intestinal antibodies in mechanical disruption of fimbrial function may provide insights relevant to ETEC vaccine development.

National Category
Biophysics Immunology Other Physics Topics Nano Technology
Identifiers
urn:nbn:se:umu:diva-108176 (URN)10.1038/srep13678 (DOI)000361806400001 ()
Funder
Swedish Research Council
Available from: 2015-09-04 Created: 2015-09-04 Last updated: 2017-12-04Bibliographically approved
5. Antibodies damage the resilience of fimbriae, causing them to be stiff and tangled
Open this publication in new window or tab >>Antibodies damage the resilience of fimbriae, causing them to be stiff and tangled
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2017 (English)In: Journal of Bacteriology, ISSN 0021-9193, E-ISSN 1098-5530, Vol. 199, no 1, e00665-16Article in journal (Refereed) Published
Abstract [en]

As adhesion fimbriae are a major virulence factor for many pathogenic Gram-negative bacteria, they are also potential targets for antibodies. Fimbriae are commonly required for initiating the colonization that leads to disease, and their success as adhesion organelles lies in their ability to both initiate and sustain bacte- rial attachment to epithelial cells. The ability of fimbriae to unwind and rewind their helical filaments presumably reduces their detachment from tissue surfaces with the shear forces that accompany significant fluid flow. Therefore, the disruption of func- tional fimbriae by inhibiting this resilience should have high potential for use as a vaccine to prevent disease. In this study, we show that two characteristic biome- chanical features of fimbrial resilience, namely, the extension force and the exten- sion length, are significantly altered by the binding of antibodies to fimbriae. The fimbriae that were studied are normally expressed on enterotoxigenic Escherichia coli, which are a major cause of diarrheal disease. This alteration in biomechanical properties was observed with bivalent polyclonal antifimbrial antibodies that recog- nize major pilin subunits but not with the Fab fragments of these antibodies. Thus, we propose that the mechanism by which bound antibodies disrupt the uncoiling of natural fimbria under force is by clamping together layers of the helical filament, thereby increasing their stiffness and reducing their resilience during fluid flow. In addition, we propose that antibodies tangle fimbriae via bivalent binding, i.e., by binding to two individual fimbriae and linking them together. Use of antibodies to disrupt physical properties of fimbriae may be generally applicable to the large number of Gram-negative bacteria that rely on these surface-adhesion molecules as an essential virulence factor.

I M P O R T A N C E Our study shows that the resiliency of colonization factor antigen I (CFA/I) and coli surface antigen 2 (CS2) fimbriae, which are current targets for vac- cine development, can be compromised significantly in the presence of antifimbrial antibodies. It is unclear how the humoral immune system specifically interrupts in- fection after the attachment of enterotoxigenic Escherichia coli (ETEC) to the epithe- lial surface. Our study indicates that immunoglobulins, in addition to their well- documented role in adaptive immunity, can mechanically damage the resilience of fimbriae of surface-attached ETEC, thereby revealing a new mode of action. Our data suggest a mechanism whereby antibodies coat adherent and free-floating bacteria to impede fimbrial resilience. Further elucidation of this possible mechanism is likely to inform the development and refinement of preventive vaccines against ETEC diar- rhea. 

Keyword
pili, IgG, vaccine, CFA/I, CS2, optical tweezers
National Category
Biophysics Immunology Other Physics Topics
Identifiers
urn:nbn:se:umu:diva-119692 (URN)10.1128/JB.00665-16 (DOI)000391288200018 ()
Funder
Swedish Research Council, 621- 2013-5379Swedish Research Council, 2012-4638NIH (National Institute of Health), RR025434
Note

Originally published in manuscript form with title "Antibodies damage fimbrial resilience, making them stiff and tangled"

Available from: 2016-04-25 Created: 2016-04-25 Last updated: 2017-11-30Bibliographically approved

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