| 1. |
- Barbercheck Epler, Chelsea R., et al.
(författare)
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Bacterial adhesion pili
- 2018
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Ingår i: Membrane protein complexes. - Singapore : Springer Publishing Company. - 9789811077579 - 9789811077562 ; , s. 1-18
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Bokkapitel (refereegranskat)abstract
- 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.
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| 2. |
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| 3. |
- Mortezaei, Narges, et al.
(författare)
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Antibodies Change the Mechanics of Adhesion Fimbriae : a Case Study of CS20 Fimbriae Expressed by Enterotoxigenic Escherichia Coli
- 2015
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Ingår i: Biophysical Journal. - : Cell Press. - 0006-3495 .- 1542-0086. ; 108, s. 602-
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Tidskriftsartikel (övrigt vetenskapligt/konstnärligt)abstract
- Enterotoxigenic Escherichia coli (ETEC) express a variety of fimbriae that mediate adhesion to host epithelial cells. It has been shown that the ability of a fimbriated bacterial cell to attach and stay attached to host cells does not merely depend on the adhesin expressed distal of the fimbriae but also the biomechanical properties of the fimbriae are vital for sustained adhesion. Fimbriae can significantly extend under a constant force when exposed to an external force and therefore reduce the load on the adhesin, which is believed to help bacteria to withstand external forces applied by various body defense systems. Thus, it is thought that the fimbrial shaft and adhesin have co-evolved for optimal function when bacteria attach to host cells. To investigate if antibodies, normally found in the intestines, affects the biomechanical properties of fimbriae, we exposed CS20 fimbriae expressed by ETEC to anti-fimbrial antibodies and measured these properties using optical tweezers force spectroscopy. Our data show a change in the force required to extend the fimbriae and that the elasticity is significantly reduced by the presence of antibodies. The reduced elasticity, likely due to cross-linking of fimbrial subunits, could thus be another assignment for antibodies; in addition to their mission in marking bacteria as foreign, our data indicate that antibodies physically compromise fimbrial function. To further confirm interaction of antibodies to their specific target we performed western blot analysis, transmission electron microscopy and immunofluoresence microscopy. In the presence of antibodies, we suggest that our assay and results will be a starting point for further studies aimed at inhibiting bacterial adhesion by antibodies.
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| 4. |
- Mortezaei, Narges, 1985-, et al.
(författare)
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Biomechanical and Structural features of CS2 fimbriae of Enterotoxigenic Escherichia coli
- 2015
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Ingår i: Biophysical Journal. - : Elsevier. - 0006-3495 .- 1542-0086. ; 109:1, s. 49-56
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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| 5. |
- Mortezaei, Narges, et al.
(författare)
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Structural and biophysical comparison of UPEC and ETEC adhesion fimbriae
- 2015
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Ingår i: Biophysical Journal. - : Cell Press. - 0006-3495 .- 1542-0086. ; 108:2, suppl 1, s. 527A-527A
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Tidskriftsartikel (refereegranskat)abstract
- Adhesion fimbriae (pili) of uropathogenic and enterotoxigenic Escherichia coli (UPEC and ETEC, respectively) facilitate adherence of the bacteria to target cells. Fimbriae are absolutely necessary for colonization and biofilm formation in the initiation of disease. The types of fimbriae expressed on the bacterial surface vary with the preferred environmental niche of the bacterial strain. For example, UPEC that express P-pili are most frequently associated pyelonephritis, an infection in the upper urinary tract, whereas bacteria that express type 1 fimbriae commonly cause cystitis through infection of the lower urinary tract. In contrast, ETEC expressing CFA/I and CS2 pili are associated with diarrheal diseases, initiating disease in the small intestines.Although expressed in different enviroments, these fimbriae share basic structural and biomechanical features. Structurally, they are all long (1-4 μm), thin (7-8 nm diameter) helix-like filaments that extend from the bacterial surface. Biomechanically, they share the ability to be extended into a thinner filament (2-3 nm diameter) by unwinding of the helical filament under a constant force. However, the force required to unwind is specific to each fimbrial type. In addition, the dependence of the force required to unwind a fimbria on the velocity of this unwinding, (that is, the kinetics of unwinding), is also type-specific and highly variable. These biomechanical parameters are dissimilar for UPEC and ETEC expressed fimbriae, separating them into two distinct groups. Using force spectroscopy data, helical reconstructions from electron microscopy data, and computational simulations, we show in this work how these pronounced biomechanical differences may be beneficial for bacterial survival in a given environment.
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| 6. |
- Mortezaei, Narges, et al.
(författare)
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Structure and function of enterotoxigenic Escherichia coli fimbriae from differing assembly pathways
- 2015
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Ingår i: Molecular Microbiology. - : John Wiley & Sons. - 0950-382X .- 1365-2958. ; 95:1, s. 116-126
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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| 7. |
- Singh, Bhupender, et al.
(författare)
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Antibodies damage the resilience of fimbriae, causing them to be stiff and tangled
- 2017
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Ingår i: Journal of Bacteriology. - 0021-9193 .- 1098-5530. ; 199:1
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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| 8. |
- Singh, Bhupender, et al.
(författare)
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Antibody-mediated disruption of the mechanics of CS20 fimbriae of enterotoxigenic Escherichia coli
- 2015
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Ingår i: Scientific Reports. - : Springer Science and Business Media LLC. - 2045-2322. ; 5
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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| 9. |
- Wiklund, Krister, et al.
(författare)
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A drag force interpolation model for capsule-shaped cells in fluid flows near a surface
- 2018
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Ingår i: Microbiology. - : Microbiology Society. - 1350-0872 .- 1465-2080. ; 164:4, s. 483-494
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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| 10. |
- Zheng, Weili, et al.
(författare)
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Cryo-EM structure of the CFA/I pilus rod
- 2019
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Ingår i: IUCrJ. - : The International Union of Crystallography. - 2052-2525. ; 6:5, s. 815-821
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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