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8 Dehydration, Embedding and Sectioning for TEM

8 Dehydration, Embedding and Sectioning for TEM

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I. Grin et al.

Fig. 16.8 The capsule of gelatine-embedded Rhodococcus bacteria can be visualized by using

DMF (dimethylformamide) as a solvent during dehydration at progressively lower temperatures

followed by low temperature embedding in Lowicryl K4M. On-section immunolabelling was

performed using a monoclonal antibody against capsular polysaccharides in combination with colloidal gold complexes. Cells provided by Thomas Neu and Karl Poralla. Bar 500 nm. See also Neu

and Poralla (1988)

layer of methyl cellulose containing uranyl acetate to stabilize structures and to

create negative contrast (Tokuyasu, 1997).

16.9 Dehydration and Critical Point Drying for SEM

In addition to the general processing steps described above, samples for SEM

inspection at ambient temperature must be critical point dried to remove organic

solvents: fixed samples are dehydrated and the organic solvent is exchanged by

liquid carbon dioxide in a pressure chamber above the critical point of carbon dioxide. Using this method, drying artifacts occurring at the transition of the liquid-gas

phase boundary are avoided. As a final step a thin metal film is sputter-coated onto

the sample to create a conductive surface and increase secondary electron emission.

For structural studies, platinum or gold alloys are used. For the detection of gold

particles in SEM by backscattered electrons immuno-labelled samples are coated

with chromium.

16.10 Affinity Labelling

Molecules can be detected by affinity labelled antibodies, lectins or other probes

which are typically visualized indirectly with the help of secondary antibodies

bound to colloidal gold complexes. If the epitopes are accessible at the sample


Electron Microscopy Techniques to Study Bacterial Adhesion


surface, pre-embedding labelling is a valuable tool, especially when the embedding

procedure abolishes the epitopes for antibody recognition. Epitopes within structures may be visualized by pre-embedding labelling of permeabilized samples or by

post-embedding labelling of thawed cryosections (Tokuyasu, 1997) or thin plastic

(in particular methacrylate) sections (Schwarz and Humbel, 2008). In all cases, the

sections can be finally stained with uranium and lead salt solutions to enhance the

structural contrast.

16.11 Cryopreparation Methods

Ice crystal growth during freezing and all subsequent preparation steps and even during imaging is a major problem in cryo-electron microscopy. Thin layers of material

with a maximal thickness of 10 μm can be vitrified by plunge freezing in liquid

ethane or propane at about 90 K. Thicker layers up to 200 μm must be frozen under

high pressure (2100 bar) to avoid ice crystal formation. Special attention must be

paid to adequate freezing at the exterior of bacterial cells: here, segregation patterns

due to ice crystal growth can easily occur and alter the native structure of surface

layers. In the worst cases, the use of non-osmotic cryoprotectants such as dextran or

bovine serum albumin may be indicated.

Thin biological samples can be directly imaged in frozen-hydrated state in a

cryo electron microscope at temperatures below 140 K, whereas thicker material

has to be sectioned. Cryosectioning of native material is prone to cutting artifacts

like compression, ruptures and crevices due to the rigid structure of frozen water.

Nevertheless it is the only method available to visualize biological structures in their

native state.

A useful compromise is the combination of cryofixation with subsequent freezesubstitution and resin embedding: here, the vitrified water is substituted by an

organic solvent at low temperatures, where the biological structure is preserved

and less organic material is extracted compared to room temperature dehydration.

Fixatives can be added to the organic solvent, and will react and stabilize the sample, while still at subzero temperatures. The polymerization of the resins is the

final step, and is done either with UV or heat activation as described above. The

resin-embedded samples can then be sectioned and imaged at ambient temperature.

16.12 Recent Developments and Future Perspectives

Even today, electron microscopy technology improves, and will probably allow us to

observe biological phenomena at even higher resolution very soon. Electron crystallography has already been mentioned in this context. Alternatively, the use of better

phase contrast imaging conditions in TEM with the introduction of phase plates

(Danev and Nagayama, 2010; Hosogi et al., 2011) and Cs-corrected lenses (Haider


I. Grin et al.

et al., 2008) will certainly improve the achievable resolution for biological specimen

and subsequent tomographic reconstructions.

Another encouraging development is X-ray microscopy using synchrotron

radiation, mainly pioneered by the group of Schmahl (in Berlin) and of Larabell (in

Berkeley) (Larabell and Nugent, 2010). In soft X-ray tomography, cells are imaged

using photons from a region of the spectrum known as the “water window”. This

results in quantitative, high-contrast images of intact, fully hydrated frozen cells

without the need to use contrast-enhancing agents. Using soft X-ray microscopy,

projection images can be collected in less than 20 min. The actual achieved resolution of the tomograms is in the range of 20–50 nm. The object can be mounted in a

capillary and therefore rotated freely without angular limitation. This is a big advantage over tilt series of thick sections in TEM tomography where the variable path

length for the electrons under different tilt angles limits the maximum tilt to +/– 70

degrees and so reduces the practical resolution of the EM tomograms (McDermott

et al., 2009).


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Chapter 17

EM Reconstruction of Adhesins: Future


Ferlenghi Ilaria and Fabiola Giusti

Abstract Both Gram-negative and Gram-positive pathogenic bacteria present a

remarkable number of surface-exposed organelles and secreted toxins that allow

them to control the primary stages of infection, bacterial attachment to host cell

receptors and colonization. The mediators of these processes, called adhesins, form

a heterogeneous group that varies in architecture, domain content and mechanism of binding. A full understanding of how adhesins mediate cellular adhesion

and colonization requires quantitative functional assays to evaluate the strength

of the binding interactions, as well as determination of the high-resolution threedimensional structures of the molecules to provide the atomic details of the

interactions. The combination of classical imaging techniques like X-ray crystallography and Nuclear Magnetic Resonance (NMR) with the emerging technique of

single-particle electron cryomicroscopy has become a tremendously helpful tool to

understand the three-dimensional structure at near atomic-level resolution of newly

discovered adhesins and their complexes. A detailed study of the structure of these

molecules, both isolated and expressed on bacterial surface is a fundamental requirement for understanding the adhesion mechanism to host cells. This chapter will

focus on the structure determination of such surface-exposed protein structures in

both Gram-negative and Gram-positive bacterial adhesins.

17.1 Introduction

17.1.1 Adhesins

Bacteria are among the most diverse living organisms and have adapted to a great

variety of ecological environments, including the human body. Pathogenic bacteria, both Gram-negative and Gram-positive, present a remarkable number of

F. Ilaria (B)

Novartis Vaccines and Diagnostics srl, 53100 Siena, Italy

e-mail: ilaria.ferlenghi@novartis.com

D. Linke, A. Goldman (eds.), Bacterial Adhesion, Advances in Experimental

Medicine and Biology 715, DOI 10.1007/978-94-007-0940-9_17,

C Springer Science+Business Media B.V. 2011



F. Ilaria and F. Giusti

surface-exposed adhesins and secreted toxins that allow them to control many different niches throughout the course of infection. Adherence to the host is a key

event in bacterial pathogenesis. The mediators of this process, called adhesins, form

a heterogeneous group that varies in architecture, domain content and mechanism

of binding. The complexity of the bacterial tools used for cell adhesion and invasion

ranges from single monomeric proteins to intricate multimeric macromolecules that

perform highly sophisticated functions.

A full understanding of how adhesins mediate cellular adhesion requires quantitative functional assays to evaluate the strength of the binding interactions, as well

as high-resolution three-dimensional structures to provide the atomic details of the

interactions. High-resolution structures of adhesins, by using the currently advanced

procedures from the observation of biological structure and for image processing combined with 3D reconstruction, help elucidate their role in pathogenicity. A

detailed study of the structure of these molecules, both isolated and expressed on the

bacterial surface is a fundamental requirement for understanding the adhesion mechanism to host cells. This chapter will focus on structure determination using electron

microscopy of such surface-exposed protein structures in both Gram-negative and

Gram-positive bacteria.

17.2 Pili and Fimbriae

Non-flagellar appendages were first observed in bacteria in the early 1950s, when

the outer–membrane surface of Gram-negative pathogens was scanned with the

electron microscope (Houwink and Iterson, 1950; Duguid et al., 1955). Pili are 1

to 3 μm-long hair-like bacterial appendages with diameters ranging from 2 to 8 nm

and are built by protein subunits called fimbrins or pilins. Pili can be classified on the

basis of physical properties, antigenic determinants, adhesion characteristics, characteristics of the major protein subunits, or assembly pathways. In the post-genomic

era, it has now become clear that Gram-negative bacteria have four major classes

of pili, based on their biosynthetic pathways: a) pili assembled by the chaperoneusher pathway (CU pili); c) Type III secretion needle; b) Type IV pili; and d) Type

IV secretion pili (not discussed below). Historically the best characterised of these

cell-surface organelles are CU pili (Duguid and Campbell, 1969; Martinez et al.,

2000) expressed by E. coli, Pseudomonas and Neisseria species (Olsen et al., 1989;

Kikuchi et al., 2005).

Under the electron microscope, CU pili appear as rigid, rod-like structures

extending in all directions from the bacterium. They are 1–2 μm in length with a

visibly flexible tip that is involved in bacterial interaction with receptors on the host

cell surface (Hahn et al., 2002; Kuehn et al., 1994; Saulino et al., 2000). Type IV pili

are similar in length but differ from CU pili in that they appear to be more flexible

and often form bundles at the pole (Craig et al., 2004; Strom and Lory, 1993). Type

III secretion-system pili were first observed in Salmonella Typhimurium as needlelike surface appendages responsible for bacterial entry into epithelial cells (Schraidt

et al., 2010). Curli are, as their name suggests, coiled structures synthesized by the


EM Reconstruction of Adhesins: Future Prospects


CU pathway as linear multi subunit pili. They are mainly found in Pseudomonas,

Haemophilus, Bordetella and Acinetobacter species (Sauer et al., 2004). All four

types are formed by non-covalent association of pilin subunits into regular polymeric structures. Pilus assembly in Gram-negative bacteria has been well studied

and involves the Sec-dependent secretion system (Wu and Fives-Taylor, 2001). A

common feature of Gram-negative pili is their role in adhesion to eukaryotic cells:

bacteria use these structures to form an initial association with host cells, which is

then followed by a more intimate attachment that brings the bacterium close to the

host-cell surface. Pili are known to adhere to components of the extracellular matrix

(ECM) (Amano, 2003), as well as to carbohydrate moieties present on glycoprotein

or glycolipid receptors (Sung et al., 2001; Schweizer et al., 1998). Receptor specificity might be important in determining the specificity and tropism of bacteria for

particular host cells (Telford et al., 2006).

So far only a few adhesin structures have been determined by three-dimensional

electron microscopy (3DEM). They are prototypes for various types of assembly:

P-pili, type I pilus and Saf pilus (CU pili), S. Typhimurium “pilus” (type III secretion

system pili), and Neisseria gonorrhoeae pilus (type IV pilus).

17.3 Structures Exported by Chaperone/Usher Pathway:

P-Pili, Type I Pili

The chaperone/usher pathway is used by a wide range of Gram-negative bacteria to expose pili on their outer surface (Hung et al., 1996). Pilus biogenesis

involves a periplasmic chaperone and an outer membrane protein called the usher

(Thanassi et al., 1998). The periplasmic chaperone aids pilus subunit folding in the

periplasm and maintains subunits in polymerization-competent folding state (Bann

and Frieden, 2004). The usher recruits chaperone-subunit complexes to the outer

membrane, facilitates their ordered polymerization by proximal addition of pilin

subunits and is responsible for pilus translocation to the outer surface.

CU pili, including P-pili, type I pili, and Hib pili are all helical structures

7–8 nm in diameter, with an axial hole of 2.0–2.5 nm in diameter, and comprising 3.0–3.5 subunits per turn of the helix. Clear depictions of the assembly and

the architecture of macromolecular complexes have been achieved by the integration of the three-dimensional (3D) reconstructions derived from cryo-electron

microscopy (cryo-EM) with atomic resolution subunit models derived from X-ray

crystallography and NMR.

FGS-chaperone assembled pili, exemplified by the type I and P-pili found in

uropathogenic Escherichia coli (UPEC), are typically rigid, rod-shaped pili with

a complex quaternary organisation made up of multiple subunit types. Type I and

P-pili are composed of two distinct subassemblies: a rigid helically wound rod of

6.8-nm diameter composed of over 1000 copies of the FimA or PapA subunits,

respectively, and a distal tip fibrillum measuring 2 nm in diameter. The tip fibrillum

is composed of a distally located adhesin (FimH in type I and PapG in P-pili, respectively) and the FimG and FimF subunits or the PapF, PapE (∼5–10 copies) and PapK


F. Ilaria and F. Giusti

subunits in type I and P-pili, respectively. These pili, also referred to as “typical CU

pili”, are readily observable as individual fibres, radiating out of the bacterial surface

in all directions (Hahn et al., 2002; Jones et al., 1995).

17.3.1 P-Pili

One of the first and best-characterised fimbria is the pyelonephritis-associated (P)

pilus, expressed by UPEC strains that colonise the urinary tract and infect the kidney. At the outer membrane the pilins are translocated through a pore-forming usher

and assembled into the pilus by proximal addition to the growing helical filament.

The pore is large enough to accommodate a single pilin subunit in its native conformation. P-pili are the appendages that pyelonephritic E. coli uses to colonise

and infect host tissues. Clear depictions of the assembly and architecture of macromolecular complexes have been achieved by the integration of 3D reconstructions

(Fig. 17.1A) from cryo-electron microscopy (cryoEM) with atomic resolution subunit models from X-ray crystallography. The three-dimensional structure of P-pili

obtained at 9.8 Å resolution from material preserved in vitreous ice and using iterative helical real space reconstruction method (IHRSR) (Egelman, 2000; Mu and

Bullit, 2006) show the P-pilus to be an elongated filament with an external diameter

of 82 Å. It has an axial channel of 25 Å in diameter running straight up the center of

the helix axis with openings that extend to the pilus surface. The P-pilus shell contains 3.28 subunits per turn of the helix turn and a 7.54 Å rise per subunit. Surface

protrusions that extend 7 Å from the filament surface with a height of 17 Å have been

also observed A homology-based model of PapA was constructed using the known

structure of the minor P-pilin, PapK (protein Data Bank ID code 1PDK) (Sauer

et al., 1999) as a template based on its 26% sequence identity with PapA. Fitting

of the modelled PapA subunit into the cryo-electron microscopy data provided a

detailed view of these pilins within the supramolecular architecture of the pilus filament. A structural hinge in the N-terminal region of the subunit is located at the site

of electron density that protrudes from the pilus surface. The structural flexibility

provided by this hinge is necessary for assembly of P-pili, illustrating one solution

to the construction of large macromolecular complexes from small repeating units.

These data support the hypothesis that domain-swapped pilin subunits transit the

outer cell membrane vertically and rotate about the hinge for final positioning into

the pilus filament.

17.3.2 Type I Pili

The structure of Type I pilus was determined in 1969 by Brinton, who used electron

microscopy, crystallography and X-ray diffraction. More recent electron microscopy

and three dimensional reconstruction studies by Hahn et al. (2002) showed that the

adhesive organelle has a composite structure based on a 6.9 nm thick and 1–2 μm

long helical rod. Briefly, an integrated approach combining information gained by

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