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3 Structures Exported by Chaperone/Usher Pathway: P-Pili, Type I Pili

3 Structures Exported by Chaperone/Usher Pathway: P-Pili, Type I Pili

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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


EM Reconstruction of Adhesins: Future Prospects






Fig. 17.1 (a) Cryo-EM density map (Mu and Bullit, 2006) of P-pili. The helical reconstruction

(left hand figure) shows a filament structure 82 Å in diameter, with surface protrusions that extend

7 Å from the filament surface, for 17 Å along the helical axis (white arrow). PapA subunits fitting

into the 3D P-pilus electron density map are visualized in different colours (right hand figure).

The subunits are inclined by an angle of 13◦ from horizontal (black arrow). (b) Three-dimensional

reconstruction of the type A Saf pilus (Salih et al., 2008). Each reconstruction is contoured at the

molecular mass expected for two SafA subunits (28.8 kDa). Structures are viewed from two orthogonal directions and show two subunits connected by a narrower stretch of density. (c) Docking of

the atomic model of MxiH into the EM density of the Shigella T3SS needle (Deane et al., 2006). (1)

Molecule A of MxiH (ribbon). (2) End-on view of a 40-Å-thick section of the assembled needle.

(3) Stereo diagram of the side view of the assembled needle

Fourier transformation, linear Markham superposition (real space) and mass-perlength measurement by scanning transmission electron microscopy was used to

analyze the helical structure of the rod-like type I pili expressed by UPEC. The

three-dimensional reconstruction showed the pili to be 6.9 nm wide, right-handed

helical tubes with a 19.31(±0.34) nm long helical repeat comprising 27 FimA

monomers associated head-to-tail in eight turns of the one-start helix. Adjacent turns

of the helix are connected via three binding sites making the pilus rod rather stiff.

In situ immuno-electron microscopy experiments showed that the minor subunit


F. Ilaria and F. Giusti

(FimH) mediating pilus adhesion to bladder epithelial cells was the distal protein

of the pilus tip, which had a spring-like appearance at higher magnification. The

subunits FimG and FimF connect FimH to the FimA rod, the sequential orientation

being FimA–FimF–FimG–FimH. The electron density map calculated at 18 Å resolution from an atomic model of the pilus rod built using the pilin domain FimH

together with the G1 strand of FimC as a template for FimA and applying the

optimal helical parameters described above was practically identical to the actual

three-dimensional reconstruction (Hahn et al., 2002; Choudhury et al., 1999).

17.3.3 Saf Pilus

In contrast to the well-established quaternary structure of typical chaperone/usher

pili, little was known about the supramolecular organisation in atypical/FGLchaperone assembled fimbriae. Salih and co-workers (2008) used negative stain

electron microscopy and single-particle image analysis to determine the threedimensional structure of the Salmonella Typhimurium Saf pilus (Fig. 17.1B). Their

results show atypical/FGL-chaperone assembled fimbriae are composed of highly

flexible linear multi-subunit fibres that are formed by globular subunits connected to

each other by short links giving a “beads on a string”-like appearance. Quantitative

fitting of the atomic structure of the SafA pilus subunit into the electron density

maps (Salih et al., 2008), in combination with linker modelling and energy minimisation, has enabled analysis of subunit arrangement and intersubunit interactions

in the Saf pilus. Short intersubunit linker regions provide the molecular basis for

flexibility of the Saf pilus by acting as molecular hinges allowing a large range of

movement between consecutive subunits in the fibre.

17.4 Type III Secretion-System Pili

Besides pili, enteropathogenic E. coli (EPEC) and enterohaemorrhagic E. coli

(EHEC) have another bacterial adhesion system (Type III secretion-related pili)

in which the bacteria provide both the ligand and the receptor with a mechanism known as “attaching and effacing”. Type III secretion-related appendages

were first observed in S. Typhimurium as needle-like surface structures responsible for bacterial entry into cultured epithelial cells (Roine et al., 1997; Kubori

et al., 1998). The assembly of these structures depends on systems now known to

form a flagellum-like secretion nanomachine known as the type III secretion system

(T3SS). T3SS assembles a complex injectisome designed to secrete effector proteins

across the bacterial and host cell envelopes. Injectisome assembly involves over

20 proteins (Cornelis, 2006). The injectisome basal structure is a large cylindrical

heterocomplex with two double rings spanning the inner and outer membranes that

are linked by a hollow structure that crosses the intermediate periplasmic space

(Kubori et al., 1998). On the cytoplasmic side, the basal body interacts with ATPases


EM Reconstruction of Adhesins: Future Prospects


and accessory proteins responsible for driving and ordering protein secretion and

filament assembly (Woestyn et al., 1994; Akeda and Galan, 2005; Müller et al.,

2006). On the outer surface, the basal body forms a hollow filamentous structure.

The basic extracellular structure, as first identified in the Salmonella SPI-1 T3SS

(Kubori et al., 1998) and later observed in detail from purified Shigella injectisomes

(Tamano et al., 2000; Blocker et al., 2001), is a short rigid hollow “needle” about

60 nm in length with an inner diameter of 2–3 nm. The basic needle is composed

of a homopolymer of 100 subunits with a RMM of 9 kDa each (PrgJ, MxiH, YscF

and Escf in Salmonella, Shigella, Yersinia and EPEC, respectively). These subunits

reveal a helix-loop-helix structure similar to the D0 portion of flagellin (Deane et al.,


Similar to flagellin, needle subunits polymerise into a hollow superhelical structure (Fig. 17.1C). The distal end of the needle carries a distinct tip complex. In

Yersinia, it is composed of the protein LcrV, one of the three translocators involved

in pore formation in the target cytoplasmic membrane (Mueller et al., 2005). LcrV

forms a dumbbell-shaped structure that has been modelled as a pentameric complex

at the tip of the needle and is thought to provide the scaffold for the additional two

translocator proteins, assembled upon contact with the target membrane (YopB and

YopD) (Derewenda et al., 2004; Deane et al., 2006). In EPEC, this needle base is

extended by a longer flexible “filament” up to 600 nm in length (Knutton et al., 1998;

Sekiya et al., 2001) and composed of an EspA homopolymer. In plant pathogens, the

needles are replaced altogether by a long flexible “pilus”, the Hrp pilus (Roine et al.,

1997; Li et al., 2002). In the Salmonella SPI-1 T3SS, a different mechanism for needle length control has been suggested based on the 3DEM reconstruction at 17 Å

resolution. Purified Salmonella injectisomes indicate the presence of an inner rod

structure, possibly composed of a specialised subunit, PrgJ, inside the basal body

of the secretion system. In the model, secretion of rod and needle subunits (PrgJ

and PrgI, respectively) occurs simultaneously. Completion of the inner rod assembly signals a halt in secretion of rod and needle subunits, thereby controlling needle

length (Marlovits et al., 2006).

17.5 Type IV Pili

Much current interest is focused in the study of type IV pili, another category of

polymeric adhesive surface structures expressed by many Gram-negative bacteria

including pathogens such as EPEC, EHEC, Neisseria gonorrhoeae and Neisseria

meningitidis. These helical arrays of thousands of copies of a single pilin subunit

such as PilA (Parge et al., 1995) and PilE (Craig et al., 2003) are extremely thin

and flexible but remarkably strong, and they have several different physiological

functions. Type IV pili are essential for host colonization and virulence for many

Gram-negative bacteria, and may also play a role in pathogenesis for some Grampositive bacteria. High resolution structures of pilin subunits have been determined

by X-ray crystallography and nuclear magnetic resonance spectroscopy both as full

length proteins and as soluble fragments (Parge et al., 1995; Craig et al., 2003).


F. Ilaria and F. Giusti

Fig. 17.2 Cryo-EM and 3D

Reconstruction of the GC

Pilus at 12.5 Å (Craig et al.,

2006) (a) Filament density

map for GC-T4P (b) Pilin

subunit match to cryo-EM

density map

These structures have been used to generate computational models of pilus filament

assemblies, guided by biophysical parameters extracted from fibre diffraction data

and electron microscopy image analysis, and by the extensive biological data. Most

recently, cryo-electron microscopy has provided an intermediate resolution (12.5 Å)

structure of a pilus filament (Fig. 17.2) showing its helical organization (Craig et al.,

2006). The pilin structures and filament models have been instrumental in advancing

our understanding of the molecular mechanisms driving pilus assembly and the role

of Type IV pili in key bacterial functions such as immune evasion, microcolony formation and DNA uptake. In very general terms the structural data point to a shared

subunit structure and filament architecture for all Type IV pili, but a comprehensive, atomic-level understanding of these filaments and their biological processes

will require additional higher resolution filament structures as well as new structural, genetic and biochemical data on the many components of the pilus assembly


17.6 Pili in Gram-Positive Bacteria

While many Gram-negative pili have been studied in detail over the last decades

(Fronzes et al., 2008), the majority of Gram-positive pili have been discovered only recently and their study, initiated by Schneewind and co-workers on

Corynebacterium diphteriae (Ton-That and Schneewind, 2003; Ton-That et al.,

2004), it is still at its infancy. Pilus-like structures on the surface of Gram-positive

bacteria were first detected in Corynebacterium renale by electron microscopy

(Yanagawa et al., 1968; Yanagawa and Otsuki, 1970). In the recent past pili have

also been characterized in all three of the principal streptococcal pathogens that

cause invasive disease in humans: Group A Streptococcus (GAS) (Mora et al.,

2005), Group B Streptococcus (GBS) (Lauer et al., 2005; Rosini et al., 2006)

and Streptococcus pneumoniae, a very important human pathogen (Barocchi et al.,


EM Reconstruction of Adhesins: Future Prospects


2006). In all cases, the pili have been shown to play a key role in adhesion and

invasion and in pathogenesis. Until a few years ago, only two types of pilus-like

structures had been identified by electron microscopy in Gram-positive bacteria. Certain Gram-positive bacteria, like Streptococcus gordonii and Streptococcus

oralis, appear to be decorated with short, thin rods or fibrils that extend between

70 and 500 nm from the bacterial surface (McNab et al., 1999; Willcox and

Drucker, 1989). Much longer (up to 3 μm) pilus-like structures that appear as flexible rods have been described in the oral pathogenic Corynebacterium species and

in pathogenic streptococci (Yanagawa et al., 1968; Yanagawa and Otsuki, 1970;

Ton-That and Schneewind, 2003; Mora et al., 2005; Thanassi et al., 2005; Rosini

et al., 2006; Barocchi et al., 2006; Jameson et al., 1995). Ton-That and Schneewind

were the first to characterize the long rod-like pili in C. diptheriae (Ton-That and

Schneewind, 2003). The general feature of these rod-like pili, as well as the rod-like

pili that have been identified in GAS (Mora et al., 2005), GBS (Lauer et al., 2005)

and S. pneumoniae (Barocchi et al., 2006), is that they have three covalently linked

protein subunits, each of which contains an LPXTG amino-acid motif or a variant of

it, which is the target of specific sortase enzymes. During pilus formation, specific

sortases catalyse the covalent attachment of the pilin subunits to each other and to

the peptidoglycan cell wall (Ton-That and Schneewind, 2003).

The molecular architecture of pilus-1 of S. pneumoniae is the only structure of a

Gram-positive pilus known so far (Hilleringmann et al., 2009). One major (RrgB)

and two minor components (RrgA and RrgC) assemble into the pilus. TEM and

scanning transmission electron microscopy (STEM) show that the native pili are

approximately 6 nm wide, flexible filaments that can be over 1 μm long. They are

formed by a single string of RrgB monomers, which give a polarity to the pilus

defined by a nose-like protrusion (Fig. 17.3). These protrusions correlate to the

Fig. 17.3 STEM analysis of

isolated TIGR4 pili

(Hilleringmann et al., 2009).

(a, b) Contrast-reversed

STEM dark-field images from

negatively stained TIGR4. A

nose-like protrusion is present

at irregular intervals. Subunit

boundaries are indicated by

white lines in panel a.

(c) Highly contoured TEM

images of RrgB–His

monomers; (d) Pilus outer

contours. Scale bar: panel A,

10 nm


F. Ilaria and F. Giusti

shape of monomeric RrgB-His, which, like RrgA-His and RrgC-His, has an elongated, multidomain structure. RrgA and RrgC are present at opposite ends of the

pilus shaft, compatible with their putative roles as adhesin and anchor to the cell

wall surface, respectively. This is the first experimental evidence that the native S.

pneumoniae pilus shaft is composed exclusively of covalently linked monomeric

RrgB subunits oriented head-to-tail.

17.7 Perspectives

The past few years have witnessed an incredible number of new discoveries. They

have not only enhanced our understanding of how adhesin structures are assembled

at the atomic and molecular level, but have also dramatically widened the number

of species known to possess adhesive structures. Thus adhesins have been found on

Gram-positive pathogens such as Streptococcus pneumoniae, the Group A and B

streptococci, and Staphylococcus aureus, complementing the wide range of Gramnegative organisms that have long been known to express adhesins. Much has also

been learned about adhesin structure and assembly: the donor-strand complementation mechanism used in the chaperone-usher assembly pathway; the assembly

of Type IV pilin subunits into complete pilus fibres; and most recently the first

structure for a Gram-positive major pilin and the structural architecture of a whole

Gram-positive pilus.

Whereas much is known about the structure and assembly of many of the adhesion machines, many questions still surround their modes of adhesion to host cells.

It is generally assumed that adhesion capability resides at the most exposed domains

of these macromolecular assembly, but this is not necessarily true for all adhesins;

there is much evidence that the pilus shaft also plays some role in adhesion in some

Gram-negative bacteria. For Gram-positive pili, for example, so-called minor pilin

subunits are necessary for binding to host cells, but it is not known how or where

these are attached to the shaft. Likewise, whereas some adhesion domains, such as

the FimH domain of Type I pili, are known to bind host-cell glycans, the molecular targets for other pili and pili-like structures are not known, and may involve

extracellular matrix components such as collagen or fibronectin.

Determination of all protein structures and their macromolecular assemblies at

high resolution is a key modern target for biological electron microscopy, representing one of the most important goals in biology and medicine. In the past two

decades, significant methodological advances have occurred in ultrastructural analysis by electron microscopy: the high resolution analysis of biological material

has been taken to a new level by the development of cryo-EM and of new efficient strategies for image processing and 3D rendering of biological structures.

After being overshadowed by the rapid advances in biochemistry, genetics, and light

microscopy, in the 1970s and 1980s, TEM has now re-attracted attention as a crucial

tool to bridge the data gained by the aforementioned techniques.

The three-dimensional methods developed to solve the different biological

problems and the different adhesion structures are based on two main groups


EM Reconstruction of Adhesins: Future Prospects


of strategies: electron tomography and single particle analysis. Cryo-electron

microscopy is a powerful tool in the study of macromolecular assemblies, especially in single particle form. Technical improvements including computer controlled microscopes equipped with field emission guns, improved specimen stages,

CCD cameras, as well as energy filters, combined with increased computational

power and improvements of image processing software, are making cryo-electron

microscopy almost routine. The popularity of this technique stems from the ability

to record the structure of radiation sensitive specimens in their native state thereby

eliminating the deleterious effects of negative stains and fixatives that mask the high

resolution components of the structure. Finally emerging methods combining cryoelectron microscopy with X-ray crystallography and NMR allow the determination

of three-dimensional structures at the atomic level for entire adhesin complexes.


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3 Structures Exported by Chaperone/Usher Pathway: P-Pili, Type I Pili

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