Structures, assembly and functions of fimbriae and pili in Gram negative bacteria

Question 1

Fimbriae and pili: definitions

Answer 1

• Appendages which protrude from the surface of the bacterium
• Consist chiefly of protein, generally polymers of pilin subunits
• Pili: Latin ‘hair’
• Fimbriae: Latin ‘thread’
• Pilin subunits align as fibres with helical symmetry along their axes
• Synthesized by addition of pilin subunits from the bottom (not like flagella, which assemble from the tip)

Question 2

Functions of fimbriae and pili

Answer 2

• DNA uptake (Conjugation and transformation)
• Adhesion
• Biofilm formation
• Antigens
• Motility

Question 3

Gram negative vs gram positive bacteria Fimbriae and pili

Answer 3

Gram negative

• pilin subunits connected non covalently
• no equivalent of a sortase
• no covalent linkage to peptidoglycan
• requirement to transverse the outer membrane through specialised pore-forming proteins

Gram positive

• pilin subunit connected covalently
• require sortase enzyme for assembly
• covalently linkage to peptidoglycan by a sortase
• no requirement to transverse

Question 4

Examples of bacterial pathogens in which pili pay an important role in infection

Answer 4

Question 5

Importance of pili in infection

Answer 5

Bind to epithelial and endothelial cells

• Participate in biofilm formation
• Promote horizontal genetic exchange (antibiotic resistance and antigenic variability)
• Are major determinants of virulence?

Question 6

Pili and fimbriae: physical properties

Answer 6

• Can extend several microns from cell surface
• Can be flexible but are also mechanically very strong
• Are synthesized by polymerization from the base (except curli)
• Can be subject to glycosylation (Type IV pili)
• Can help to determine pathogen tissue tropism
• Can be involved in motility (Type IV pili) • NOT related to flagella

Question 7

Principal classes of fimbriae and pili in Gram negative bacteria

Answer 7

Based on assembly mechanisms:

1. Chaperone-usher
2. Curli
3. Type IV pili
4. Type III secretion
5. Type IV secretion/conjugative pili

Question 8

Type I and P Pili

Answer 8

Type I pili make a major contribution to bladder infections (cystitis) by uropathogenic E. coli (UPEC)

• UPEC adhere to urinary tract cells- pili prevent clearance by urine flow
• FimH tip adhesin binds to mannose groups on uroplakins, glycoproteins on bladder epithelial cells
• pyelonephritis-associated (P) pilus, expressed by E. coli strains that colonize the urinary tract
• Assemble by chaperone-usher pathway

All the pilin subunits, FimA, F, G and H, form immunoglobulin (beta) folds. This turns out to be extremely important in the way the pilus fibre is assembled. A domain from FimH (right) is shown bound to a mannose oligosaccharide

Type I pili form by sequential addition of pilin subunits in a helical manner (left). Critically, each FimA pilin is joined to its neighbour by donating a beta strand to the next pilin in the sequence, thus completing the fold (see strands in red, left). This is known as donor strand exchange Donor strand exchange between adjacent pilin subunits This occurs in type I and P pili In the CU pathway, the chaperone (FimC) binds to the pilin protein (FimH) in the periplasm and delivers it to the OM This is a way of regulating the polymerization process and ensures that it occurs in the right place

Question 9

Fibre assembly at the outer membrane

Answer 9

• Chaperone-pilin complexes in the periplasm are dissociated by the usher protein
• The folded pilin passes through the usher channel (and hence crosses the OM)
• The pilin fibre is formed on the outer surface of the OM by donor strand exchange

Question 10

Curli

Answer 10

• Highly aggregated flexible fibres
• Found on Enterobacteriaceae
• Virulence factors
• Enhance adhesion and invasion
• Important in sepsis & septic shock
• Not known to phase vary

Question 11

Type IV pili (TFP): Major functions

Answer 11

Adhesion eg EPEC (enterotoxigenic) type IV bundle-forming pili attach to brush border cells; P. aeruginosa bind to asialo-GM1 and -GM2 gangliosides on epithelial cells

• Immune evasion (antigenic variability)
• Motility (twitching)
• Natural Transformation

Question 12

Antigenic variation in TFP

Answer 12

• TFP form helical structures made up of pilin subunit. this forms an alpha/beta fold fundamentally different from type 1 and P pili
• The majority of antigenic variation occurs in a surface exposed loop region in the pilin subunit
• TFP are subject to glycosylation
• meisseria acitve pilin pilE gene recombines with silent PilS locus the generates a new pilE varient

Question 13

Two distinct types of surface structres on gram positives

Answer 13

pili 3-4nm long

fimbrils are shorter

Question 14

Model for pilus mediated adherence of gram + cells.

Answer 14

1. tip protein adhesion
2. ancilary pilus protein in a zipper like adhesion mechanism
3. high affinity surface adhesions + pilus aggregation

Question 15

pili in gram + bacteria

Answer 15

All so far peritrichous

covalently attached to peptidoglycan

sortase mediated biogenesis

responsable for host tissue adhesion

Question 16

fimbrils on Gram + bacteria

Answer 16

Much shorter then pili

localised or peritrichous

covalently attached to peptidoglycan comprised of one protein or glycoprotein molecule

multifunctional (adhesion to host proteins-fimbronectin, hostcells, coaggregation, salivary aggregation

Question 17

Sortase enzyme

Answer 17

Links LPxTG motif to peptidoglycan

sortase (SrtA: for surface protein sorting A)

cleaves the LPxTG between T and G

Links thr (T) to the amino group of pentaglycine cross bridge in peptidoglycan hense transpeptidase

Question 18

Role of sortases in pilus assembly

Answer 18

Also function by making covalent linkages to adjacent subunit in the pilus fimbre

pilus assemly sortases are frequently encoded within the same operon as the pilin subunit itself

Question 19

Pilin fibre assembly

Answer 19

and finally the transpeptidase reaction occurs to bind pilin to peptidoglycan