Lecture 3

Question 1

What is the common structure of pili?

Answer 1

An ‘adhesion on a stalk’

The stalk is a homopolymer of pilin or a major pilus protein this a tip pilin which s typically an adhesion

Question 2

What are the various functions of pili?

Answer 2

Cell adhesion, aggregation, biofilm formation, DNA transfer and gliding motility
They are often an essential virulence factor for pathogens

Question 3

What are the groups of pili and how are they determined?

Answer 3

Pili are grouped based on assembly mechanism adn there are four main families
Chaperon-Usher pathway assembed pili
Type IV pili
Nucleation precipitation pathway assembled pili
Alternative chaperone usher pathway assembled pili

Question 4

What is the chaperon usher pathway for the assembly of pili?

Answer 4

Proteins are secreted out via a sec translocon to the periplasma
A chaperon stabilizes the protein
An usher forms a channel guiding the proteins out of the cell
The pilin monomers can come together without any need for energy

Question 5

What are the proteins involved in type I pili formation?

Answer 5

The sec translocon as the secretion system to the periplasma
FimC as the chaperon protein
Fim D as the usher protein
DsbA periplamsa protein which forms disulphide bonds in FimA to allow for greater structural integrity
FimA as the pilin monomer
FimH as an adhesion (Lechtin which binds to oligo-mannose)

Question 6

What pili are fromed formed via the chaperone-usher pathway for the assembly of pili?

Answer 6

Type I pili
Pyelonephritis Associated Pili of UPEC
S Pili
Hif Pilus
PMF

Question 7

What is UPEC E.Coli?

Answer 7

E.Coli that infects the urinary tract

Question 8

What is the function of type 1 pili?

Answer 8

Pili which are common on enteric bacteria and is the most common pili on UPEC E.Coli the adhesion allows it to bind to uroplakin UP1a (bladder epithelium)
beta1alpha3 integrins (urothelium)
CD48 (mast cells, macrophages)
laminin

Question 9

What is the function of Pyelonephritis Associated Pili of UPEC?

Answer 9

this is a right handed helical cylinder made of the PapA pilin as a monomer with a tip made up of PapE, PapG, and PapF
The Pap G binds to specific sugar residues containing galactose alpha 1-4 Galactose bind
this is found in GbO3 on uroepithelial cells, GbO4 glycolipid receptor in kidney
GbO5 globopentosylceramide associated with cystitis
This allows the E.Coli bacteria to colonize the kidney

Question 10

What is the function of S pili?

Answer 10

In E.coli it plays a role in sepsis and UTIs

The tip adhesion (Sfas) binds to kidney and baldder epithelial cells

Question 11

What is the function of Hif pilus?

Answer 11

In haemophilus influenzae plays a role in otitis media

tip adhesion HifE binds to nasopharyngeal cells

Question 12

What is the function of PMF?

Answer 12

Plays a role in UTIs caused by proteys mirabilis

tip adhesion PmfF binds to kidney and bladder epithelial cells

Question 13

How are the genes for chaperone-usher assembled pili arranged?

Answer 13

In an operon often with different tip adhesions that can be swapped out to allow the bacteria to adapt

Question 14

How are type 4 pili assembled?

Answer 14

Made of monomers which contain a signalling peptide and a hydrophobic helix which can bury itself in the membrane with a head which sticks in the periplasma
They then go to a complex ATPase which uses ATP from the cell to form the pili from the monomers
The pili is then passed out through a gated channel in the outer membrane

Question 15

How can type 4 pili allow for movement?

Answer 15

The pili can be extended and retracted via ATPase mechanisms allowing for gliding motility

Question 16

What are the proteins involved in type 4 pili formation?

Answer 16

PilE which is the pilin monomer
PilC which may be the adhesion
PilD which is the prepilinpeptidase
PilB which is the assembly ATPase
PilG which is the ATPase associated protein
PilQ which is the secretin forming the gated channel
PilT which is the retraction ATPase

Question 17

How can type 4 pili add to antigenic variation?

Answer 17

They can undergo gene conversion and use different pilin proteins

Question 18

How do the pilins of chaperone-usher assembled proteins combine together?

Answer 18

Are made up entirely of beta sheet but contain one missing strand, this is intially replaced by the stabilizing chaperone protein but in the completed pilus the strand is replaced by an extending strand from the other monomer

Question 19

What are examples of type IV pili?

Answer 19

GCP/MCP

Bundle forming pilus

Question 20

What is the function of GCP/MCP?

Answer 20

Type 4 pili which are used by Neisseria gonorrhoaeae and N.Meningitiidis to bind to endothelial and epithelial cells, formation of microcolonies, extensive antigenic variation, phase variation, post-translational modification of PilE pilin

Question 21

What is the fucntion of bundle forming pilus?

Answer 21

Used by enteropathogenic E. Coli
forms long intertwined rope-like aggregates
microcolonies on epitheliavia localised adherence and organisation into higher order cables

Question 22

How can type 4 pilus affect bacterial genomes?

Answer 22

Can be used to pick up DNA which can then be brought into the cell which may recombine with the bacterias DNA to add new genes

Question 23

What is the difference in motility gained by flagella and type 4 pili?

Answer 23

Type 4 pili give a twitching motility to the bacteria while flagella give a swarming motility
The motive force of type 4 pili is retraction while flagella use a proton motif force
Type 4 pili also facilitate attachment and are a major virulence factor

Question 24

What are the differences between chaperone-usher assembled pili and type 4 pili?

Answer 24

Type 4 pili have the ability to aggregate into bundles
Type 4 pili are anchored to the inner rather than outer membrane and thus have contact with the cytosol
Type 4 pili have access to intracellular ATP which chaperon-usher assembled proteins cannot access as there is no ATP in the periplasma where they are assembled
There are no chaperon proteins required by type 4 pili
Type 4 pili have more functional possiblities due to there ability to aid motility and pick up DNAand phages

Question 25

What are the features of pili in gram positive bacteria?

Answer 25

They have only recently been discovered as they are very thin not even visible by electron microscopy
They are involved in cell adhesion and biofilm formation
The structure and assembly mechanisms are different to gram negative bacteria however it appears to be conserved in gram positive bacteria
Pilus proteins are covalently linked by pilus sortase
pilus is linked covalently to bacterial cell wall by housekeeping sortase A
do not confer motility

Question 26

What are examples of gram positive bacteria which have pili?

Answer 26

Corneybacterium
Actinomyces
several pathogenic streptococci

Question 27

What are the key structural features of a gram positive pilus?

Answer 27

Collegen binding protein at the extremity of the pilus
a major pilin to make up the stalk
an adaptor pilin to attach the pilus to the cell

Question 28

How does the M1 pilus in Streptococcus pyogenes become assembled?

Answer 28

Sortase C recognizes the VVPTG motif in Cpa and makes a cut between T and G and forms an amide bond between COOH group of terminal T in Cpa and NH2 side chain of internal lysine in pilin it then recognises the EVPTG motif in pilin and cuts between T and G forming and amide bond this tie between COOH group of the terminal T in pilin and the NH2 side chain of the internal lysine in the next pilin
The process of joining pilins via sortase C repeats about 100 times
Sortase C then forms and amide bond between COOH of terminal T in pilin and internal lysin in adaptor pilin
Sortase A then recognises LPXTG motif in the adaptor protein and cuts between the T and G froming an amide bond between the COOH group of the terminal T in the adaptor protein and the N terminal NH2 group of a peptide on peptidoglycan