9 - Pilus Biogenesis

Question 1

What is the function of pili?

Answer 1

Bacterial pili in pathogenic organisms, such as pathogenic E.coli, are used for host recognition and attachment to host membranes. Interaction typically causes the target cell membrane to invaginate the bacterial cell.

Question 2

Typically where do type 1 pilus attach?

Answer 2

To the bladder, conducive of UTIs and cystitis.

Question 3

Typically where do the P pilus of bacteria attach?

Answer 3

To the epithelial layer of the kidneys, this can lead to pyelonephritis.

Question 4

What class of pilus do the P pilus and type 1 pilus belong?

Answer 4

Class I

Question 5

Briefly describe the structure of a pilus?

Answer 5

Composed of a thin tip section, the fibrillum, this controls the recognition and binding (PapG, adaptor/initator PapF, 10-15 PapE and adaptor/initator PapK), this is held in an open helical conformation. This extends into the longer and less flexible pilus rod, composed entirely of coiled chains of PapA.
They are typically helical structures of 7-8nm in diameter with an axial hole of 2-2.25nm, comprising 3 subunits per turn.

Question 6

What does PapG adhere to?

Answer 6

Gal-(a)1,4 - Gal diagalctoside

Question 7

Describe the Pap Gene cluster

Answer 7

I-B-A-H-C-D-J-K-E-F-G

Question 8

What is missing from each subunit of the pilus, besides PapH

Answer 8

They take up a typical IG fold, however they are missing 10-15 C Terminal residues that usually make up the B-strand labelled the G1 strand, in an IG this is held anti-parallel to the F strand, however it is held in parallel in Donor Strand Exchange.

Question 9

Briefly describe donor exchange.

Answer 9

The ability of proceeding subunits to ‘donate’ a G1-strand in parallel to the preceding subunit, in which there is a hydrophobic groove where a G1 strand should be.

Question 10

Describe the structure of PapD (chaperone) binding a subunit.

Answer 10

A subunit, say PapK, binding to PapD alters the PapD structure significantly, the tip of its F1-G1 loop undergoes a flap motion of about 11A, such that residues 101-105 become a part of the G1 Beta strand that inserts into the Ig fold of PapK. Through shielding this hydrophobic core of the subunit PapD contributes to its stabilisation.

Question 11

How is PapD then replaced in a subunit?

Answer 11

PapD will be replaced in PapK by the proceeding subunit PapA through a zip-in-zip-out mechanism. The base of PapA’s N terminal Extension (Nte) will insert residue P5 into the P5 pocket in the channel of PapK, binding to the groove and insertion extends vectorially towards the tip of the Nte, slowly replacing the chaperone strand, residue by residue (P4 > P3 > P2 > P1).
This was analysed using real time MS.

Question 12

What is DSE dependent on?

Answer 12

The formation of the transient ternary complex consisting of the chaperone-subunit complex and the Nte of incoming subunit.

Question 13

Briefly describe assembly.

Answer 13

Following translation the pilus subunits will enter the periplasmic space via YEG transporter and attach via DSE to PapD (chaperone). The first subunit, PapG, consisting of two immunoglobulin folds, inserts itself into the usher complex - PapC. Through zip-in-zip-out the chaperone will be replaced by proceeding subunit PapF, pushing PapG upwards through the usher complex.
F > 10-15 E subunits > K > 1000s A subunits > PapH.

Question 14

Describe the function of the terminator PapH.

Answer 14

The terminator PapH doesnt hold the hydrophobic groove as it holds a complete Ig Fold, thus nothing will be added onto it.

Question 15

What happens as the subunits enter the extracellular space.

Answer 15

They will rotate perpendicularly about their axis, specifically in PapA the hinge region provides a pivot point for rotation after exciting PapC, allowing for coil formation.

Question 16

Describe the Translocation Domain of FimD.

Answer 16

FimH holds a large beta barrel (24-stranded beta barrel), which when not producing a pilus contains a mid-barrel coded domain that plugs the pore in the membrane. This must be displaced for FimD:FimC:FimH complex formation and thus biogenesis of the pilus.

Question 17

Provide a brief description of the initiation of pilus biogenesis in Type I pili.

Answer 17

1. The usher plug domain of FimD is flipped/displaced from the barrel lumen to the periplasm, concomitant with a marked conformation change in the beta barrel, following FimH insertion
2. FimH is positioned within TM pore, with FimC (chaperone) still attached through strand donation complexing
3. FimG binds to the ‘free’ NTD, superimposition of its structure onto usher indicates that this would position its groove in the preceding subunit. Forcing the chaperone to zip-out, FimG swings to the CTD freeing NTD for next subunit
4. Following DSE the FimC is binds next subunit whose complex binds to the NTD, this has been shown to push FimH:FimG upwards, thus freeing the NTD to recruit the next subunit, correctly positioning FimC:subunit complex

Question 18

Why would an Ab targetting pili be so useful?

Answer 18

There is great advantage to targeting the pili of pathogenic bacteria as an antibiotic treatment; unlike most antibiotics it is not dangerous to the cell itself, interfering with no crucial cellular functions but instead removing their virulence factors. This means that it targets only the pathogen and not the other symbiotic intestinal flora as these do not use pili for support, as well as being much slower to cause resistance as it creates no direct selection pressure.

Question 19

What are pillicides?

Answer 19

These novel antibiotics are called pilicides, and have been shown to be effective ex vivo – with a molecule noted as ‘pilicide 1’ causing full depilation at 3.6mM concentrations. A wide variety of slightly different molecules have been tested in vivo, some of which have – like Np048 – been shown to reduce both adherence and biofilm presence greatly by targeting the ‘donor strand exchange’ subunit recruitment mechanism. Np048 specifically binds to the part of the subunits by which it would be recruited to the NTD, preventing it from being incorporated into the pilus by the usher.