Mimm 465: Lecture 11 Moual

Front 1

How do virulence factors help bacteria?

Back 1

1) Colonize host: multiply and send virulence factors
2) Invade the host: have access to tissues
3) Cause disease
4) Evade host defenses

Front 2

What are some virulence factors?

Back 2

Flagella
Adherence factors
Invasion factors
Capsules
Siderophores
Endotoxins (LPS)
Exotoxins
Secretion systems

Front 3

Which ptn is required for adhesion between bacterial cells and receptors on host cells?
Where are they found?

Back 3

Adhesins
-Found on both bacteria and receptors

Front 4

What is an essential 1st step to colonization and invasion by the bacteria?

Back 4

Adherence of bacteria to specific epithelial surfaces

Front 5

Why is adhesion important for bacteria?

Back 5

Bacteria can resist physical removal by fluids (saliva/urine), peristalsis (gut) and sneezing (resp tract)

Front 6

What is specific adhesion associated with?

Back 6

Host and tissue tropism
(Only colonize tissue if the receptor is present)

Front 7

What kind of other factors are some adhesins?

Back 7

Invasion factors (Invasins)

Front 8

What are invasins?

Back 8

Signalling event that will trigger bacterial uptake

Front 9

What are the 2 types of adhesins?

Back 9

1) Pili or fimbriae
2) Non-fimbrial adhesins

Front 10

What are pili/fimbriae adhesins?

Back 10

Hair like fibers
Have multiple subunits (more then 1 ptn)
(Longer than non-fimbrial adhesins)

Front 11

What are non-fimbrial adhesins?

Back 11

Only have one ptn
Shorter than pili

Front 12

What are the type of fimbrial/pili adhesins?

Back 12

Type 1 Pili
P Pili
(these are the 2 types focused on but there are Type I-IV pili)

Front 13

What kind of genes do Type I pili have?

Back 13

fim genes

Front 14

What kind of genes do P pili have?

Back 14

pap genes

Front 15

What kind of adhesins are there for G- bacteria?

Back 15

-Afimbrial adhesion: bacteroids, stick to cells using OM ptns
-Pap fimbriae
-Type IV (bundle forming pilus)
-Curli

Front 16

What is the use of pili?

Back 16

Enable bacteria to adhere to epithelia
-> Tip of pili recognize a specific receptor

Front 17

What type of pili do 80% of uropathogenic E. coli produce?

Back 17

Type I

Front 18

How many fimbriae does the typical type I fimbriates bacterium have?

Back 18

200-5000

Front 19

What is the major subunit of a single type 1 fimbria?

Back 19

FimA

Front 20

How many copies of FimA is in a single typ 1 fimbria?

Back 20

1000 copies

Front 21

What type of infection are uropathogenic E.coli (UPEC) ass't with?

Back 21

Cystitis
Pyelonephritis

Front 22

How are UPEC infectious?

Back 22

They attach to and invade bladder epithelial cells
(invade epithelial cells to find shelter)

Front 23

What form do UPECs take when they invade and get to a protective environment?

Back 23

Biofilms
(in the cytoplasm)

Front 24

What does infection by UPEC trigger?

Back 24

Inflammation and exfoliation of infected epithelial cells

Front 25

What does Caspase activation lead to?

Back 25

Apoptosis of infected cell

Front 26

What is a negative regulator of proliferation?

Back 26

Bmp4

Front 27

Does exfoliation completely stop continued infection?

Back 27

No, because the bacteria has time between inflammation and exfoliation to move to adjacent cells
Even if the top layer of cells is exfoliated, still some bacteria in lower layer
Will cause continued inflammation

Front 28

What do the adhesins at the UPEC OM include?

Back 28

1) Type 1 pili (fim genes)
2) Pyelonephritis-ass't (P) pili (pap genes)
3) S pili
4) Dr adhesins

Front 29

What do type 1 pili (fim genes) bind?

Back 29

D-mannose moiety of glycoptns or glycoptns

Front 30

What do P pili (pap genes) bind to?

Back 30

alpha-D-galactopyranosyl-(1-4)-beta-D-galactopyranoside moiety on glycolipids

Front 31

What do S pili bind?

Back 31

Sialic acid residue

Front 32

Look at pic pg 3

Back 32

look at picture pg 3

Front 33

What is the organization of a type 1 pilus operon?

Back 33

Has 9 Fim genes
B-E----A---I----C-----D------F--G--H(mannose binding adhesin)

Front 34

What is the organization of a type 1 pilus? (not the operon)

Back 34

FimA- ---Fim F-- Fim G-- Fim H

Front 35

Which Fim genes are for regulation?

Back 35

Fim B & E

Front 36

Which Fim gene is the major pilus subunit?

Back 36

Fim A

Front 37

What does Fim C do?

Back 37

Periplasmic chaperone

Front 38

What does Fim D do?

Back 38

Outer mb usher (exports)

Front 39

What are Fim F & G?

Back 39

Adaptors
Initiators
Terminators

Front 40

What does Fim H do?

Back 40

Recognizes the receptor on the host
(Mannose binding adhesin)

Front 41

Which genes makes up the type I pilus tip?

Back 41

FimF--- Fim G-- Fim H

Front 42

What is the organization of the P pilus operon?

Back 42

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

Front 43

Which pap genes are involved in regulation?

Back 43

Pap I and B

Front 44

Which pap gene is the major component of the pilus rod?

Back 44

Pap A

Front 45

Which gene is the pap anchor?

Back 45

Pap H

Front 46

Which paps are part of the pilus assembly machinery?

Back 46

Pap C
Pap D

Front 47

What is Pap C?

Back 47

Outer mb usher
(opp of Type 1)

Front 48

What is Pap D?

Back 48

Periplasmic chaperone (opp from Type 1)

Front 49

What do periplasmic chaperones do?

Back 49

Prevents aggregation of major subunit in cytoplasm

Front 50

What are the pilus tip fibrillium subunits for a P pilus?

Back 50

Pap K
Pap E
Pap F
Pap G

Front 51

What is Pap K and F?

Back 51

Adaptor/initiators

Front 52

What is Pap E?

Back 52

Major component on TIP fibrilliium

Front 53

What is Pap G?

Back 53

Real adhesin that recognizes the receptor
Gal-a(1-4) Gal binding adhesin

Front 54

What is the difference between a Type 1 pilus tip and a P pilus tip?

Back 54

P pilus tip can be longer
Pap E on P pilus can have 10-100 copies

Front 55

Describe UPEC type 1 pili.

Back 55

Rigid helical rod: Repeating FimA subunits
Distal tip: Fim F and Fim G (adaptors) and Fim H (adhesin)

Front 56

How many domains does FimH have?
Describe

Back 56

2 domains
Each have Ig-like folds
Adhesin domain has a binding pocket that can accomodate D-mannose

Front 57

How wide is the tip of a type 1 pilus?

Back 57

3 nm

Front 58

How long is the rod/shaft?

Back 58

1 um long
7 nm wide

Front 59

What is teh adhesin of Pap genes (P pilus)?

Back 59

Pap G

Front 60

What does pap G bind?

Back 60

Globosides
(Globoside binds the side of PapG

Front 61

What is a globoside? (GbO4)

Back 61

Tetrasaccharide
GalNacβ1-3Galα1-4Galβ1-4Glc link to ceramide (glycopeptide linked to host)

Front 62

How do pilus subunits avoid ptn aggregation in the periplasm?

Back 62

Pilus subunits interact with a chaperone (PapD or FimC)

Front 63

How do the pilus subunits oligomerize?

Back 63

Through complementation
-Nte in donor-strand exchange
-> Have 1 free β-strand that is associated with the next subunit (donor-strain exchange)

Front 64

What happens if bacteria wants a pilus with a tip?

Back 64

Express tip ptn
Then express adaptors
Then the major ptns
This is controlled at the transcriptional level
->Usher ptns (PapC/FimD) allow export of the ptns
to the outside of the bacteria

Front 65

Describe the assembly of P pili via the chaperone-usher path (similar for type 1 path)

Back 65

All components are secreted into the periplasmic space
Pap D chaperne prevents premature oligomerization in the periplasmic space by directly interacting with Pap A, G, F and K subunits
Pap D delivers these subunits (A, G, F, K) to the OM assembly site (PapC usher)
Assembly is timely regulated

Front 66

How many β barrels are there in this adhesion?

Back 66

2 but only one used for the pilus formation
Other one used to recognize the next subunit
(form 2pores, but only one used for secretion)

Front 67

Which Fim ptn mediates bacterial invasion?

Back 67

Fim H

Front 68

Describe the experiment that show FimH adhesin mediates bacterial invasion.

Back 68

-Coat latex beads with FimC-FimH complexes
these are easily internalized by bladder epithelial cells grown in vitro
-Beads coated only with FimC (chaperone) alone or with BSA are not internalized
-**FimH (adhesin) is sufficient to mediate bacterial uptake by bladder epithelial cells

Front 69

What are the fcts of FimH?

Back 69

Adhesin
Invasin

Front 70

What happens when FimH binds to host cell receptors?

Back 70

Activates host signal transduction pathways that trigger bacterial uptake
.: Without FimH, bacteria can't be internalized

Front 71

What pili system do most G- bacteria have?

Back 71

Type IV

Front 72

What is a Type IV pilus system important for?

Back 72

Adhesion and auto-aggregation
Surface motility (twitching motility)
Microcolony and biofilm formation
Phage attachment
DNA uptake by natural transformation

Front 73

What is twitching motility?

Back 73

Motion of bacteria over surfaces without the use of flagellum (need type IV pili)

Front 74

How are pili used in twitching motility?

Back 74

Pili extend from the front of the cell
Contact of pili with a surface induces retraction, which results in cell movement
(this is how bacteria glide on the surface)

Front 75

When were pili first visualized in G+ bacteria?

Back 75

1968
-> Immunogold e- microscopy showed pilus-like structures on Streptococcal surface

Front 76

Which G+ bacteria definitely have pili?

Back 76

Streptococcus
Corynebacterium
Actinomyces

Front 77

How is the pilus assembled in G+ bacteria? (different from G-)

Back 77

Pilus subunits exported and assembled into pilus
Put into p'g with enzyme SORTASE

Front 78

What is the sequence recognized by sortase?

Back 78

L P S P G
->cuts at L

Front 79

What happens if you inhibit the sortase?

Back 79

Have G+ bacteria without pili at their surface

Front 80

Which bacteria use non-fimbrial adhesins?

Back 80

Yersinia

Front 81

What are the enteropathogenic Yersinia species?

Back 81

Y. pseudotuberculosis
Y. enterocolitica

Front 82

How do Yersinia bacteria invade the body?

Back 82

Bacteria translocated across M cells (like epithelial cells) into Peyer's patches of small intestine
->once they cross the M cells, will then go into the mb and cause disease

Front 83

How is internalization of Yersinia promoted?

Back 83

Interaction between the bacterial invasin ptn and β1 integrin receptors (which is expressed on the luminal side of M cells)

Front 84

Why do they invade M cells?

Back 84

Receptor for the invasin is only in the M cells (not the epithelial cells)

Front 85

Describe the Yersinia invasin

Back 85

986-residue ptn
Elongated ptn (18nm rod)
N-terminal part is a β-barrel that spans the OM
C-terminal part (extracellular) consists of Ig-like domains (D1-4)
Domains D4 and D5 bind β1 integrins
(look at figure pg 11 (6))

Front 86

What is the Yersinia invasin (what system)?

Back 86

Autotransporter
-> Type V secretion (can sometimes be cleaved)

Front 87

Describe the Type V secretion system (autotransporter)

Back 87

Inserts into OM through β-barrel domain
Passenger domain goes through β-barrel and forms outside
-> Invasin will then bind to the β1 integrin

Front 88

Describe the β1 integrins on host cells that invasin binds

Back 88

-*Heterodimeric (α &β) integral mb ptns
-α3β1, α4β1, α5β1, α6β1 integrins

Front 89

What do the β1 integrins bind in absence of Yersinia?

Back 89

ECM ptns
(i.e. fibronectin)

Front 90

How come the β1 integrins can bind Yersinia if already bound to ECM ptns?

Back 90

Invasin binds β1 integrins with a much higher affinity than fibronectin

Front 91

What are the signalling paths that lead to Yersinia invasion?

Back 91

-Engagement of β1 integrins by invasin leads to FAK autophosphorylation
-Src is recruited by FAK
- FAK-Src interaction leads to Rac1 activation
- Rac1 might activate WAVE
- WAVE can bind Arp2/3 complex
-->Actin polymerization and bacterial engulfment

Front 92

Look at figures

Back 92

Look at figure on last page