urogenitary tract infections

Question 1

uropathogenic E.coli

Answer 1

-gram-negative, motile (flagella), rods
- they do facultative metabolism - they can survive in environments with or without oxygen

Question 2

uropathogenic E.coli reservoir

Answer 2

• The reservoir for e.coli is humans or other animals intestines.
  ○ So they live as commensals within the intestine.

Question 3

uropathogenic E.coli disease

Answer 3

Certain strains of E. Coli can cause disease and the diseases associated with E.coli include diarrhoea, dysentery, urinary tract infections, hemolytic uremic disease, septicaemia, pneumonia and meningitis

Question 4

UPEC and UTIs

Answer 4

account for over 90% of community-acquired urinary tract infections.
- they are most commonly associated with cystitis, which is inflammation of the bladder.

• These infections are almost always ascending infections - they ascend from the urethra up into the bladder rather than coming from the kidneys and down.
• the infections are usually from the gut Flora. So it’s contamination of the urethra from the gut Flora that leads to these ascending infections

most community-acquired infections occur in females below age of 10 or between 20 and 40

Question 5

the pathogenesis of UPEC

Answer 5

by expressing different fimbriae UPEC E.coli can:
- they contaminate the urethral area from the gut
- they’re able to then colonise different cell types with different receptors in the urethra and they migrate up into the bladder
- once they’re in the bladder they’re able to colonize and also invade the bladder epithelial cells and they do this by Using pilli and other adhesins

Question 6

what happens when UPEC are in the bladder

Answer 6

when they’re in the bladder they cause inflammation and you get a neutrophil infiltration- the neutrophils can start to clear the infection, but now you’re getting inflammation.
in the case of catheterized patients you start to get fibrinogen accumulation in the catheter, which can block the catheter.

Question 7

how can some UPEC bacteria evade the immune system once in the bladder

Answer 7

some bacteria are able to continue to multiply and evade the immune system.
- And this is by forming biofilms. But also they can invade the epithelial cells
- you get epithelial damaged by toxin release and proteases

Question 8

what happens if bladder infection is not treated

Answer 8

it can start to ascend up into the kidneys
- in the kidneys They’re able to colonise and cause damage there
- and then if they’re able to break out of the kidneys, it can lead to bacterial septicemia
- the bacteria get into the bloodstream and then you can get septicemia meningitis
- So the fimbriae are really important for UPEC infections.

Question 9

why are the fimbriae really important for UPEC infections

Answer 9

○ They allow them to colonise the bladder And then they use different Pili to colonise the kidneys and they can switch between their pili to allow them to move to the different niches,

Question 10

uropathogenic E.coli adhesins - type 1 fimbriae

Answer 10

• specific adhesion: FimH
  -bind to mannose residues on bladder glycoproteins (uroplakins)
• bind Tamm-Horsfall glycoprotein (washed out of bladder)
• associated diseases: cystitis, sepsis, meningitis

Question 11

uropathogenic E.coli adhesins - P fimbriae

Answer 11

(pyelonephritis - associated pili)
- specific adhesin: PapG
- many different antigenic types
- all bind alpha-D-Gal-(1,4)-alpha-D-Gal (globobiose)

Question 12

how can E.coli switch between the expression of type 1 and P fimbriae

Answer 12

• they can switch them on and off by a mechanism Called phase variation
• this is switching gene expression on and off.

Question 13

phase and antigenic variation

Answer 13

,this is a strategy used by pathogens to evade our adaptive immunity and to be able to Target different niches and there’s different mechanisms of the phase and antigenic variation.
- So phase variation is switching on and off
-antigenic variation is where you change the sequence.

Question 14

different mechanisms of phase and antigenic variation

Answer 14

1. DNA inversion (e.g. phase variation of UPEC type 1 pili)
2. DNA methylation (e.g. phase variation of UPEC P-pili)
3. complex recombination systems - e.g. phase and antigenic variation of Neisseria gonorrhea type 4 pili protein pilE by homologous recombination
4. slipped strand mispairing - involves repetitive DNA: can result in frame shifts that affects translation or promoter activity

Question 15

type 1 fimbriae

Answer 15

• a helical array of a protein called the pillin protein which in this case is FimA
• so you getrepeating subunits of FimA that form the big Rod shaped structure
• Then you’ve got a short 3 NM wide tip fibrillum containing the adhesin FimH and two adaptor proteins FimF and FimG that attach that adhesion to the main Rod shape structure.
• variation of FimH causes distinct adhesive properties.
• In the UPEC strains they have undergone a mutation that allows them to recognize mono mannose residues, whereas the majority of the commensal bacteria just to express FimH that can recognize tri Manosse
• allows adherence to epithelial cells, but also mediates cell invasion.

Question 16

type 1 fimbriae-mediated attachment to the bladder epithelium

Answer 16

type 1 fimbriae bind to mannose attached to uroplakins
-Attachment causes invasion
-Association of the bacteria with the epithelial cells causes signalling in the host epithelial cells to engulf the bacteria
- Part of our defense against that is then to rapidly slough off those cells and they get excreted in urine
But some of the bacteria stay dormant with these cells and doesn’t trigger the slewing so they are able to remain dormant

Question 17

the advantage to bacteria being dormant within these cells

Answer 17

The advantage to the bacteria is that they’re protected from antibiotics whilst within these cells
- but at a later time, they can become active again start replicating and give rise to a new infection.
this is why people with urinary tract infections often get repeat infection, so they get treated the symptoms go but then the bacteria that have been dormant can come back and Get repeat infections

Question 18

DNA inversion: type 1 pili phase variation

Answer 18

the promoter region is an invertible switch
- So at either end of the promoter is an inverted repeat
- These two genes encode recombinases and they bind to these repeats and flip the orientation of that DNA.
-This is important because to be able to get the type 1 fimbria, the promoter has to be pointing in the right direction.
○ Phase on - correct orientation, downstream genes will be transcribed and then get translated and you will get your type 1 fimbriae
○ If they flip the other way then that promoter is not active and then you don’t have the type
one fimbria produced
- within a clonal population This is happening all the time. So they’re flipping on and off between expressing the type 1 fimbria and that allows them to Target different niches.

Question 19

P. fimbriae structure

Answer 19

these are involved in the colonization of the kidneys
- very similar to the type one pillI but instead of FimA the main structural part of the Rod structure is PapA
- short 2nm wide, flexible tip fibrillum containing adhesin PapG and PapE with 2 adaptors PapK and Pap F
- and you get variation of Pap G by the addition of one or 2 N-acetyl- galactosamine sugars affects specificity

Question 20

organisation and regulation of pap genes

Answer 20

all of the genes For the synthesis and the structure are encoded within a single operon from a single promoter Upstream.
- and again these undergo phase variations - gets switching on and off of expression
- . But in this case, it’s due to selective methylation

Question 21

methylation

Answer 21

when DNA gets synthesized there’s a short period before that DNA gets methylated and DNA in bacteria gets methylated by an enzyme called dam methylase.

Question 22

at what site does Dam methylase methylate

Answer 22

• Dam methylase methylate at gatc site
• and within the promoter of the pap genes are two demethylase sites called:
• distal because it’s further away from the main genes
• and proximal because its next to the operon

you get selective or differential methylation of these sites

Question 23

what happens if the distal site or proximal site gets methylated

Answer 23

• if the distal site gets methylated, you don’t get expression of this Operon so you don’t get P Fimbriae produced
• if the other one gets methylated, then you do

Question 24

regulatory protein lrp and demethylase

Answer 24

○ there’s a regulatory protein called lrp, and it has to bind to an unmethylated gatc site
- and if it binds to the Proximal site it acts as a repressor and switches off gene expression.

Whereas if it binds to the distal site, which is unmethylated it acts as an activator and switches on expression.

• So this competition between lrp and demethylase determines whether you get the p fimbriae produced or not.

Question 25

Neisseria species

Answer 25

• nonmotile, gram-negative diplococci

2 pathogenic species:
- Neisseria gonorrhoeae: gonorrhea (inflammation and purulent discharge)
- Neisseria meningitidis: epidemic meningitis

Question 26

Neisseria - reservoir

Answer 26

humans

Question 27

Neisseria treatment

Answer 27

antibiotics

• vaccine for some serotypes of N.meningitidis

Question 28

how does Neisseria gonorrhoea spread

Answer 28

not stable outside the human host
- spread by intimate sexual contact (or direct transmission during birth)
- infection of the urethra (men) or cervix (women)
- cannot colonise vagina
- can also infect rectum, throat and conjunctiva of the eye

Question 29

complications of Neisseria gonorrhoea

Answer 29

• bacteria can ascend from the cervix to the uterus and fallopian tubes - cause salpingitis or PID
• infection of newborn during passage down birth canal - inflammation of the eyes, leading to blindness
• system infection - localisation of bacteria in joints, acute arthritis

Question 30

gonococcal pathogenesis

Answer 30

1. intimate sexual contact/ direct transmission
   - evades slgA by secreting lgA protease
2. attach to apical epithelial surfaces as microcolonies
3. dispersal over epithelial surface
4. intimate attachment of individual diplococci with epithelial cell membrane
5. invasion of epithelial cells
6. transcytosis and exit
7. inflammation and cytotoxicity

Question 31

how does N. gonorrhoea evade host cell responses

Answer 31

• phase and antigenic variation are major mechanisms of neisserial pathogenesis

phase variation: the control of expression is binary: ON or OFF
antigenic variation: changes in gene sequence leading to changes in amino acid sequence of a protein

Question 32

type 4 pili

Answer 32

-Responsbile for initial interactions with the epithelial cells. initiate infection by attaching to host cells
-Bind to a receptor called CD46 which allows them to bind to the epithelial cells and also promotes the aggregation
- mediate twitching motility over epithelial surface
- composed primarily of pilin
- PilC involved in assembly and adhesion
- both PilE and Pile undergo phase and antigenic variation - host is unable to generate a protective immuner response leading to repeat infections

Question 33

example of antigenic variation: PilE

Answer 33

Within this gene there is hyper variable regions called micro cassettes
- There are lots of copies of PILLE - of the same gene on the chromosome, but only one is expressed
- The fact you have got all these other copies means you can get homology DNA exchange between them
- so you have got conserved regions and variable regions
- The conserved regions allow genetic exchange - constantly gettting swapping of this information between them and this causes variation
- So because this is always happening you can constantly change the sequence of the pilli

Question 34

example of phase variation: PilC

Answer 34

Repeats are associated with slipped strand mispairing
You get a run of Gs in the sequence for PilC

What happens is during DNA replication where you get repeats you sometimes get errors
in the replication.
- So you might get an extra G added or taken away just because of slippage of the Strand
because DNA codons are read in triplets, If you add or take away a g it will change the reading frame.
- So depending on the number of Gs, you might hit a premature stop codon or you might get the whole Gene transcribed and translated.