Lecture 5

Question 1

Where does Helicobacter pylori colonize?

Answer 1

The gastric Mucosa

Question 2

What occurs due to infection by H.pylori?

Answer 2

Stomach Ulcers; Gastritis

Question 3

How does H.pylori achieve this?

Answer 3

Secretion of Urease; which hydrolyses Urea to ammonia, creating micro environments at higher pH. Modifying the environment to allow its own growth

Question 4

What other pathogens have urease? What do they use it for?

Answer 4

Proteus mirabilis and Klebsiella use urease when urea is the carbon source. Presence of urea and lack of nitrogen respectively

Question 5

What are PAMP’s?

Answer 5

Pathogen Associated Molecular Patterns Generic molecules that will generate an innate immune response

Question 6

Give some example PAMP’s

Answer 6

Lipid tail of LPS on Gram -ve bacteria recognised by TLR-4 Peptidoglycan of gram +ve bacteria recognised by TLR-2

Question 7

What effect does a capsule have on a bacteria ability to evade the innate immune system

Answer 7

Physical prevention against ingestion by macrophage

Question 8

How does salmonella enterica use its capsule?

Answer 8

Stops neutrophils being able to use chemotaxis to extended towards the bacterium

Question 9

Give one way in which pathogens can use host-like compounds to hide?

Answer 9

Presenting sialic acid makes the pathogen appear as ‘self’

Question 10

What three things can lead to a population of cells being different to each other?

Answer 10

Stochastic variation, antigenic variation and phase variaiton

Question 11

What is phase variation?

Answer 11

Within a population a gene can be on/off and hence cause a difference in the population

Question 12

What is antigenic variation?

Answer 12

Antigenic variants of each protein exist, and each cell is not identical due to ability to express each variant

Question 13

How is antigenic variation used to evade the immune system?

Answer 13

Once the immune system is able to elicit a sufficient response to one antigenic variant and remove it from the population, another antigenic variant can grow into the space now available and the immune system has to start again. This is repeated for each antigenic variant

Question 14

What type of proteins commonly show antigenic variation?

Answer 14

Cell surface structures and proteins

Question 15

What type of plasmids exist?

Answer 15

Expression plasmids and storage plasmids

Question 16

Why are storage plasmids important in antigenic variation?

Answer 16

Hold the protein variants. These loci can undergo recombination into the expression plasmid

Question 17

Give some examples of antigenic surface structures

Answer 17

Flagells, outer membrane proteins, LPS and fimbriae

Question 18

What is the difference between transcriptional and translational fusion?

Answer 18

Transcriptional fusion you will tag a reporter after a promoter of interest and will be able to see when that promoter is active and hence when the downstream gene is being transcribed

Translational fusion you tag the protein of interets and can see where, and how much of, the protein is actually in the cell

Question 19

What effect can short sequence repeats have on DNA replication?

Answer 19

May cause DNA polymerase to make a mistake incorporating too few or too many of the repeated nucleotide

Question 20

How can short sequence repeats then have transcriptional effect?

Answer 20

1. The activator may not be able to make contact with the RNAPolymerase
2. Changes to the spacing between the -10 and -35 locations can stop RNA polymerase being able to bind
3. Stability of the RNA may be compromised
4. Missense or nonsense mutation if happens in coding region

Question 21

What is classified as a short sequence repeat?

Answer 21

Mono-, di-, tri- or tetra-nucleotide repeats between 30 and 50 bp long

Question 22

What causes site-specific recombination?

Answer 22

Inverted repeat recombinase recognition sequences either side of DNA sequence can be recognised by a recombinase and the DNA can be inverted.

Question 23

Outline an example of site-specific recombination

Answer 23

Type 1 fimbriae

DNA inversion in Fim operon

Controlled by site-specific recombinases: FimE and FimB

FimE promotes ON to OFF transition

FimB promotes both directions equally

Question 24

Outline an experiment which investigated if phase variation is a virulence factor

Answer 24

E.coli on murine bladder model

Locked ON and locked OFF Type 1 Fim

Locked ON survived as well as wild type, but locked OFF did not

Doesn’t prove that phase variation is virulence factor only that the fimriae was important for survival

Question 25

How can phase variation been controlled at the non-DNA sequence level?

Answer 25

DNA Methylation

Question 26

What protein is imporant in DNA methylation?

Answer 26

Deoxyadenosine methylase - which methylates adenine of GATC sequence

Question 27

What are the substares of DAM?

Answer 27

Both unmethylated and hemimethylated DNA

Question 28

Why is the methylation irreverisble in E.coli?

Answer 28

There is no demethylating enzyme in E.coli

Question 29

How is DAM activity regulated?

Answer 29

Regulatory protein binding blocks DAM access

But once DNA is methylated the regulatory protein can no longer bind and DAM can continue to methylate

Question 30

How can DNA switch between methylated and unmethylated?

Answer 30

During DNA replication

Question 31

Outline phase variation by Lrp and DAM

Answer 31

Lrp can bind to two sites on Pap operon - when they are not methylated

Binding to one site due to other site being methylated causes pilin production to be off

Binding to other site due to first site being methylated causes pilin production to be on

Question 32

How is a positive feedback loop involved in the Pap operon phase variation?

Answer 32

Binding of Lrp to site which causes pilin production requires PapI

PapI is made due to the binding of PapB, and CAP, which is made due to Lrp binding to correct site

Question 33

How can sRNA be found to be sequenced?

Answer 33

Pull down of Hfq- a factor associated with sRNA for stability

Question 34

Who wrote the paper from the lecture?

Answer 34

Khandige

Question 35

What effect was Hfq shown to have in bladder cell infection?

Answer 35

Knockout of Hsq lead to less adhesion and invasion of cells