genetic approaches to studying bacterial pathology

Question 1

goal of genetic approaches

Answer 1

identify bacterial genes encoding virulence factors in order to
develop new and better ways of preventing or treating infections

Question 2

genetic complementation goal/steps

Answer 2

GOAL; ID REGION/GENE RESPOBSIBLE FOR INVASIVENESS

1. isolate microbe of interest, then isolate and cut the DNA
2. introduce DNA into plasmids and donate to E. Coli
3. enrich and select for invasive colonies with growth, Ab treatment and washing several times
4. analyze plasmid seq for invasion gene
5. mutate plasmid via recombination=loss of function mutation/ suicide plasmid
6. introduce plasmid to microbe via sex pili
7. test microbe mutants to prove they can no longer invade

Question 3

plasmid creation in genetic complementation

Answer 3

restriction enxymes used to cut DNA of microbe which is then incorporated into plasmids
many possible variations produced that are then introduced to E coli

Question 4

selection mechanism in genetic complementation

Answer 4

Gentamycin kills E. coli that do not invade cells
Gentamycin does not penetrate mammalian cells.
lysis of cells after treatment and washing to extract invading E coli
Positive selection for E coli capable of invasion due to genes

Question 5

what is done in genetic complementation after the invasive factor is ID’d

Answer 5

After identification of gene for invasion factor: Manipulate gene further to
prove that invasin really does promote cell invasion.
Generate DNA sequence = inv gene
Deduce protein coding region = invasin protein

“Loss-of-function mutation” produced for suidice plasmid that replicates in E coli

Question 6

Transposons

Answer 6

small, mobile elements of DNA

two kinds: simple and composite

Question 7

simple transposons

Answer 7

a core area with gene for transposition flanked by inverted repeat sequences then direct repeats

Question 8

complex transposons

Answer 8

core area contains multiple genes for things such as drug resistance

Question 9

Insertion of a transposon in a gene most often creates a?

Answer 9

loss of function

Question 10

Transposon marks the site of?

Answer 10

the mutation (sequence and antibiotic resistance)

Question 11

Tn-phoA mutagenesis purpose/ steps

Answer 11

phoA gene Encodes a periplasmic phosphatase>engineered phoA gene
lacks N-terminus so expression depends on fusion to an adjacent gene after transposition
1. introduce pho-A/KMr transposon onto suicide plasmid
2. incorporate plasmid to microbial chromosome
3. select for KMr
4. Identify colonies also expressing phoA, indicates expression of a periplasmic protein
5. measure PhoA activity after growth in variable mediums to determine the environment this protein is expressed in
6. inject mouse with mutated pathogen, virulence should be decreased due to likely LOF mutation.

Question 12

Genetic screen versus genetic selection.

Answer 12

screen: examine individual bacteria for desirable trait

selection: only bacteria with desirable trait grow (Gentamycin selection in
complementation assay)

Question 13

Signature-tagged mutagenesis steps

Answer 13

transposon based, negative trait selection

1. transposons with KMr core and DNA tags flanking in E coli and variable seq
2. mate E.coli with other microbe to transfer transposons and create a library of mutants
3. pool mutants and inject mouse but also extract DNA from pool for PCR, tagging and membrane probing
4. extract sample from mouse and plate= recovered pool
5. both mutant pool and recovered pool are plated and compared, ONLY EXAMINE THE POPULATIONS THAT ARE NO LONGER PRESENT IN RECOVERED POOL

Question 14

Promoter-trapping/ IVET goal/steps

Answer 14

Look for genes of S. typhimurium that are expressed in infection but not in the laboratory

1. digested chromo combined with plasmid containing reporter genes PurA and LacZY= library created
2. propagation of these plasmid occurs in E coli
3. plasmids transferred to salmonella/ integrated into chromo
4. recombination events occur, markers remain
5. bacteria injected into mouse and survival dependent on the plasmid which req PurA function
6. remove sample from mouse/ plate with ampicillin (resistance gene in plasmid)
7. screen for colonies not expressing LacZY, no dye effect= promoter only expressed in vivo

Question 15

DFI: Differential fluorescence induction steps

Answer 15

cell sorting with fluorescence

1. ligate chromo fragments to the gfp plasmid that is then incorporated into salmonella
2. salmonella mixed with macrophages and allowed to invade
3. invaded pages separated based on the florescence of the bac inside them, will light up if genes of plasmids being expressed due to gfp expression
4. lyse phages, grow bac on media and sort again with FACS
5. infect phages with non gfp bac then sort again based on fluorescence with FACS
6. analyze sequences fluorescent in bac but not in media

Question 16

IVIAT: In vivo-induced antigen technology steps

Answer 16

Antibody-based approach, specific to pro of the pathogen

1. genome fragments of the bac used to create an expression library for bac phage
2. phage infect bac and produce phage factory that cause cell bursting, continuing cycle
3. plaques produced
4. pt sample taken, Ab extracted for specificity (remove Ab that bind factors from organisms grown on medium)
5. perform immunoassay with Ab and plaques
6. examine plaques interacting with Ab to determine DNA seq of pathogen

Question 17

microarray

Answer 17

transcription analysis, determine up/down regulations of different genes

1. RNA isolation from both pt sample and lab sample of microbe
2. create cDNA and label with marker sensitive to dye for different genes
3. hybridize both populations of cDNA on a microarray
4. color of microarray can indicate relative expression of the genes

Question 18

whole genome sequencing

Answer 18

DNA based approach
ID virulence factors and compare pathogenic strains to non pathogenic
unique sequences may represent virulence factor

Question 19

RNA sequencing

Answer 19

cell/tissue sample taken, directly sequenced no need for cDNA
can look at mixed samples between host/pathogen (unlike microarrays)

Question 20

RNA seq compared to microarray

Answer 20

RNA sequencing is more sensitive and has a wider dynamic range than microarray analysis.