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Molecular Microbiology > Virulence Factors > Flashcards

Virulence Factors Flashcards

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1
Q

Describe some gram positive rod bacteria

A

One membrane stained purple:

  • Bacillus anthracis: anthrax
  • Corynebacterium diphtheriae: diptheria
  • clostridium botulinum: botulism
  • clostridium tetani: tetanus
  • clostridium perfringens: gangrene
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2
Q

Describe some gram positive round cocci bacteria

A
  • staphylococcus aureus
  • s.epidermidis
  • s.pyogenes: tonsillitis
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3
Q

What symptoms are associated with s.aureus?

A

If skin infected it can cause an abcess, pneumonia and toxic shock syndrome.
MRSA occurs where s.aureus has become resistant to multiple antibodies like methicillin

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4
Q

Describe gram negative bacteria

A

Two membranes; cell wall is between the inner and outer membrane that cannot be stained.

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5
Q

Describe some gram negative ROD bacteria

A
  • Salmonella enterica: food poisoning
  • Shigella dysenteriae: dysentery
  • E.coli
  • yersinia pestis: black death
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6
Q

Describe some gram negative Cocci bacteria

A
  • Neisseria gonorrhoeae

- N.meningingitidis

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7
Q

Describe some gram negative Curved/spiral bacteria

A
  • Vibrio chloerae: shaped like a comma which causes cholera via a cholera toxin
  • Helicobacter pylori: stomach ulcers
  • Treponema pallidum: syphillis
  • Borrelia burgdoferi: lyme disease
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8
Q

Describe some gram negative Coccobacilli bacteria (between cocci and rods/elongated)

A

-Bordetella pertussis: whooping cough via pertussis toxin

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9
Q

What are virulence factors?

A

Products/structural components that allow the organism to enter and alter host function and cause disease

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10
Q

List the virulence factors

A
  • Adhesions: stick to host cell receptors and anchor bacteria
  • Toxins: into environmen or directly into host cells
  • Invasins: adhesion molecules that activate cytoskeleon and enable bacteria entry via phagocytosis
  • Protein secretion systems: like toxins and enzymes
  • Iron uptake systems
  • polysaccharide capsule to evade recognition via immune system
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11
Q

Describe the need for iron scavenging in bacteria

A

It is due to bacterial survival. Bacteria need it for respiration and as a enzyme co-factor.

Free Iron is limited in the host as it bound to protein usually like haemoglobin. So pathogens need scavenging system

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12
Q

What are Molecular Koch’s postulates?

A

set of experimental criteria that show that a gene found in pathogen encodes for a product that contributes to the disease.
It addresses how a particular gene contributes to virulence
Genes that satisfy Koch are virulence factors.

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13
Q

Describe genes that satisfy molecular Koch postulates/virulence factors

A

1) Gene found only of strains/genus of bacteria that cause disease not in non-pathogenic bacteria
2) Gene isolated by cloning
3) Disruption of genes in virulent strain should reduce virulence; reversion/allelic replacement of muted gene leads to pathogenicity
4) Gene expressed by bacterium during infectious process in animal or human

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14
Q

How do you identify virulence factors?

A

1) Cloning
2) Transposon mutagenesis
3) Transcriptional fusions
4) In vivo expression technology (IVET)
5) Signature-tagged mutagenesis (STM)

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15
Q

Describe cloning identification

A
  • take genomic DNA from pathogen
  • digest with restriction enzymes and insert into plasmid
  • Transform in E.coli so there are library of different plasmids containing different inserts.
  • look for genes that make E.coli virulent.

However, there is the possibility of making a new pathogen.

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16
Q

Describe transposon mutagenesis identification

A

-take individual transposon mutants and test them to see if they have lost virulence via infection tests

However, there needs to be lots of mutants as there are so many genes in a bacterium; 4000 mutants needed in E.coli and so it is time consuming.

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17
Q

What is a transposon?

A

a peice of DNA that gets inserted at random into chromosomal DNA; transposon encodes resistant to antibiotic and so chromosome that have taken this up are resistant.

18
Q

Describe transcription fusion identification

A

This is an indirect method aiming to find the switched on genes during infection.

  • fuse a reporter gene like GFP to all the promoters on the genome/ on a plasmid transformed into a pathogen
  • infect the host with the pathogen
  • look for strains that express the gene in the host environment
19
Q

Describe IVET

A

Positive selection

  • place all of promoters in front of a gene like purA gene in purine synthesis that can be used as positive selection.
  • in mouse bacteria will only live if PurA gene has jumped in front of the active promoter; if promoter is no activated, purA not expressed and bacteria dies.
  • use strain of mouse where bacteria deficient in purine synthesis (negative mutant)
  • prepare transposon that contains purA gene without a promoter and insert the transposon randomly into chromosome of bacterium
  • it will hop into genes and the expression of purA will be regulated by which ever gene it has jumped into.
  • cells with PurA will live after days of infection
  • 3 days, mice killed and bacteria harvested
  • all bacteria contain active purA gene in front of promoter; post mouse cells grown on plates.
  • replicate colonies onto a purine containing media and a non-purine containing media
  • cells that grow on nonpurine media means promoters are active, purA switched on under infection conditions
  • Sequence these transposons and identify which genes are next to it
  • knock out these genes to determine if they are involved in virulence
20
Q

What is the difference between growing bacteria with purA gene in lab and in host?

A

in lab, there are free purines in growth media so mutants with no promoter can still survive; there are no free purines in host so only bacteria mutant with PurA gene inserted in front of another promoter that is expressed survives.

21
Q

Describe signature-tagged mutagenesis identification

A

This is negative screening where a high throughput technique screens millions of mutants at the same time; it most direct and most powerful to identify virulence genes
-make a large pool of mutants each with ind. transposon of variabe sequence and invarient sequences
make transposon library by growing them on well plates
-inject host with each mutant from library/replicate each onto filters
-mutants that die in host are essential for virulence
-3 days kill mouse harvest/plate bacteria
-PCR reaction with labelled oligonucleotides to amplify all the tags of starting pool and from surviving colonies in undiseased mice finishing pool.
-PCR probe used to probe filter; input pool should light up, in finishing pool probes disappear as they are in genes needed for virulence shown by a blank space on filter
-Therefore determine which gene was knocked out.

22
Q

What use are filters in terms of mutant library?

A

Filters can be dried out and lyse bacteria, then probe with DNA.

23
Q

Why is PCR easy in signature tagged muagenesis?

A

This is easy because of the way the DNA bar codes are designed, they all have an identical region shown in black so you can use the same oligonucleotides to amplify across all of the different bar codes in the population.

24
Q

Give an example of signature tagged mutagenesis

A

In salmonella typhimurium virulence. 28 genes for virulence were found.
STM is used across a wide range of organisms like meningitis, tuberculosis

25
Q

What is TRADIS

A

Transposon directed insertion site; the most recent development of STM

  • make transposon library of pathogen
  • determine insert sites using next generation sequencing
  • grow under selective conditions
  • sequence survivors
  • those that do no survive are essential for survival under conditions
26
Q

What are genetic elements encoding pathogenicity factors?

A

1) Bacteriophage like choler/shiga/botulinum toxin
2) Plasmids like Salmonella enterica
3) Transposons containing virulence genes
4) Pathogenicity islands (PAI)

27
Q

How are new pathogens developing

A

through horizontal gene transfer

28
Q

What can identify a transposon?

A

Transposons have signatures: inverted repeats and the presence of gene coding for transposase that allows the transposon to move around.

29
Q

What are pathogenicity islands?

A

They are in the genomes of all bacteria,large or small.
They can be identified due to a different ratio of bases in them to the genome average; stretch of DNA different from genome average it suggests that DNA could come from another bacterium via horizontal transfer.

transfered DNA could code for virulence genes.

30
Q

Streptomyces

A

some bacteria have higher ratio of GC than AT; 70% GC

31
Q

Staph aureus

A

Has more AT bases: 67% AT

32
Q

Ecoli

A

has same amounts of A,T,G and C

33
Q

What are the common features of PAIs?

A
  • Carry genes encoding virulence factors
  • present in pathogenic organisms but absent from genomes of non-pathogenic of same species
  • relatively large regions bigger that 10kb-20kb
  • different G/C content percentage.
  • flanked by DR sequences
  • inserted at tRNA genes
  • carry cryptic or functional genes encoding mobility factors
  • represent unstable DNA regions
  • represents integrated plasmids/transposons/bacteriophage
34
Q

What techniques can help detect PAIs?

A

bioinformatic programmes which calculate the average % of GC in the genome; they go along sequence in short steps of 500bp and compare this to the average.

35
Q

What are DR sequences?

A

Direct repeats of DNA which is related to the integration process; they are attached to remnants of previous donor plasmid.

36
Q

Why do PAIs get inserted at a tRNA gene

A

tRNA gene has a conserved secondary structure that provides a structural motif to help the integration of foreign DNA by integrases

37
Q

Where do you find PAIs?

A

In almost all bacteria but especially shown in Enterobacteriaceae (E.coli)
-also in Vibrio cholerae, Pseudomonas syringae, Listeria spp, Saphylococcus aureus

38
Q

Describe E.Coli detection of PAIs using a genetic map.

A

there blue regions where both pathogen and harmless e.coli match and red regions which are only in pathogens; these regions cluster and show differing GC content of these regions.

39
Q

In exam: experimental approaches to identify virulence factor genes

A

-genome comparisons using genetic map also a valid approach.

40
Q

What kind of genes can be found on a PAI?

A
  • Adhesion
  • Secretion system
  • toxins
  • Iron uptake system
  • O antigen synthesis
41
Q

Describe the development of different pathotypes of E.coli of different diseases

A

The uptake of mobile genetic elements like phages, PAI, plasmids and loss of chromosome regions in different E.coli lineages has enable the evolution of separate clones

Molecular Microbiology (4 decks)

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