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MICB 308 > Finals: Midterm 2 Content > Flashcards

Finals: Midterm 2 Content Flashcards

1
Q

toxins (2)

A
  • kill cells
  • alter host-cell functions without killing cells directly
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2
Q

type I toxins

A
  • act extracellularly
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3
Q

type II toxins (3)

A
  • act on the cell membrane and destroy cell membrane
  • cytolytic
  • can be enzymatic or non-enzymatic
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4
Q

type III toxins

A
  • classical A/B toxins
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5
Q

cytolytic

A
  • damage to membranes usually causes host cell lysis or death
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6
Q

type II toxins: non-enzymatic (2)

A
  • form large pores/channels in membrane
  • cholesterol-dependent cytolysins
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7
Q

type II toxins: how are non-enzymatic pores formed (2)

A
  • toxin monomers can bind cholesterol and assemble on surface to form a pre-pore and then insert
  • toxin monomer binds cholesterol and inserts into membrane, triggering monomers to bind and form a large pore
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8
Q

why does cell/phagosome lysis occur after non-enzymatic pore formation

A
  • water enters the cell/phagosome which causes swelling
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9
Q

how does LLO function as a type II, non-enzymatic toxin (2)

A
  • change in pH causes conformational change in the protein
  • change allows toxin to insert into phagosome membrane
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10
Q

what do type III toxins do (2)

A
  • alter metabolism of the host cell
  • exploit or subvert normal host cell processes
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11
Q

what are A/B toxins (2)

A
  • B is the Binding component of the toxin
  • A is the enzymatically Active component of the toxin that binds to target inside host
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12
Q

what kinds of toxins are A/B toxins (5)

A
  • toxins that target protein synthesis
  • toxins that alter signal transduction
  • toxins that alter actin polymerization
  • neurotoxins
  • anthrax toxins
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13
Q

A/B toxin: forms of B component (3)

A
  • single unit that binds to receptor
  • multi-meric structure that is preformed
  • mulit-meric structure that forms on the membrane
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14
Q

type II toxins: enzymatic damage (3)

A
  • caused by phospholipases
  • enzyme removes polar head groups from phospholipid (PlcC activity)
  • causes damage to the membrane, and instability leads to lysis
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15
Q

what is one way that toxins can alter signal transduction

A
  • toxins can target or alter cAMP production
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16
Q

what is the purpose of the techniques for studying virulence factors

A
  • they are used to investigate whether something is actually a virulence factor
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17
Q

Koch’s Postulate: First Postulate

A
  • the microorganism must be found in abundance in all organisms suffering from the disease, but should not be found in healthy organisms
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18
Q

Koch’s Postulates: Second Postulate

A
  • the microorganism must be isolated from a diseased organism and grown in pure culture
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19
Q

Koch’s Postulate: Third Postulate

A
  • the cultured microorganism should cause disease when introduced into a healthy host
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20
Q

Koch’s Postulates: Fourth Postulate

A
  • microorganism must be re-isolated from the inoculated diseased experimental host and identified as being identical to the specific causative agent
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21
Q

Molecular Version of Koch’s Postulates: First Postulate

A
  • gene for virulence should be present in the strain of bacteria that cause disease and absent in avirulent strains
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22
Q

Molecular Version of Koch’s Postulates: Second Postulate

A
  • (i) knocking out or disruption the gene should reduce virulence, and (ii) introduction of the cloned gene into an avirulent strain should render the avirulent strain virulent
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23
Q

Molecular Version of Koch’s Postulates: Third Postulate

A
  • expression of the gene should be demonstrated in human or a relevant model
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24
Q

Molecular Version of Koch’s Postulates: Fourth Postulate

A
  • antibodies or a cell-mediated immune response to a virulence factor should be protective
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25
Q

what are two categories of techniques used to study virulence factors (2)

A
  • biochemical; developed first
  • genetic
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26
Q

supernatant

A
  • liquid lying above a solid residue after crystallization, precipitation, centrifugation, or other process
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27
Q

what is the basis for biochemical techniques (2)

A
  • to purify and identify the virulence factor (toxins, adhesins, etc)
  • relies on having functional assay for the virulence trait
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28
Q

what are the disadvantages of using biochemical techniques (3)

A
  • purified molecule may be missing co-factor that was removed during purification
  • molecule may not have same function in the test tube compared to the cell
  • does not tell us if our purified protein is the sole contributor to disease/virulence
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29
Q

what virulence factors work well in biochemical techniques (2)

A
  • protein-based macromolecules
  • factors that can be targeted for hydrophobicity, charge, mass, ligand binding, etc
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30
Q

what are some examples of biochemical techniques (6)

A
  • centrifugation
  • ion exchange
  • size exclusion
  • immunoprecipitation
  • ligand binding
  • Ni2+ affinity/6xHis
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31
Q

biochemical techniques: centrifugation (2)

A
  • harvest bacteria by centrifugation
  • separate supernatant into fractions and test for toxicity in animal model/cultured cell assay
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32
Q

biochemical techniques: ion exchange (2)

A
  • use of positively or negatively charged beads in a column
  • proteins attracted to the beads will remain in the column, while those repelled with leaves through the bottom of the column
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33
Q

biochemical techniques: size exclusion (2)

A
  • use of beads with small aqueous channels in a column
  • large molecules pass quickly, but small molecules are slow as they spend more time in the channels
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34
Q

biochemical techniques: immunoprecipitation (4)

A
  • use of antibodies attached to beads in a column
  • proteins are loaded in pH 7 buffer; proteins recognized by antibody bond to beads and other proteins remain in solution
  • column washed with pH 7 buffer to remove proteins in solution
  • column washed with pH 3 buffer to disrupt protein-antibody bonds and elute proteins
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35
Q

biochemical techniques: ligand binding (4)

A
  • use of affinity bead with ligand attached in a column
  • proteins are loaded; proteins recognized by ligand bond to beads and other proteins remain in solution
  • column washed with buffer to remove proteins in solution
  • column washed with low pH buffer or soluble ligand to elute proteins
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36
Q

biochemical techniques: Ni2+ affinity/6xHis (4)

A
  • use of NTA-coated agarose bead coordinate Ni2+ ions in a column
  • tagged and untagged proteins are loaded; tagged proteins bind to Ni2+ and other proteins remain in solution
  • column washed with buffer to remove proteins in solution
  • column washed with low pH buffer or imidazole (binds Ni2+) to elute proteins
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37
Q

how would you prove that a putative virulence factor (purified protein) caused disease (3)

A
  • isolate toxin and investigate effects in other cells
  • sequence toxin and knockout toxin to see if disease occurs or investigate for its presence in avirulent strain
  • identify immune response in model to show that virulence factor grants immunity
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38
Q

molecular genetic techniques (2)

A
  • gain of function experiments
  • loss of function experiments
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39
Q

gain of function techniques (2)

A
  • look for putative virulence factors by doing gain of function in an avirulent strain
  • observing for restoration of virulence
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40
Q

loss of function techniques (2)

A
  • knock out genes and look for reduction in virulence
  • can be done through directed mutants or transposon mutants
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41
Q

gene expression experiments (3)

A
  • reporter assays - promoter traps
  • hybridization-based experiments
  • sequencing (RNA-seq)
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42
Q

advantages of genetic approaches (3)

A
  • allows us to find novel genes by screening mutants
  • connection with virulence is established at the beginning of the experiment
  • relatively biased
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43
Q

disadvantages of genetic approaches (2)

A
  • usefulness of the information (results) depends on the screen
  • you may not be able to identify the function of the gene product
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44
Q

disadvantages of gain of function experiments (3)

A
  • there may be other accessory factors or genes involved
  • many different restriction enzymes may need to be used
  • virulence may not be expressed in an avirulent strain
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45
Q

GoF Salmonella invasion experiment: what are the steps to create the library (5)

A
  1. isolate virulent Salmonella DNA
  2. cut DNA with a restriction enzyme
  3. run on gel to separate DNA fragments according to size
  4. DNA between 1000-3000bp are isolated from the gel
  5. these DNA are cloned into plasmids
  6. plasmids are transformed into avirulent E. coli to create a library
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46
Q

GoF Salmonella invasion experiment: what are the steps to test the library against an assay (6)

A
  1. library of E. coli clones is used to infect tissue cells
  2. cells are washed and incubated
  3. gentamycin is added; kills extracellular bacteria, but not intracellular bacteria
  4. wash to remove gentamycin
  5. lyse the host cells to release the surviving bacteria
  6. plate surviving bacteria on lab media
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47
Q

GoF Salmonella invasion experiment: why is DNA between 1000-3000bp isolated only

A
  • average size of genes, so scientists are hoping to find intact genes in this region
48
Q

GoF Salmonella invasion experiment: what is gentamycin (2)

A
  • an antibiotic
  • does not penetrate host cells
49
Q

GoF Salmonella invasion experiment: what kind of assay is used (2)

A
  • an invasion assay
  • the gentamycin protection assay
50
Q

GoF Salmonella invasion experiment: what are the steps to identify the possible virulence factor gene (3)

A
  1. plasmids are isolated from each colony
  2. the inserted Salmonella DNA is sequenced using Sanger’s Sequencing
  3. the sequence is ran through databases, such as BLAST, to identify the gene
51
Q

Sanger Sequencing steps (3)

A
  1. DNA sequence for chain terminator PCR
  2. size separation by gel electrophoresis
  3. gel analysis of determination of gene sequence
52
Q

Illumina Sequencing steps (4)

A
  1. sample prep
  2. clustering
  3. sequencing
  4. data analysis
53
Q

sanger sequencing: templates

A
  • usually one template
54
Q

sanger sequencing: output

A
  • one sequence
55
Q

sanger sequencing: typical usage (3)

A
  • plasmids
  • PCR products
  • gDNA sequence
56
Q

illumina sequencing: templates

A
  • many barcoded templates
57
Q

illumina sequencing: output (2)

A
  • millions of barcoded sequencing
  • cluster = one sequence
58
Q

illumina sequencing: usage

A
  • can multiplex (combine) experiments
59
Q

transposon mutagenesis (3)

A
  • mobile genetic elements
  • “random” mutagenesis approach
  • all contain an antibiotic resistance marker
60
Q

loss of function experiments (4)

A
  • uses transposon mutagenesis
  • virulent bacteria is “infected” in transposon
  • plates are selected with appropriate antibiotic
  • collection of mutants is the library
61
Q

what are the characteristics of the bacteria in a loss of function experiment library (2)

A
  • mostly random mutants
  • Tn is in their chromosome
62
Q

what are some disadvantages of loss of function experiments (3)

A
  • only generates mutants in non-essential genes
  • transposons have transcriptional terminators within them
  • assays can sometimes be laborious
63
Q

loss of function experiments: how do the terminators within Tn affect the experiment (2)

A
  • bacterial mRNA is polycistronic
  • can lead to the “polar” effect
64
Q

how can you identify a mutated gene without sequencing the entire genome (7)

A
  1. insert Tn into bacterial chromosomes to create the random mutations
  2. use restriction enzymes to randomly cut the chromosome
  3. clone the fragments into E. coli
  4. subject E. coli to antibiotics; the surviving E. coli contain a mutated gene
  5. identify insertion site of transposon using prime from plasmid and primer from Tn
  6. use Sanger’s sequencing to identify gene disrupted by Tn
  7. BLAST against the database
65
Q

why do we use two primers for Sanger’s Sequencing in plasmids (2)

A
  • this method has a limit of ~800bp
  • running sequencing both ways allows scientist to obtain more length of the desired DNA
66
Q

signature tagged mutagenesis (2)

A
  • negative selection for virulence gene identification
  • involves generating strains using Tn libraries
67
Q

how does signature tagged mutagenesis work (5)

A
  1. mutate gene with a Tn carrying a unique tag
  2. introduce Tn mutants into chosen disease model
  3. harvest bacteria after certain amount of time
  4. use hybridization to identify tags
  5. isolate genomic material from clone and use Sanger’s sequencing to find which gene the transposon was inserted into
68
Q

signature tagged mutagenesis: what is the purpose of looking for identity tags in the last step (2)

A
  • looking for tags that were present in initial inoculum that are absent in recovered fraction
  • identifies mutants that did not grow as they were mutated for a gene that represents a potential virulence factor
69
Q

what are the advantages of signature tagged mutagenesis (2)

A
  • can look at many mutants at a time using a few animals
  • can identify gene important for growth and survival directly in vivo
70
Q

signature tagged mutagenesis: disadvantage (2)

A
  • usual Tn issues
  • polar effect and insertion bias
71
Q

polar effect (2)

A
  • when a Tn is inserted into DNA that in transcripted into a polycistronic mRNA
  • describes how the insertion may remove more genes that expected
72
Q

how can we test for the polar effect (2)

A
  • do complementary experiments to restore function
  • do targeted deletions in each gene
73
Q

Tn Seq

A
  • looks for genes important to survival by comparing two conditions
74
Q

what are some required elements for Tn Seq (2)

A
  • high coverage of the Tn insertion sites; need to create a saturating library to determine all non-important genes
  • use of the Himar Mariner Tn
75
Q

Himar Mariner Tn (3)

A
  • contains an antibiotic resistance marker
  • contain MmeI restriction enzyme recognition sites within the Tn, but cut sites occur 20bp away from site (outside of Tn)
  • MmeI leaves two-bp overhang for adaptor ligation
76
Q

Tn Seq steps (5)

A
  1. mutanagize bacteria with Himar Mariner Tn several times to cover all possible genes
  2. digest chromosomal bacteria with MmeI
  3. ligate adaptors (MmeI cut site and Tn) for Illumina Sequencing
  4. compare read counts for each insertion site under different conditions
  5. identify loci required for growth under different conditions
77
Q

IVET (2)

A
  • in vivo expression technology
  • promoter trap for in vivo expressed genes
78
Q

IVET: reporter gene (2)

A
  • β-Galactosidase protein
  • promoter-less lacZ gene
79
Q

how can we used IVET to identify Salmonella virulence factors: starting components (2)

A
  • Salmonella genomic DNA
  • engineered plasmid
80
Q

how can we used IVET to identify Salmonella virulence factors: salmonella genomic DNA processes

A
  • DNA is partially digested by Sau3AI restriction enzyme
81
Q

how can we used IVET to identify Salmonella virulence factors: origin of replication

A
  • it functions in E. coli, not in Salmonella
82
Q

how can we used IVET to identify Salmonella virulence factors: experimental set-up (2)

A
  • use a model system
  • find avirulent strain that does not infect host model
83
Q

how can we used IVET to identify Salmonella virulence factors: first 1/2 of steps (purA importance) (5)

A
  1. ligate Sau3AI-digested Salmonella DNA and Bgl II-digested pIVET plasmid into E. coli
  2. activate mob gene to transfer into LoF purA Salmonella via conjugation
  3. pIVET will integrate into Salmonella purA auxotroph via homologous recombination, creating a library of Salmonella clones
  4. pool library and inject into mice
  5. mash up spleen and collect surviving bacteria (PurA+ phenotype) into a petri dish
84
Q

how can we used IVET to identify Salmonella virulence factors: what are the characteristics of the surviving bacteria from the mouse spleen (2)

A
  • fragments successfully recombined with a promoter
  • gene with promoter turned on is thought to be necessary for survival in the mouse
85
Q

how can we used IVET to identify Salmonella virulence factors: second 1/2 of steps (lacZ importance) (4)

A
  1. plate bacteria on media containing substrate for β-galactosidase (X-gal)
  2. if β-galactosidase is expressed, it will cleave X-gal and the colony will appear blue
  3. collect colonies that appear white and discard colonies that appear blue
  4. sequence insertion to find the gene that was expressed in white colonies
86
Q

how can we used IVET to identify Salmonella virulence factors: importance of white colonies (3)

A
  • β-galactosidase does not cleave X-gal; no blue appears
  • these colonies are expressed in vivo, but not in vitro
  • these are genes thought to be needed for survival specifically in the mouse model
87
Q

how can we used IVET to identify Salmonella virulence factors: importance of blue colonies (2)

A
  • β-galactosidase cleaves X-gal; blue appears
  • these colonies are expressed in vivo and in vitro
88
Q

how can we used IVET to identify Salmonella virulence factors: what should happen after the experiment

A
  • LoF or biochemical experiment is needed to test the virulence/essential-ness of the gene
89
Q

how can we used IVET to identify Salmonella virulence factors: why might genes that are only expressed in vivo have no effect on virulence (3)

A
  • several genes may be under the same promoter
  • there may be redundant factors (eg. 10 redundant adhesion factors); knocking out one does not affect the virulence as compensation by other factors will occur
  • gene may be involved in house-keeping functions
90
Q

IVET: disadvantages (2)

A
  • genes expressed in vivo are not confirmed to be virulence factors; subsequent testing must be done
  • IVET does not tell us where or when the genes were turned on
91
Q

DFI (3)

A
  • differential fluorescence induction
  • promoter trap technology
  • uses fluorescence-based selection for intracellularly-induced genes
92
Q

DFI: reporter construct (2)

A
  • promoterless gfp gene
  • green fluorescence proteins
93
Q

DFI steps: rough collection (6)

A
  1. make library of random fragments of genomic DNA
  2. insert upstream of the promoterless gfp gene in a plasmid
  3. infect host cell (eg. macrophage) with plasmod
  4. sort and collect fluorescing macrophages
  5. lyse macrophages and plate bacteria on lab media
  6. sort and collect bacteria (from lab media) with lowest fluorescence
94
Q

DFI steps: refined collection/observation (5)

A
  1. use collected bacteria to infect macrophages again
  2. see enrichment of fluorescing macrophages
  3. lyse macrophages and collect bacteria
  4. study each bacterium in greater detail, paying attention to timing, levels and location of expression
  5. sequence cloned fragments and identify the gene
95
Q

hybridization-based techniques (3)

A
  • used for gene expression studies
  • used to compare the relatedness of 2 bacterial strains
  • involves dsDNA -> ssDNA
96
Q

subtractive hybridization

A
  • used to identify gene sequences found in virulent strains, but not avirulent ones
97
Q

subtractive hybridization steps (6)

A
  1. label DNA from “avirulent” strain with biotin (after adding linkers)
  2. combine all the fragments from the virulent and avirulent strain
  3. denature and reanneal DNA
  4. use streptavidin to remove biotinylated DNA
  5. what’s left (fragments without streptavidin) are fragments unique to the virulent strain
  6. clone fragment into plasmid and sequence the fragment
98
Q

subtractive hybridization: denature and reanneal steps (3)

A
  1. mix avirulent + virulent DNA
  2. avirulent + biotin strands in excess
  3. complementary strands will hybridize: all virulent fragments, avirulent fragments, and shared virulent/avirulent strands will hybridize
99
Q

gene expression experiments (2)

A
  • exposure of virulent strains to two different conditions
  • compare virulent vs avirulent strains
100
Q

how can we use subtractive hybridization for gene expression experiments (4)

A
  • one strain (virulent) exposed to two different conditions
  • collect mRNA
  • convert to cDNA; reverse transcriptase and random primers
  • do subtractive hybridization experiment
101
Q

DNA microarrays

A
  • used for gene expression studies or strain (DNA) comparisons
102
Q

microarray (2)

A
  • miniaturized device containing short single-stranded DNA oligonucleotide probes attached to solid substrate
  • $100 per chip
103
Q

microarray probes (2)

A
  • designed to have sequences complementary to segments of one or more target organism genomes
  • 20,000 probes
104
Q

microarray probe synthesis (4)

A
  • mechanically spotted
  • sprayed
  • using an ink jet print head
  • synthesized using photochemical reactions
105
Q

gene expression using microarrays: steps (7)

A
  1. expose virulent strain to 2 different conditions
  2. collect mRNA from each conditions
  3. convert mRNA into cDNA
  4. label cDNA with fluorescent probes, using different fluorescence for each condition
  5. mix and use to hybridize to an array
  6. array has every gene from the organism
  7. compare expression patterns (fluorescence colours)
106
Q

microarrays to compare 2 strains (2)

A
  • collect DNA from strain A and use it to hybridize to array from strain B
  • if there are spots that don’t fluoresce on array, the genes are missing from strain A
107
Q

DNA microarray: disadvantages to comparing two strains

A
  • won’t identify any unique genes from hybridized strain
108
Q

pangenome array

A
  • DNA microarray with genes from many, many bacteria
109
Q

RNA Seq (2)

A
  • used to detect and quantify mRNA levels
  • will also find small (non coding) RNA (sRNA)
110
Q

RNA Seq: steps (7)

A
  1. extract RNA
  2. deplete rRNA and tRNA
  3. fragment the RNA
  4. convert to cDNA
  5. add adaptors, amplify library, and do Illumina sequencing
  6. align sequences to genome using bioinformatics
  7. the number of sequence reads is roughly proportional to amount of mRNA for a particular gene
111
Q

what does each cluster on an Illumina sequencing chip represent when doing RNA Seq (2)

A
  • one mRNA transcript for gene X in the original sample
  • a cluster is the bridge-amplified produce of a single transcript
112
Q

how do we measure RNA expression using RNA seq (3)

A
  1. count up number of identical sequences from each cluster on the chip
  2. map sequences to genome
  3. compare experimental conditions
113
Q

RNA seq: advantages (3)

A
  • can use RNA seq to compare bacteria grown under different conditions
  • may discover novel genes
  • small non coding RNA and antisense RNA can be found
114
Q

dual RNA seq (2)

A
  • can be used to find in vivo expressed genes
  • can sequence bacterial and host cell transcripts (cDNA) at the same time
115
Q

dual RNA seq: steps (5)

A
  1. infect cells with fluorescently labeled bacteria
  2. sort for infected cells to get a homogenous starting population
  3. extract RNA, depleting rRNA and tRNA
  4. convert to cDNA and sequence
  5. align to host genome and the bacterial genome separately
116
Q

protein/proteome microarrays: steps before array (5)

A
  1. start with bacterial genome
  2. amplify all open reading frames (genes)
  3. clone into an expression plasmid
  4. express the proteins using an in vitro transcription and translation system
  5. do not need to purify the proteins
117
Q

protein/proteome microarray: steps after array (4)

A
  1. use array with nickel-NTA (Ni++) which binds 6xHis tags
  2. anti-V5 tag antibody confirms expression of the construct
  3. add serum from patients or from experimental model
  4. if antibody binds to protein, a signal will be produced

MICB 308 (22 decks)

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