MCB4203 - Exam #3 Latest Update
Expression in Avirulent Strain - Answer - Best applied to closely-related species
- Can be time consuming
- Can only analyze small segments of genes (cloning limitation of 30 kb)
Transposons - Answer Advantages: High frequency, relatively-stable interaction, amr
genes, random insertion into target DNA, the disrupted gene of interested can be
identified through PCR
Disadvantages: Can carry terminators that have polar effects on downstream gene
expression, can only be used to identify non-essential genes
Transcriptional Fusion - Answer Where/when a virulence gene promoter is active
Translational Fusion - Answer Where/when a virulence gene is translated
TnSeq - Answer Advantages: Bypasses need for in vitro screen
Disadvantages: Competition experiment; trans complementation effect
In vivo induced antigen technology (IVIAT) - Answer Advantages: Identifies genes
expressed during human infection, avoids excessive use of animal models, in vivo
expressed antigens
Disadvantages: Requires IRB approval and may not identify all virulence determinants
Infant Mice - Answer Immature immune system, more susceptible to infection
Irradiated mice - Answer Immunocompromised because their immune cells have been
destroyed, so they are more susceptible to infection
Neutropenic mice - Answer Defective in the ability to produce neutrophils
Nude Mice - Answer Absence of the thymus and consequently a marked deficiency of T
cells and cell-mediated immunity.
SCID Mice - Answer No B cells or T cells
Transgenic Mice - Answer Genetically defective in specific immune cells; more
susceptible to disease
Gnotobiotic animals - Answer Germ-free
•Poorly developed immune systems
•Very susceptible to infection by pathogens
SPF Animals - Answer Raised in environment free of particular pathogens but exposed
,to other microbes
Laser-capture microdissection - Answer Can be used to extract specific/localized
regions of interest within a tissue
RNA-seq - Answer Can be used to compare host gene expression changes in response
to infection
Proteomic profiling - Answer Can be used to compare changes in global protein
production and/or indicator proteins (biomarkers; proinflammatory cytokines)
RNA interference (RNAi) - Answer Can be used to turn off/silence specific host genes
CRISPR - Answer Edit host genomes
DICER - Answer RNAse activity that processes dsRNA -> siRNA when doing RNAi
Which of the following techniques would be best for assessing the contribution of a
specific C. elegans immune gene to its immune response to S. aureus infection: -
Answer Compare S. aureus virulence in parallel infection models using wild-type C.
elegans in the presence and absence of RNA interference directed against the immune
gene of interest
A bacterial ___ is comprised of two or more ___ that are all regulated by a single
transcription factor. - Answer regulon; operons
Which of the following methods would be the best one to understand the response of
mouse intestinal Peyers patches to Salmonella infection? - Answer RNAseq comparison
of mouse gene expression between RNA isolated from LCM-exised Peyers patches from
infected intestines and uninfected mouse intestines.
Which of the following are not advantages to using TnSeq to identify bacterial virulence
genes important for pathogenesis in vivo? - Answer Since TnSeq is essentially a
competition experiment between many mutants growing in a single host, trans
complementation effects could be observed.
Which of the following is not an advantage of using a transposon mutagenesis strategy
to study bacterial virulence genes important for pathogenesis in vivo? - Answer
Transcriptional terminators located on the transposon can disrupt expression of genes
located downstream of the inserted transposon
Which of the following experiments could be used to test the hypothesis that V. birdsii
infection of mammals is a toxin-mediated disease. i.e. the disease symptoms in the host
are caused primarily a result of ToxA protein. - Answer Clone and express the toxA gene
in a related avirulent strain of Vibrio, followed by inoculation and monitoring for disease
symptoms in mice.
Which of the following mutants should not be used in an in vivo competition model (i.e.
measuring competitive index between wild-type and mutant strains)? - Answer toxA
, Which of the following experiments could be used to study the promoter activity of the
toxA gene during a mouse infection model? Keep in mind that the same strategy(s)
could also be used to study the promoter activity of sigY and orf2559. - Answer - Clone
the promoter region of the toxA gene in front of the lux operon in wild-type V. birdsii,
followed by testing of lux expression in vivo using biophotonic imaging.
- Clone the promoter region of the toxA gene in front of the gfp gene in wild-type V.
birdsii, followed by testing of gfp expression in vivo using biophotonic imaging.
Which of the following "follow-up" experiments would be the best one to ensure that
each of the three mutations identified in the V. birdsii case study experiment did not
result in a polar effect on expression of the genes located downstream of the
transposon insertion? For simplicity sake the toxA transposon mutant is used as the
example in the answers below, but the same strategy could also be applied to the other
mutants as well. - Answer Complement the toxA transposon mutant by introducing a
cloned toxA gene back into the mutant strain (on a plasmid or by integrating it
elsewhere on the chromosome), followed by in vivo testing for attenuated virulence.
Acyl homoserine lactone (AHL) - Answer Small, hydrophobic, and membrane-diffusible.
Mostly used by gram negative bacteria for intraspecies communication.
Autoinducing peptide (AIP) - Answer Small, hydrophilic, transported across the
membrane. Mostly used by gram positive bacteria for intraspecies communication.
AI2 - Answer Furanosyl borate diester, common in all bacteria. Characteristics of AHLs
but binding method of AIPs.
Vibrio cholerae - Answer Gram-negative, motile, halophilic
The causative agent of cholera
V. cholerae QS - Answer High levels of ci-di-GMP increase biofilm formation, increase
CT, decrease virulence genes involved in exiting host
S. aureus Agr QS system - Answer - Effector of system is an untranslated RNA molecule
"RNAiii"
- Regulates at post-translational level
- When activated, it increases exotoxin production and decreases surface protein
production
Streptococcus pneumoniae - Answer Gram positive diplococci
- Has over 83 types of polysaccharide capsule
PPSV-23 Vaccine - Answer Affects 23 capsule types and is recommended for those over
2 years but specifically the elder population
Sublytic concentrations of pneumolysin - Answer Induction of host cell apoptosis,
Expression in Avirulent Strain - Answer - Best applied to closely-related species
- Can be time consuming
- Can only analyze small segments of genes (cloning limitation of 30 kb)
Transposons - Answer Advantages: High frequency, relatively-stable interaction, amr
genes, random insertion into target DNA, the disrupted gene of interested can be
identified through PCR
Disadvantages: Can carry terminators that have polar effects on downstream gene
expression, can only be used to identify non-essential genes
Transcriptional Fusion - Answer Where/when a virulence gene promoter is active
Translational Fusion - Answer Where/when a virulence gene is translated
TnSeq - Answer Advantages: Bypasses need for in vitro screen
Disadvantages: Competition experiment; trans complementation effect
In vivo induced antigen technology (IVIAT) - Answer Advantages: Identifies genes
expressed during human infection, avoids excessive use of animal models, in vivo
expressed antigens
Disadvantages: Requires IRB approval and may not identify all virulence determinants
Infant Mice - Answer Immature immune system, more susceptible to infection
Irradiated mice - Answer Immunocompromised because their immune cells have been
destroyed, so they are more susceptible to infection
Neutropenic mice - Answer Defective in the ability to produce neutrophils
Nude Mice - Answer Absence of the thymus and consequently a marked deficiency of T
cells and cell-mediated immunity.
SCID Mice - Answer No B cells or T cells
Transgenic Mice - Answer Genetically defective in specific immune cells; more
susceptible to disease
Gnotobiotic animals - Answer Germ-free
•Poorly developed immune systems
•Very susceptible to infection by pathogens
SPF Animals - Answer Raised in environment free of particular pathogens but exposed
,to other microbes
Laser-capture microdissection - Answer Can be used to extract specific/localized
regions of interest within a tissue
RNA-seq - Answer Can be used to compare host gene expression changes in response
to infection
Proteomic profiling - Answer Can be used to compare changes in global protein
production and/or indicator proteins (biomarkers; proinflammatory cytokines)
RNA interference (RNAi) - Answer Can be used to turn off/silence specific host genes
CRISPR - Answer Edit host genomes
DICER - Answer RNAse activity that processes dsRNA -> siRNA when doing RNAi
Which of the following techniques would be best for assessing the contribution of a
specific C. elegans immune gene to its immune response to S. aureus infection: -
Answer Compare S. aureus virulence in parallel infection models using wild-type C.
elegans in the presence and absence of RNA interference directed against the immune
gene of interest
A bacterial ___ is comprised of two or more ___ that are all regulated by a single
transcription factor. - Answer regulon; operons
Which of the following methods would be the best one to understand the response of
mouse intestinal Peyers patches to Salmonella infection? - Answer RNAseq comparison
of mouse gene expression between RNA isolated from LCM-exised Peyers patches from
infected intestines and uninfected mouse intestines.
Which of the following are not advantages to using TnSeq to identify bacterial virulence
genes important for pathogenesis in vivo? - Answer Since TnSeq is essentially a
competition experiment between many mutants growing in a single host, trans
complementation effects could be observed.
Which of the following is not an advantage of using a transposon mutagenesis strategy
to study bacterial virulence genes important for pathogenesis in vivo? - Answer
Transcriptional terminators located on the transposon can disrupt expression of genes
located downstream of the inserted transposon
Which of the following experiments could be used to test the hypothesis that V. birdsii
infection of mammals is a toxin-mediated disease. i.e. the disease symptoms in the host
are caused primarily a result of ToxA protein. - Answer Clone and express the toxA gene
in a related avirulent strain of Vibrio, followed by inoculation and monitoring for disease
symptoms in mice.
Which of the following mutants should not be used in an in vivo competition model (i.e.
measuring competitive index between wild-type and mutant strains)? - Answer toxA
, Which of the following experiments could be used to study the promoter activity of the
toxA gene during a mouse infection model? Keep in mind that the same strategy(s)
could also be used to study the promoter activity of sigY and orf2559. - Answer - Clone
the promoter region of the toxA gene in front of the lux operon in wild-type V. birdsii,
followed by testing of lux expression in vivo using biophotonic imaging.
- Clone the promoter region of the toxA gene in front of the gfp gene in wild-type V.
birdsii, followed by testing of gfp expression in vivo using biophotonic imaging.
Which of the following "follow-up" experiments would be the best one to ensure that
each of the three mutations identified in the V. birdsii case study experiment did not
result in a polar effect on expression of the genes located downstream of the
transposon insertion? For simplicity sake the toxA transposon mutant is used as the
example in the answers below, but the same strategy could also be applied to the other
mutants as well. - Answer Complement the toxA transposon mutant by introducing a
cloned toxA gene back into the mutant strain (on a plasmid or by integrating it
elsewhere on the chromosome), followed by in vivo testing for attenuated virulence.
Acyl homoserine lactone (AHL) - Answer Small, hydrophobic, and membrane-diffusible.
Mostly used by gram negative bacteria for intraspecies communication.
Autoinducing peptide (AIP) - Answer Small, hydrophilic, transported across the
membrane. Mostly used by gram positive bacteria for intraspecies communication.
AI2 - Answer Furanosyl borate diester, common in all bacteria. Characteristics of AHLs
but binding method of AIPs.
Vibrio cholerae - Answer Gram-negative, motile, halophilic
The causative agent of cholera
V. cholerae QS - Answer High levels of ci-di-GMP increase biofilm formation, increase
CT, decrease virulence genes involved in exiting host
S. aureus Agr QS system - Answer - Effector of system is an untranslated RNA molecule
"RNAiii"
- Regulates at post-translational level
- When activated, it increases exotoxin production and decreases surface protein
production
Streptococcus pneumoniae - Answer Gram positive diplococci
- Has over 83 types of polysaccharide capsule
PPSV-23 Vaccine - Answer Affects 23 capsule types and is recommended for those over
2 years but specifically the elder population
Sublytic concentrations of pneumolysin - Answer Induction of host cell apoptosis,
