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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
activation of host complement, induction of proinflammatory reaction in the immune cell.
High lytic concentrations - Answer Potential for widespread direct cellular and tissue damage via pore forming properties.
Sec system in +ve bacteria - Answer Mature protein folds as it emerges through SecYEG and is released to the surfaces of the bacteria
Sec system in -ve bacteria - Answer Transported protein folds in the periplasmic space, where is may remain or be transported across the outer membrane
Sec B - Answer Targeting factor that prevents the premature folding of the precursor protein
SRP - Answer Signal recognition particle, recognizes signal sequence as it emerges from ribosome and causes a pause in translation
FstY - Answer SRP receptor protein
Sec A - Answer Molecular motor (ATPase-dependent)
SecYEG - Answer Protein conduction channel
Yersinia pestis - Answer Gram negative rod that is non-motile
- Type III secretion is one of the most studied
- LPS lacks O antigent (anti=phagosytosis)
- Capsule (anti-phagosytosis)
- Pla protease (dissemination from flea bite)
Bubonic plague - Answer - Infected flea bite
- Sudden onset of fever, headache, chills, and weakness
- 80% of US cases is this type
Septicemic plague - Answer - Infected flea bite or handling of infected animal
- May develop from untreated bubonic plague
Pneumonic plague - Answer - Only form that can be spread from person to person
- Most serious but more rare
T3SS +Calcium - Answer - LcrG blocks secretion pore
- YopN blocks secretion pore
T3SS -Calcium - Answer - Pore is opened and Yops is secreted
What is true of the V. cholerae quorum-sensing system? - Answer - Production of c-di-GMP is inhibited at high cell density
- Production of c-di-GMP can be inhibited by both AHL and AI-2 quorum sensing systems
- Inhibition of c-di-GMP production results in increased expression of genes involved in virulence/transmission
Pneumonic plague is most common type of Yersinia pestis infection encountered in the present-day United States. - Answer False
Which of the following statements are true regarding the Yersinia pestis type 3 secretion system (T3SS)? - Answer - The injectosome (aka Yop secretion machinary) directly delivers effector Yop proteins into target cells.
- Expression of genes encoding the Yop secretion machinery (aka "injectosome") is induced at 37 C.
- Yop proteins do not require Sec-dependent secretion signals to be delivered to host cells by the T3SS.
- Secretion of Yop proteins through the injectosome is energy-dependent
Glycopeptides - Answer Mechanism of action: Inhibit transglycosylation and transpeptidation
Resistance mechanisms: -vs outer membrane alterations, modifing target (substitute -lactate or -ala)
Spectrum: +ve
Examples: Vancomycin, Teicoplanin
Aminoglycosides - Answer Mechanism of action: Bind 16sRNA in 30S subunit
Spectrum: Bacteriocidal
Examples: Kanamycin
Tetracyclins - Answer Mechanism of action: Bind 16s rRNA in 30S subunit
Resistance mechanisms: Inactivation of antibiotic, mutation of 16s rRNA
Spectrum: Broadly bacteriostatic
Examples: Tetracyclin, Doxycyclin
Macrolides - Answer Mechanism of action: Bind 23S rRNA in 50S subunit
Resistance mechanisms: Methylation of target
Spectrum: Bacteriostatic for most, bacteriocidal in some +ve
Examples: Erythromycin, Lincomycin
Fluoroquinolones - Answer Mechanism of action: Bind DNA gyrase, inhibiting DNA replication
Spectrum: Broadly bactericidal
Examples: Ciprofloxacin, norfloxacin
Rifampin - Answer Mechanism of action: Binds beta subunit of bacterial RNA polymerase
Spectrum: Broadly antibacterial
Examples: Rifampin
Golden era for antibiotic discovery - Answer 1940s to Late 1970s
Average $ for Market Release - Answer $873 M
Antiporters - Answer Uptake of H+/Na+ provides energy to pump out toxic compounds
ABC transporters - Answer ATP hydrolysis provides energy to pump out toxic compounds
Mechanisms of tetracycline resistance - Answer 1. Efflux pump: Intracellular tetracycline remains too low, prevents binding to 16s rRNA
- Ribosome modification: TetM GTPase activity perturbs helix in 16S region bound by tetracycline; preventing binding to 16S rRNA
Regulation of tetB (efflux pump) expression - Answer Normally: TetR binding to promoter repressed transcription
Tetracycline present: Magnesium and tetracycline lead to TetR binding which makes it fall off the promoter, allowing tetB transcription
Enterococcus faecalis - Answer (+)ve facultative anaerobe that is the causative agent of UTIs
VRE (vancomycin-resistant enterococcus) - Answer - About 30% of all hospital-acquired Enterococcus infections
- Horizontal Gene Transfer is a concern because VRE can become MRSA = VRSA
Mechanism of vancomycin resistance - Answer 1. vanS - Kinase sense vancomycin and phosphorylates VanR
Which of the following is NOT an example of a disinfectant: - Answer Antimicrobial peptides (AMPs)
Which of the following is an example of a bacterial factor that has been shown to promote the development and spread of drug-resistance in bacteria? - Answer Inter- and intra-species horizontal gene transfer of bacterial antibiotic resistance genes
Horizontal gene transfer of vancomycin resistance genes from ___ to ___ has been documented in only a few clinical infections thus far. - Answer Enterococcus; Staphylococcus aureus
MRSA resistance - Answer Methicillin-resistance encoded by the mecA gene -> alternative penicillin binding protein PBP2a resists B-lactam antibiotics
USA400 Strain - Answer Highly associated with necrotizing pneumonia or pulmonary abscesses and sepsis
USA300 Strain - Answer Skin soft-tissue infection
Community MRSA transmission - Answer Direct contact with an infected wound, sharing personal items
S. aureus virulence factors - Answer Surface proteins are released during exponential-growth phase
Secreted toxins are released during stationary phase
Regulation of agr quorum-sensing - Answer argD forms small cyclic peptide that is exported and processed by argB. When it reaches a quorum it binds to argC and elicits a phosphorelay to argA.
Arg is a regulator of biofilm development - Answer Accumulation of AIP induces biofilm dispersal
S. aureus intracellular lifestyle - Answer 1. Immune clearance
- Cytotoxic effects
- Small-colony variants (SCVs)
- Immune evasion and viable reservoir
- Escape and re-internalization
- Escape and dissemination
S. aureus methicillin‐resistance is encoded by the mecAgene, the product of which ___ - Answer encodes an alternative penicillin-binding protein that is no longer recognized by methicillin and related antibiotics.
AgrC - Answer sensor kinase that binds secreted AIP when it reaches a threshold concentration
AgrA - Answer response regulator that, in its phosphorylated form, activates high-level expression of the agrACDB and RNAIII transcripts
AgrD - Answer encodes the precursor AIP peptide
S. aureus expresses secreted toxins/tissue-degrading enzymes during exponential growth phase. - Answer False
Inactivation of the Agr quorum sensing system prevents biofilm dispersal. - Answer True
Bioterrorism - Answer The deliberate spread of pathogenic organisms into a community to cause widespread illness, fear, and panic
Dual-use agents - Answer Biological agents (bacteria, virues) that are used non-maliciously (therapeutic development, educational purposes) but could also be
Edema factor - Answer Raises cAMP levels, causes fluid secretion, tissue swelling and disruption of intracellular signaling pathways
Lethal factor - Answer Zinc metalloprotease selectively inactivates mitogen-activated protein kinase kinases (MAPKKs) that cleaves protein kinases and blocks immune system from attacking
B anthracis strains - Answer Strains lacking either pXO1 or pXO2 display greatly reduced virulence
B. anthracis attenuated live vaccine - Answer Developed by Sterne in 1937, lacks pXO and is there negative for the Capsule toxin
Anthrax toxins - Answer Heptameric complex binds up to 3 EF/LF subunits and are considered AB toxins
Biofilm formation by S. pneumoniae - Answer in vitro biofilm formation enhanced in mutants lacking capsular polysaccharide (CPS)
CPS as a primary virulence factor - Answer - Resists opsonophagocytosis
- CPS production is required for efficient in vivo colonization because it is resistant to host defenses
Adult vaccination S. pneumoniae - Answer Vaccine consists of 23 most common antigenic capsular types and is especially effective in elderly
Child vaccination S. pneumoniae - Answer A protein conjugated-capsule vaccine used in children under 2.
SpxB - Answer Stimulates cytokine production by surrounding host cells
LTA - Answer Modified with phosphocholine groups
Surface Adhesins - Answer Adherence to epithelial cells
Pneumolysis - Answer Assembles in the cholesterol-containing membranes of the host cells, large transmembrane pore that inhibit ciliary movement
Which of the following strains of anthracis, developed by Sterne in 1937 is still used today as an attenuated live vaccine that is the basis of most anthrax vaccines for livestock? - Answer A strain in which the capsule plasmid has been removed, leaving only the toxin plasmid within the strain.
Why is the botulinum neurotoxin included in the bioterrorist's repertoire of select agents that can be used for biological warfare? - Answer - Its potential to be introduced into the water or food supply
- The relative ease of its acquisition
B. anthracis lethal factor and edema factor toxins are considered "simple" AB toxins. - Answer False, not simple
The PPSV-23 vaccine is highly effective in preventing S. pneumoniae disease in infants. - Answer False
Which of the following S. pneumoniae virulence factors is important for niche competition and energy production? - Answer SpxB
In the Brackman et al. research paper (Week 12 assigned reading), which of the following invertebrate models of infection were used to study the effect of quorum-sensing inhibitors on antibiotic treatment of bacterial infection in vivo: - Answer
- C. elegans survival assay
- G. mellonella survival assay
- The agr mutant displays decreased virulence in a mouse model of abscess infection.
- The agr mutant displays decreased dispersal when grown as a biofilm.
A bacterial ___ is comprised of two or more ___ that are all regulated by a single transcription factor. - Answer regulon; operons
Secreted auto-inducing peptides (AIPs) are hydrophilic and therefore need to be actively transported outside of the bacterial cell. - Answer True
Transcription of which of the following antibiotic resistance genes is repressed by a DNA-binding protein when the antibiotic is not present? - Answer tetB and blaZ
RNA interference (RNAi) can be used to mutate a specific host gene in C. elegans (nematode). - Answer False
Which of the following is NOT a characteristic of a transposon that makes it a useful genetic tool for making a mutant library? - Answer Transposon is unstable once it has "hopped" into genomic DNA
Which of the following statements concerning V. cholerae quorum-sensing is FALSE? - Answer Inhibition of c-di-GMP production results in decreased expression of genes involved in shedding/transmission
SecY - Answer Portion of the Sec protein-conducting channel
SecA - Answer Provides energy to Sec transport system ("molecular motor")
Which of the following is NOT a characteristic of an antiseptic: - Answer often contains concentrated disinfectants
S. aureus methicillin‐resistance is encoded by the mecA gene, the product of which ___ -
Answer encodes an alternative penicillin-binding protein that is no longer recognized by methicillin and related antibiotics.
Which of the following statement is NOT TRUE regarding the Yersinia pestis type 3 secretion system (T3SS)? - Answer Expression of genes encoding the Yop secretion machinary (aka "injectosome") is induced at 28 C.
Alcohols - Answer Denature proteins
Alkylating agents - Answer Forms epoxide bridge that disrupts proteins
Phenols - Answer disrupt membranes, denature proteins
