Midterm Exam Flashcards
How many americans die from sepsis?
210,000
How many americans are affected with sepsis every year?
700,000
What is requred for RNA polymerase to begin transcribing DNA?
Sigma subunit
where does sigma subunit bind?
promoter sequence
Sigma subunit leaves RNA polymerase at what point?
When polymerase begins transcribing
Amino acids attach to the ____ end of a tRNA
3’
what is the modified amino acid corresponding to the start codon in bacteria?
f-MET (formyl methionine)
the first tRNA in translation binds in the ___ site of the ribosome
P site
what form of energy is used for translation?
GTP (hydrolysis)
Infection-
Colonization of the body by an organism
capable of causing disease
Disease-
infection that causes symptoms
Colonization
: A bacterium occupies and multiplies in the human body
carrier
infected but asymptomatic
Pathogenecity
Capacity of a bacterium to cause disease
virulence
Often used interchangeably with
pathogenicity. Can also refer to the degree of
disease a bacterium has the capacity to cause
(e.g., highly virulent)
Virulence (pathogenicity) factor
A component of a
bacterium that is required for disease
Who studied anthrax?
Robert Koch
What is Koch’s first postulate?
The microbe must be associated with symptoms of the disease and must be present at the site of infection
What is Koch’s second postulate?
The microbe must be isolated from the lesions of disease and grown as pure culture
What is Koch’s third postulate?
a pure culture of the microbe, when inoculated into a susceptible host, must reproduce the disease in the experimental host
What is Koch’s fourth postulate?
the microbe must be reisolated in pure culture from the experimentally infected host
What is the proposed fifth postulate for Koch?
Elimination of the disease-causing microbe from the infected host or prevention of exposure of the host to the microbe should eliminate or prevent disease.
Molecular Koch’s first postulate
The gene (or its product) should be found
only in strains of a bacterium that cause
disease and not in strains that are avirulent
Molecular Koch’s second postulate
The gene should be isolating by cloning
Molecular Koch’s third postulate (a and b)
3a. Disrupting the gene in a virulent strain should reduce (attenuate) its virulence.
3b. Introducting the cloned gene into an
avirulent strain should render the strain
virulent.
What approaches are used to identify new virulence factors?
Biochemical
Molecular genetics
What does the Biochemical approach to identifying new virulence factors entail?
purify the factor (e.g., a toxin) and demonstrate that it can reproduce disease symptoms.
What four ways can you identify new virulence factors using the molecular genetics approach?
a. Clone genes from pathogen into avirulent host (e.g., Escherichia coli) and show that the resulting strain is now virulent.
b. Mutagenize pathogen with a transposon and screen the resulting random insertion mutants for loss of virulence.
c. Measure virulence gene regulation using gene fusions. ( Fuse the promoter region of a given gene (or operon) to a reporter gene, which can be used to more easily measure gene expression)
d. Identify genes that are required for survival in the host.
Transposons experimentally introduced into cells often have _______ genes in order to isolate colonies with the transposon.
antibiotic resistance genes (selectable marker)
Transposon mutagenesis is a straightforward method to create a library of ________ of a strain of bacteria.
random insertion mutants
Transposon mutagenesis is only useful for studying ________ genes, because ___________
non-essential
insertion into a gene usually inactivates it
transposon Insertions may be polar, meaning they ______________
knock out expression of downstream genes in an operon
location of a transposon may be determined with certain types of _____
PCR
B-galactosidase is an example of a reporter gene product that can be useful for measuring gene expression. B-galactosidase cleaves 3 things: ________
B- galactosides, galactopyranoside (X-gal), and ONPG
B-galactosidase cleaves X-gal to form a _______colored product
blue
B-galactosidase cleaves ____ to create a yellow product. This technique is especially useful in that it helps ____________
quantify the amount of gene expression occurring, rather than simply telling you the gene is being expressed. (it is quantified on a spectrophotometer)
When B-galactosidase is fused to a virulence gene, it is regulated the same way/a different way as the virulence gene?
the same way
Gene fusions can be constructed:
i. _______
ii. ________
i. Using cloned promoter region sequence of gene of interest.
ii. In the pathogen using a transposon derivative that contains a reporter gene (note: this creates a mutant and a gene fusion simultaneously).
Strains containing gene fusions can be further mutagenized to _____________
isolate regulatory mutants
Signature-tagged mutagenesis combines _______ with ______ using an animal model of infection to screen for mutants that _______.
Signature-tagged mutagenesis (STM)
combines transposon mutagenesis with an in vivo selection using an animal model of infection to screen for mutants that do not grow in the host.
instead of using a single transposon to generate a library of mutants, STM uses ________.
a mixture of transposon variants generated from a single transposon.
In STM, each transposon variant has its own unique “_______” or “______”
“tag” or “barcode”
Endotoxins-
toxic integral bacterial membrane components, including lipopolysaccharide (LPS), lipotechoic acid (LTA) and phosphatidylglycerol (PG), that are released into the host environment typically upon cell lysis.
LPS contains 3 structural regions:
O-specific chain, core, and Lipid A
LPS comes from _______ in bacteria
the outer cell membrane
Sepsis results from ________
septicemia
Septicemia-
systemic disease in which bacteria multiply in the blood or are continuously seeded into the bloodstream
The stages of sepsis in order are:
SIRS, sepsis, severe sepsis, septic shock, MODS
When septicemia occurs, circulating bacteria release ______, which triggers the _________ that leads to sepsis
endotoxin
systemic immune response
The innate immune system consists of _____
Cells and proteins that are always present
and ready to respond to foreign invaders,
including bacteria.
The cells of the innate immune system include:
Phagocytes, Natural killer cells, Mast cells
Define Phagocytes-
cells that ingest and kill
bacteria
Name the 4 phagocytic types of cells of the innate immune system:
- polymorphonuclear leukocytes (aka PMNs, neutrophils)
- monocytes
- macrophages
- dendritic cells (DCs).
Which two phagocytes migrate constantly throughout the bloodstream?
PMN’s and monocytes
Dendritic cells localize to _______
tissues that are in contact with the external environment or the bloodstream
When monocytes leave the bloodstream to enter a tissue, they differentiate into _______
macrophages (more phagocytic)
Define Natural Killer cells-
kill host cells containing intracellular bacteria
Where do mast cells accumulate?
around blood vessels
What is the purpose of a mast cell?
when a foreign invader is detected, mast cells release histamine (vasodilator that makes blood vessels leaky) in order to facilitate the exit of PMNs and monocytes from the bloodstream to the site of infection
The three classes of proteins in the innate immune system are the:
cytokines, chemokines, and complement proteins
define cytokines and provide 4 examples
glycoproteins produced by a number of cell types including macrophages
- TNF-alpha
- IL-1
- IFN- gamma
- IL-8
how big is a cytokine in kDal?
8-30 kDal
define chemokines-
peptides produced by same cells as cytokines, but are smaller than cytokines
The binding of cytokines and chemokines to surface receptors on target cells initiates __________
a signal transduction cascade that modifies the function of the target cells
define complement proteins-
a set of proteins produced
by the liver that circulate in blood and have
the capability to enter tissue
complement proteins become active through ______
proteolytic cleavage
what are the 3 functions complement proteins are involved in?
a. Enhancing phagocytosis of invaders.
b. Chemotaxis of macrophages, PMNs.
c. Rupturing membranes of invaders
what are the 3 pathways involved in complement activation?
mannose-binding lectin pathway, classical pathway, and alternative pathway
define the mannose-binding lectin pathway
mannose-binding lectins are produced in the liver and bind specifically to mannose residues
on the surface of bacteria.
define the classical pathway
involves antibodies attached to bacteria.
define the alternative pathway
involves binding of bacterial surface components such as LPS and teichoic acids by complement component C3b.
the act of sticking stuff to a bacterial cell to make it less slippery and easier for a WBC to catch is called:
opsonization
when a phagocyte ingests a bacteria, it endocytizes it into a vesicle called a _______, which it then fuses with a ______ to destroy the bacteria. this fused vesicle is called a ______
phagosome
lysosome
phagolysosome
creation of a phagosome involves the rearrangement of __________
the actin cytoskeleton
The phagocyte engulfs bacteria by extending ______
pseudopods
a lysosome contains 7 things:
lysozyme, proteases, nucleases, anti-microbial peptides, membrane permeabilizing proteins, phospholipases, myeloperoxidase
as a result of the oxidative burst in a phagolysosome, these 4 products are formed:
peroxide, superoxide, hypochlorous acid, nitric oxide
The enzyme _____ is present in the phagosome membrane, and converts oxygen and NADPH into superoxide. ______ then converts the superoxide into _______, which is then converted into hydroxide radicals in the ________ reaction or hypochlorous acid by the enzyme _______ (present in the lysosome).
The enzyme NADPH oxidase is present in the phagosome membrane, and converts oxygen and NADPH into superoxide. Superoxide dismutase then converts the superoxide into H2O2, which is then converted into hydroxide radicals in the Fenton reaction or hypochlorous acid by the enzyme Myeloperoxidase (present in the lysosome).
Phagocytes become activated (produce _______) when what happens?
(cytokines and chemokines) when they recognize certain conserved structural components of bacteria
Name 4 conserved structural components of bacteria that phagocytes illicit a response to. What are they known as collectively?
LPS, lipotechoic acid, flagella, and CpG-rich DNA. They are collectively known as PAMPs (pathogen-associated molecular patterns)
how do PAMPs trigger cytokine/chemokine release?
Toll like receptors on host cell membranes recognize these PAMPs and subsequently initiate cytokine/chemokine release via signal transduction.
Toll was first identified as a _________ required for development and immune function in Drosophila
transmembrane receptor protein
We must know cyto/chemokine release pathway for the Lipid A receptor. Fill in the blanks below: When LPS dissociates from a bacterium such as E. coli, _____ will bind to it and transfer the LPS to ______ on the cell membrane, which then transfers LPS to ______, also on the cell membrane. Via signal transduction, ______ is activated and translocated to the nucleus, where it induces _____ and ______ genes.
We must know cyto/chemokine release pathway for the Lipid A receptor. Fill in the blanks below: When LPS dissociates from a bacterium such as E. coli, LBP will bind to it and transfer the LPS to CD14 on the cell membrane, which then transfers LPS to TLR-4, also on the cell membrane. Via signal transduction, NF-kB is activated and translocated to the nucleus, where it induces inflammatory and immune response genes.
NF-kB is important in regulating cellular responses because it belongs to the category of “________” ___________ i.e. transcription factors that are present in cells in an inactive state and do not require new protein synthesis to be activated (think about c-Jun, Fos, etc. These are in the same category) This fact allows NF-kB to be a first responder to ________
“rapid acting” primary transcription factors
harmful cellular stimuli
Phagocytes become activated (produce _______) when what happens?
(cytokines and chemokines) when they recognize certain conserved structural components of bacteria
Name 4 conserved structural components of bacteria that phagocytes illicit a response to. What are they known as collectively?
LPS, lipotechoic acid, flagella, and CpG-rich DNA. They are collectively known as PAMPs (pathogen-associated molecular patterns)
how do PAMPs trigger cytokine/chemokine release?
Toll like receptors on host cell membranes recognize these PAMPs and subsequently initiate cytokine/chemokine release via signal transduction.
Toll was first identified as a _________ required for development and immune function in Drosophila
transmembrane receptor protein
We must know cyto/chemokine release pathway for the Lipid A receptor. Fill in the blanks below: When LPS dissociates from a bacterium such as E. coli, _____ will bind to it and transfer the LPS to ______ on the cell membrane, which then transfers LPS to ______, also on the cell membrane. Via signal transduction, ______ is activated and translocated to the nucleus, where it induces _____ and ______ genes.
We must know cyto/chemokine release pathway for the Lipid A receptor. Fill in the blanks below: When LPS dissociates from a bacterium such as E. coli, LBP will bind to it and transfer the LPS to CD14 on the cell membrane, which then transfers LPS to TLR-4, also on the cell membrane. Via signal transduction, NF-kB is activated and translocated to the nucleus, where it induces inflammatory and immune response genes.
NF-kB is important in regulating cellular responses because it belongs to the category of “________” ___________ i.e. transcription factors that are present in cells in an inactive state and do not require new protein synthesis to be activated (think about c-Jun, Fos, etc. These are in the same category) This fact allows NF-kB to be a first responder to ________
“rapid acting” primary transcription factors
harmful cellular stimuli
Cytokines and chemokines fully activate killing capacity of _____ upon entrance into tissue, and cause ______ to differentiate into ______.
PMNs
monocytes into macrophages
inflammation is characterized by 4 things
redness, swelling, pain, heat
What 5 cytokines recruit phagocytes, thus initiating the inflammatory response?
TNF-a, IL-1, IL-8, PAF, IL-6 (they are produced at the site of infection)
_____ induces mast cells to produce vasoactive compounds at site of infection, increasing vascular permeability.
PAF
Increased permeability of blood vessels at site of infection results in increased blood flow to infected area, causing ____ and ____, as well as leakage of fluid into tissue, causing ______.
redness and heat
swelling
During killing of bacteria at site of infection, what is released from phagocytes.
some lysosomal enzymes (even more are released as phagocytes die)
The lysozymes released into tissues at sites of infection will _________
damage and kill surrounding tissue cells
this occurrence is possibly an attempt to kill hidden bacterial cells
The pro-inflammatory response is down regulated by _______
anti-inflammatory cytokines
Name 4 anti-inflammatory cytokines
IL-1 receptor antagonist, IL-4, IL-10, IL-13
anti-inflammatory cytokines reduce production of _________, and reduce killing capacity of __________
inflammatory cytokines
phagocytes
Exotoxins-
Protein toxins produced by bacteria that are usually released into the host environment during growth
Effector proteins (exoenzymes)-
bacterial toxins delivered directly into host cells by bacterial secretion systems
Superantigens (type I toxins)-
peptide
toxins that cause T cells (part of adaptive
immune response) to become overstimulated and release large of amounts of cytokines.
Membrane-disrupting toxins (type II toxins)-
lyse host cells (or compartment within host cell) by disrupting the integrity of their plasma membranes
What are the two types of membrane disrupting toxins (type 2 toxins)?
Proteins that form channels (pores) in the
plasma membrane and Enzymes that compromise the integrity of
the plasma membrane phospholipids.
What are the two classes of exotoxins?
Superantigens and membrane-disrupting toxins
alpha hemolysin is what kind of toxin?
a type 2 pore forming exotoxin
If pore forming proteins are one class of type II exotoxins, then what would the other class be?
phospholipases
Type III toxins are also called:
A-B toxins
Type III toxins-
Consist of two
functional subunits. The A subunit has enzymatic (toxin) function, while the B subunit binds to a receptor on host cells.
Single-chain A-B toxins have
one A subunit and one B subunit.
What type does diptheria toxin fall under?
Type III
Diptheria toxin is a ______ chain A-B toxin
single
Diptheria toxin receptor on cell membrane is:
HB-EGF (heparin binding epidermal growth factor)
The biological activity of the diptheria toxin is:
ADP-ribosyltransferase
Diptheria toxin:
- Outside the cell, how are the two subunits associated?
- Once they are endocytized, the reducing environment causes what to happen?
- they are joined by a disulfide bond
- The disulfide bond breaks, and the B subunit acts as a pore for the A subunit to enter the cytosol
Diptheria toxin: once the A subunit has entered the cytosol, what reaction does it catalyze?
the formation of ADP-ribose from NAD+ and transfers it to a target protein.
What is the target protein for diptheria toxin?
binds ADP-ribose to target protein
EF-2
- Diptheria bacteria do not produce the toxin unless what happens?
- If they do produce the toxin, it is under what conditions?
- a bacteriophage containing the toxin gene undergoes the lysogenic cycle in the bacteria
- low-iron conditions
Cholera toxin is a ______ subunit A-B toxin
multi
Cholera causes _____ deaths per year
100,000-200,000
What is the ratio of subunits in a multi-subunit A-B toxin?
one A subunit and multiple B subunits. The B units may or may not be identical
The B subunits of Cholera toxin bind to what receptor?
GM1 gangliosides
The target protein of Cholera toxin is:
Gs protein
What does cholera toxin do that fucks with you?
It prevents hydrolysis of GTP from the Gs protein, causing excessive cAMP production
Anthrax toxin is a _____ subunit ______ toxin
- multi
- A-B
Humans and animals are susceptible upon infection by B. anthracis spores.
3 forms of infection depending on route of
infection-
subcutaneous, gastrointestinal,
and inhalation anthrax.
Anthrax toxin
Consists of 3 plasmid-encoded proteins:
Lethal factor (LF) Edema factor (EF) Protective antigen (PA)
Lethal factor (LF)-
a protease that inactivates MAP kinase kinases.
Edema factor (EF)-
a calmodulin-dependent adenylate cyclase.
Protective antigen (PA)-
delivers LF and EF to the cytosol.
First anthrax vaccine was developed by who when?
Louis Pasteur in 1881
Sec system secreted proteins are synthesized in ______ form with a signal (leader) sequence at the _____
- precursor
- N-terminus
Signal sequence is cleaved by a _____ during transport across the
cytoplasmic membrane.
-leader peptidase
Gram positive/negative. Is sec system found in one (which one) or both?
both
____ brings a Sec dependent protein to _____. ______ uses ATP hydrolysis to undergo a conformational change to push the protein through ______. ______ stabilizes this process.
- Sec B
- Sec A
- Sec A
- SecYEG
- SecDF-YajC
Integral membrane (IM) proteins inserted into the cytoplasmic membrane via ________.
- the Sec system
SRP is/is not necessary for the Sec system?
is not
As IM proteins begin to exit the ribosome their signal sequence is recognized by________
the signal recognition particle.
Secretion of IM protein occurs _______ (as it is being synthesized)
co-translationally.
TAT stands for:
Twin-arginine transport
TAT transports proteins that are ______
already folded
Proteins have N-terminal twin-arginine
signal sequence, which includes the
motif _____
-SRRXFLK.
SRP, attached to signal recognition sequence is recognized by _____, which somehow transfers the protein to ____
- FtsY
- SecA
In the TAT system, the TAT signal sequence on the protein binds to ______. ______ is recruited to the complex, inducing transport of the protein across the membrane, also resulting in _______.
- TatBC
- TatA
- cleavage of the signal sequence
Does TAT system involve ATP hydrolysis?
no
What secretion systems are Sec-dependent?
T2SS and T5SS
What secretion systems are specific to gram negative bacteria?
T1,2,3,4,5,6 secretion systems
T2SS proteins are secreted across the outer membrane through:
a pore consisting of 12-14 proteins.
Proteins secreted via T5SS export themselves, they are also called ________
autotransporters
The ____ domain of a T5SS inserts itself in the outer membrane, forming a pore for the rest of the protein to be secreted through
Beta
T5SS uses __________ to cross the inner membrane, then ______ forms a pore for the rest of the protein to cross the outer membrane. Then, the protein undergoes ______ to release the _______ from ______
- general Sec dependent system
- beta domain
- autoproteolytic cleavage
- alpha domain (mature protein)
- the beta domain (pore)
Which types of gram-negative secretion systems are Sec-independent?
T1,3,4,6 secretion system
Type 1 secretion system (T1SS) involves a complex of ______ that span
_________________.
- 3 proteins
- the cytoplasmic membrane, periplasm and outer membrane.
Type 1 secretion system (T1SS) secreted proteins have a _____ signal
sequence containing the _____-rich
motif _____ repeated many times. This signal sequence is/is not cleaved during export?
- C-terminal
- glycine
- GGXGXD
- is not
The E. coli hemolysin a T1SS system consists of a IM spanning _______ transporter, a periplasmic connecting protein ______, and an OM spanning pore protein called _____
- ABC transporter (HlyB)
- accessory factor HlyD
- TolC
Type 3 secretion system (T3SS) has a contact-dependent mechanism
involving an elaborate complex of over
20 proteins that forms a ________
delivery apparatus (injectisome).
Is ATP required for T3SS involving an injectisome?
yes
What kind of proteins are secreted in T3SS?
Effector proteins, because they are directly injected into the host cell
What genus species uses T3SS?
Pseudomonas aeruginosa
What toxins are secreted by P. aeruginosa?
ExoS,T,U,Y
What activity do ExoS and T have?
ADP-ribosyltransferase and Rho GTPase-activating protein domain
-ADPRT and GAP
What activity does ExoU have?
Phospholipase A2
What activity does ExoY have?
Adenylate cyclase
What does the injectisome resemble?
flagella
P. aeruginosa is a pathogen of:
humans, animals and plants
Pseudomonas aeruginosa survives on:
a variety of surfaces including on skin.
Pseudomonas aeruginosa is an __________ pathogen, which is capable of causing infections in ______(4 places)
- opportunistic and nosocomial
- the pulmonary tract, urinary tract, and burn and wound sites.
A type 3 secretion system can be divided
into five components:
Effector proteins Chaperones Translocation apparatus Regulatory proteins Needle complex and other proteins
Many pathogenic bacteria use type 3 secretion, often with distinct specific effects on host cells. The specific effects are due to the \_\_\_\_\_\_\_\_\_\_\_\_
particular secreted effectors.
the injectisome consists of what components?
the translocation apparatus and the needle complex with other proteins
GAP activity-
promotes GTP hydrolysis of small G proteins.
G proteins targeted by ExoS and T GAP activity are:
-What do these G proteins regulate?
-Rho, Rac, and Cdc42.
-These regulate a variety of cellular
processes including cytoskeletal
structuring.
Activated Rho stimulates ___________
contractile actin-
myosin filament assembly.
Activated Rac triggers:
actin-rich surface protrusions (lamellipodia)
Activated Cdc42 triggers:
finger-like projections (filopodia)
Inactivation of the various G proteins by ExoS and T results in _____________
dramatic changes in cell shape.
An endothelial cell with active Rac is round, but one without activated Rac looks like ______
sharp, sickle shaped cells
Upon injection, a number of host cell
proteins are targeted for __________
ADP-ribosylation
ExoS-mediated ADP ribosylation results in
_____________(4 things)
actin cytoskeleton disruption, inhibition of
DNA synthesis, vesicular trafficking and
endocytosis, and eventually cell death.
ExoT-mediated ADP ribosylation results in
_____________(4 things)
actin cytoskeleton disruption, and inhibition
of cell migration, adhesion, and proliferation.
ExoU’s phospholipase domain is also called _______ and has _______ activity
- Patatin-like domain
- PLA2
ExoU-
A potent phospholipase that can cause cell death rapidly.
ExoU substrates include: (3 things)
phospholipids, lysophospholipids, and neutral lipids.
T3SS chaperones maintains newly synthesized effector proteins in a _________ state in the cytosol and guides them to ______
- translocation-competent (unfolded)
- the injectisome.
SpcS is the chaperone for ________
SpcU is the chaperone for _________
No chaperone yet identified for _______
- ExoS and ExoT.
- ExoU.
- ExoY.
_____ is the chaperone for ExoS and ExoT
_____ is the chaperone for ExoU
_____ is the chaperone for ExoY
- SpcS
- SpcU
- none identified
where do chaperones bind to the effector proteins ExoS,T,U?
on the CBD (chaperone binding domain)
Does ExoY have a CBD?
No
Type 3 secretion in P. aeruginosa is regulated at 2 levels:
The 2 levels are _____.
1) transcription of type III secretion genes.
2) initiation of the secretion process itself.
- linked.
Normally the T3SS is turned off until what happens?
contact with host cell
T3SS
Upon contact with host cell ____ begins to be secreted into host cell, depleting it in the bacteria, which leaves ____ unbound. It may now bind ___, which is inhibiting ___.
- ExsE
- ExsC
- ExsD
- ExsA
Once ExsD is bound to C, ExsA can act as a _____________
-transcription activator of T3SS secretions.
For reasons that are not clear, nearly all aeruginosa strains have either the ____ or the ____ gene but not both
- exoS
- exoU
exoU gets ______ in host cells, but the effect is unknown
ubiquitylated
The T3SS of aeruginosa is most closely related to that of _____
Yersinia.
The aeruginosa T3SS most likely evolved for ______ instead of for _______
- defense against predators
- invasion of a host
