Clostridia toxins
Treatments for botulism in food
heating up canned food to denature and destroy proteins
1. Group A is sensitive to bacitracin, Group B is resistant
2. Surface carbohydrate on bacteria, serotyping with an Ab that recognizes Group A and Group B sugars
A = activating, enzymatic activity, ADP ribosyl-transferase
B = binding, determines tissue specificity
The main function of Opa proteins are...
adhesins that bind to specific receptors on the host cell or to proteins in the extracellular matrix and allows for bacteria to bind to host cells and internalize by host cells
Protein A virulence factor mechanism...
binds to Fc portion of antibodies, the antibodies are essential bound backwards to the bacteria, Fc portion isn't available to be recognized by Fc receptors on phagocytic cells
TLR are..
a type of pattern recognition receptor (PRR) and recognize molecules that are broadly shared by pathogens but distinguishable from host molecules, collectively referred to as pathogen-associated molecular patterns (PAMPs)
How would you test the hypothesis that strains isolated from CSF originated from the normal flora of individuals suffering from meningitidis?
1. Serotyping polysaccharide capsules: Incubate bacteria from CSF with Ab against different capsule types and look for clumping, if same Ab causes clumping of both bacteria they are the same serotype and possible the same strain
2. RFLP typing via pulsified gel electrophoresis: isolate genomic DNA from each strain via restriction enzyme digest, run digests on pulsified gel, if banding patterns are the same strain the strains are the same and infection may have been caused by normal flora
Treatments that could be used in botulism or tetanus cases...
Inactivate the toxin with anti-toxin antibodies that are injected, vaccinate with an inactive version of the botulinum neurotoxin or tetanus toxin to induce production of the anti-toxin antibodies, or prevent binding of the toxin to its receptor on cells by injecting a molecule that binds to the toxin receptor – this could be an antibody or small molecule – that outcompetes the toxin to bind the receptors
Hyaluronic capsule helps GAS evade complement by...
preventing lysis by MAC (already resistant since it's Gram-positive), C3b is not efficiently deposited on the surface
The cholera toxin causes diarrhea because...
ADP ribosyl transferase leads to increased adenylate cyclase, causes activation of cAMP which leads to Cl- ion gated channels open, Cl- ions leak out, and water leaves the ciliated cells through osmosis
The two ways to test for syphilis from serum of a patient with Treponema pallidum bacteria...
1. Cardiolipin test: serum from the patient is mixed with particles coated in cardiolipin, if particles aggregate the Ab is present
2. Ab against bacteria: incubate serum with whole bacteria, then add a fluorescent secondary antihuman Ab, bacteria will light up if Ab against the bacteria are present
Super antigens (SAgs) trigger inflammation by...
binding to the V-alpha chain on TCRs, trigger inflammation by spontaneously binding to T cells and APCs, causing massive cytokine release
S. typhi evades TLRs in two major ways:
1. Decreases expression of flagellin proteins after it invades host tissues so that it doesn't stimulate TLR5
2. Expresses Vi capsule protein that masks the LPS of the outer membrane from detection by TLR4
The virulent factor of the isolated CSF strain may have a thicker capsule because...
Thicker capsules are associated with duplication of genes that encode for polysaccharide synthesis, thicker capsule helps bacteria avoid opsonization by complement and lysis via MAC attack
Botulinum bacteria can cause infection in infants because..
infants still have developing microbiome, adults need to ingest inactivated neurotoxin
C5a helps GAS evade complement by...
cleaves C5a, prevents C5a gradient around bacteria cell that would attract innate immune cells such as neutrophils
The pertussis toxin targets a different G protein compared to the cholera toxin, these different targets are...
Pertussis toxin targets G inhibitory protein, cholera toxin targets G activating proteins, both are ADP ribosyl-transferases
N. gonorrhoeae uses Type IV pili for...
attachment to host cells, twitching motility, DNA uptake for transformation, aggregation of bacteria in microcolonies
Anti-toxin Abs work against superantigens by...
binding to them (SAgs) and preventing them from attaching to TCRs or MHC Class II
S. typhimurium invades intestinal tissues through these two ways:
1. Invasion through M cells on Peyer's Patches, doesn't require specific virulence factors, M cells are constantly sampling the intestine
2. Invasion of intestinal epithelial cells via Type III secretion system (SPI-1) which injects effector proteins into host cells which causes the membrane to ruffle, allows for uptake of bacteria into phagosome
IgA proteases assist with infection by...
cleave IgA at the hinge region, separates Fab region of Ab that can bind to Ag from the Fc portion that's bounded by phagocytic cells, even if Ab binds to surface of pathogen it won't be recognized by Fc receptor on phagocytes because the Fc portion won't be attracted to the Ab
The botulinum neurotoxin targets...
proteins in the SNARE complex in neurons that are involved in fusion of vesicles containing neurotransmitter with the neuronal cell membrane at the neuro-muscular junction
Factor H helps evade immune system by...
prevents complement activation on host cells, stimulates proteolysis of C3b that may be deposited on host cell surface to an inactive form (prevents opsonization), also can prevent accumulation of active C3b on bacterial surface
pertussis toxin ADP ribosylates Gia causes Gia to be locked in its inactive form, adenylate cyclase can't be inhibited which leads to the constant activation of adenylate cyclase
Describe antigen variation in the pilE gene that encodes for Type IV pili
genetic information from many different silent pilS genes can be recombined into the pilE expression site, causes the amino acids of the pilE protein structure exposed on the outside of the pilus to change, new pilE variants can't be recognized by previously generated Ab and avoid opsonization
Decoy receptors protect against SAgs by...
trapping circulating SAgs, stops them from activating T cells/APCs
N. meningitidis crosses the blood-brain barrier by...
Binding to endothelial cells in blood vessels, causes endothelial cells to rearrange actin cytoskeleton to wrap around bacterial cells, this disrupts tight junctions between endothelial cells which allows bacteria to move through the spaces
Tetanus and Botulinum neurotoxins cause different effects because...
It has the opposite effect as botulinum neurotoxin because it is acting in the inhibitory neuron instead of in the motor neuron. It gets to the inhibitory neuron by retrograde transport and transcytosis from the motor neuron.
Distinguishing between C. difficile and S. aureus, GAS...
C. difficile is rod-shaped
Tracheal cytotoxin mechanism...
interacts with NOD receptors on host cells, leads to activation of NO synthase, ciliated cells are sensitive to NO and die
Compare and contrast antigenic variation in pilin proteins of N. gonorrhoeae vs phase variation in Opa proteins of N. gonorrhoeae
Antigenic variation requires RecA protein and other proteins involved in homologous recombination, the overall sequence of the pilin protein changes
Phase variation in Opa proteins doesn't require RecA protein, the overall gene sequence doesn't change, and it only changes if the full length of the protein is expressed, removal of or introduction of repeats in reading frame can result in premature stop codons
Variance between different S. aureus SAgs due to...
them being encoded on phage related pathogenicity islands (SaPIs)
N. meningitidis bead agglutination assay involves...
1. Isolating CSF sample
2. Add CSF to beads that have Ab conjugated to them that are specific for a given polysaccharide capsule
3. If beads agglutinate then the capsule Ag is present in the CSF
Botulinum neurotoxin causes paralysis because...
in intoxicated neurons the vesicles cannot fuse with the cell membrane and so neurotransmitter is never released, and muscles are never stimulated to contract
Distinguishing between GAS and S. pneumoniae
GAS is beta-hemolytic, S. pneumoniae is alpha-hemolytic
The target for the diphtheria toxin is...
elongation factor 2 that's required for protein synthesis, ADP-ribosylation inhibits protein synthesis which leads to cell death
SaPIs move between lysogenic phages like...
requires a helper phage that provides capsid proteins for packaging SaPIs, integrate into other genomes like a lysogenic phage using integrase
N. meningitidis capsule interferes with phagocytosis because...
polysaccharides prevents C3b from being deposited onto cell surface, capsule also increases size of bacteria
The toxin required for tetanus targets...
inhibitory neurons and cleaves synaptobrevin (involved in SNARE complex), this prevents the release of inhibitory NT glycine at these synapses, prevents inhibition of motor neuron that controls muscle contraction, the muscle contracts spastically
Distinguishing between GAS and S. aureus
GAS is catalase positive, S. aureus is catalase negative
Vaccination with inactivated diphtheria toxin protect against diphtheria disease by...
creates antibodies against the DT protein that neutralize the toxin and prevent it from getting into cells.
The adaptive immune system can counter the action of capsules by...
generation of Ab against capsule, C3b can then be deposited on the Ab for opsonization and activation of complement
Clostridia perfringens entertoxin is...
a pore-forming toxin that binds to the tight junction proteins found in epithelial cells, causes diarrhea by disrupting tight junctions which leads to the death of epithelial cells
What reagents are needed for an ELISA to test for antibodies to pertussis toxin in a patient with a severe cough?
Purified pertussis toxin protein to coat the plate. Blood from the patient. Anti-human Ig antibody conjugated to a fluorescent molecule or enzyme for detection.
Clostridia perfringens alpha-toxin
a phospholipase that removes the charged head group from phospholipids, causes the disruption of the cell membrane and lysis of the cell
What positive and negative controls would you do? What results would you expect for a positive test?
Positive control: blood from a patient who has had pertussis. Negative control: blood from an uninfected person. There would be a color change (or fluorescence) in wells of the ELISA plate where there are antibodies in the blood that can bind to the toxin
What reagents are necessary to test for the presence of Bordetella pertussis by PCR in nasal secretions of a patient suspected of having whooping cough?
Primers against either B. pertussis 16S rRNA gene or to some gene that is specific to Bordetella (like the genes encoding the virulence factors pertussis toxin or filamentous hemagglutinin, etc).
What positive and negative controls would you do?
Positive control: purified DNA from B. pertussis, or nasal secretion from a patient known to be infected. Negative control: nasal secretion from a person known to not be infected.
How are Bordetella pertussis virulence factors regulated?
By the two-component signal transduction system BvgAS. BvgS phosphorylates and transfers the phosphate to BvgA which is a DNA binding response regulator that binds to DNA in the promoters of the genes that encode the virulence factors and activates transcription.
How is an inactivated diphtheria toxin created
The inactivated DT can either be a mutant version of the protein that has no enzymatic activity or the protein that has been treated with formalin to eliminate its enzymatic activity