Title: Blood test revealed evidence of infection with S' pyogenes
1Case 1
- Blood test revealed evidence of infection with
S. pyogenes - (blood test is ASOT, antibody titers against
Streptolysin-O)
2Streptococcus pyogenes
3Soft tissue infections caused by S. pyogenes
4Streptolysin O
- Single chain, multidomain protein
- Secreted by Streptococcus pyogenes
- Binds cholesterol and oligomerizes x 7
- After formation of the heptamer, a
conformational change is presumed to cause
penetration of the cytoplasmic membrane - SLO is not internalized and is not enzymatically
active - Allows ion and water leakage, ultimately causing
cell death - SLO pore is also a channel for translocation of
Spn (and possibly other virulence factors) - Result is cell death and tissue destruction -
presumably facilitates spread and resistance to
phagocytosis
5Pore-forming Mechanism
- TMHs contain 3 ?-helices
- Unfold to form transmembrane a-hairpins
- Oligomer (40-50 monomers) forms large b-barrel
pore - SLO, PFO similar mechanisms
TMH1
TMH2
Adapted from Rajesh Ramachandran et al. Nature
Structural Biology 9, 823 - 827 (2002)
6Cholesterol-DependentCytolysins
Adapted from Rossjohn et al. Cell, Vol.
89, 685 - 692 (1997)
- Pore-forming proteins
- Extensive family 20 molecules from
Gram-positives (Arcanobacterium, Bacillus,
Clostridium, Listeria, Streptococcus) - 30-60 amino acid identity
- Cholesterol-dependency for prepore to pore
conversion - 20-30 nm diameter pores
7Streptococcus pyogenes - Pathophysiology
Fe
3
Streptolysin S Streptolysin O Streptokinase Hyalur
onidase DNAase Pyrogenic exotoxins
M protein F protein
Capsule M protein C5a peptidase
8Case 2
Baby R.A. 3 mos infant with 1 week of
constipation, decreased activity, weak cry, and
now seems floppy. Other history parents
sometimes put honey on his pacifier.
- Noted to have decreased muscle tone in all
extremities, poor feeding, no bowel movements. - Muscle weakness increased over next week.
- Noticed to look a littlebluish, blood gases
revealed low oxygen, high CO2 required
mechanical ventilation. - Stool culture positive for
Clostridium botulinum
9Infantile Botulism
10Clostridium botulinum
11Botulinum Toxin
- The most toxic agent known. Medically useful.
Potential biowarfare agent. - Secreted by Clostridium botulinum, either after
ingestion or innoculation - skin trauma. - 7 serologically distinct family members BoNT A
to G. - Comprised of heavy and light chain linked by
disulfide bond (A-B toxin). - Heavy chain mediates receptor binding. Light
chain is enzymatically active. - Bind to gangliosides (low affinity) and
synaptobrevins (high affinity) - Target cell is peripheral motor neuron
(stimulates muscle contraction by release of
acetylcholine into neuromuscular junction).
12Botulinum Toxin (2)
- Internalized by receptor-mediated endocytosis.
- Upon acidification, light chain (A domain) is
translocated to cytoplasm. - Light chain is a zinc metalloprotease with high
specificity for synaptobrevin (SNAP-25). - Prevents fusion of synaptic vesicles with
membrane, prevents acetylcholine release. - Results in weakness progressing to paralysis.
- (Note that tetanus toxin TeNT has almost
identical structure and enzymatic activity, but
it is transported to inhibitory neurons in the
spinal cord - blocking their inhibitory effect on
motor neurons. Results in tetany - spastic
paralysis)
13Structure of Botulinum Toxin
Protease domain (blue)
Translocation domain (green)
Binding domains (red and yellow)
14Synaptic vesicle fusion with nerve terminus via
SNARE proteins
15Effect of botulinum toxin on synaptic vesicle
fusion
16Case 3
D.R. - 5 y.o. admitted with bloody diarrhea x 1
week--gt anemia, low platelet count, decreased
urine output. Family cooked hamburgers two days
before D.R. became ill.
- Acute kidney failure - required dialysis
- RBC damage (hemolytic anemia) - required multiple
transfusions - Platelet consumption - bleeding from IV lines
- Pancreatitis - weeks of IV nutrition
- Inflammation of the brain - hallucinations,
decreased level of consciousness - Stool culture positive for
E. coli O157H7
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18Shiga-like Toxins
- Released by enterohemorrhagic E. coli (E.H.E.C.),
especially E. coli O157H7, ingested in
contaminated foods. - AB5 structure
- Toxin released only upon lysis of bacterium
- Binds to glycolipid receptor on host cells
- Endothelial cells are predominant target cells -
esp. in kidneys
19Shiga-like Toxins (2)
- Transported in retrograde direction to the ER
- A subunit translocated into cytoplasm
- RNA-N-glycosylase activity depurinates 28S
ribosome - results in inhibition of protein
synthesis - Ultimately causes cell death
- Since endothelial cells are predominantly
damaged, coagulation cascade is activated and
numerous small thrombi form
20Hemolytic Uremic Syndrome
Fibrin deposition -shearing of RBC
Hemolytic anemia
Tiny blood clots -ischemia
Damage to bowel Damage to kidneys
21Hemolytic Uremic Syndrome
- anemia, low platelet count, and kidney failure
22Case 4
XX. 48 year old cattle rancher from Texas
admitted with difficulty breathing, chest pain
and coughing up blood. Within 12 hrs he
developed rapidly progressive hypotension and
coma. The only exposure history was disposal of
a cow found dead on his farm 10 days ago.
- There was blood and PMN in his bronchial fluid.
Chest X-ray did not show pneumonia, but he had an
enlarged lymph node in chest. - Laboratory investigations revealed he had
meningitis and gram stain was positive for long,
thin Gram negative organisms. - Blood and spinal fluid cultures subsequently grew
Bacillus anthracis
23Pulmonary Anthrax
24Anthrax Lethal Factor
- One of two anthrax toxin A domains that share a
common B domain. The other A domain is edema
factor (EF) - Released by B. anthracis along with PA and EF
- PA binds receptor as a monomer, is
proteolytically cleaved (to 63 kD) and
oligomerizes x 7 - PA receptor is a widely expressed 369 aa protein
- initially called ATR (anthrax toxin receptor),
now identified as tumor endothelial marker-8 (
TEM8 ) - Recently, a second cellular receptor for PA was
identified as the human capillary morphogenesis
protein 2 (CMG2) - LF (or EF) binds to PA heptamer
25Anthrax Lethal Factor (2)
- LF-PA7 is internalized by receptor mediated
endocytosis - Upon acidification of endocytic vesicle, LF is
translocated thru PA channel - LF is a Zn-metalloprotease - cleaves MAPKK and
possibly other substrates - Major cellular target is macrophage (target
MAPKK, rather than ATR) - Upon MAPKK cleavage, inflammatory cytokine
expression is markedly upregulated, and
macrophage dies. - Resuls is septic shock and impaired phagocytosis
of B. anthracis
26Internalization of LF and PA
Heptamer of protective antigen (PA)
27Pathogenesis of Anthrax
28Case 4
- M.V. 17 yr old girl with 2 days of fever,
malaise, and bright red rash. Muscles ache all
over. Felt dizzy when got out of bed. - In ER, had blood pressure of 70/30.
- Admitted to hospital. Hypotension did not
respond to IV fluids. BP dropped to 56 systolic. - Transferred to the ICU. Unable to keep oxygen
saturation above 85 by mask. - Intubated and paralyzed to assist ventilation and
started on pressor medications to support blood
pressure. - When placing a urinary catheter, the nurses
noticed a thick vaginal discharge. Cultures were
obtained.
29Stapylococcus aureus
TSST-1
30Toxic Shock Syndrome Toxin-1
- Single-chain, multi-domain protein (but NOT a
pore-forming toxin) - Secreted by some isolates of S.aureus - may only
be colonizing host - Silmultaneously binds MHC class II and T-cell
receptor (TCR) - Target cells are macrophages (and other antigen
presenting cells) T-cells - Causes crosslinking and non-specific activation
of T-cells (not internalized, is not
enzymatically active) - Secretion of inflammatory cytokines
- Vasodilation and capillary leak cause rash,
conjunctivitis, hypotension, fluid in lungs - Inflammation and dysregulated blood flow causes
abnormal function of essentially all organs
31Toxic Shock Syndrome Toxin-1
TSST-1
32Case 6
- A.H. was a 16 year old girl who presented to
another hospital with a one day history of fever
and malaise (feeling tired and bad). Examination
was unremarkable. - Bloodwork, including blood culture, was obtained.
She was observed for four hours then discharged
to home late in the evening. - The next morning her father found her without
pulse or respiration and she could not be
resuscitated. There was a diffuse rash
consisting of large purple macules. - The initial blood culture was subsequently
positive for
Neisseria meningitidis
33Neisseria meningiditis
34(No Transcript)
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36Endotoxin
- LPS released by gram negative organisms
- Binds LPS-binding protein (LBP) and presented to
CD14 which complexes with TLR-4. LPS can also
bind directly to TLR-4 - LPS is not internalized (and is not
enzymatically active) - Activation of TLR-4 ultimately results in
activation and translocation of NF-KB into
nucleus - Expression of inflammatory cytokines TNFa, IL-1b,
and others - Like TSST-1, causes vasodilation, abnormal blood
flow to organs, resulting in septic shock,
multi-organ system failure - Endothelial cells particularly affected, activate
abnormal thrombosis and multiple small clots
(especially in the skin, which causes the rash)
37Structure of Gram-negative LPS Endotoxin
38Pathogenesis of Septic Shock
ARDS adult respiratory distress
syndrome DICdiffuse intravascular coagulation
(multiple tiny blood clots) MOSFmulti-organ
system failure (esp. kidney, liver, intestinal
failure)
39N. meningitidis - Pathophysiology
Fe
3
Adherence
Invasion
Intracellular
Nutrient
Immune
Toxin
survival
acquisition
evasion
production
Iron scavengers
Endotoxin (LPS)
Capsule IgA proteases
Opa proteins
Pilus
40Biologic Effects of Bacterial Toxins
- Damage membranes
- Inhibit protein synthesis
- Activate second messenger pathways
- Damage cytoskeleton
- Arrest cell cycle
- Inhibit neurotransmitter release
- Induce inflammation
41Mechanisms of Bacterial Toxin Action
- Pore formation
- ADP-ribosyltransferase activity
- Glucosyltransferase activity
- N-glycosidase activity
- Adenylcyclase activity
- Protease activity
- Deamidase activity
- DNA damage
- Superantigen properties
- Biological effect depends on target molecule.
- Clinical effect depends on target cell