Lecture 10 BIOL 533 1
Staphylococci and Streptococci
BIOL 533
Lecture 10
Medical Microbiology
Lecture 10 BIOL 533 2
Staphylococci
? Important human pathogen
– Causes both relatively minor and serious
diseases
? One of the hardiest of the non-
sporeforming bacteria
– Can exist on dry surfaces for a long period
– Relatively heat-resistant; temperature range
of 18° - 40° C
Lecture 10 BIOL 533 3
Staphylococci
? Morphology
– Gram+ grape-like cluster,but in clinical
specimens,can be a single cocci or diplococci
? General physiological characteristics
– Nonmotile
– Facultatively anaerobic
– Catalase +
– Grows in media containing 10% NaCl
Lecture 10 BIOL 533 4
Staphylococci
? Relationship to disease (only 3 important)
– S,aureus—causes a number of diseases
– S,epidermidis—present in normal flora
(normally benign,except when introduced via
catheters,etc.)
– S,saprophyticus—causes uninary tract
infections
Lecture 10 BIOL 533 5
Staphylococci
? Microbial physiology and structure
– Capsule may not be found growing on media,
but it is usually present in vivo
– Teichoic acids are phosphate containing
polysaccharides bound to both peptidoglycan
and cytoplasmic membrane
? Species specific
? Poor immunogens,but when bound to
peptidoglycan,get an antibody response
Lecture 10 BIOL 533 6
Pathogenesis of S,aureus
? Features typical of staphylococci
infections,
– Initial lesion is normally mild and localized
– Results in a boil—normally,it is self-limiting
– Can result in systemic infection
Lecture 10 BIOL 533 7
Pathogenesis of S,aureus
? Stage I,encounter—humans are major
reservoir for S,aureus
– Colonize nose and are found in about 30% of
individuals
– Transiently found on skin,oropharynx,and
feces
– Transmitted via,
? Hand contact
? Aerosols from pneumonia patients
Lecture 10 BIOL 533 8
Pathogenesis of S,aureus
? Stage I,continued
– Certain occupations are more prone to
colonization
? Physicians,nurses,hospital workers
– Certain classes of patients are more prone to
colonization
? Diabetics,hemodialysis patients,and drug abusers
Lecture 10 BIOL 533 9
Pathogenesis of S,aureus
? Stage II,entry—not normally through
unbroken skin
– Can enter if large numbers have accumulated
through poor hygiene
Lecture 10 BIOL 533 10
Pathogenesis of S,aureus
? Stage III,spread and multiplication
– Survival depends on
? Number of organisms
? Site involved
? Speed with which inflammatory response is
mounted
? Immunological competence of host
– If inoculum is small and host immunologically
competent,infection normally defeated
Lecture 10 BIOL 533 11
Pathogenesis of S,aureus
? Stage IV,damage
– Local infection leads to formation of abscess
(collection of pus)
? In skin,boils or furuncles
? Interconnected abscesses are called carbuncles
– May also spread in subcutaneous or
submucosal tissues—cellulitis
Lecture 10 BIOL 533 12
Pathogenesis of S,aureus
? Stage IV,continued
– Development—involves both host and
bacterial factors
? Acute inflammatory reaction
– Proportion of bacteria survive and are capable of lysing
neutrophils that engulfed them
? Outpouring of lysosomal enzymes that damage
surrounding tissues
– Inflammatory area surrounded by fibrin clot
Lecture 10 BIOL 533 13
Virulence Factors of S,aureus
? Stage IV,continued
– Virulence factors—most designed to avoid
phagocytosis or survive once ingested
? Wall components
– Surrounded by capsule,not as effective as
pneumococcus or meningococcus
– Cell wall murein activates complement by alternative
pathway
– Teichoic acid also activates and involved in adherence
– Protein A interferes with opsonization by binding with Fc
region of Ab—complement activated primary pathway
Lecture 10 BIOL 533 14
Virulence Factors of S,aureus
? Stage IV,continued
– Secretion of enzymes
? Catalase—hydrogen peroxide to water and oxygen
(all staphylococci produce)
? Coagulase—makes fibrin clot (wbc penetrate
badly; only S,aureus)
? Hylauronidase—degrades connective tissues
(facilitates spread; 90% of S,aureus strains)
? Fibrinolysin (staphylokinase)—dissolve fibrin clots
(virtually all S,aureus)
Lecture 10 BIOL 533 15
Virulence Factors of S,aureus
? Stage IV,continued
– Secretion of enzymes
? Lipases—required for invasion into cutaneous and
subcutaneous tissues (found in all S,aureus and
30% of others)
? Nuclease—heat stable (role is uncertain;
S,aureus)
? Penicillinase
Lecture 10 BIOL 533 16
Virulence Factors of S,aureus
? Stage IV,continued
– Secretion of toxins
? Cytolytic (membrane-damaging by pores)
– Alpha,beta,(sphingomyelinase C),delta,gamma,
leukocidin (cannot lyse red blood cells)
– Others lyse rbc and leukocytes (referred to previously as
hemolysins)
– Cause lysis of neutrophils leading to massive lysosomal
enzyme secretion
Lecture 10 BIOL 533 17
Virulence Factors of S,aureus
? Stage IV,continued
– Secretion of toxins
? Exfoliative toxin (scalded skin syndrome)—
extrachromosomal
? Toxic Shock Syndrome toxin-1 (enterotoxin F)—
exotoxin secreted during growth
– Some produce enterotoxin B instead (role not clear)
Lecture 10 BIOL 533 18
Virulence Factors of S,aureus
? Stage IV,continued
– Secretion of toxins
? Enterotoxins (A-E)—found in both S,aureus and
S,epidermidis
– Resistant to hydrolysis by gastric and jejunal enzymes
– Stable to heating at 100° C for 30 minutes
– Mechanism of toxin activity not understood; no
satisfactory animal model
? Stimulate intestinal peristalsis and have CNS effect;
intense vomiting
Lecture 10 BIOL 533 19
Pathogenesis of S,aureus
? Treatment
– Antibiotics
? Types,
– Methicillin,oxacillin,nafcillin,and dicloxacillin
(semisynthetic penicillins resistant to ?-lactam
hydrolysis)
– Majority of patients can be treated,but 10-15%
S,aureus and 40% coagulase-negative staphylococci are
resistant; treat with vancomycin
Lecture 10 BIOL 533 20
Pathogenesis of S,aureus
? Treatment
– Antibiotics
? Resistance,
– Plasmid-borne (hydrolysis of ?-lactam ring)
– Chromosomal—change in structure of penicillin-binding
proteins
Lecture 10 BIOL 533 21
Streptococci
? Fermentative (oxygen tolerant) Gram+
cocci in chains
? Sensitive to penicillins
? Human reservoir—passed from person to
person
Lecture 10 BIOL 533 22
Streptococci
? Properties of Lancefield Groups
(CHO antigens on wall—see handout)
– Group A,cross-reaction can lead to,
? Rheumatic fever
? Glomerulonephritis
Lecture 10 BIOL 533 23
Streptococci
? Recent Group A Streptococcus virulence
factors
– M-like proteins—bind IgM IgG (protease
inhibitor) and alpha2 macroglobulin
– F protein—adherence to epithelial cells
– C5a peptidase—degrades C5A pyrogenic
exotoxins; previously called erythrogenic
toxins
Lecture 10 BIOL 533 24
Staph and Strep Toxins
? S,aureus toxic shock TSST-1
? S,pyogenes toxic shock TSSL-1
? S,pyogenes scarlet fever SPE-1
(children,not adults; immunity)
Lecture 10 BIOL 533 25
Staph and Strep Toxins
? S,aureus, Toxic Shock Syndrome
– Fever,diffuse rash
– Exfoliation of skin on palms and soles of feet
– Normally doesn’t compete well in relatively
anaerobic vaginal area
Lecture 10 BIOL 533 26
Staph and Strep Toxins
? S,aureus, Toxic Shock Syndrome
– Super-absorbent tampon,
? Created aerobic pockets
? Removed Mg?producing toxin
– After removed tampon,cases declined; did
not disappear
– Still associated with wounds,rare nasal
surgery
Lecture 10 BIOL 533 27
Staph and Strep Toxins
? S,pyogenes,Toxic Shock-Like Syndrome
– Skin or wound infection ---> bloodstream
– Death rate ~30%; over 10-fold higher than
TSST
? Seen in immunocompromised people
? Also other infections occurred,soft tissue infection
with influenza symptoms
– High fatality rate because rapid development
of shock and multiple organ failure
Lecture 10 BIOL 533 28
Staph and Strep Toxins
? S,pyogenes,Toxic Shock-Like Syndrome
– Features in common with scarlet fever
? Occur in healthy people
? Both associated with high fatality rate
? Produce same exotoxin,streptococcal pyrogenic
exotoxin (Spe)
– Similar in mechanism to TSST-1
Lecture 10 BIOL 533 29
Staph and Strep Toxins
? Comparing TSLS-1 and TSST-1,
– Rash,fever,shock,multiple organ failure;
resemble endotoxin septic shock
– Both toxins superantigens
– Same mechanisms of action
– Limited similarity at amino acid sequence level
Lecture 10 BIOL 533 30
Staph and Strep Toxins
? TSLS-1 related to erythrogenic toxin
(scarlet fever; SpeA)
– Serotypes,
? Spe A—TSLS or invasive S,progenes
? Spe B
? Spe C
– Some strains don’t produce Spe A,so Spe B
or Spe C also has role
Lecture 10 BIOL 533 31
Staph and Strep Toxins
? How do TSST-1 and SPE cause shock and
multiple organ failure?
? Hypotheses not mutually exclusive
Lecture 10 BIOL 533 32
Shock and Organ Failure
? First Hypothesis,same as LPS triggering
release cytokines IL1,TNF?
– Consistent with role as superantigen
? Promote association between macrophage and
helper T cells?proliferation of T cells producing
high IL2 level
– Secondarily produce IL1 TNF?
? Inject TSST-1 into rabbits; elevated levels IL1
TNF?
Lecture 10 BIOL 533 33
Shock and Organ Failure
? Second hypothesis,increase body’s
sensitivity to LPS; consistent with,
– Acts synergistically with LPS to amplify toxic
effects in vitro and in animals
– Conceivable—low levels leaching into blood
due to lysis of resident microflora
? Normally no effect
? Presence of Spe or TSST-1 causes an effect
Lecture 10 BIOL 533 34
Shock and Organ Failure
? Evidence to support role for LPS in TSST
and TSLS
– Injecting TSST-1 or Spe is lethal to rabbits
– Injecting exfolatin and concanavalin A not
lethal to rabbits
? Both elicit T cell proliferation,but don’t enhance
sensitivity to LPS
Lecture 10 BIOL 533 35
Shock and Organ Failure
? Evidence to support role for LPS in TSST
and TSLS
– TSST-1 not lethal to gnotobiotic animals
– Wouldn’t expect leakage,but still T cell
response
– Therefore,both suggest T cell proliferation
not as important as synergy of LPS
? Not conclusive; difficult to prove same level T cell
stimulation,proliferation occurred in all cases
Lecture 10 BIOL 533 36
Shock and Organ Failure
? Third hypothesis,
– TSST-1 can act directly on endothelial cells
? Damage causes malfunction in circulatory system,
which creates hypotension
? Data,swelling associated with massive leakage of
fluid from capillaries is marked symptom of both
TSST and TSLS
? Could also be result of action of blood vessels by
cytokines,coagulation,or complement cascade
Lecture 10 BIOL 533 37
Staph and Strep Toxins
? Mortality of S,pyogenes vs,S,aureus
– TSLS higher than TSS
– TSLS strains enter bloodstream
– TSS,only the toxin circulates
? S,pyogenes known to be invasive; killed
by PMNs and macrophage if ingested
Lecture 10 BIOL 533 38
S,pyogenes Invasiveness
? Strategies for evading phagocytosis; (1),
– M protein binds H factor better than factor B
? Leads to degradation of C3b
? Therefore,prevents opsonization by C3b and
formation of C3 convertase
Lecture 10 BIOL 533 39
S,pyogenes Invasiveness
? Strategies for evading phagocytosis
– Data supporting,
? M— mutants yield more susceptible to
phagocytosis; less virulent than wild type
? Ab against M protective
– 80 serotypes of M; possibly evades host
antibodies by changing serotype; however,no
data to support this hypothesis
Lecture 10 BIOL 533 40
S,pyogenes Invasiveness
? Strategies for evading phagocytosis; (2),
– Protease cleaves C5a
? Chemoattractant stimulates oxidative burst
? Some activation of complement could occur in
spite of M protein because
– Lysis releases wall components that activate complement
– Streptococci could protect themselves- C5a peptidase
– Data supporting,
? C5a mutants less virulent that wild type in animals
Lecture 10 BIOL 533 41
S,pyogenes Invasiveness
? Strategies for evading phagocytosis; (3),
– M— like proteins
? Sequence and structural similarity to M
? COOH embedded; NH2 exposed
? Most similar to M and each other at carboxy end
? These proteins bind Fc portion of IgG and IgA
Lecture 10 BIOL 533 42
S,pyogenes Invasiveness
? Strategies for evading phagocytosis
– M— like proteins; possible roles
? Coat with host protein—less likely detected as
invader by complement and immune system
? Adherence for body cells that contain Ab on
surface
– Also can bind host protease inhibitor such as ?2
macroglogulin
– Host uses protease inhibitor to protect against proteases
released by phagocytes
Lecture 10 BIOL 533 43
S,pyogenes Invasiveness
? Strategies for evading phagocytosis; (4),
– F protein—binds fibronectin
? Adherence of bacteria to tissues
? Evasion of immune system
– Summary of invasiveness
? No direct evidence M-like proteins involved in
virulence
? Found in impetigo strains,not always in severe
invasive strains
? Need mutant studies to answer questions
Lecture 10 BIOL 533 44
S,pyogenes Virulence
? Regulation of S,pyogenes virulence genes
– Expression M,C5a peptidase,and some M-like
proteins; regulated at transcriptional level
– Responds to CO2 levels
? Increased CO2 causes increased production
Lecture 10 BIOL 533 45
S,pyogenes Virulence
? Regulation of S,pyogenes virulence genes
– Regulatory gene
? mry transcriptional activator; sequence analysis
shows it is part of two-component system
– Sensor—not found
– Activator
? Also known that speA gene on temperate phage
Lecture 10 BIOL 533 46
S,pyogenes Pathogenesis
? Treatment and prevention
? TSST,TSLS are medical emergencies
– Surgical debridement of wounds prevents
further production of toxin
– Antibiotics; penicillin
– Toxic effects TSST-1 countered by
intravenous rehydration; counter hypotension
Lecture 10 BIOL 533 47
Streptococcal Treatment
? Prevention
– Vaccine possible
– Target against M
? Possible problems
– # serotypes,but severe invasive disease
caused by few
– AB against M cross-reacts with heart
Lecture 10 BIOL 533 48
Streptococcal Sequellae
Hypotheses
? First,Autoimmune theory
– Epitopes that cross react with epitopes on
cardiac myosin and sarcolemmal membrane
proteins
? Thus,T cells or antibodies could attack tissue
? Inflammatory response damages heart valves
Lecture 10 BIOL 533 49
Streptococcal Sequellae
Hypotheses
? Glomerulonephritis
– High levels Ab to streptococcal Ag circulating
in blood stream causes AgAb complexes to
accumulate in kidney
– Inflammatory response attacks kidney
interfering with kidney function
Lecture 10 BIOL 533 50
Streptococcal Sequellae
Hypotheses
? Data supporting
– Ag-Ab complexes visible in people with
glomerulonepheritis—glomeruli
– Decrease in C3 and other complement
components also seen; supports hypothesis
that inflammatory response is occurring
? Second,Toxins cause sequellae
Lecture 10 BIOL 533 51
Streptococcal Sequellae
Hypotheses
? Main argument against
– Time lag between initial infection and
development of rheumatic fever (RF; several
weeks) or glomerulonephritis (10 days)
– Normally,if due to toxin,within a week
– Candidates for toxin most likely to cause
glomerulonephritis,streptococcal O,
streptokinase,or Spe
Lecture 10 BIOL 533 52
Glomerulonephritis Hypotheses
? Streptococcal O cytotoxin
– Mechanism and aa sequence similarity to
pneumolysin
– Pore-forming toxin
? Injected into lab animals; damages heart
– Therefore,may have role in RF
? Also,very immunogenic; maybe Ab damage
Lecture 10 BIOL 533 53
Glomerulonephritis Hypotheses
? Streptokinase
– Plasminogen?plasmin
– Therefore causes symptoms similar to
glomerulonephritis in animals
? Interesting,but not proven
Lecture 10 BIOL 533 54
Rheumatic Fever Hypotheses
? Spe
– RF strains produce Spe; others don’t
– Enhances cardiotoxicity caused by
Streptococcal O in animals
? Haven’t explained long time lag
Lecture 10 BIOL 533 55
Rheumatic Fever Hypotheses
? Mysterious feature of RF unexplained
– Treated with antibiotics for as late as 9 days
after symptoms,still protected against RF
? After 9 days,toxic products should be circulating
and immune response underway
– Recurrence of disease
? Normally,infection results from different strain
? Some result from same strain
– RF symptoms take as long to develop as in original
Lecture 10 BIOL 533 56
Rheumatic Fever Hypotheses
? Mysterious feature of RF unexplained
– If caused by autoimmune response,would
expect faster response
? Possible explanation,previously exposed produces
primed immune system
Lecture 10 BIOL 533 57
Streptococcal Sequellae
? Treatment and prevention
– Strep throat self-limiting
– Treat with antibiotics and prevent RF and
glomerulonephritis
? Only ? S,pyogenes strains cause RF or G
? Not all people colonized actually contract RF or G
– Because of the seriousness,treat any strain
with antibiotics
Lecture 10 BIOL 533 58
Streptococcal Sequellae
? Tests
– Blood agar
? Hemolysis
? Bacitracin sensitivity
– Rapid test and culture test both yield high
number of false negatives
? For patients who are allergic to penicillin,
use erythromycin
Lecture 10 BIOL 533 59
Streptococcal Sequellae
? Previous damage
– Even heart murmur
– Dentists recommend prophylactic penicillin
before dental work
? Kill bacteria escaping into blood stream from
mouth (oral bacteria are susceptible to penicillin)
? Reduces chance of colonization
Lecture 10 BIOL 533 60
Staphylococcal Enterotoxins
? Normal enterotoxins cause diarrhea
– Water loss from small intestine mucosa
– Cause c-GMP or c-AMP levels in mucosal cells
to rise
Lecture 10 BIOL 533 61
Staphylococcal Enterotoxins
? Staph toxins operate in a different mode;
hypotheses include,
– 1) Stimulates vagus nerve endings in stomach
lining that control emetic (vomiting) response
? If hypothesis correct,it’s a neurotoxin,not
enterotoxin
– 2) Superantigen?IL2
? Administering IL2 to human volunteers produces
many of the same symptoms (nausea,vomiting,
fever,malaise)
Lecture 10 BIOL 533 62
Staphylococcal Enterotoxins
? Neither hypothesis conclusively proven
– Could be a combination of both
? Seven serotypes,SEA,SEB,SEC1,SEC2,
SEC3,SED,SEE
? 30 kDa proteins share considerable aa
similarity and single internal S-S
Lecture 10 BIOL 533 63
Staphylococcal Enterotoxins
? SE closely related to Spe
– Genes coding for SEC1 and Spe A have 60%
nucleotide identity
? SE produced in different amounts by
different strains
– entA produces much less toxin than entB
? Differences in promoter strength
Lecture 10 BIOL 533 64
Staphylococcal Enterotoxins
? SEB on integrated plasmid
? SEA on lysogenized phage
– One strain,entB,entC,plasmid-borne
? SED,entD on 27.6 kb plasmid
Lecture 10 BIOL 533 65
Lecture 10
? Questions?
? Comments?
? Assignments..,
Staphylococci and Streptococci
BIOL 533
Lecture 10
Medical Microbiology
Lecture 10 BIOL 533 2
Staphylococci
? Important human pathogen
– Causes both relatively minor and serious
diseases
? One of the hardiest of the non-
sporeforming bacteria
– Can exist on dry surfaces for a long period
– Relatively heat-resistant; temperature range
of 18° - 40° C
Lecture 10 BIOL 533 3
Staphylococci
? Morphology
– Gram+ grape-like cluster,but in clinical
specimens,can be a single cocci or diplococci
? General physiological characteristics
– Nonmotile
– Facultatively anaerobic
– Catalase +
– Grows in media containing 10% NaCl
Lecture 10 BIOL 533 4
Staphylococci
? Relationship to disease (only 3 important)
– S,aureus—causes a number of diseases
– S,epidermidis—present in normal flora
(normally benign,except when introduced via
catheters,etc.)
– S,saprophyticus—causes uninary tract
infections
Lecture 10 BIOL 533 5
Staphylococci
? Microbial physiology and structure
– Capsule may not be found growing on media,
but it is usually present in vivo
– Teichoic acids are phosphate containing
polysaccharides bound to both peptidoglycan
and cytoplasmic membrane
? Species specific
? Poor immunogens,but when bound to
peptidoglycan,get an antibody response
Lecture 10 BIOL 533 6
Pathogenesis of S,aureus
? Features typical of staphylococci
infections,
– Initial lesion is normally mild and localized
– Results in a boil—normally,it is self-limiting
– Can result in systemic infection
Lecture 10 BIOL 533 7
Pathogenesis of S,aureus
? Stage I,encounter—humans are major
reservoir for S,aureus
– Colonize nose and are found in about 30% of
individuals
– Transiently found on skin,oropharynx,and
feces
– Transmitted via,
? Hand contact
? Aerosols from pneumonia patients
Lecture 10 BIOL 533 8
Pathogenesis of S,aureus
? Stage I,continued
– Certain occupations are more prone to
colonization
? Physicians,nurses,hospital workers
– Certain classes of patients are more prone to
colonization
? Diabetics,hemodialysis patients,and drug abusers
Lecture 10 BIOL 533 9
Pathogenesis of S,aureus
? Stage II,entry—not normally through
unbroken skin
– Can enter if large numbers have accumulated
through poor hygiene
Lecture 10 BIOL 533 10
Pathogenesis of S,aureus
? Stage III,spread and multiplication
– Survival depends on
? Number of organisms
? Site involved
? Speed with which inflammatory response is
mounted
? Immunological competence of host
– If inoculum is small and host immunologically
competent,infection normally defeated
Lecture 10 BIOL 533 11
Pathogenesis of S,aureus
? Stage IV,damage
– Local infection leads to formation of abscess
(collection of pus)
? In skin,boils or furuncles
? Interconnected abscesses are called carbuncles
– May also spread in subcutaneous or
submucosal tissues—cellulitis
Lecture 10 BIOL 533 12
Pathogenesis of S,aureus
? Stage IV,continued
– Development—involves both host and
bacterial factors
? Acute inflammatory reaction
– Proportion of bacteria survive and are capable of lysing
neutrophils that engulfed them
? Outpouring of lysosomal enzymes that damage
surrounding tissues
– Inflammatory area surrounded by fibrin clot
Lecture 10 BIOL 533 13
Virulence Factors of S,aureus
? Stage IV,continued
– Virulence factors—most designed to avoid
phagocytosis or survive once ingested
? Wall components
– Surrounded by capsule,not as effective as
pneumococcus or meningococcus
– Cell wall murein activates complement by alternative
pathway
– Teichoic acid also activates and involved in adherence
– Protein A interferes with opsonization by binding with Fc
region of Ab—complement activated primary pathway
Lecture 10 BIOL 533 14
Virulence Factors of S,aureus
? Stage IV,continued
– Secretion of enzymes
? Catalase—hydrogen peroxide to water and oxygen
(all staphylococci produce)
? Coagulase—makes fibrin clot (wbc penetrate
badly; only S,aureus)
? Hylauronidase—degrades connective tissues
(facilitates spread; 90% of S,aureus strains)
? Fibrinolysin (staphylokinase)—dissolve fibrin clots
(virtually all S,aureus)
Lecture 10 BIOL 533 15
Virulence Factors of S,aureus
? Stage IV,continued
– Secretion of enzymes
? Lipases—required for invasion into cutaneous and
subcutaneous tissues (found in all S,aureus and
30% of others)
? Nuclease—heat stable (role is uncertain;
S,aureus)
? Penicillinase
Lecture 10 BIOL 533 16
Virulence Factors of S,aureus
? Stage IV,continued
– Secretion of toxins
? Cytolytic (membrane-damaging by pores)
– Alpha,beta,(sphingomyelinase C),delta,gamma,
leukocidin (cannot lyse red blood cells)
– Others lyse rbc and leukocytes (referred to previously as
hemolysins)
– Cause lysis of neutrophils leading to massive lysosomal
enzyme secretion
Lecture 10 BIOL 533 17
Virulence Factors of S,aureus
? Stage IV,continued
– Secretion of toxins
? Exfoliative toxin (scalded skin syndrome)—
extrachromosomal
? Toxic Shock Syndrome toxin-1 (enterotoxin F)—
exotoxin secreted during growth
– Some produce enterotoxin B instead (role not clear)
Lecture 10 BIOL 533 18
Virulence Factors of S,aureus
? Stage IV,continued
– Secretion of toxins
? Enterotoxins (A-E)—found in both S,aureus and
S,epidermidis
– Resistant to hydrolysis by gastric and jejunal enzymes
– Stable to heating at 100° C for 30 minutes
– Mechanism of toxin activity not understood; no
satisfactory animal model
? Stimulate intestinal peristalsis and have CNS effect;
intense vomiting
Lecture 10 BIOL 533 19
Pathogenesis of S,aureus
? Treatment
– Antibiotics
? Types,
– Methicillin,oxacillin,nafcillin,and dicloxacillin
(semisynthetic penicillins resistant to ?-lactam
hydrolysis)
– Majority of patients can be treated,but 10-15%
S,aureus and 40% coagulase-negative staphylococci are
resistant; treat with vancomycin
Lecture 10 BIOL 533 20
Pathogenesis of S,aureus
? Treatment
– Antibiotics
? Resistance,
– Plasmid-borne (hydrolysis of ?-lactam ring)
– Chromosomal—change in structure of penicillin-binding
proteins
Lecture 10 BIOL 533 21
Streptococci
? Fermentative (oxygen tolerant) Gram+
cocci in chains
? Sensitive to penicillins
? Human reservoir—passed from person to
person
Lecture 10 BIOL 533 22
Streptococci
? Properties of Lancefield Groups
(CHO antigens on wall—see handout)
– Group A,cross-reaction can lead to,
? Rheumatic fever
? Glomerulonephritis
Lecture 10 BIOL 533 23
Streptococci
? Recent Group A Streptococcus virulence
factors
– M-like proteins—bind IgM IgG (protease
inhibitor) and alpha2 macroglobulin
– F protein—adherence to epithelial cells
– C5a peptidase—degrades C5A pyrogenic
exotoxins; previously called erythrogenic
toxins
Lecture 10 BIOL 533 24
Staph and Strep Toxins
? S,aureus toxic shock TSST-1
? S,pyogenes toxic shock TSSL-1
? S,pyogenes scarlet fever SPE-1
(children,not adults; immunity)
Lecture 10 BIOL 533 25
Staph and Strep Toxins
? S,aureus, Toxic Shock Syndrome
– Fever,diffuse rash
– Exfoliation of skin on palms and soles of feet
– Normally doesn’t compete well in relatively
anaerobic vaginal area
Lecture 10 BIOL 533 26
Staph and Strep Toxins
? S,aureus, Toxic Shock Syndrome
– Super-absorbent tampon,
? Created aerobic pockets
? Removed Mg?producing toxin
– After removed tampon,cases declined; did
not disappear
– Still associated with wounds,rare nasal
surgery
Lecture 10 BIOL 533 27
Staph and Strep Toxins
? S,pyogenes,Toxic Shock-Like Syndrome
– Skin or wound infection ---> bloodstream
– Death rate ~30%; over 10-fold higher than
TSST
? Seen in immunocompromised people
? Also other infections occurred,soft tissue infection
with influenza symptoms
– High fatality rate because rapid development
of shock and multiple organ failure
Lecture 10 BIOL 533 28
Staph and Strep Toxins
? S,pyogenes,Toxic Shock-Like Syndrome
– Features in common with scarlet fever
? Occur in healthy people
? Both associated with high fatality rate
? Produce same exotoxin,streptococcal pyrogenic
exotoxin (Spe)
– Similar in mechanism to TSST-1
Lecture 10 BIOL 533 29
Staph and Strep Toxins
? Comparing TSLS-1 and TSST-1,
– Rash,fever,shock,multiple organ failure;
resemble endotoxin septic shock
– Both toxins superantigens
– Same mechanisms of action
– Limited similarity at amino acid sequence level
Lecture 10 BIOL 533 30
Staph and Strep Toxins
? TSLS-1 related to erythrogenic toxin
(scarlet fever; SpeA)
– Serotypes,
? Spe A—TSLS or invasive S,progenes
? Spe B
? Spe C
– Some strains don’t produce Spe A,so Spe B
or Spe C also has role
Lecture 10 BIOL 533 31
Staph and Strep Toxins
? How do TSST-1 and SPE cause shock and
multiple organ failure?
? Hypotheses not mutually exclusive
Lecture 10 BIOL 533 32
Shock and Organ Failure
? First Hypothesis,same as LPS triggering
release cytokines IL1,TNF?
– Consistent with role as superantigen
? Promote association between macrophage and
helper T cells?proliferation of T cells producing
high IL2 level
– Secondarily produce IL1 TNF?
? Inject TSST-1 into rabbits; elevated levels IL1
TNF?
Lecture 10 BIOL 533 33
Shock and Organ Failure
? Second hypothesis,increase body’s
sensitivity to LPS; consistent with,
– Acts synergistically with LPS to amplify toxic
effects in vitro and in animals
– Conceivable—low levels leaching into blood
due to lysis of resident microflora
? Normally no effect
? Presence of Spe or TSST-1 causes an effect
Lecture 10 BIOL 533 34
Shock and Organ Failure
? Evidence to support role for LPS in TSST
and TSLS
– Injecting TSST-1 or Spe is lethal to rabbits
– Injecting exfolatin and concanavalin A not
lethal to rabbits
? Both elicit T cell proliferation,but don’t enhance
sensitivity to LPS
Lecture 10 BIOL 533 35
Shock and Organ Failure
? Evidence to support role for LPS in TSST
and TSLS
– TSST-1 not lethal to gnotobiotic animals
– Wouldn’t expect leakage,but still T cell
response
– Therefore,both suggest T cell proliferation
not as important as synergy of LPS
? Not conclusive; difficult to prove same level T cell
stimulation,proliferation occurred in all cases
Lecture 10 BIOL 533 36
Shock and Organ Failure
? Third hypothesis,
– TSST-1 can act directly on endothelial cells
? Damage causes malfunction in circulatory system,
which creates hypotension
? Data,swelling associated with massive leakage of
fluid from capillaries is marked symptom of both
TSST and TSLS
? Could also be result of action of blood vessels by
cytokines,coagulation,or complement cascade
Lecture 10 BIOL 533 37
Staph and Strep Toxins
? Mortality of S,pyogenes vs,S,aureus
– TSLS higher than TSS
– TSLS strains enter bloodstream
– TSS,only the toxin circulates
? S,pyogenes known to be invasive; killed
by PMNs and macrophage if ingested
Lecture 10 BIOL 533 38
S,pyogenes Invasiveness
? Strategies for evading phagocytosis; (1),
– M protein binds H factor better than factor B
? Leads to degradation of C3b
? Therefore,prevents opsonization by C3b and
formation of C3 convertase
Lecture 10 BIOL 533 39
S,pyogenes Invasiveness
? Strategies for evading phagocytosis
– Data supporting,
? M— mutants yield more susceptible to
phagocytosis; less virulent than wild type
? Ab against M protective
– 80 serotypes of M; possibly evades host
antibodies by changing serotype; however,no
data to support this hypothesis
Lecture 10 BIOL 533 40
S,pyogenes Invasiveness
? Strategies for evading phagocytosis; (2),
– Protease cleaves C5a
? Chemoattractant stimulates oxidative burst
? Some activation of complement could occur in
spite of M protein because
– Lysis releases wall components that activate complement
– Streptococci could protect themselves- C5a peptidase
– Data supporting,
? C5a mutants less virulent that wild type in animals
Lecture 10 BIOL 533 41
S,pyogenes Invasiveness
? Strategies for evading phagocytosis; (3),
– M— like proteins
? Sequence and structural similarity to M
? COOH embedded; NH2 exposed
? Most similar to M and each other at carboxy end
? These proteins bind Fc portion of IgG and IgA
Lecture 10 BIOL 533 42
S,pyogenes Invasiveness
? Strategies for evading phagocytosis
– M— like proteins; possible roles
? Coat with host protein—less likely detected as
invader by complement and immune system
? Adherence for body cells that contain Ab on
surface
– Also can bind host protease inhibitor such as ?2
macroglogulin
– Host uses protease inhibitor to protect against proteases
released by phagocytes
Lecture 10 BIOL 533 43
S,pyogenes Invasiveness
? Strategies for evading phagocytosis; (4),
– F protein—binds fibronectin
? Adherence of bacteria to tissues
? Evasion of immune system
– Summary of invasiveness
? No direct evidence M-like proteins involved in
virulence
? Found in impetigo strains,not always in severe
invasive strains
? Need mutant studies to answer questions
Lecture 10 BIOL 533 44
S,pyogenes Virulence
? Regulation of S,pyogenes virulence genes
– Expression M,C5a peptidase,and some M-like
proteins; regulated at transcriptional level
– Responds to CO2 levels
? Increased CO2 causes increased production
Lecture 10 BIOL 533 45
S,pyogenes Virulence
? Regulation of S,pyogenes virulence genes
– Regulatory gene
? mry transcriptional activator; sequence analysis
shows it is part of two-component system
– Sensor—not found
– Activator
? Also known that speA gene on temperate phage
Lecture 10 BIOL 533 46
S,pyogenes Pathogenesis
? Treatment and prevention
? TSST,TSLS are medical emergencies
– Surgical debridement of wounds prevents
further production of toxin
– Antibiotics; penicillin
– Toxic effects TSST-1 countered by
intravenous rehydration; counter hypotension
Lecture 10 BIOL 533 47
Streptococcal Treatment
? Prevention
– Vaccine possible
– Target against M
? Possible problems
– # serotypes,but severe invasive disease
caused by few
– AB against M cross-reacts with heart
Lecture 10 BIOL 533 48
Streptococcal Sequellae
Hypotheses
? First,Autoimmune theory
– Epitopes that cross react with epitopes on
cardiac myosin and sarcolemmal membrane
proteins
? Thus,T cells or antibodies could attack tissue
? Inflammatory response damages heart valves
Lecture 10 BIOL 533 49
Streptococcal Sequellae
Hypotheses
? Glomerulonephritis
– High levels Ab to streptococcal Ag circulating
in blood stream causes AgAb complexes to
accumulate in kidney
– Inflammatory response attacks kidney
interfering with kidney function
Lecture 10 BIOL 533 50
Streptococcal Sequellae
Hypotheses
? Data supporting
– Ag-Ab complexes visible in people with
glomerulonepheritis—glomeruli
– Decrease in C3 and other complement
components also seen; supports hypothesis
that inflammatory response is occurring
? Second,Toxins cause sequellae
Lecture 10 BIOL 533 51
Streptococcal Sequellae
Hypotheses
? Main argument against
– Time lag between initial infection and
development of rheumatic fever (RF; several
weeks) or glomerulonephritis (10 days)
– Normally,if due to toxin,within a week
– Candidates for toxin most likely to cause
glomerulonephritis,streptococcal O,
streptokinase,or Spe
Lecture 10 BIOL 533 52
Glomerulonephritis Hypotheses
? Streptococcal O cytotoxin
– Mechanism and aa sequence similarity to
pneumolysin
– Pore-forming toxin
? Injected into lab animals; damages heart
– Therefore,may have role in RF
? Also,very immunogenic; maybe Ab damage
Lecture 10 BIOL 533 53
Glomerulonephritis Hypotheses
? Streptokinase
– Plasminogen?plasmin
– Therefore causes symptoms similar to
glomerulonephritis in animals
? Interesting,but not proven
Lecture 10 BIOL 533 54
Rheumatic Fever Hypotheses
? Spe
– RF strains produce Spe; others don’t
– Enhances cardiotoxicity caused by
Streptococcal O in animals
? Haven’t explained long time lag
Lecture 10 BIOL 533 55
Rheumatic Fever Hypotheses
? Mysterious feature of RF unexplained
– Treated with antibiotics for as late as 9 days
after symptoms,still protected against RF
? After 9 days,toxic products should be circulating
and immune response underway
– Recurrence of disease
? Normally,infection results from different strain
? Some result from same strain
– RF symptoms take as long to develop as in original
Lecture 10 BIOL 533 56
Rheumatic Fever Hypotheses
? Mysterious feature of RF unexplained
– If caused by autoimmune response,would
expect faster response
? Possible explanation,previously exposed produces
primed immune system
Lecture 10 BIOL 533 57
Streptococcal Sequellae
? Treatment and prevention
– Strep throat self-limiting
– Treat with antibiotics and prevent RF and
glomerulonephritis
? Only ? S,pyogenes strains cause RF or G
? Not all people colonized actually contract RF or G
– Because of the seriousness,treat any strain
with antibiotics
Lecture 10 BIOL 533 58
Streptococcal Sequellae
? Tests
– Blood agar
? Hemolysis
? Bacitracin sensitivity
– Rapid test and culture test both yield high
number of false negatives
? For patients who are allergic to penicillin,
use erythromycin
Lecture 10 BIOL 533 59
Streptococcal Sequellae
? Previous damage
– Even heart murmur
– Dentists recommend prophylactic penicillin
before dental work
? Kill bacteria escaping into blood stream from
mouth (oral bacteria are susceptible to penicillin)
? Reduces chance of colonization
Lecture 10 BIOL 533 60
Staphylococcal Enterotoxins
? Normal enterotoxins cause diarrhea
– Water loss from small intestine mucosa
– Cause c-GMP or c-AMP levels in mucosal cells
to rise
Lecture 10 BIOL 533 61
Staphylococcal Enterotoxins
? Staph toxins operate in a different mode;
hypotheses include,
– 1) Stimulates vagus nerve endings in stomach
lining that control emetic (vomiting) response
? If hypothesis correct,it’s a neurotoxin,not
enterotoxin
– 2) Superantigen?IL2
? Administering IL2 to human volunteers produces
many of the same symptoms (nausea,vomiting,
fever,malaise)
Lecture 10 BIOL 533 62
Staphylococcal Enterotoxins
? Neither hypothesis conclusively proven
– Could be a combination of both
? Seven serotypes,SEA,SEB,SEC1,SEC2,
SEC3,SED,SEE
? 30 kDa proteins share considerable aa
similarity and single internal S-S
Lecture 10 BIOL 533 63
Staphylococcal Enterotoxins
? SE closely related to Spe
– Genes coding for SEC1 and Spe A have 60%
nucleotide identity
? SE produced in different amounts by
different strains
– entA produces much less toxin than entB
? Differences in promoter strength
Lecture 10 BIOL 533 64
Staphylococcal Enterotoxins
? SEB on integrated plasmid
? SEA on lysogenized phage
– One strain,entB,entC,plasmid-borne
? SED,entD on 27.6 kb plasmid
Lecture 10 BIOL 533 65
Lecture 10
? Questions?
? Comments?
? Assignments..,
