Lecture 30 - Coxiella and Rickettsia Flashcards
Type of evolution that Rickettsia and Coxiella have undergone
Reductive evolution
Why are Coxiella and Rickettsia hard to study?
They are obligate parasites
Can Coxiella and Rickettsia synthesise their own ATP?
Yes, though they encode proteins that efficiently transport ATP from host cell
General features of both Coxiella and Rickettsia
1)
2)
3)
1) Small, obligate, Gram - bacteria
2) Intracellular pathogens
3) Zoonoses
What does Coxiella burnetii cause?
Q fever
Coxiella genome size
2MB
Rickettsia genome size
1.1-1.3 MB
How is Coxiella spread? 1) 2) 3) 4)
1) Ticks are reservoir, but don’t transmit to humans
2) Ticks transmit to livestock
3) When livestock get pregnant, Coxiella divides in placenta, can cause abortion
4) Bacteria from placenta enter soil. When soil dries, becomes dust, aerosolised bacteria can be inhaled, cause infection
Peak time of Coxiella infections
Summer
After spring, when livestock are pregnant
When it’s hot and dry, and dust forms
Acute Q fever symptoms
1)
2)
3)
1) Influenza-like illness
2) Complications such as pneumonia, hepatitis
3) Broad range of symptoms, so often misdiagnosed
How much of Coxiella seroconversion is asymptomatic?
50%
Chronic Q fever symptoms
1)
2)
1) Commonly endocarditis, a damaged heart valve
2) Poor prognosis, as need to be on antibiotics for several years (doxycycline)
Proportion of people who develop chronic Q fever
2%
Proportion of people who develop long-term complications from Coxiella
~15%
People most at rick of Coxiella infection
Vets, farmers, abattoir workers
Coxiella vaccine
Formalin-inactivated whole-cell vaccine
Why is Coxiella a potential bioweapon? 1) 2) 3) 4)
1) Extremely contagious
2) Environmentally persistent
3) Easily spread
4) Incapacitating agent
Netherlands outbreak of Coxiella 1) 2) 3) 4)
1) Dense goat populations near human populations
2) 2006-2007 - Increased goat abortions, but farmers didn’t need to notify public health authorities
3) 2008-2009 - Number of cases increased each year, peaking in summer.
4) ~2,300 human cases in 2009
Two forms of Coxiella
1) Small-cell variant
2) Large-cell variant
Small-cell variant 1) 2) 3) 4) 5)
1) Environmental form of Coxiella
2) 0.2-0.5 micrometer
3) Condensed chromatin
4) Resistant to heat, desiccation, osmotic shock, UV light, disinfectants
5) Metabolically inactive
Large-cell variant 1) 2) 3) 4) 5)
1) Form taken when inside host phagosome
2) Can exceed 1 micrometer
3) Pleomorphic
4) Metabolically active
5) Activated at low pH
How can Coxiella survive in the phagosome? 1) 2) 3) 4)
1) Not completely understood
2) Unusually basic proteome (average pH=8.25)
3) Production of acid phosphatase that might inhibit cellular NADPH oxidase, which prevents ROS formation
4) DNA repair system upregulated under oxidative stress
Difference between Coxiella and Legionella vacuoles
Coxiella vacuole interacts with early, late endosomes, phagosome. pH drops.
Legionella vacuole evades endocytic vacuoles, interacts with secretory vesicles
Secretion system used by Coxiella
Dot/ICM
How can Coxiella be grown outside a host cell? 1) 2) 3) 4) 5)
1) ACCM: Acidified citrate cysteine medium
2) 37C
3) 5% CO2
4) 2.5% O2
5) 4.75 pH
Effect of transposon mutagenesis of Coxiella Dot/ICM
Coxiella mutant can’t replicate intravacuolarly.
Growth curve similar to Coxiella treated with chloramphenicol
Reporter assay
A test which determines if a protein is secreted by a secretion system
Reporter assay used to test Coxiella 1) 2) 3) 4)
1) pBlaM
2) Transposon with a beta-lactamase placed in Coxiella
3) Host cell has CCF2-AM placed in it
4) If transposon inserts into secreted gene, then CCF2-AM will emit blue light (not green) when stimulated
CCF2-AM
A molecule that if stimulated with light, will emit green light.
Has a beta-lactamase cleavage site.
If cleaved, will emit blue light when stimulated with light
pBlaM
A reporter assay used to test Coxiella proteins
Amount of time before Coxiella begins translocating effectors
16-24 hours
This is the amount of time that it takes for phagosome to acidify, Coxiella to become large-cell variant
Number of effector proteins so far identified in Coxiella
~130
How have Coxiella effector proteins been identified? 1) 2) 3) 4)
1) Homology to Legionella proteins
2) PmrA promotor region (PmrA is a promotor for ~40 genes)
3) Eukaryotic motifs
4) Digestion of Coxiella genome, insertion into a plasmid with a reporter
Potential function of Coxiella effector proteins 1) 2) 3) 4)
1) Prevent host cell apoptosis
2) Promote vacuole fusogenicity
3) Mediate vacuole interaction with autophagosomes
4) Mediate cholesterol acquisition
How can we test effector functions? 1) 2) 3) 4)
1) Express effector in eukaryotic cells and examine localisation and influence on cell pathways
2) Produce neutralising antibodies to effector and see what happens to Coxiella
3) Biochemical and protein function studies
4) Identification of eukaryotic binding partners
Genera included in Rickettsiae
1)
2)
3)
1) Rickettsiae
2) Ehrlichia
3) Orientia
How are Rickettsiae transmitted to humans?
Arthropod vectors
Found in faeces of arthropods, cause irritation of the skin. If the skin is scratched, can introduce bacteria in the body
Three biogroups of Rickettsiae
1) Spotted fever
2) Typhus
3) Scrub typhus
Spotted fever biogroup
1)
2)
3)
1) Rocky Mountain spotted fever (rickettsii)
2) Mediterranean spotted fever (conorii)
3) Flinders Island spotted fever (honei)
Typhus biogroup
1)
2)
1) Epidemic typhus (prowazekii)
2) Murine typhus (typhi)
Scrub typhus
Orientia tsutsugamushi
Rocky mountain spotted fever mortality rate
~25%
~4% with early treatment
Rocky mountain spotted fever symptoms
1) a, b, c, d
2) a
3)
1) Early, non-specific symptoms
a) Sudden onset of fever
b) Headache
c) Vomiting
d) Abdominal pains
2) Rash occurs 2-5 days after onset of symptoms
a) Begins on hands, spreads to trunk (centripetal spread)
3) If not treated, can lead to multiple organ failure
Rocky mountain spotted fever vector
American dog tick
Adult female transmits to humans
Rickettsia rickettsii transmission within ticks
1)
2)
1) Trans-ovarial - Bacteria transmit from parent arthropod to offspring
2) Trans-stadial - Bacteria persist through all tick life-cycle stages
Rickettsia prowazekii transmission within lice
Not trans-ovarial, as R. prowazekii kills host tick
Rickettsia prowazekii reservoir
Flying squirrels (in the USA)
What is Rickettsia prowazekii associated with?
Unsanitary conditions (war, famine)
Epidemic typhus symptoms
1)
2)
3)
1) Sudden onset of fever, chills, myalgia
2) Rash
a) First presents on trunk, then spreads to extremities (centrifugal spread)
3) Complications include myocarditis, stupor, delirium
Rickettsial infection that leads to stupor and delirium
Rickettsia prowazekii (epidemic typhus)
Difference in rash between R. rickettsii and R. prowazekii
R. rickettsii rash has centripetal spread
R. prowazekii has a centrifugal spread
Brill-Zinsser disease
1)
2)
3)
1) Caused by R. prowazekii
2) Recrudescent, milder form
3) Associated with immunosuppression, often years after exposure to R. prowazekii
How do Spotted Fever biogroup Rickettsia species enter endothelial cells?
1)
2)
1) OmpB binds Ku70 on target cell
2) Cholesterol-dependent cell entry
3) Bacterium is phagocytosed
Which cells do Rickettsial species invade?
Endothelial cells of blood vessels
What do typhus biogroup Rickettsia do in host cell?
1)
2)
1) Escape phagosome using membranolytic phospholipase D and haemolysin C
2) Replicate in cytosol until host cell lyses
What do spotted fever Rickettsia do in host cell?
1)
2)
1) Escape phagosome using membranolytic phospholipase D and haemolysin C
2) Recruit host cell actin using either RickA or ScaA, burst into adjacent cells
How do Spotted fever Rickettsia recruit host cell actin?
1)
2)
1) RickA - C-terminal WH2 domain (WASP-homology 2) binds actin monomers, and arp2/3 polymerises them
2) ScaA - Can nucleate unbranched actin filaments
Why might RickA not be spotted fever Rickettsia's only mode of intracellular motility? 1) 2) 3) 4)
1) There are naturally-occurring spotted fever Rickettsia species that lack RickA
2) No arp2/3 observed in Rickettsia actin tails
3) Rickettsia are still motile when arp2/3 is inhibited
4) Unbranched organisation of tails indicates an arp2/3-independent mode on motility
Rickettsia virulence factors
1)
2)
1) Poorly-understood, as can’t grow in a lab, like Coxiella can be
2) Type IVa secretion system (different to Dot/ICM system)
Type IVa secretion system
1) Used by other bacterial species for transfer of protein and DNA
2) Present in Rickettsia spp.
