Lecture 30 - Coxiella and Rickettsia

Question 1

Type of evolution that Rickettsia and Coxiella have undergone

Answer 1

Reductive evolution

Question 2

Why are Coxiella and Rickettsia hard to study?

Answer 2

They are obligate parasites

Question 3

Can Coxiella and Rickettsia synthesise their own ATP?

Answer 3

Yes, though they encode proteins that efficiently transport ATP from host cell

Question 4

General features of both Coxiella and Rickettsia
1)
2)
3)

Answer 4

1) Small, obligate, Gram - bacteria
2) Intracellular pathogens
3) Zoonoses

Question 5

What does Coxiella burnetii cause?

Answer 5

Q fever

Question 6

Coxiella genome size

Answer 6

2MB

Question 7

Rickettsia genome size

Answer 7

1.1-1.3 MB

Question 8

How is Coxiella spread?
1)
2)
3)
4)

Answer 8

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

Question 9

Peak time of Coxiella infections

Answer 9

Summer
After spring, when livestock are pregnant
When it’s hot and dry, and dust forms

Question 10

Acute Q fever symptoms
1)
2)
3)

Answer 10

1) Influenza-like illness
2) Complications such as pneumonia, hepatitis
3) Broad range of symptoms, so often misdiagnosed

Question 11

How much of Coxiella seroconversion is asymptomatic?

Answer 11

50%

Question 12

Chronic Q fever symptoms
1)
2)

Answer 12

1) Commonly endocarditis, a damaged heart valve

2) Poor prognosis, as need to be on antibiotics for several years (doxycycline)

Question 13

Proportion of people who develop chronic Q fever

Answer 13

2%

Question 14

Proportion of people who develop long-term complications from Coxiella

Answer 14

~15%

Question 15

People most at rick of Coxiella infection

Answer 15

Vets, farmers, abattoir workers

Question 16

Coxiella vaccine

Answer 16

Formalin-inactivated whole-cell vaccine

Question 17

Why is Coxiella a potential bioweapon?
1) 
2) 
3) 
4)

Answer 17

1) Extremely contagious
2) Environmentally persistent
3) Easily spread
4) Incapacitating agent

Question 18

Netherlands outbreak of Coxiella 
1)
2)
3)
4)

Answer 18

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

Question 19

Two forms of Coxiella

Answer 19

1) Small-cell variant

2) Large-cell variant

Question 20

Small-cell variant 
1)
2)
3)
4)
5)

Answer 20

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

Question 21

Large-cell variant 
1) 
2) 
3) 
4) 
5)

Answer 21

1) Form taken when inside host phagosome
2) Can exceed 1 micrometer
3) Pleomorphic
4) Metabolically active
5) Activated at low pH

Question 22

How can Coxiella survive in the phagosome?
1)
2)
3)
4)

Answer 22

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

Question 23

Difference between Coxiella and Legionella vacuoles

Answer 23

Coxiella vacuole interacts with early, late endosomes, phagosome. pH drops.

Legionella vacuole evades endocytic vacuoles, interacts with secretory vesicles

Question 24

Secretion system used by Coxiella

Answer 24

Dot/ICM

Question 25

How can Coxiella be grown outside a host cell?
1) 
2) 
3) 
4) 
5)

Answer 25

1) ACCM: Acidified citrate cysteine medium
2) 37C
3) 5% CO2
4) 2.5% O2
5) 4.75 pH

Question 26

Effect of transposon mutagenesis of Coxiella Dot/ICM

Answer 26

Coxiella mutant can’t replicate intravacuolarly.

Growth curve similar to Coxiella treated with chloramphenicol

Question 27

Reporter assay

Answer 27

A test which determines if a protein is secreted by a secretion system

Question 28

Reporter assay used to test Coxiella
1)
2)
3)
4)

Answer 28

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

Question 29

CCF2-AM

Answer 29

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

Question 30

pBlaM

Answer 30

A reporter assay used to test Coxiella proteins

Question 31

Amount of time before Coxiella begins translocating effectors

Answer 31

16-24 hours

This is the amount of time that it takes for phagosome to acidify, Coxiella to become large-cell variant

Question 32

Number of effector proteins so far identified in Coxiella

Answer 32

~130

Question 33

How have Coxiella effector proteins been identified?
1)
2)
3) 
4)

Answer 33

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

Question 34

Potential function of Coxiella effector proteins
1)
2)
3)
4)

Answer 34

1) Prevent host cell apoptosis
2) Promote vacuole fusogenicity
3) Mediate vacuole interaction with autophagosomes
4) Mediate cholesterol acquisition

Question 35

How can we test effector functions?
1) 
2) 
3) 
4)

Answer 35

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

Question 36

Genera included in Rickettsiae
1)
2)
3)

Answer 36

1) Rickettsiae
2) Ehrlichia
3) Orientia

Question 37

How are Rickettsiae transmitted to humans?

Answer 37

Arthropod vectors
Found in faeces of arthropods, cause irritation of the skin. If the skin is scratched, can introduce bacteria in the body

Question 38

Three biogroups of Rickettsiae

Answer 38

1) Spotted fever
2) Typhus
3) Scrub typhus

Question 39

Spotted fever biogroup
1)
2)
3)

Answer 39

1) Rocky Mountain spotted fever (rickettsii)
2) Mediterranean spotted fever (conorii)
3) Flinders Island spotted fever (honei)

Question 40

Typhus biogroup
1)
2)

Answer 40

1) Epidemic typhus (prowazekii)

2) Murine typhus (typhi)

Question 41

Scrub typhus

Answer 41

Orientia tsutsugamushi

Question 42

Rocky mountain spotted fever mortality rate

Answer 42

~25%

~4% with early treatment

Question 43

Rocky mountain spotted fever symptoms
1) a, b, c, d
2) a
3)

Answer 43

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

Question 44

Rocky mountain spotted fever vector

Answer 44

American dog tick

Adult female transmits to humans

Question 45

Rickettsia rickettsii transmission within ticks
1)
2)

Answer 45

1) Trans-ovarial - Bacteria transmit from parent arthropod to offspring
2) Trans-stadial - Bacteria persist through all tick life-cycle stages

Question 46

Rickettsia prowazekii transmission within lice

Answer 46

Not trans-ovarial, as R. prowazekii kills host tick

Question 47

Rickettsia prowazekii reservoir

Answer 47

Flying squirrels (in the USA)

Question 48

What is Rickettsia prowazekii associated with?

Answer 48

Unsanitary conditions (war, famine)

Question 49

Epidemic typhus symptoms
1)
2)
3)

Answer 49

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

Question 50

Rickettsial infection that leads to stupor and delirium

Answer 50

Rickettsia prowazekii (epidemic typhus)

Question 51

Difference in rash between R. rickettsii and R. prowazekii

Answer 51

R. rickettsii rash has centripetal spread

R. prowazekii has a centrifugal spread

Question 52

Brill-Zinsser disease
1)
2)
3)

Answer 52

1) Caused by R. prowazekii
2) Recrudescent, milder form
3) Associated with immunosuppression, often years after exposure to R. prowazekii

Question 53

How do Spotted Fever biogroup Rickettsia species enter endothelial cells?
1)
2)

Answer 53

1) OmpB binds Ku70 on target cell
2) Cholesterol-dependent cell entry
3) Bacterium is phagocytosed

Question 54

Which cells do Rickettsial species invade?

Answer 54

Endothelial cells of blood vessels

Question 55

What do typhus biogroup Rickettsia do in host cell?
1)
2)

Answer 55

1) Escape phagosome using membranolytic phospholipase D and haemolysin C
2) Replicate in cytosol until host cell lyses

Question 56

What do spotted fever Rickettsia do in host cell?
1)
2)

Answer 56

1) Escape phagosome using membranolytic phospholipase D and haemolysin C
2) Recruit host cell actin using either RickA or ScaA, burst into adjacent cells

Question 57

How do Spotted fever Rickettsia recruit host cell actin?
1)
2)

Answer 57

1) RickA - C-terminal WH2 domain (WASP-homology 2) binds actin monomers, and arp2/3 polymerises them
2) ScaA - Can nucleate unbranched actin filaments

Question 58

Why might RickA not be spotted fever Rickettsia's only mode of intracellular motility?
1)
2)
3)
4)

Answer 58

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

Question 59

Rickettsia virulence factors
1)
2)

Answer 59

1) Poorly-understood, as can’t grow in a lab, like Coxiella can be
2) Type IVa secretion system (different to Dot/ICM system)

Question 60

Type IVa secretion system

Answer 60

1) Used by other bacterial species for transfer of protein and DNA
2) Present in Rickettsia spp.