Legionella pneumophila

1
Legionella pneumophila

• Ubiquitous in soil and water
• causative agent of Legionnaires disease
• Only recognized within last 20 years

2
L. pneumophila

• Only affects immune-compromised

• Air-conditioning cooling towers
• good habitat
• disseminate aerosolized bacterium

3
Parasitizes Amoebae
Intracellular
4
Invades Human Macrophages in Lung
Parasitizes Amoebae
Intracellular
5
Invades Human Macrophages in Lung
Parasitizes Amoebae
Intracellular
Extracellular
Lives in Soil or Water
6

• How does L. pneumophila adapt between
  extracellular and varying intracellular
  environments?

7
More about L. pneumophila
8
More about L. pneumophila

• Amino Acids sole source of Carbon and Energy

9
More about L. pneumophila

• Amino Acids sole source of Carbon and Energy

Cells starved of amino acids
10
More about L. pneumophila

• Amino Acids sole source of Carbon and Energy

Cells starved of amino acids

• Virulent
• Slower growth

11

• Why become virulent when starved?

12

• Why become virulent when starved?

SICK
SICK
Starvation due to sick hosts
13

• Why become virulent when starved?

SICK
SICK
Starvation due to sick hosts
Escape sick hosts
14

• Why become virulent when starved?

SICK
SICK
Starvation due to sick hosts
Escape sick hosts
Disperse via Soil or Water
15

• Why become virulent when starved?

SICK
SICK
Starvation due to sick hosts
Escape sick hosts
Disperse via Soil or Water
Finds new host cells
16

• Why become virulent when starved?

SICK
SICK
Starvation due to sick hosts
Escape sick hosts
Disperse via Soil or Water
Invades healthy hosts
Finds new host cells
17

• If signal to escape is low amino acids...

18

• If signal to escape is low amino acids...

Low amino acids
?
stringent response
19

• If signal to escape is low amino acids...

Low amino acids
?
stringent response
?
virulence and slower growth
20
In E. coli and M. xanthus
amino acids
(p)ppGpp
Response (less protein synthesis, sporulation)
21
Maybe in L. pneumophila
amino acids
(p)ppGpp
virulence and slower growth
22
Maybe in L. pneumophila
amino acids
Question 1
(p)ppGpp
virulence and slower growth
23
Maybe in L. pneumophila
amino acids
Question 1
(p)ppGpp
Question 2
virulence and slower growth
24
Focus of experiment
(p)ppGpp
Question 2
virulence and slower growth
25
SICK
SICK
Starvation due to sick hosts
Escape sick hosts
26
SICK
SICK
Starvation due to sick hosts
Signal ?amino acids?
27
SICK
SICK
Starvation due to sick hosts
Signal ?amino acids?
?
?(p)ppGpp
28
SICK
SICK
Starvation due to sick hosts
Signal ?amino acids?
?
Virulence
?(p)ppGpp
?
29
SICK
SICK
Starvation due to sick hosts
Escape sick hosts
Signal ?amino acids?
?
Virulence
?(p)ppGpp
?
30
SICK
SICK
Starvation due to sick hosts
Escape sick hosts
Signal ?amino acids?
Virulence
?
?(p)ppGpp
?
31
Experimental Concept
Change in Growth Rate?
(p)ppGpp
32
Experimental Concept
Change in Growth Rate?
(p)ppGpp
Change in virulence?
33
Experimental Concept
Change in Growth Rate?
(p)ppGpp
Change in virulence?
Hammer et. al.(1999) Co-ordination of Legionella
pneumophila virulence with entry into stationary
phase by ppGpp. Molecular Microbiology. 33
721-731.
34
Experimental Concept
Change in Growth Rate?
(p)ppGpp
Change in virulence? Hypothesis Induction of
virulence
35
Experimental Concept
Change in Growth Rate? Hypothesis ? How are
growth and virulence related?
36
Experimental Concept
Change in Growth Rate?
(p)ppGpp
Change in virulence?
37
Experimental Concept
Can measure growth directly (OD)
Need virulence marker
38
FlaA - Virulence Marker
Flagellar synthesis appears co-regulated with
virulence
39
PflaA/gfp fusion
PflaA
flaA
gfp
PflaA
gfp
40
PflaA/gfp fusion
When FlaA expressed by cell...
41
PflaA/gfp fusion
When FlaA expressed by cell...
Observe GFP
42
Experimental Concept
Change in Growth Rate?
(p)ppGpp
Change in virulence marker expression?
43
How do we get (p)ppGpp?
44
Ptac/relA fusion
Ptac
relA
(Using E. colis relA)
45
Ptac/relA fusion
Ptac
relA
(Using E. colis relA)
RelA expression inducible with IPTG
46
Ptac/relA fusion
Ptac
relA
(Using E. colis relA)
RelA expression inducible with IPTG
RelA
(p)ppGpp
IPTG
47
Strain

• Wt Chromosome
• inducible RelA
• GFP virulence reporter

48
Results No IPTG
1.0
10
OD (log scale)
Relative Fluorescence
1
.5
24
24
0
0
time (hrs)
time (hrs)
49
Results IPTG
1.0
10
OD (log scale)
Relative Fluorescence
1
.5
24
24
0
0
time (hrs)
time (hrs)
50
Results Compare
1.0
10
OD (log scale)
Relative Fluorescence
1
.5
24
24
0
0
time (hrs)
time (hrs)
51
Conclusions
SICK
SICK
Starvation due to sick hosts
Escape sick hosts
Signal ?amino acids?
Virulence and Slow Growth
?
?(p)ppGpp
YES
52
Stringent Response still candidate for virulence
and growth control
53
Stringent Response still candidate for virulence
and growth control
Remaining Questions
54
Stringent Response candidate for virulence and
growth control
Remaining Questions

• Other signals?

55
Stringent Response candidate for virulence and
growth control
Remaining Questions

• Other signals?

• Can you separate virulence and growth rate?

56
Stringent Response candidate for virulence and
growth control
Remaining Questions

• Other signals?

• Can you separate virulence and grwoth rate?

• Does L. pneumophila have the stringent response
  system?

57
Native relA?
58
Native relA?

• BLAST unfinished genome with E. coli relA

59
Native relA?

• BLAST unfinished genome with E. coli relA

• Sequence 1 E value 0.0
• Identities 44, Positives 63, Gaps 0

60
Native relA?

• BLAST unfinished genome with E. coli relA

• Sequence 1 E value 0.0
• Identities 44, Positives 63, Gaps 0

• Sequence 2 E value 3e-83
• Identities 34, Positives 52, Gaps 1

61
Future Directions
SICK
SICK
Starvation due to sick hosts
Escape sick hosts
Signal ?amino acids?
?
Virulence
?(p)ppGpp
?
62
Future Directions

• Does (p)ppGpp accumulate when low a.a.?

63
Future Directions
relA knockout mutant
Loss of virulence?
Effect on growth rate?