What

1
Whats a Legionnaire?We Didnt Know Either.
courtesy of http//legion.org
2
Bob
Courtesy of www.vodcasonic.com/dylan
3
Bobs story

• Bob went to the Legionnaires Conference
• in 1976
• 3 days later Bob felt sick and went to the
  Doctors
• What Bob caught was Legionnaires disease

4
Legionnaires Disease

• Legionnaires Disease is a severe form of
  pneumonia
• 1976 convention was the first major outbreak
• 221 cases of pneumonia
• 34 died
• Dr. Joseph McDade, from CDC, isolated bacterium
  from lung specimen

5
Current Trends

• 8,000 18,000 cases reported in the U.S. per
  year
• This number is only 5-10 of estimated occurrence

6
Legionella pneumophila

• Causes Legionnaires disease, a severe form of
  pneumonia
• Lives naturally in freshwater streams, rivers,
  lakes
• Optimal temperature 35-37 degrees Celsius
• Infects and reproduces in some protozoa and
  mammals

Courtesy of diseaseworld.com/legion.htm
7
How Do Humans Get This Bacteria?

• Inhalation of water vapor containing L.
  pneumophila
• Man-made habitats of proliferation
• Cooling towers (in air conditioning units)
• Water distribution systems (showerheads)
• Saunas

Courtesy of www.q-net.net.au/legion
8
Bobs Symptoms

• Early symptoms (24-48 hrs)
• Fever, yucky feeling, muscle ache, loss of
  appetite, headache
• Mild cough progressing to occasional phlegm
• Late symptoms
• Chest pain, vomiting, diarrhea, nausea, abdominal
  pain, neurological symptoms
• Cough with phlegm containing blood

9
High Risk Patients

• Elderly
• Smokers
• Those with repressed immune systems

Courtesy of www.vodcasonic.com/dylan
10
A day (or more) in the life of L. pneumophila
water droplets
Attachment to host cell
L. pneumophila
Human lungs
Entry via pseudopod coil
Nuclear apoptosis begins
Evasion of lysosome Replication
Cell Death and release of bacteria into the
extracellular fluid
11
WHATS NEW in the day (or more) in the life of L.
pneumophila
water droplets
Attachment to host cell
L. pneumophila
Human lungs
Entry via pseudopod coil
Nuclear apoptosis begins
Evasion of lysosome Replication
Cell Death and release of bacteria into the
extracellular fluid
12
Getting in the door

• Attachment of opsonin
• Complement receptors fix to MOMP
• Phagocytosis of L.pneumophila is SPECIAL

13
Oooooh, pseudopod coil
Courtesy of Infectious Diseases, 1990
14
Signal Transduction during Legionella pneumophila
Entry into Human Monocytes

• Patricia Y. Coxen, James T Summersgill, Julio A.
  Ramirez, AND Richard D. Miller
• Department of Immunology and Division of
  Infectious Disease, Department of Medicine,
  University of Louisville, Louisville, Kentucky
• Infection and Immunity, June 1998

15
Role of Kinases in Bacterial Entry

• Kinase inhibitors inhibit host cell invasion in
  dose dependant manner
• Do not inhibit attachment to host cell once
  removed, the bacteria invade normally
• Do not affect cellular replication

Courtesy of Infection and Immunity, 1998
16
Proteins get Phosphorylated

• Specific bands showed increased phosphorylation
  upon infection
• Baseline-level fluorescence in kinase inhibited
  cells
• Phosphorylation is essential to bacterial entry

Courtesy of Infection and Immunity, 1998
17
Actin gets Polymerized

• Cellular actin levels correlate with phagocytic
  entry of L. p.
• Kinase inhibitors also inhibit actin
  polymerization
• Role of kinase signaling in this event

Courtesy of Infection and Immunity, 1998
18
Localization of Actin
Courtesy of Infection and Immunity, 1998
19
WHATS NEW in the day (or more) in the life of L.
pneumophila
water droplets
Attachment to host cell
L. pneumophila
Human lungs
Entry via pseudopod coil
Nuclear apoptosis begins
Evasion of lysosome Replication
Cell Death and release of bacteria into the
extracellular fluid
20
Activation of Caspase 3 during Legionella
pneumophila- Induced Apoptosis

• Lian- Yong Gao and Yousef Abu Kwaik
• University of Kentucky Chandler Medical Center
• Infection and Immunity, September 1999

21
L. Pneumophila induces apoptosis

• Apoptosis of macrophages after just 2-3 hours
  post infection
• Biphasic model of cell death by L. p.
• Apoptosis low levels of L. p., early infection
• Necrosis post exponential growth, late stage
  infection, results from cytotoxicity

22
Caspase Cascade Leads to Apoptosis

• L. p. activates caspase 3 in macrophages
• Leads to nuclear apoptosis

Courtesy of Infection and Immunity, 1999
23
How Does L. p. Activate Caspase 3?
Courtesy of Pac Land, Pacman Web Ring

• Can occur outside of macrophage
• Three proposals
• Translocation of factor into cell through DOT/ICM
  channel
• Factor bind to death receptor on cell surface
• Bacteria binds to death receptor with factor on
  surface

24
Why Apoptosis So Early In Infection?

• Four Theories
• Blocks endocytic fusion activity
• Inhibits anti-bacterial mechanisms
• Method of release after reproduction in host cell
• Reduces inflammation

25
WHATS NEW in the day (or more) in the life of L.
pneumophila
water droplets
Attachment to host cell
L. pneumophila
Human lungs
Entry via pseudopod coil
Nuclear apoptosis begins
Evasion of lysosome
Cell Death and release of bacteria into the
extracellular fluid
Replication
26
Early Events in Phagosome Establishment Are
Required for Intracellular Survival of Legionella
pneumophila

• L.A. Wiater, K. Dunn, F.R. Maxfield, and H.A.
  Shuman
• College of Physicians and Surgeons, Columbia
  University
• Infection and Immunity, September 1998

27
Mutations in dotA and icm

• Row 1
• Wild-type L.p.
• after 6 hrs., no PLF
• Row 2
• Mutated icmX
• after 0.5, PLF
• Row 3
• Mutated dotA
• after 6 hrs., PLF
• Row 4
• Mutated icmE
• after 6 hrs., PLF

courtesy of Infection and Immunity, 1998
28
Just incase that wasnt clear . . .
Wild-Type
Wild-Type
dotA
dotA
icmX
icmE
icmE
icmB
icmU
icmX
icmU
icmR
icmB
icmR
0.5 hrs.
6 hrs.
Adapted from Infection and Immunity, 1998
29
Whats This All Mean?

• dotA and icm genes play a role in dodging PLF
• no PLF increases life-span of L.p. within host
• may lead to more favorable conditions for
  replication

phagosome
lysosome
30
WHATS NEW in the day (or more) in the life of L.
pneumophila
water droplets
Attachment to host cell
L. pneumophila
Human lungs
Entry via pseudopod coil
Nuclear apoptosis begins
Evasion of lysosome
Cell Death and release of bacteria into the
extracellular fluid
Replication
31
Different fates of Legionella pneumophila pmi and
mil mutants within macrophages and alveolar
epithelial cells

• L.Y. Gao, B.J. Stone, J.K. Brieland Y.A. Kwaik
• Deparment of Microbiology and Immunology,
  University of Kentucky Chandler Medical Center
• Unit for Laboratory Animal Medicine, University
  of Michigan
• Microbial Pathogenesis, September 1998

32
Messages from Mutants

• 121 mutant L. pneumophila could not kill
  macrophages
• 30/121 mutant L. pneumophila could not kill
  alveolar epithelial cells
• 91/121 mutant L. pneumophila could kill alveolar
  epithelial cells

Macrophages Alveolar epithelial cells
wild type
Adapted from Microbial Pathogenesis, 1998
33
Mice experiments
Mutant 1 cannot kill macrophages cannot
alveolar epithelial cells
Mutant 2 cannot kill macrophages can kill
alveolar epithelial cells
injected into trachea
injected into trachea
24 hrs
24 hrs
No replication, bacteria killed
Bacterial replication similar to wild-type
34
Why cant some mutant L. pneumophila replicate in
host cells?

• If you cant recruit rough ER, you cant replicate

• Wild type bacteria in phagosome
• Many ribosomes

• Mutant bacteria in phagosome
• No ribosomes

Courtesy of Microbial Pathogenesis, 1998
35
What does all this mean?

• Most other research has focused on macrophages
• Infection of alveolar epithelial cells is
  different
• Infection of alveolar epithelial cells leads to
  replication of L. pneumophila in vivo
• Recruitment of rough ER is needed for replication
• It is important to study alveolar epithelial cells

36
What About Bob?

• Bob presents symptoms that suggest pneumonia

37
X-ray, Day 1
Courtesy of Lattimer, 1981
38
DNA probe
Courtesy of Lattimer, 1981
39
X-ray, Post-treatment
Courtesy of Lattimer, 1981
40
Future Research
Receptors?
Attachment to host cell
water droplets
L. pneumophila
Human lungs
Entry via pseudopod coil
Nuclear apoptosis begins
Evasion of lysosome Replication
Cell Death and release of bacteria into the
extracellular fluid
41
Future Research
Attachment to host cell
water droplets
L. pneumophila
Human lungs
Which proteins?
Entry via pseudopod coil
Nuclear apoptosis begins
Evasion of lysosome Replication
Cell Death and release of bacteria into the
extracellular fluid
42
Future Research
Attachment to host cell
water droplets
L. pneumophila
Human lungs
Death Receptors?
Entry via pseudopod coil
Nuclear apoptosis begins
Evasion of lysosome Replication
Cell Death and release of bacteria into the
extracellular fluid
43
Future Research
Attachment to host cell
water droplets
L. pneumophila
Human lungs
Entry via pseudopod coil
Nuclear apoptosis begins
Big Fish?
Evasion of lysosome Replication
Cell Death and release of bacteria into the
extracellular fluid
Courtesy of Printmaster Online Graphics
44
Future Research
Attachment to host cell
water droplets
L. pneumophila
Human lungs
Entry via pseudopod coil
Nuclear apoptosis begins
Why arent they friends?
Evasion of lysosome Replication
Cell Death and release of bacteria into the
extracellular fluid
45
Future Research
Attachment to host cell
water droplets
L. pneumophila
Human lungs
Entry via pseudopod coil
Nuclear apoptosis begins
Evasion of lysosome Replication
Cell Death and release of bacteria into the
extracellular fluid
Epithelial Cells?
46
Future Research
Why us?
Water treatment
Receptors?
Attachment to host cell
water droplets
L. pneumophila
Human lungs
Death Receptors?
Which proteins?
Immunizations
Entry via pseudopod coil
Nuclear apoptosis begins
Big Fish?
Why arent they friends?
Evasion of lysosome Replication
Cell Death and release of bacteria into the
extracellular fluid
Epithelial Cells?
47
Acknowledgements

• Dr. DebBurman
• Lisa Herron
• Shea Hogan and Annette Sieg
• Bob