1. Intracellular Infection: a Journey from Cell biology to the Design of New Chemotherapeutic Agents
Paul M. Tulkens, MD, PhD
Cellular and Molecular Pharmacology Unit
Catholic University of Louvain, Brussels, Belgium

2. What is Intracellular Infection ?
The Cell
(black box)
antibiotic
bacteria

3. These they are… OBLIGATORY OR MAINLY INTRACELLULAR:
respiratory infections (pneumopathies):
Chlamydia pneumoniae: 10% in children
Legionella pneumophila: frequent if immunosuppression
Mycobacterium spp.: frequent if immunosuppression
sexually transmitted diseases
Chlamydia trachomatis: most common pathogen
CNS infections + other sites:
Listeria monocytogenes: pregnant women; immunosuppression
FACULTATIVE OR MAINLY EXTRACELLULAR:
digestive tract infections
Salmonella spp., Shigella spp.
respiratory, cutaneous, etc…tract infections
Streptococcus spp., Staphylococcus spp.

4. You need to consider (at least) the following questions:
1. where are the bacteria ?
2. which antibiotics accumulate in cells ?
3. where are antibiotics located ?
4. what is the intracellular expression of activity ?
5. what is the bacterial responsiveness ?
6. cooperation with the cell own defenses and
cytokines?

5. 1st question (and answer) : where do intracellular bacteria sojourn and thrive ?
cytosol
endosomes
Listeria hly+,
Shigella
phagolysosomes
phagosomes
S. aureus
Salmonella,
M. leprae, ...
Legionella
Chlamydia, ...

6. Antibiotics and Intracellular Bacteria : a Bit of Theory
Eur. J. Microbiol Infect. Dis. 10: , 1991

7. 2d question (and answer): which antibiotics do accumulate in cells ?
beta-lactams:  1x
aminoglycosides: <1 to 2 x
ansamycins: 2-3 x
tetracyclines: 2-4 x
fluoroquinolones: x
macrolides: 4 to > 100 x

8. 3d question (and answer) : where are intracellular antibiotics located ?
cytosol
fluoroquinolones
beta-lactams
ansamycins
endosomes ?
phagolysososomes
macrolides
aminoglycosides
phagosomes ?

9. 4th question: what are the antibiotic intracellular expressions of activity
fluoroquinolones: yes
ansamycins: excellent
beta-lactams: yes
endosomes
pH = 7
pH < 6
phagosomes
macrolides: reduced
aminoglycosides: very reduced

10. 5th question: what is the bacterial responsiveness to the antibiotics ?
in cytosol:
excellent
in endosomes: ?
pH = 7
pH < 6
in phagolysosomes:
very variable
in phagosomes
likely to excellent

11. 6th question [1 of 2]: cooperation with the cell own defenses ?
in cytosol:
unlikely
in endosomes:
likely
pH = 7
pH < 6
O2. -
NO.
in phagolysosomes:
very variable
in phagosomes
likely
myeloperoxydase
acidity, cationic proteins...

12. 6th question [2 of 2]: cooperation or antagonism with the actions of cytokines ?
The very black box
interferons
ILx
growth fact.
TNF
...
antibiotic

13. Listeria monocytogenes
A first story : Listeria in phagosomes
antibiotics:
ampicillin
sparfloxacin
azithromycin
interferon
gamma
Listeria monocytogenes
hly+ and hly-

14. INTRACELLULAR INFECTION CYCLE OF Listeria monocytogenes hly+
from Portnoy et al.

15. Listeria and Interferon : confocal microscopy
Bacteria are
stained with
fluorescein
(FITC) [green]
and cell actin
with phalloidin
rhodamin
[red];
hly+: virulent
hly-: non virulent

16. Following the intracellular fate of Listeria m. in EM
A: phagocytosis
B: escape from vacuole
C: surrounded by actin
D,E: cells pre-treated with
interferon-gamma, L. m. hly+
remains in vacuoles.
F,G, H: L.m. hly- remains
constantly in vacuoles
A = 1 h post infection;
B, C, D, F, G = 3 h post infection
E, H = 5 h post infection

17. Demonstration of the expression of IFN receptors on THP-1 cells
control
IFN receptor
mouse AB
+ goat anti-mouse
FITC IgG
Upper panel : expression of
the IFN receptor as
determined by
FACS
Lower panel : binding
of 125I-IFN
Inset: Scatchard plot .

18. Characterization of the effect of IFN on the intracellular growth of L. monocytogenes hly+. A: dose-dependency; B: specifity
control
control
IFN 50 U/ml
IFN 100 U/m
+ anti IFN  IgG
IFN 100 U/ml
IFN 100 U/ml

19. The growth containment of L
The growth containment of L. monocytogenes caused by IFN is due to NO and H202 production...

20. Cytokines and host defenses: Interferon 
changes of intracellular traffic
+ INF 
stimulation of natural host defenses (NO and H2O2
(Ouadrhiri et al, 1999)

21. IFN  and antibiotics... LMMA/catalase Intracellular activity:
5 h
0 h
log10
control
+ IFN
+ IFN  and
LMMA/catalase

22. Listeria, ampicillin and Interferon
1. Ampicillin is poorly active against
intracellular Listeria;
2. Ampicillin activity is completely defeated when IFN  is present
(IFN makes the job !)
Why do you keep ampicillin ?
extracellular bacteria
get activity with very large doses

23. Listeria, macrolides and Interferon
azithromycin
T = 5h
T = 5h
AZI
AZI +
INF CT
AZI
AZI +
INF
AZI + INF
+ LMMA + catalase

24. Listeria, fluoroquinolones and Interferon
sparfloxacin
T = 5h
T = 5h SP SP +
INF
SP + INF
+ LMMA + catalase CT SP SP +
INF
SP + INF
+ LMMA + catalase
(Ouadrhiri et al, 1999)

25. A few among many unanswered questions ...
beta-lactams
can you enhance drug accumulation ?
macrolides
what with ketolides ?
fluoroquinolones
can you make VERY active compounds ?
can you eradicate ?

26. Where published ?
Scorneaux et al., Antimicrob. Agents Chemother. 40: ,
Ouadrhiri et al.,
Antimicrob. Agents Chemother. 43: , 1999
J. Infect. Dis. 180: , 1999

27. A story with beta-lactamsN R 2 CH 3 H CH 3 C N C N O O
COOH
COOH
D-alanine
beta-lactam

28. H20 DD-peptidase / transpeptidase Beta-lactamase Inactive enzymeO
ENZ-SerOH
ENZ-SerOH
COOH
Inactive
enzyme R R C HN C HN O O O
O-Ser
ENZ
H20
ENZ
COOH
COOH
ENZ-SerOH
ENZ-SerOH
Inactive
antibiotic C HN
COOH OH O R C HN
COOH OH O R

29. ALL beta-lactam have (and must have) a free carboxyl group
COOH
ALL beta-lactam have (and must have)
a free carboxyl group
(or an equivalent acidic group [electron donor])
at appropriate distance from the C — N bound

30. Why do beta-lactams not accumulate in cells ?
exatracellular
pH  7.4
cytosol
pH  7.2
phagolysososomes
pH  5-6

31. Why beta-lactams do not accumulate in cells ?AH A- AH A- AH A- H+ H+ H+

32. Why beta-lactams do not accumulate in cells ?AH A- AH A- AH A- H+ H+ H+
Proton pump

33. What if you are basic ? B
BH+ B
BH+ B
BH+ H+ H+ H+
Proton pump

34. To get accumulation, make a basic beta-lactam...R C N O
- NH2
CO- R
To get accumulation, make a basic beta-lactam...

35. N-(3-dimethylaminopropyl)benzylpenicillamide...S R
CH3
CH3 C N O H
CO- NH-CH2-CH2-CH2-N (CH3)2
N-(3-dimethylaminopropyl)benzylpenicillamide...

36. N-(3-dimethylaminopropyl) benzylpenicillamide…
get accumulated in cells (5 to 10-fold
concentrates in lysosomes
but…
is inactive because the amide linkage is not splitted by lysosomal enzymes...

37. C so ALL enzymes will probably be inactive !!
CH3
CH3 C N C O H
CO-R
This C adjacent to the C=O is of a D configuration…
so ALL enzymes will probably be inactive !!

38. Not Splitted !! Splitted !! D-proline L-proline C C CH3 CH3 CH3 N CH3
CO-NH-CH-COOH
CO-NH-CH-COOH |
CH3 |
CH3
Not Splitted !!
Splitted !!

39. You need to make a chemically labile ester...
CO - O - CH2 - X - NH2
You need to make a chemically labile ester...

40. What you can do with old pivampicillin...
pH 7
pH < 6 NH 2 + H NH N 3 C S CH 3 H N O C N CH S CH 3 - 3 O
COO O N CH 3
ampicillin O
COOH + NH 3 NH 2 H N H C S CH 3 C N S CH 3 O N CH 3 O CH O N CH O 3 3 O CH C H O 3 2 C C C CH C H 3 2 O O O C C C CH 3 O CH O O 3 CH 3
pivampicillin

41. Old pivampicillin does bring ampicillin in cells...
T = 24h
AMPI FROM PIVA CT
AMPI
PIVA
(Fan et al, 1997)

42. New compounds...?

43. Where published ? Renard et al., J. Med. Chem. 29:1291-1293, 1986
Renard et al., Antimicrob. Agents Chemother. 31: , 1987
Fan et al., Bioorg. Med. Chem. Letters 24: , 1997

44. Thanks to the crew ... Zubida Bumkhalla (beta-lactams [chemistry])
Marie-Béatrice Carlier (macrolides, fluoroquinolones)
Huajuan Fan (betlactams [chemistry] )
Christine Renard (beta-lactams)
Yousef Ouadrhiri (Listeria, interferon, IL6, fluoroquinolones)
Isabelle Paternotte (beta-lactams [chemistry], efflux mech.)
Bernard Scorneaux (Listeria, interferon, fluoroquinolones)
Etienne Sonveaux (beta-lactams [chemistry] )
Andrée Zenebergh (lincosaminides, macrolides)

45. But where do they work ?

46. But, where is that ? Cellular and Molecular Pharmacology Unit
Catholic University of Louvain, Belgium