Intracellular Antibiotics :
what does it (really) mean ?
Paul M. Tulkens, MD, PhD
Cellular and Molecular Pharmacology Unit
Catholic University of Louvain, Brussels, Belgium
&
International Society of Antiinfective Pharmacology
www.isap.org
www.md.ucl.ac.be/facm
Melbourne, Victoria
April 6th 2001
Intracellular antibiotics (ASA)
April 5th, 2001
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What is Intracellular Infection ?
antibiotic
The Cell
bacteria
(black box)
Intracellular antibiotics (ASA)
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2
Intracellular antibiotics: the issues
the bug
1. which bacteria and where ?
2. which antibiotics accumulate ?
3. influx vs efflux ? gist
lo
o
c
in cells ?
3. where are antibiotics
a
rm
a
h expression of activity ?
4. intracellular
p
e
th
5. bacterial responsiveness ?
6. cooperation with host defenses ?
7. any toxicity ?
the toxicologist
Intracellular antibiotics (ASA)
April 5th, 2001
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Intracellular antibiotics: the issues
1. which bacteria and where ?
2. which antibiotics accumulate ?
3. influx vs efflux ?
3. where are antibiotics in cells ?
4. intracellular expression of activity ?
5. bacterial responsiveness ?
6. cooperation with host defenses ?
7. any toxicity ?
Intracellular antibiotics (ASA)
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Which bacteria … and which diseases ...
➨ Obligatory or mainly intracellular:
➘respiratory infections (pneumopathies):
Chlamydia pneumoniae:
Legionella pneumophila:
Mycobacterium spp.:
10% in children
frequent if immunosuppression
frequent if immunosuppression
➘sexually transmitted diseases
Chlamydia trachomatis:
most common pathogen
➘ CNS infections + other sites:
pregnant women; immunosuppression
Listeria monocytogenes:
➨ Facultative or mainly extracellular:
➘ digestive tract infections
Salmonella spp., Shigella spp.
➘ respiratory, cutaneous, etc…tract infections
Streptococcus spp., Staphylococcus spp.
Intracellular antibiotics (ASA)
April 5th, 2001
etc...
5
Question :
where do intracellular bacteria sojourn and thrive ?
somewhere in the vacuolar
apparatus, but also outside ...
Intracellular antibiotics (ASA)
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where do intracellular bacteria sojourn and thrive ?
cytosol
endosomes
phagolysosomes
phagosomes
lysosomes
Intracellular antibiotics (ASA)
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where do intracellular bacteria sojourn and thrive ?
cytosol
endosomes
phagolysosomes
phagosomes
Legionella
Chlamydia, ...
lysosomes
Intracellular antibiotics (ASA)
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where do intracellular bacteria sojourn and thrive ?
cytosol
endosomes
phagolysosomes
phagosomes
S. aureus
Salmonella,
M. leprae, ...
Legionella
Chlamydia, ...
lysosomes
Intracellular antibiotics (ASA)
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where do intracellular bacteria sojourn and thrive ?
cytosol
Listeria hly+,
Shigella
endosomes
phagolysosomes
S. aureus
Salmonella,
M. leprae, ...
lysosomes
Intracellular antibiotics (ASA)
April 5th, 2001
phagosomes
Legionella
Chlamydia, ...
10
where do intracellular bacteria sojourn and thrive ?
cytosol
Listeria hly+,
Shigella
endosomes
phagolysosomes
S. aureus
Salmonella,
M. leprae, ...
Intracellular antibiotics (ASA)
April 5th, 2001
phagosomes
Legionella
Chlamydia, ...
11
Finally the answer to the question...
(where do intracellular bacteria sojourn and thrive ?)
The subcellular localization of bacteria may be
highly variable according to
• their type
Very different environments...
• phagosomal
pH ≈ 6
• phagolysosomal
pH ≈ 5
• cytosolic
pH ≈ 7
• the state of their journey in cell
Dynamics is essential ...
Intracellular antibiotics (ASA)
April 5th, 2001
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2d question (and answer):
which antibiotics do or do not accumulate in cells ?
•
•
•
•
•
•
•
beta-lactams: ≤ 1x
aminoglycosides: <1 to 2 x
ansamycins: 2-3 x
tetracyclines: 2-4 x
fluoroquinolones: 5 - 20 x
macrolides: 4 to > 100 x *
glycopeptides: 1 to 400 x !! **
* azithromycin, ketolides
** LY 333328
Intracellular antibiotics (ASA)
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3d question (and answer) :
where are intracellular antibiotics located ?
cytosol
• fluoroquinolones
• beta-lactams
• ansamycins
• macrolides (1/3)
endosomes
?
? phagolysososomes
phagosomes
• macrolides (2/3) lysososomes
• aminoglycosides
Intracellular antibiotics (ASA)
April 5th, 2001
?
14
3d question bis (and answer) :
what is the subcellular mobility of antibiotics ?
High
Fair
FQ / Ansamyc. / cytosol. ML
Intracellular antibiotics (ASA)
April 5th, 2001
Nil
lysosom. ML / AG
15
3d question bis (and answer) :
what is the subcellular mobility of antibiotics ?
Fluoroquinolones
move easily
across
membranes
Intracellular antibiotics (ASA)
April 5th, 2001
FQ
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3d question bis (and answer) :
what is the subcellular mobility of antibiotics ?
AG
Aminoglycosides
and lysosomal macrolides
reamain largely if not totally sequestered
Intracellular antibiotics (ASA)
April 5th, 2001
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4th question (and answer): what is the antibiotic
intracellular expression of activity
In cytosol:
• fluoroquinolones: yes
• ansamycins: excellent
• beta-lactams: yes
endosomes
pH = 7
pH 6
in phagolysosomes
• macrolides: reduced
• fluroquinolones: fair
• ansamycin: excellent
Intracellular antibiotics (ASA)
April 5th, 2001
phagosomes
• macrolides: fair
• fluoroquinolones:
good
in lysosomes (pH 5) :
• aminoglycosides: very reduced
18
5th question (and answer) : what is the bacterial
responsiveness to the antibiotics ?
in cytosol:
excellent
pH = 7
in endosomes:
?
pH < 6
in phagolysosomes:
very variable
Intracellular antibiotics (ASA)
April 5th, 2001
in lysosomes:
don’t know …
But, is it important ?
in phagosomes
likely to excellent
19
6th question [1 of 2] (and possible answer) :
cooperation with the cell own defenses ?
in cytosol:
unlikely
pH = 7
pH < 6
in endosomes:
likely
O2. .
NO
in phagolysosomes:
in phagosomes
very variable
likely
Myeloperoxydase,
acidity, cationic proteins...
Intracellular antibiotics (ASA)
April 5th, 2001
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6th question [2 of 2]: cooperation or antagonism
with the actions of cytokines ?
antibiotic
The very black box
Intracellular antibiotics (ASA)
April 5th, 2001
interferons
ILx
growth fact.
TNF
...
21
cooperation with cytokines :
one limited answer...
Gamma-Interferon stimulates ORS and NRS production
pH = 7
pH < 6
O2. .
NO
higher killing
of bactaria
in phagosomes
Intracellular antibiotics (ASA)
April 5th, 2001
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Intracellular antibiotics: the issues
1. which bacteria and where ?
2. which antibiotics accumulate ?
3. influx vs efflux ?
3. where are antibiotics in cells ?
4. intracellular expression of activity ?
5. bacterial responsiveness ?
6. cooperation with host defenses ?
7. any toxicity ?
the toxicologist
Intracellular antibiotics (ASA)
April 5th, 2001
23
antibiotics:
• ampicillin
• sparfloxacin
• moxifloxacin
• azithromycin
Intracellular antibiotics (ASA)
April 5th, 2001
Listeria monocytogenes
hly+ and hly-
gamma
interferon
24
Intracellular infection cycle of Listeria monocytogenes hly+
from Portnoy et al.
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Listeria m. and γ-interferon : confocal microscopy
γ-interferon maintains
L. m. hly+ in non-cytosolic
compartment...
Bacteria
are
stained with
fluorescein
(FITC) [green]
and cell actin
with phalloidin
rhodamin
[red];
hly+: virulent
hly-: non virulent
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Following the intracellular fate
of Listeria m. by 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
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γ - IFN blocks the apparent growth of L. m.
A: dose-dependency; B: specifity
control
Control
IFNγγ 50 U/ml
IFNγγ 100 U/m
+ anti IFN γ IgG
IFNγγ 100 U/ml
Intracellular antibiotics (ASA)
April 5th, 2001
IFNγγ 100 U/ml
28
The lack of apparent growth of L. m. with γ-IFN is due to
NO and H202 production...
Intracellular antibiotics (ASA)
April 5th, 2001
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Cooperation between γ-IFN and antibiotics...
Intracellular activity:
log10
CFU @ 5 h
sparfloxacin
Bacterial kill
CFU @ 0 h
azithromycin
control
ampicillin
+ γ-IFN
+ γ-IFN γ and
LMMA +
catalase
Intracellular antibiotics (ASA)
April 5th, 2001
Bacterial growth
30
Listeria m., ampicillin and γ-interferon
1. ampicillin is poorly active against intracellular
Listeria m. ;
2. ampicillin does NOT cooperate and is actually
antogonized by γ-IFN
Why do you keep ampicillin ?
! extracellular bacteria
! get activity with very large doses
Intracellular antibiotics (ASA)
April 5th, 2001
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Listeria m., azithromycin and γ-interferon
1. azitromycin accumulates much more than ampicillin
but is not much more active ...
2. azithromycin in indifferent to host defenses* …
“Intracellular” antibiotics are NOT the panacea
Oh, yeah, but, did you test the ketolides ?
Sorry ...
Intracellular antibiotics (ASA)
April 5th, 2001
Avez-vous évalué les kétolides ?
32
γ-interferon
Listeria m., fluoroquinolesanand
d
1.
2.
ell y pH
c
e db
h
t
te
t
a
u
)
ho defe
…
g
s
u ot
o
r
o
r
u
n active agent (intrinsically
hmost
Sparfloxacin IS the
!! )
E
t
s
i
n
e
i
…
v ity
s ize
o
n
v
i
o
m
r
t
against intracellular
Listeria
m.
;)
s
p
c
Q
a
a
F ir
re big
e
e
th
th ( a
ow n ??
n
k tio
Sparfloxacin activity is enhanced
by the presence of
t
’
s
n
o ge
d
ugcooperate !)
γ-IFN (γ-IFN and SpWeDO
s
y
n
A
Why don’t you use fluoroquinolones ?
! too low intrinsic activity ...
This is where we are ...
Intracellular antibiotics (ASA)
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Where was this stuff published ?
Scorneaux et al.,
Antimicrob. Agents Chemother. 40:1225-30, 1996
Ouadrhiri et al.,
Antimicrob. Agents Chemother. 43:1242-51, 1999
Ouadrhiri et al.,
J. Infect. Dis. 180:1195-204, 1999
see also:
Renard et al., AAC (1987) 31:410-416
Carlier et al., JAC (1990) 26(B):27-39.
Tulkens, Eur. J. Clin. Microbiol. Infect. Dis. (1991) 10:100-106.
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Thanks to the crew ...
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Intracellular antibiotics (ASA)
Zubida Bumkhalla (beta-lactams [chemistry])
Marie-Béatrice Carlier (macrolides, fluoroquinolones)
Séphane Carryn (méropenème, moxifloxacine, …)
Huajuan Fan (betalactams [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)
April 5th, 2001
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But where do they work ?
Catholic University of Louvain
Faculty of Medicine
Brussels, Belgium
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But, where the hell is that University?
Cellular and Molecular Pharmacology Unit
Catholic University of Louvain, Brussels, Belgium
http://www.md.ucl.ac.be/facm
Intracellular antibiotics (ASA)
April 5th, 2001
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But don’t forget ISAP...
population
pharmacokinetics
tissue
concentrations
efficacy/toxicity ratios
postantibiotic
effect...
AUIC
intracellular
pharmacodynamics
http://www.isap.org
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April 5th, 2001
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Let’s hope it helps you in Australia
South
tissue
concentrations
postantibiotic effect...
efficacy/toxicity ratios
population
pharmacokinetics
intracellular
pharmacodynamics
AUIC
North
Intracellular antibiotics (ASA)
April 5th, 2001
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