soutenance thèse octobre 2012
DESCRIPTION
Présentation de la soutenance de thèse pour l'obtention du grade de docteur de l'Université de Caen Basse-Normandie et de l'Université de Kiel. Thèse soutenue le 19 octobre 2012 à Caen.TRANSCRIPT
Therapeutic potential of equine antimicrobial peptides against Rhodococcus equi and other major horse
pathogens
Margot Schlusselhuber19th of October 2012
Thesis supervisors:Pr. Joachim GRÖTZINGER,
Christian-Albrechts-Universität zu Kiel, Biochemistry InstituteDr. Claire LAUGIER,
Anses, Dozulé laboratory for equine diseasesPr. Roland LECLERCQ,
Université de Caen Basse-Normandie, Unité de Recherche Risques Microbiens (URRM)
Advisor: Dr. Julien CAUCHARD,
Anses, Dozulé laboratory for equine diseases
Introduction
Objectives
Results
General conclusion, discussion and perspectives
2
3
INTRODUCTION
Rhodococcus equi
• Causative agent of rhodococcosis• Susceptible hosts: 1-6 month-old foals
• Clinical aspects:– bronchopneumonia– digestive lesions– musculoskeletal lesions
• Facultative intracellular pathogen infecting macrophages
Very few antibiotics are effective in vivo
• Treatment: Combination macrolide-rifampicinProblematic: ethic problem, fatal side effects, cost of the treatment, antibiotic-resistance*
4
M. Schlusselhuber, Anses
C. Laugier, Anses
*Giguère et al., 2010
R. equi bronchopneumonia: associated pathogens
5
R. equi alone
Mixed infection
Adapted from Mauger, 2009 DVM thesis « retrospective study of equine rhodococcosis observed on 1617 foals necrospied at the « LERPE » (AFSSA, DOZULE) from 1986 to 2006 ».
S. zooepidemicus: ~17% RifampicinR, ~23% macrolideR
K. pneumoniae: 100% RifampicinR and macrolideR
Possible failure of the treatmentNeed a treatment active against the associated pathogens
Type of infection Risk factors
Salmonella enterica subsp enterica
Adults: colitis
Foal: septicemiaStress, congregation, marked changes in diet, disease, medication
Streptococcus zooepidemicusLung infectionEndometritis
Viral infection, rhodococcosis
Alteration of the genital flora
Klebsiella pneumoniaeEndometritisLung infectionSepticemia
Alteration of the genital flora
Viral infection, rhodococcosis
Neonatal foal
Escherichia coli
EndometritisSepticemiaLung infectionWound infection
Alteration of the genital flora
Neonatal foal
Viral infection, rhodococcosis
Wounds
Pseudomonas aeruginosaEndometritisWound infectionSepticemia
Alteration of the genital flora
Wounds
Neonatal foal
Staphylococcus aureus Wound infections Surgery, wounds
6
Common problem for equine veterinarians regarding these opportunists: antibiotic-resistance*
Other major bacterial pathogens of the horse: opportunists
* Van den Eede et al., 2009; Vo et al., 2007; Singh et al., 2007
Antibiotic-resistance and the « One Health perspective »
7
Joint FAO/WHO/OIE expert meeting on Critically Important Antimicrobials (CIAs), 2007
« Conclusions on the impact of antimicrobial resistance in the human health sector and in the veterinary sector » (Council of the European Union, June 2012)
“Stresses the need to be restrictive in both the human and veterinary use of CIAs and newly developed antimicrobials, eventually with the aim in the future
to reserve CIAs as much as possible for human use”
Need to develop alternative to antibiotics especially for equids!
8
Antimicrobial peptides (AMPs)
Emerging as particularly promising anti-infectives in alternative to antibiotics
Development of complete resistance is proposed to be unlikely
Produced virtually by all living organisms
High structural diversity
Small size (12-50 aa), positive net charge (pH 7), amphipathic
Usually derive from a larger and neutral precursor
Peptide antibiotics
Ribosomally synthesized peptides (Antimicrobial peptides, AMPs)
Generally amphipathic, positively charged, have at least 50% hydrophobic
residues
9
Other organisms (animal and plant kingdoms)
Ex: defensins, cathelicidins, indolicidin, cecropins …
Broad spectrum of action
Bacteria (G+ & G-)Ex: bacteriocins (also include
proteins)Generally able to kill specific
bacterial competitors
Non-Ribosomally synthesized peptides
(antibiotics)
Bacteria, fungi and streptomycetesEx: gramicidins, polymyxins, bacitracins, glycopeptides, …
Hancock et al., 1999, Peptides antibiotics, Antimicrobial Agents and Chemotherapy
Peptide antibiotics
Ribosomally synthesized peptides (Antimicrobial peptides, AMPs)
Generally amphipathic, positively charged, have at least 50% hydrophobic
residues
10
Other organisms (animal and plant kingdoms)
Ex: defensins, cathelicidins, indolicidin, cecropins …
Broad spectrum of action
Bacteria (G+ & G-)Ex: bacteriocins (also include
proteins)Generally able to kill specific
bacterial competitors
Non-Ribosomally synthesized peptides
(antibiotics)
Bacteria, fungi and streptomycetesEx: gramicidins, polymyxins, bacitracins, glycopeptides, …
Hancock et al., 1999, Peptides antibiotics, Antimicrobial Agents and Chemotherapy
Antimicrobial peptides of the horse
11Bruhn et al. Veterinary Research 2011 42:98© Virbac
34 AMPs identified
Mode of action: attraction
12
* cytoplasmic membrane: phospholipids such as cardiolipin, phosphatidilglycerol, and phosphatidylserine
The McGraw-Hill Companies, Inc© (Reproduced with permission)
Anionic compounds
*
Gram+
Gram-
13
Mode of action: insertion and targets
Secondary targets : intracellular molecules
Adapted from Brogden et al., 2005
Carpet model Barrel stave model Torroidal pore model
Primary target : cytoplasmic membrane
DNA, RNA and proteins
14
OBJECTIVES
HippoKAMP project
15
3 persons enrolled to work on this project:PhD student (France/Germany): nov. 2009-2012
Researcher (France): 2010-2013PhD student (Germany): nov. 2012-?
Scientific partners
Industrial partner
PhD thesis
16
First approach
17
Second approach
18
* PhD student (Germany)
• Synthesis, refolding and characterization of new equine AMPs – Opportunity in 2011 (1 month)– Project of the PhD student in Germany
19
Opportunity
Opportunity
• Production in large amount (2nd objective of the HippoKAMP project)– Opportunity in 2010 (6 months): Bioproduction in plants– Project of the French researcher
20
21
PERSONAL WORK
Therapeutic potential of equine antimicrobial peptides against Rhodococcus equi and other major horse pathogens
I. Comparison of the in vitro cytotoxicity and activity of DEFA1 and eCATH1 against R. equi and associated pathogens
II. Therapeutic potential of eCATH1 against rhodococcosis
III. In vitro activity of eCATH1 against antibiotic-resistant bacterial pathogens of the horse
IV. Selection of new equine AMPs
22
Therapeutic potential of equine antimicrobial peptides against Rhodococcus equi and other major horse pathogens
I. Comparison of the in vitro cytotoxicity and activity of DEFA1 and eCATH1 against R. equi and associated pathogens
In vitro activityIn vitro cytotoxicity
II. Therapeutic potential of eCATH1 against rhodococcosis
III. In vitro activity of eCATH1 against antibiotic-resistant bacterial pathogens of the horse
IV. Selection of new equine AMPs
23
24
In vitro activity against Rhodococcus equi and associated pathogens
MIC
CLSI standard microdilution method with modifications*
Peptide + 0.2% BSAMaterial: polypropylene microplate, round bottomIncubation: 40h, 37°C (R. equi) 24h, 37°C (associated pathogens)
*as outlined by R.E.W. Hancock Laboratory for AMP testing
25
100 µg/ml D
EFA1 – 5 m
inC
ontr
olIn vitro activity of DEFA1 against Rhodococcus equi
X 10,000
X 30,000
X 10,000
X 30,000
Collaboration with Dr D. Goux
26
X 30,000
In vitro activity of DEFA1 against Rhodococcus equi
Rapid action by
destabilization of the
bacterial envelope
27
100 µg/ml eC
ATH
1– 5 min
Con
trol
In vitro activity of eCATH1 against Rhodococcus equi
X 350 X 10,000
X 30,000
X 10,000
X 30,000
28
X 30,000
Scanning Electron MicroscopyIn vitro activity of eCATH1 against Rhodococcus equi
Rapid action by
destabilization of the
bacterial envelope
29
In vitro cytotoxicity
DEFA1 is toxic for
mammalian cells
below 100 µg/ml
(≠ eCATH1)
C+ C-peptide
123
Hemolysis assay
LDH release assay
No hemolysis of sheep and horse
erythrocytes up to 100 µg/ml (<1%)
Both peptides:- Have an antibacterial activity against R. equi and the major associated pathogen, S. zooepidemicus- Rapid action by destabilization of the bacterial envelop- Do not have hemolytic activity on horse and sheep erythrocytes below 100 µg/ml
30
Conclusion
eCATH1 - Does not harm mammalian cell line below 100 µg/ml
- Is active against all major associated pathogens
DEFA1- Is toxic for mammalian cell line below 100 µg/ml
- Is not active against K. pneumoniae
≠
Therapeutic potential of equine antimicrobial peptides against Rhodococcus equi and other major horse pathogens
I. Comparison of the in vitro cytotoxicity and activity of DEFA1 and eCATH1 against R. equi and associated pathogens
II. Therapeutic potential of eCATH1 against rhodococcosisSalt toleranceAcquisition of resistanceInteraction with other drugsActivity against intracellular R. equiEffectiveness in the treatment of an experimental rhodococcosis induced miceIn vivo cytotoxicity
III. In vitro activity of eCATH1 against antibiotic-resistant bacterial pathogens of the horse
IV. Selection of new equine AMPs
31
Salt tolerance
32
•Activity is not hampered by a
physiological salt concentration
• Bactericidal activity (MBC ~ MIC = 4
µg/ml): highly desirable mode of action
33
RifampicinR
ErythroR Days
0 25 50 75 100
Subculture at subinhibitory concentration of peptide over 90 days
1 passage
Peptide-free medium
Resistance tooks much longer to select than conventional antibiotics, was modest and was only transient
Selection of resistance by R. equi
Interaction with other drugs
34
C-
C+
C+
R. equi ATCC33701 P+
[drugA]
[dru
gB]
Positive interaction with rifampicin
FIC = A/MICA + B/MICB
synergy : ≤ 0.5indifferent: 0.5-4antagonism : ≥ 4
Nagaoka et al., 2000; Cassone et al., 2010
35
Cell line: J774.2 mouse macrophage
Bacteria: R. equi ATCC 33701 P+
Treatment: 45 min after infection20 µg/ml of eCATH1 24 hours
Staining: LIVE/DEAD BaclightPermeabilization of macrophages mb by saponin
+ eCATH1 w/o eCATH1
Activity against intracellular R. equi cells, in vitro study
x400 x400
x1000x1000 Nucleus of macrophages appear red
36
Collection of peritoneal macrophages
2h treatment with ATB to kill extracellular bacteria
InfectionMice: Female BALB/c
Bacteria: 107-108 R. equiInjection: intraperitoneal
CFU counting
8 hours
Treatment with i) eCATH1 (20 µg/ml)ii) Rifampicin (5 µg/ml)
iii) eCATH1 + rifampicin
24h post-infectionLyse macrophages
Activity against intracellular R. equi, ex vivo study
Collaboration with Pr M. Sanguinetti (Italy)
Control
eCATH1 + Rifampicin
eCATH1
Rifampicin
***
***
****
20 µg/ml
5 µg/ml
0 1000
2000
3000
4000
37
Activity against intracellular R. equi, ex vivo study
↓ ~60%
↓~90%
eCATH1 is active against
intracellular R. equi
Rhodococcus cells/105 macrophages
38
Effectiveness in the treatment of an experimental rhodococcosis induced in mice
InfectionMice: Female BALB/c
Bacteria: sublethal dose R. equiInjection: intravenous
Treatment (daily, subcutaneous)i) eCATH1 (1 mg/kg ≈ 20 µg/mouse)
ii) Rifampicin (10 mg/kg ≈ 200 µg/mouse)iii) eCATH1 + rifampicin
24 hours
CFU counting
Organs homogenized
SacrificeAt days 1, 4 and 8
post infection(5 mice per time point
per group)
Collaboration with Pr M. Sanguinetti (Italy)
39
No drug
eCATH1Rifampicin eCATH1 + rifampicin
Liver Spleen
Effectiveness in the treatment of an experimental rhodococcosis induced in mice
Combination therapy:
• Significant decrease of the bacterial load
(°, p<0.05)• Stronger effect / single
drug treatment
Single-drug therapy:• Significant decrease of the bacterial load (*, p<0.05)• 20 µg of eCATH1 decreased by >99% CFU (~2 log10)• Similar result with 200 µg of rifampicin• eCATH1 is more active than rifampicin (ex vivo ≠ in vivo)
No drug
Rifampicin eCATH1eCATH1 + rifampicin
In vivo cytotoxicity
40
5 non-infected mice (Female BALB/c)
Organs removed(intestines, spleens, livers, lungs, kidneys
and stomachs)
Sacrifice
7 daysTreatment:
1 mg/kg eCATH1, daily, subcutaneous injections
Histopathology
Monitoring: behavioral changes,
survival and drug-related adverse effects
Collaboration with Pr M. Sanguinetti (Italy)
41
In vivo cytotoxicity, histopathology
a, lung; b, kidney; c, spleen; d, small intestine; e, stomach; f, liver
Group receiving daily 1 mg/kg eCATH1
d
e
c
a
f
b
Untreated group
d
e
c
a
f
b
Analyzed by pathologists from LNPC (LeNet Pathology Consulting, Amboise)
In vivo cytotoxicity
Throughout the experimental period:– No death– No difference of behavior (food consumption, …)– No detectable adverse drug-related effects (local
signs of inflammation, weight loss, diarrhea, …)
42
No detectable toxicity or
adverse drug-related effects
Therapeutic potential of equine antimicrobial peptides against Rhodococcus equi and other major horse pathogens
I. Comparison of the in vitro cytotoxicity and activity of DEFA1 and eCATH1 against R. equi and associated pathogens
II. Therapeutic potential of eCATH1 against rhodococcosis
III. In vitro activity of eCATH1 against antibiotic-resistant bacterial pathogens of the horse
IV. Selection of new equine AMPs
43
In vitro activity against antibiotic-resistant bacterial pathogens
MIC, µg/ml
Pseudomonas spp.
Reference strain (ATCC 10145) 4
Gentamicin-resistant (2) 1-4
TIM-resistant (10) 1-8
Ceftazidim-resistant (3) 1-4
Ticarcillin-resistant (10) 1-8
Escherichia coli
Reference strain (ATCC 25922) 4
Cephalosporin (C1G, C3G)-resistant (11) 1-16
Ticarcillin-resistant (13) 1-16
Gentamicin-resistant (9) 1-16
Colistin-resistant (1) 1-2
SXT-resistant (11) 1-16
44
MIC, µg/ml
Klebsiella pneumoniae
Reference strain (ATCC 13883) 2
Cephalosporin (C1G, C3G)-resistant (4) 2-4
Gentamicin-resistant (4) 2-4
Colistin-resistant (1) 4
SXT-resistant (4) 2-4
Salmonella enterica serovar Typhimurium
Reference strain (ATCC 14028) 2
Cephalosporin (C1G, C3G)-resistant (7) 0.5-16
Ticarcillin-resistant (7) 0.5-16
Gentamicin-resistant (6) 0.5-2
Colistin-resistant (1) 2
SXT-resistant (8) 0.5-16
TIM, ticarcillin + clavulanate SXT, trimethoprim-sulphamethoxazole (X), number of clinical isolates
Gram-negative
No cross-resistance
45
MIC, µg/ml
Rhodococcus equi
Reference strain (ATCC 33701) 4
Macrolide-resistant (17) 1-4
Rifampicin-resistant (20) 0.5-4
Ceftiofur-resistant (2) 2-4
SXT-resistant (7) 1-4
MIC, µg/ml
Staphylococcus aureus
Reference strain (ATCC 29213) >32
Gentamicin-resistant (2) >32
Oxacillin-resistant (2) >32
Chloramphenicol-resistant (2) >32
SXT-resistant (4) 1->32
In vitro activity against antibiotic-resistant bacterial pathogens
Gram-positive
No cross-resistance
Not active against S.
aureus
Intrinsic resistance ?
SXT, trimethoprim-sulphamethoxazole (X), number of clinical isolates Collaboration with Pr S. Giguère (USA)
Therapeutic potential of equine antimicrobial peptides against Rhodococcus equi and other major horse pathogens
I. Comparison of the in vitro cytotoxicity and activity of DEFA1 and eCATH1 against R. equi and associated pathogens
II. Therapeutic potential of eCATH1 against rhodococcosis
III. In vitro activity of eCATH1 against antibiotic-resistant bacterial pathogens of the horse
IV. Selection of new equine AMPs
46
47
Selection
α-defensins
β-defensinsβ-defensin 103β-defensin 1β-defensin 2β-defensin 3
CathelicidinseCATH1eCATH2eCATH3
OthersNK-lysinPsoriasin 1eNAP-1eNAP-2Equinins (5)Hepcidin
Mature peptide sequence homologyNeighbor joining tree
Objective: selection of 12 candidates
48
α-defensins
β-defensinsβ-defensin 103β-defensin 1β-defensin 2β-defensin 3
Step 1: elimination of already known AMPs
Mature peptide sequence homologyNeighbor joining tree
Selection
49
β-defensinsβ-defensin 103β-defensin 1β-defensin 2β-defensin 3
OthersNK-lysinEquininsHepcidin
Step 2: Selection (x6)
β-defensin 103 (BDEF103):- Shares high identity (75%) and similarity (84%) with hBD3- hBD3 known to be salt tolerant, non cytotoxic for eukaryotic cells, has a broad spectrum of activity, activity on biofilms, antiviral activity, chemo-attractive activity (Dhople et al., 2006; Hazrati et al., 2006; Huang et al., 2012)
β-defensin 2 (BDEF2):- High similarity with BDEF1 and BDEF3 (75-95%)- BDEF2 has a positive net charge and a hydrophobic ratio
slightly higher
NK-lysin:- Shares high identity (68%) and similarity (76%) with porcine NK-lysin- Fragment NK-2 from porcine NK-lysin has activity against parasites (Jacobs et al., 2003)- Selection of 4 equine fragments (confidential sequences)
Selection
50
Step 2: Selection of 6 α-defensins
DEFA18
Mature peptide sequence homologyNeighbor joining tree
Selection
51
General conclusion, discussion and perpectives
eCATH1, a promising drug against rhodococcosis
Activity against R. equi and associated pathogens (independently of antibiotic-resistant profile)
Kills R. equi inside macrophages without harming the host cell
Activity in a model of rhodococcosis without inducing toxicity in doses compatible for a medical use (by extrapolation: 6-16 g depending on the animal weight)
Positive interaction with rifampicin
Short term perspective: Set up a method of peptide production in large amount and low cost to go further
52
Hypothesis
• How eCATH1 reaches intramacrophage rhodococci ?
53
phagosome
Early endosome
fusion
Macrophage
Multiplication of R. equi inside macrophages
Schlusselhuber, Anses
Fernandez-Mora et al., 2005
R. equi multiplying inside murine macrophages
Hypothesis
• How eCATH1 reaches intramacrophage rhodococci ?
“macrophages are able to internalize neutrophil
antimicrobial peptide HNP from apoptotic cells and traffick
peptide to early endosomes.” Tan et al., 2004
“[…] granule contents traffic to early endosomes, and
colocalize with mycobacteria.” Tan et al., 2006
“[…] results in decreased viability of intracellular M.
tuberculosis.”Tan et al., 2006
phagosome
Early endosome
fusion
lyse
Macrophage
54
Hypothesis• Why eCATH1 appears more effective in vivo than in vitro ?
Probable combination of direct and indirect activities (immunomodulatory properties)
- Yang et al., 2000: “LL-37 utilizes a receptor to chemoattract human peripheral blood neutrophils, monocytes, and T cells ”- Zheng et al., 2007: “LL-37 induces the release of human alpha-defensins from neutrophils”
Similarly:- Paget et al., 2012: “Bovine cathelicidin induce differential effects on neutrophil activity”
And:- Martens et al., 2005: “Protective role of neutrophils in mice experimentally infected with Rhodococcus equi”- Tan et al. 2006: “Macrophages acquire neutrophil granules for antimicrobial activity against intracellular pathogens”
Short-term perspective: study of the immunomodulatory properties of eCATH1
55
eCATH1
Resistance:– Proved to be effective against various MDR bacterial pathogens
of the horse (no cross-resistance with antibiotics including colistin)
Conlon et al., 2012; Saugar et al., 2006; Wang et al., 2011; Fedders et al., 2010
– Selection resistance: Lower rate than conventional antibiotics, modest and not stable
Zhang et al., 2005; Steinberg et al., 1997
56
Stable resistance to eCATH1 is unlikely
Short-term perspectives: evaluation of the frequency of emergence of resistance and cross-resistance with
equine AMPs
(but not impossible)
Other equine AMPs
• β-defensin 103• β-defensin 2• 4 NK-lysin fragments• α-defensin 5• α-defensin 8• α-defensin 16• α-defensin 18• α-defensin 21• α-defensin 29
57
12 new equine AMPs selected and
chemically synthetized
Short-term perspectives: refolding, characterization and in vitro antimicrobial activity
Long-term perspectives
58
Clinical trial of eCATH1 in foals infected with R.
equi
Pre-clinical development of eCATH1
Extend tests to
equine parasites &
viruses and
pathogens of
other animal
species
Structures-
Mechanism of action
Optimization of cost of
production by decreasing size
of peptides
Potential of equine AMPs (eCATH1) against septic shock
Potential of equine AMPs
againstBiofilms
Optimization of cost
of treatment by
increasing in vivo
stability of eCATH1
Acknowledgments
Dozulé laboratory for equine diseases, Anses
(France)
Anthémis horse breeding(France)
Institute of Biochemistry, Christian-Albrechts Universität zu
Kiel (Germany)Dr. Sascha Jung, Dr. Doreen Floss
Unité de Recherche Risques microbiens,
Université de Caen-Basse-Normandie (France)
Director: Pr. Alain Rincé
College of Veterinary Medicine, University of
Georgia (USA)Pr. Steeve Giguère,
Kristen Guldbech
Centre de Microscopie Appliquée à la Biologie,
SFR ICORE, Université de Caen Basse-Normandie
(France)Dr. Didier Goux
Institute of Microbiology, Catholic University of
Sacred Heart (Italy)Pr. Maurizio Sanguinetti,
Dr. Riccardo Torelli, Cecilia Martini
Department of Zoophysiology, Zoological
Institute, Christian-Albrechts-Universität zu
Kiel (Germany)Pr. Matthias Leippe
Institute of Experimental and Clinical
Pharmacology (Germany)Dr. Oliver Bruhn
Thank you for your attention !
61Anthémis horse breeding