soutenance thèse octobre 2012

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



Neonatal foal

Escherichia coli

EndometritisSepticemiaLung infectionWound infection


Neonatal foal


Wounds

Pseudomonas aeruginosaEndometritisWound infectionSepticemia


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

http://www.veterinaryresearch.org/content/42/1/98/figure/F1

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



In vitro activityIn vitro cytotoxicity




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

≠



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



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)


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


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






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


Cephalosporin (C1G, C3G)-resistant (11) 1-16

Ticarcillin-resistant (13) 1-16


Colistin-resistant (1) 1-2

SXT-resistant (11) 1-16

44

MIC, µg/ml

Klebsiella pneumoniae


Cephalosporin (C1G, C3G)-resistant (4) 2-4


Colistin-resistant (1) 4


Salmonella enterica serovar Typhimurium


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


Macrolide-resistant (17) 1-4

Rifampicin-resistant (20) 0.5-4

Ceftiofur-resistant (2) 2-4


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)






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


Step 1: elimination of already known AMPs


Selection

49


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


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

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Thank you for your attention !

61Anthémis horse breeding

soutenance thèse octobre 2012

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