Hunting intracellular bacteria with antibiotics

Karmanshah, Iran, 24 May 20111st International and 12th Iranian Congress of Microbiology 1

Hunting intracellular bacteria with antibiotics

Paul M. Tulkens, MD, PhD

Cellular and Molecular Pharmacology Unit & Centre for Clinical Pharmacy

Louvain Drug Research Institute

Catholic University of Louvain,Brussels, Belgium


But before that, where are you from ?

slides are available on www.facm.ucl.ac.be Lectures

Belgium

Brussels

The medical campus of theUniversité catholique de Louvain

The Cellular and Molecular Pharmacology Group


Intracellular infection: bacterial strategies

Carryn et al. Infect Dis Clin North Am. 2003;17:615-34.


Intracellular infection and recurrence/relapses: the case of S. aureus (as an example)


Intracellular infection and recurrence/relapses: the case of S. aureus (as an example)

Phagocytic and non phagocytic cells in mastitis

Brouillette et al., Microb. Pathog. (2003) 35:159-68


A real need... Pharmacochemistryand Biologyto your help !

• Staphyloxanthin and intracellular survival

• Activity of availailable antibiotics

• What for the future ?


Settting up the model …

Seral et al. Antimicrob. Agents Chemother. 2003 47:2283-2292

0 6 12 18 24

0

2

4 extraintra

time (h)

lo

g C

FU

fro

m t

ime

0 h


S. aureus virulence factors

EbpS

ClfA ClfB

FnBPA FnBPB

SpA

IgG

vWF

elastin

fibrinogen

fibronectin

Cnacollagen

staphyloxanthin Diseases-related

toxins

Hemolysins

Hla

Hlb

Hlg

tissues necrosis and inflammation

Hld

Coa

Degradatives enzymes

S. aureus

Eukaryotic cell

internalization

α5β1integrin

nucleases

proteases

lipases

collagenase

hyaluronidase

prothrombin

KatA

H2O2

H2O

Adhesion – Invasion (early steps of infection)

Protection vs. host defenses

Colonization - Degradation - Nutrition (late steps of infection)

PVL

SEs

ETs

TSST-1 Toxic shock syndrome

Food poisoning

Skin infections & necrotizing pneumonia

Scald skin syndrome &impetigo

Olivier et al., Role of virulence factors in S. aureus infections. Submitted


S. aureus virulence factors

A.C. Olivier. Intracellular fate of Staphylococcus aureus in human phagocytic and non phagocytic cells and role of virulence factors. PhD Thesis, 2008


Staphyloxanthinsynthesis


Our material….

2 isogenic strainsrsbu- and rsbu+ inhibitors of staphyloxanthin synthesis *

Olivier et al. J. Infect. Dis. (2009) 200:1367-1370* made by E. Oldfield, Champaign, IL


The results….

Olivier et al. J. Infect. Dis. (2009) 200:1367-1370


And with other strains….

• the more staphyloxanthin is produced, the more successful is the strain for intracellular growth …

• blocking staphyloxanthin synthesis makes all strain equally ill-successful…

Olivier et al. J. Infect. Dis. (2009) 200:1367-1370


Antibiotics and intracellular infection…

Carryn et al. Infect Dis Clin North Am. 2003 Sep;17(3):615-34.


-lactams: a pharmacokinetic paradox

• beta-lactams: 1x• aminoglycosides: <1 to 2 x• ansamycins: 2-3 x• tetracyclines: 2-4 x• fluoroquinolones: 10-20 x• macrolides: 4 to > 100 x

Accumulation of antibiotics in macrophages

Tulkens PM, Intracellular distribution and activity of antibiotics. Eur J Clin Microbiol Infect Dis. 1991; 10:100-6


-lactams and S. aureus... (MSSA)

Lemaire et al., JAC 55:897-904, 2005

extracellular intracellular

Emax

Cs

Emax

Cs


The intracellular pharmacodynamic paradox of -lactams…

Hint:pH in vacuoles is acidic

Lemaire et al., AAC (2007) 51:1627-1632


Back to the future…


Biochemistry comes to your help…


Acid pH favors penicillin binding to PBP2a

Lemaire et al., JBC (2008) 283:12769-12776


Acid pH favors oxacillin-induced conformational change in PBP2a

Lemaire et al., JBC (2008) 283:12769-12776


Screening available antibiotics

Barcia-Macay et al. Antimicrob. Agents Chemother. 2006, in press


New glycopeptides (oritavancin,telavancin, dalbavancin)…

Phase III, Theravance Astellas

Awaiting FDA approval, Vicuron Pfizer

Van Bambeke, Current Opinion in Pharmacology (2004) 4:471-478

Hemi-synthetic derivatives derived from

vancomycin teicoplanin

Phase III, Intermune Targenta


Telavancin (and oritavancin) new modes of action …

• Possibility of dimerization potential increase of intrinsic activity against

D-Ala-D-Ala displaying organisms (MSSA, MRSA, VISA)

• Membrane destabilization effects… strong concentration-dependent bactericidal effect

(all strains ...) *

Beauregard et al., AAC 1995; 39:781-85


Telavancin …

lipophilic side chain

polar group

VAN

TLV

this causes increase in bacterial membrane permeability

Higgins et al., Antimicrob Agents Chemother. (2005) 49:1127-34


Telavancin intracellular accumulation and subcellular disposition

Van Bambeke et al., unpublished


Intracellular activity of telavancin vs. vancomycin: MSSA MRSA

Barcia-Macay et al., JAC, in the press

24h CFU at Cmax: • vanco: 0.5 log• TLV: 2 log


Barcia-Macay et al., JAC, in the press

24h CFU at Cmax: • vanco: static• TLV: 1.2 log

Intracellular activity of telavancin vs. vancomycin: VISA VRSA


Telavancin intracellular activity in comparison with other drugs…

Lemaire et al., ISSSI 2006 – Sept. 3-6, 2006


SCVs of S. aureus as a cause of persistent foreign body infection


Intracellular SCVs…


Intracellular SCVs and antibiotics …

it's hard to exceed 2 log

drop…Perhaps due to membrane-detabilization effect …• Domenech et al. Biochim Biophys Acta. 2009 1788:1832-1840• Baudoux et al. ICAAC 2009 [C1-1354]

Nguyen et al., Antimicrobial Agents and Chemotherapy (2009) 53:1434–1442


Will this be successful ?


And now Listeria…

Carryn et al. Infect Dis Clin North Am. 2003;17:615-34.


As if you were there …

phagocytosis escape from

vacuole

in cytosol

Ouadrhiri et al. Antimicrob Agents Chemother. 1999; 43:1242-51.


A surprise …

ampicillin is MORE active intracellularly

Lemaire et al. J Antimicrob Chemother. 2005; 55:897-904.

Emax


A potential explanation … through proteomics

Van de Velde et al. Isolation and 2D-DIGE Proteomic Analysis of Intracellular Listeria monocytogenes in comparison with its extracellular form. Proteomics (2009) 9:5484-5496.


What may change in intracellular Listeria…

pyruvate

citratefatty acids

glutamate

Synthesis of thiamine precursors

synthesis of dTDP-rhamnoseUDP-glucose

hexose transportersABC transporters

teichoic acidlipoteichoic acid

Oxidative stress response

nucleotide synthesis

syn

the

sis

of

pe

ptid

og

lyca

n

metabolism of sugars/aminosugars/glycerolipids

pentose phosphate pathway

glyceraldehyde-3-P

D-alanine

D-A

la-D

-Ala

Van de Velde et al. Isolation and 2D-DIGE Proteomic Analysis of Intracellular Listeria monocytogenes in comparison with its extracellular form. Proteomics (2009) 9:5484-5496.


Anyway, the intracellular infection Anyway, the intracellular infection kept us busy and happy …kept us busy and happy …

Cristina Cristina SeralSeral Hoang Hoang

Anh Anh NguyenNguyen

Maritza Maritza Barcia-Barcia-MacayMacay

Pierre Pierre BaudouxBaudoux

SandrineSandrineLemaireLemaire

AurélieAurélieOlivierOlivierSébastienSébastien

Van de VeldeVan de Velde

FrançoiseFrançoiseVan BambekeVan Bambeke

In collaboration also with :In collaboration also with : Y. Glupczynski, Cliniques universitaires de l'UCL à Mont-Godinne, Yvoir, Belgium A. Vergison, O. Denis, M. Struelens, Hôpital Erasme, ULB, Brussels, Belgium P. Appelbaum, Hershey Medical Center, Hershey, PA, USA and many others…

and many and many othersothers

Hunting intracellular bacteria with antibiotics

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