Hunting intracellular bacteria with antibiotics Paul M Tulkens

Hunting intracellular bacteria with antibiotics Paul M. Tulkens, MD, Ph. D Cellular and Molecular Pharmacology Unit & Centre for Clinical Pharmacy Louvain Drug Research Institute Catholic University of Louvain, Brussels, Belgium 1 st International and 12 th Iranian Congress of Microbiology Karmanshah, Iran, 24 May 2011 1

But before that, where are you from ? Belgium The medical campus of the Université catholique de Louvain Brussels The Cellular and Molecular Pharmacology Group slides are available on www. facm. ucl. ac. be Lectures 1 st International and 12 th Iranian Congress of Microbiology Karmanshah, Iran, 24 May 2011 2

Intracellular infection: bacterial strategies Carryn et al. Infect Dis Clin North Am. 2003; 17: 615 -34. 1 st International and 12 th Iranian Congress of Microbiology Karmanshah, Iran, 24 May 2011 3

Intracellular infection and recurrence/relapses: the case of S. aureus (as an example) 1 st International and 12 th Iranian Congress of Microbiology Karmanshah, Iran, 24 May 2011 4

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 1 st International and 12 th Iranian Congress of Microbiology Karmanshah, Iran, 24 May 2011 5

Pharmacochemistry and Biology to your help ! A real need. . . • Staphyloxanthin and intracellular survival • Activity of availailable antibiotics • What for the future ? 1 st International and 12 th Iranian Congress of Microbiology Karmanshah, Iran, 24 May 2011 6

Settting up the model … Seral et al. Antimicrob. Agents Chemother. 2003 47: 2283 -2292 1 st International and 12 th Iranian Congress of Microbiology Karmanshah, Iran, 24 May 2011 7

Adhesion – Invasion (early steps of infection) S. aureus virulence factors fibrinogen Ig. G Clf. A Clf. B fibronectin v. WF Sp. A elastin internalization Ebp. S α 5β 1 integrin S. aureus Fn. BPA Fn. BPB collagen Cna staphyloxanthin Protection vs. host defenses Diseases-related toxins Coa Kat. A PVL SEs ETs TSST-1 H 2 O 2 Degradatives enzymes H 2 O nucleases Eukaryotic cell proteases lipases collagenase Hemolysins Skin infections & necrotizing pneumonia Food poisoning Scald skin syndrome & impetigo Toxic shock syndrome prothrombin Hla Hlb Hlg tissues necrosis and inflammation Hld hyaluronidase Colonization - Degradation - Nutrition (late steps of infection) 1 st International and 12 th Iranian Congress of Microbiology Karmanshah, Iran, 24 May 2011 Olivier et al. , Role of virulence factors in S. aureus infections. Submitted 8

S. aureus virulence factors A. C. Olivier. Intracellular fate of Staphylococcus aureus in human phagocytic and non phagocytic cells and role of virulence factors. Ph. D Thesis, 2008 1 st International and 12 th Iranian Congress of Microbiology Karmanshah, Iran, 24 May 2011 9

Staphyloxanthin synthesis 1 st International and 12 th Iranian Congress of Microbiology Karmanshah, Iran, 24 May 2011 10

Our material…. 2 isogenic strains rsbu- and rsbu+ inhibitors of staphyloxanthin synthesis * Olivier et al. J. Infect. Dis. (2009) 200: 1367 -1370 * made by E. Oldfield, Champaign, IL 1 st International and 12 th Iranian Congress of Microbiology Karmanshah, Iran, 24 May 2011 11

The results…. Olivier et al. J. Infect. Dis. (2009) 200: 1367 -1370 1 st International and 12 th Iranian Congress of Microbiology Karmanshah, Iran, 24 May 2011 12

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 1 st International and 12 th Iranian Congress of Microbiology Karmanshah, Iran, 24 May 2011 13

Antibiotics and intracellular infection… Carryn et al. Infect Dis Clin North Am. 2003 Sep; 17(3): 615 -34. 1 st International and 12 th Iranian Congress of Microbiology Karmanshah, Iran, 24 May 2011 14

-lactams: a pharmacokinetic paradox Accumulation of antibiotics in macrophages • • • beta-lactams: 1 x aminoglycosides: <1 to 2 x ansamycins: 2 -3 x tetracyclines: 2 -4 x fluoroquinolones: 10 -20 x macrolides: 4 to > 100 x Tulkens PM, Intracellular distribution and activity of antibiotics. Eur J Clin Microbiol Infect Dis. 1991; 10: 100 -6 1 st International and 12 th Iranian Congress of Microbiology Karmanshah, Iran, 24 May 2011 15

-lactams and S. aureus. . . (MSSA) Cs Cs Emax intracellular extracellular Lemaire et al. , JAC 55: 897 -904, 2005 1 st International and 12 th Iranian Congress of Microbiology Karmanshah, Iran, 24 May 2011 16

The intracellular pharmacodynamic paradox of -lactams… Hint: p. H in vacuoles is acidic Lemaire et al. , AAC (2007) 51: 1627 -1632 1 st International and 12 th Iranian Congress of Microbiology Karmanshah, Iran, 24 May 2011 17

Back to the future… 1 st International and 12 th Iranian Congress of Microbiology Karmanshah, Iran, 24 May 2011 18

Biochemistry comes to your help… 1 st International and 12 th Iranian Congress of Microbiology Karmanshah, Iran, 24 May 2011 19

Acid p. H favors penicillin binding to PBP 2 a Lemaire et al. , JBC (2008) 283: 12769 -12776 1 st International and 12 th Iranian Congress of Microbiology Karmanshah, Iran, 24 May 2011 20

Acid p. H favors oxacillin-induced conformational change in PBP 2 a Lemaire et al. , JBC (2008) 283: 12769 -12776 1 st International and 12 th Iranian Congress of Microbiology Karmanshah, Iran, 24 May 2011 21

Screening available antibiotics Barcia-Macay et al. Antimicrob. Agents Chemother. 2006, in press 1 st International and 12 th Iranian Congress of Microbiology Karmanshah, Iran, 24 May 2011 22

New glycopeptides (oritavancin, telavancin, dalbavancin)… Hemi-synthetic derivatives derived from vancomycin teicoplanin Phase III, Intermune Targenta Awaiting FDA approval, Vicuron Pfizer Phase III, Theravance Astellas Van Bambeke, Current Opinion in Pharmacology (2004) 4: 471 -478 1 st International and 12 th Iranian Congress of Microbiology Karmanshah, Iran, 24 May 2011 23

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 1 st International and 12 th Iranian Congress of Microbiology Karmanshah, Iran, 24 May 2011 24

Telavancin … VAN lipophilic side chain TLV polar group this causes increase in bacterial membrane permeability Higgins et al. , Antimicrob Agents Chemother. (2005) 49: 1127 -34 1 st International and 12 th Iranian Congress of Microbiology Karmanshah, Iran, 24 May 2011 25

Telavancin intracellular accumulation and subcellular disposition Van Bambeke et al. , unpublished 1 st International and 12 th Iranian Congress of Microbiology Karmanshah, Iran, 24 May 2011 26

Intracellular activity of telavancin vs. vancomycin: MSSA MRSA 24 h CFU at Cmax: • vanco: 0. 5 log • TLV: 2 log Barcia-Macay et al. , JAC, in the press 1 st International and 12 th Iranian Congress of Microbiology Karmanshah, Iran, 24 May 2011 27

Intracellular activity of telavancin vs. vancomycin: VISA VRSA 24 h CFU at Cmax: • vanco: static • TLV: 1. 2 log Barcia-Macay et al. , JAC, in the press 1 st International and 12 th Iranian Congress of Microbiology Karmanshah, Iran, 24 May 2011 28

Telavancin intracellular activity in comparison with other drugs… Lemaire et al. , ISSSI 2006 – Sept. 3 -6, 2006 1 st International and 12 th Iranian Congress of Microbiology Karmanshah, Iran, 24 May 2011 29

SCVs of S. aureus as a cause of persistent foreign body infection 1 st International and 12 th Iranian Congress of Microbiology Karmanshah, Iran, 24 May 2011 30

Intracellular SCVs… 1 st International and 12 th Iranian Congress of Microbiology Karmanshah, Iran, 24 May 2011 31

Intracellular SCVs and antibiotics … Nguyen et al. , Antimicrobial Agents and Chemotherapy (2009) 53: 1434– 1442 it's hard to exceed 2 log drop… 1 st International and 12 th Iranian Congress of Microbiology Perhaps due to membrane-detabilization effect … • Domenech et al. Biochim Biophys Acta. 2009 1788: 1832 -1840 • Baudoux et al. ICAAC 2009 [C 1 -1354] Karmanshah, Iran, 24 May 2011 32

Will this be successful ? 1 st International and 12 th Iranian Congress of Microbiology Karmanshah, Iran, 24 May 2011 33

And now Listeria… Carryn et al. Infect Dis Clin North Am. 2003; 17: 615 -34. 1 st International and 12 th Iranian Congress of Microbiology Karmanshah, Iran, 24 May 2011 34

As if you were there … phagocytosis escape from vacuole in cytosol Ouadrhiri et al. Antimicrob Agents Chemother. 1999; 43: 1242 -51. 1 st International and 12 th Iranian Congress of Microbiology Karmanshah, Iran, 24 May 2011 35

A surprise … Emax Lemaire et al. J Antimicrob Chemother. 2005; 55: 897 -904. 1 st International and 12 th Iranian Congress of Microbiology Karmanshah, Iran, 24 May 2011 ampicillin is MORE active intracellularly 36

A potential explanation … through proteomics Van de Velde et al. Isolation and 2 D-DIGE Proteomic Analysis of Intracellular Listeria monocytogenes in comparison with its extracellular form. Proteomics (2009) 9: 5484 -5496. 1 st International and 12 th Iranian Congress of Microbiology Karmanshah, Iran, 24 May 2011 37

What may change in intracellular Listeria… ABC transporters synthesis of d. TDP-rhamnose UDP-glucose hexose transporters metabolism of sugars/aminosugars/glycerolipids glyceraldehyde-3 -P pentose phosphate pathway teichoic acid lipoteichoic acid Synthesis of thiamine precursors nucleotide synthesis fatty acids D-Ala-D-Ala synthesis of peptidoglycan glutamate D-alanine 1 st International and 12 th Iranian Congress of Microbiology citrate pyruvate Oxidative stress response Van de Velde et al. Isolation and 2 D-DIGE Proteomic Analysis of Intracellular Listeria monocytogenes in comparison with its extracellular form. Proteomics (2009) 9: 5484 -5496. Karmanshah, Iran, 24 May 2011 38

Anyway, the intracellular infection kept us busy and happy … Cristina Seral Pierre Baudoux Sandrine Lemaire Françoise Van Bambeke In collaboration also with : Hoang Anh Nguyen Sébastien Van de Velde Maritza Barcia. Macay Aurélie Olivier and many others § 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… 1 st International and 12 th Iranian Congress of Microbiology Karmanshah, Iran, 24 May 2011 40