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INTRODUCTION

The Gram positive cell wall is formed by a thick peptidoglycan layer, decorated with proteins, polysaccharides and anionic polymers made of alternating phosphate and alditol groups called teichoic acids (TAs). Protonated D-alanyl ester residues are covalently linked to TAs and provide counterions to phosphate groups, which determine the net anionic charge of TAs.D-Ala-deficient mutants in various Gram positive species have been found to exhibit a variety of phenotypic changes, that could be linked to the resulting charge modification of their TAs. In L. lactis, the characterization of such mutants has revealed a role of D-alanyl teichoic acid synthesis in UV sensitivity, autolysis and protein secretion (Duwat et al. 1997; Nouaille et al. 2004; Steen et al. 2005).

The aim of this study was to examine the impact of D-alanylation of L. lactis TAs on the physicochemical properties of the bacterial surface and on the bacterial adhesion to solid surfaces.

Variations in the D-alanylation degree of teichoic acids in Lactococcus lactis alter resistance to cationic antimicrobials but have no effect on bacterial surface hydrophobicity and charge

Efstathios Giaouris1,2†, Romain Briandet2, Mickaël Meyrand1, Pascal Courtin1 and Marie-Pierre Chapot-Chartier1*

1Unité de Biochimie Bactérienne, UR477, INRA, Jouy-en-Josas, France, 2Unité mixte de Recherche en Bioadhesion et Hygiène des Matériaux, UMR 763, INRA-AgroParisTech, Massy, France. *e-mail: Marie-Pierre.Chapot@jouy.inra.fr

†Present address: Laboratory of Microbiology and Biotechnology of Foods, Department of Food Science and Technology, Agricultural University of Athens, Athens, Greece.

D-Ala incorporation machinery & L. lactis mutant strains

CONCLUSIONS �A correlation between the D-alanylation degree of teichoic acids and the resistance to cationic antimicrobials was observed in L. lactis in agreement with the data obtained for several other Gram positive bacteria (Peschel et al. 1999; Kristian et al. 2005; Kovacs et al. 2006; Perea Velez et al. 2007).

�The variations of the degree of D-alanine substitution of L. lactis TAs obtained in this study, do not modify the global surface physicochemical properties and bacterial adhesion to inert surfaces.

�Our results suggest that, concerning L. lactis, the decrease/increase of D-alanylation is not high enough to modify the global surface properties, or that the modification of the TAs’ structure by the substitution with D-alanine is not exposed at the cell surface.

�The modification of the cationic antimicrobial resistance probably results from variations of negative charges borne by TAs that are embedded inside the cell wall, rather than to the modification of the global bacterial surface charge.

References:• Bellon-Fontaine, M.-N., J. Rault, and C.J. van Oss. 1996. Microbial adhesion to solvents: a novel method to determine the electron donor/electron acceptor or Lewis acid-base properties of microbial cells. Colloids Surf. B: Biointerfaces 7:47-53.• De Ruyter,P.G., O.P. Kuipers and W.M. De Vos. 1996. Controlled gene expression systems for Lactococcus lactis with the food-grade inducer nisin. Appl. Environ. Microbiol. 62:3662-3667.• Duwat, P., A. Cochu, S.D. Ehrlich, and A. Gruss. 1997. Characterization of Lactococcus lactis UV-sensitive mutants obtained by ISS1 transposition. J. Bacteriol. 179:4473-4479.• Kovacs, M., A. Halfmann, I. Fedtke, M. Heintz, A. Peschel, W. Vollmer, R. Hakenbeck, and R. Bruckner. 2006. A functional dlt operon, encoding proteins required for incorporation of D-alanine in teichoic acids in Gram-positive bacteria, confers resistance to cationic antimicrobial peptides in Streptococcus pneumoniae. J. Bacteriol. 188:5797-5805.

• Kristian, S.A., V. Datta, C. Weidenmaier, R. Kansal, I. Fedtke, A. Peschel, R.L. Gallo, and V. Nizet. 2005. D-alanylation of teichoic acids promotes group A Streptococcus antimicrobial peptide resistance, neutrophil survival, and epithelial cell invasion. J. Bacteriol. 187:6719-6725.• Lambert, R.J.W., and R. Lambert. 2003. A model for the efficacy of combined inhibitors. J. Appl. Microbiol. 95:734-743.• Neuhaus, F.C., and J. Baddiley. 2003. A continuum of anionic charge: structures and functions of D-alanyl-teichoic acids in Gram-positive bacteria. Microbiol. Mol. Biol. Rev. 67:686-723.• Nouaille, S., J. Commissaire, J.J. Gratadoux, P. Ravn, A. Bolotin, A. Gruss, Y. Le Loir, and P. Langella. 2004. Influence of lipoteichoic acid d-alanylation on protein secretion in Lactococcus lactis as revealed by random mutagenesis. Appl. Environ. Microbiol. 70:1600-1607. • Perea Velez, M., T.L.A. Verhoeven, C. Draing, S.V. Aulock, M. Pfitzenmaier, A. Geyer, I. Lambrichts, C. Grangette, B. Pot, J. Vanderleeyden, and S.C.J. De Keersmaecker. 2007. Functional analysis of D-alanylation of lipoteichoic acid in the probiotic strain Lactobacillus rhamnosus GG. App. Environ. Microbiol. 73:3595-3604.

• Peschel, A., M. Otto, R.W. Jack, H. Kalbacher, G. Jung, and F. Gotz. 1999. Inactivation of the dlt operon in Staphylococcus aureus confers sensitivity to defensins, protegrins, and other antimicrobial peptides. J. Biol. Chem. 274:8405-8410.• Steen, A., E. Palumbo, M. Deghorain, P. Sandro Cocconcelli, J. Delcour, O.P. Kuipers, J. Kok, G. Buist and P. Hols. 2005. Autolysis of Lactococcus lactis is increased upon D-alanine depletion of peptidoglycan and lipoteichoic acids. J. Bacteriol. 187:114-124.

Sensitivity to cationic antimicrobials

E. G. was recipient of a Marie Curie fellowship of the LABhealthEST project (contract MEST-CT-2004-514428).

Quantification D-Ala esterified to teichoic acids

Bacterial surface physicochemical properties

Adhesion on solid surfaces (3 h in 1,5 mM NaCl at 25oC)

dltA dltB dltCdltD

4274 bp

thiE mgtA

�Two different constructions were made in order to overexpress the dlt operon in L. lactis MG1363 and NZ9000 (MG1363, pepN::nisRK):

�Release of D-Ala from whole cells by alkaline hydrolysis, as reported previously for group A Streptococcus (Kristian et al. 2005). �Quantification of released D-Ala by HPLC (after its derivatizationwith Marfey’s reagent).

�dltD gene inactivation results in the almost absence of released D-Ala.

� Both dlt overexpressing strains exhibit an increase of the quantity of D-Ala released, compared with their control strains (1.2- and 1.5-fold increase respectively).

�MIC values for nisin and lysozyme were determined using an optical density modeling method, described by Lambert and Lambert (2003).

� The MICs of nisin and lysozyme for the dltD negative mutant are reduced compared to those of MG1363.� dlt overexpressing strains are more resistant to both nisin and lysozyme, compared to their respective control strains.

�The hydrophobic/hydrophilic character and the Lewis acid/base characteristics were assessed by the Microbial Adhesion To Solvents (MATS) (Bellon-Fontaine et al. 1996).

� The dlt operon of L. lactis MG1363 comprises four genes (dltA, dltB, dltC and dltD), that catalyze the incorporation of D-alanine residues into TAs:

�cloning the promoterless dlt operon under the control of nisin-inducible promoter of pNZ8048 plasmid (de Ruyter et al., 1996) ���� NZ9000(pNZdlt) mutant strain.� Control strain: NZ9000(pNZ), containing the empty pNZ plasmid.

�cloning the dlt operon with its own promoter into the high copy number vector pILN13:���� MG1363(pILNdlt) mutant strain.� Control strain: MG1363(pILN), containing the empty pILN13 plasmid.

�MG1363dltD mutant strain (kind gift from A. Gruss, UBLO, INRA, Jouy-en-Josas) It was previously obtained by mutagenesis with the transposition vector pGhost9::ISS1 (Duwat et al. 1997)

� L. lactis MG1363 strain deficient in D-alanylation:

� No significant differences at the overall net surface charge areobserved between each mutant and its respective control strain.

�Adhesion of bacteria on polystyrene microplates was quantified by crystal violet staining and OD575nm measurements. � Adhesion of bacteria on glass slides was quantified by acridine orange staining and estimation of surface covered by bacteria by epifluorescence microscopy.

� No significant differences in adhesion to hydrophobic polystyrene and hydrophilic glass are observed between the strains. �This result correlates with the absence of differences between strains regarding their cell surface physicochemical properties.

MG1363(pILNdlt)MG1363(pILNdlt)

MG1363(pILN)

MG1363(pILN)

MG1363

MG1363

MG1363dltD

MG1363dltD

NZ9000(pNZdlt)

NZ9000(pNZdlt)

NZ9000(pNZ)

NZ9000(pNZ)

LYSOZYME (µg/ml)NISIN (ng/ml)

MG1363

MG1363dltD

MG1363(pILN)

MG1363(pILNdlt)

NZ9000(pNZ)

NZ9000(pNZdlt)

nmol

/mg

of d

ried

cel

ls

STRAINS POLYSTYRENE (OD575nm) GLASS (% SURF. COVER.)MG1363 0,02 ± 0,01 31,0 ± 5,8

MG1363dltD 0,02 ± 0,01 25,6 ± 1,9MG1363(pILN) 0,03 ± 0,01 28,9 ± 2,4

MG1363(pILNdlt ) 0,03 ± 0,01 33,4 ± 4,2NZ9000(pNZ) 0,03 ± 0,01 31 ± 2,5

NZ9000(pNZdlt ) 0,03 ± 0,01 27,7 ± 2

ADHESION

�Model for incorporation of D-alanyl ester residues into TAs (from Neuhaus and Baddiley, 2003):

�The D-alanyl carrier protein ligase (Dcl, encoded by dltA) activates D-Ala by use of ATP and ligates it to the D-alanyl carrier protein (Dcp, encoded by dltC).

�DltB is a transmembrane protein proposed to be involved in the secretion of the activated D-alanyl-Dcp complex outside the cytoplasmic membrane where D-alanylation occurs.

�DltD is a membrane-anchored protein that facilitates the ligation of Dcp with activated D-Ala and removes the mischarged Dcp proteins.

�The electrical properties of the bacterial surfaces were assessed by electrophoretic mobility (E.M.) measurements.

� No significant differences between strains regarding their surface hydrophobicity and polarity.

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MG1363

MG1363dltD

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MG1363(pILN)

MG1363(pILNdlt)

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NZ9000(pNZ)

NZ9000(pNZdlt)

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MG1363

MG1363dltD

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MG1363(pILN)

MG1363(pILNdlt)

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NZ9000(pNZ)

NZ9000(pNZdlt)