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, Mickal Meyrand1, Pascal Courtin1 and Marie-Pierre Chapot-Chartier1*
1Unit

de Biochimie Bactrienne, UR477, INRA, Jouy-en-Josas, France, 2Unit mixte de Recherche en Bioadhesion et Hygine des Matriaux, UMR 763, INRA-AgroParisTech, Massy, France. *e-mail: Marie-Pierre.Chapot@jouy.inra.fr
address: Laboratory of Microbiology and Biotechnology of Foods, Department of Food Science and Technology, Agricultural University of Athens, Athens, Greece.

Present

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.

D-Ala incorporation machinery & L. lactis mutant strains

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: thiE dltA dltB dltC
mgtA dltD
70

Sensitivity to cationic antimicrobials

MIC values for nisin and lysozyme were determined using an optical density modeling method, described by Lambert and Lambert (2003).
NISIN (ng/ml)
MG1363 MG1363(pILNdlt) NZ9000(pNZdlt)
400 350 300

LYSOZYME (g/ml)
MG1363(pILNdlt) NZ9000(pNZdlt)

4274 bp

60 50

MG1363 MG1363(pILN)

Model for incorporation of D-alanyl ester residues into TAs (from Neuhaus and Baddiley, 2003):
The D-alanyl carrier protein ligase (Dcl, encoded by

40 30 20 10 0

MG1363(pILN) MG1363dltD

250 200 150

NZ9000(pNZ) MG1363dltD

NZ9000(pNZ)

100 50 0

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-alanylDcp 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 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.

Bacterial surface physicochemical properties

The hydrophobic/hydrophilic character and the Lewis acid/base characteristics were assessed by the Microbial Adhesion To Solvents (MATS) (Bellon-Fontaine et al. 1996). No significant differences between strains regarding their surface hydrophobicity and polarity. The electrical properties of the bacterial surfaces were assessed by electrophoretic mobility (E.M.) measurements.
0 2 -1 3 4

L. lactis MG1363 strain deficient in D-alanylation:

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)

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

pH
5 6 7 8

0 2 -1 3 4

pH
5 6 7 8

pH 0 2 -1 3 4 5 6 7 8

MG1363 MG1363dltD

MG1363(pILN) MG1363(pILNdlt)

NZ9000(pNZ) NZ9000(pNZdlt)

-2

-2

-2 E.M. -3 -4 -5

-3

Quantification D-Ala esterified to teichoic acids

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 derivatization with Marfeys reagent).
25
MG1363(pILNdlt) NZ9000(pNZdlt)

-4

-5

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

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

nmol/mg of dried cells
20
MG1363 MG1363(pILN) NZ9000(pNZ)

15

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.
ADHESION STRAINS POLYSTYRENE (OD575nm) GLASS (% SURF. COVER.) MG1363 0,02 0,01 31,0 5,8 MG1363dltD 0,02 0,01 25,6 1,9 MG1363(pILN) 0,03 0,01 28,9 2,4 MG1363(pILNdlt ) 0,03 0,01 33,4 4,2 NZ9000(pNZ) 0,03 0,01 31 2,5 NZ9000(pNZdlt ) 0,03 0,01 27,7 2

10

5
MG1363dltD

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).

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.

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