Cell surface of Lactococcus lactis is covered by a protective polysaccharide pellicle

Abstract

In Gram-positive bacteria, the functional role of surface polysaccharides (PS) that are not of capsular nature remains poorly understood. Here, we report the presence of a novel cell wall PS pellicle on the surface of Lactococcus lactis. Spontaneous PS-negative mutants were selected using semi-liquid growth conditions, and all mutations were mapped in a single chromosomal locus coding for PS biosynthesis. PS molecules were shown to be composed of hexasaccharide phosphate repeating units that are distinct from other bacterial PS. Using complementary atomic force and transmission electron microscopy techniques, we showed that the PS layer forms an outer pellicle surrounding the cell. Notably, we found that this cell wall layer confers a protective barrier against host phagocytosis by murine macrophages. Altogether, our results suggest that the PS pellicle could represent a new cell envelope structural component of Gram-positive bacteria.

FIGURE 1.

Phase contrast (A–C) and transmission electron micrographs (D–F) of WT L. lactis control strain MG1363 (A and D) and of two long-chain mutants VES5748 (B and E) and VES5751 (C and F). The outer pellicle indicated by arrows (D) is not visible in the mutants (E and F). The scale bar represents 0.1 μm.

FIGURE 2.

Partial heteronuclear ¹H-¹³C chemical shift correlation (HSQC) spectrum of the L. lactis MG1363 polysaccharide (A) and OS1 fragment (B). The corresponding one-dimensional ¹H NMR spectrum of OS1 is also shown (B). C-H signals appear in black; CH₂ signals are in red (A) and green (B). Residues are indicated as in Table 2.

FIGURE 3.

Structure of L. lactis MG1363 PS and the oligosaccharide products of its partial hydrolysis (OS1 to OS3). A and C, Glc residues; B, Galf; E and F, GlcNAc, and D, Rha; see also Table 2.

FIGURE 4.

AFM deflection images for single L. lactis WT (A) and its pellicle-negative derivative VES5748 (B) cells during the course of the division process.

FIGURE 5.

Phagocytosis of L. lactis WT (A) and its pellicle-negative derivative VES5748 (B) by murine macrophages RAW 264.7 and J774 is shown. Micrographs of bacteria after 30 min of incubation with RAW 264.7 (A and B) and phagocytosis efficiency (C) are shown.