Cell Wall Glycans Mediate Recognition of the Dairy Bacterium Streptococcus thermophilus by Bacteriophages

Abstract

Receptors on the cell surfaces of bacterial hosts are essential during the infection cycle of bacteriophages. To date, the phage receptors of the industrial relevant dairy starter bacterium Streptococcus thermophilus remain elusive. Thus, we set out to identify cell surface structures that are involved in host recognition by dairy streptococcal phages. Five industrial S. thermophilus strains sensitive to different phages (pac type, cos type, and the new type 987), were selected to generate spontaneous bacteriophage-insensitive mutants (BIMs). Of these, approximately 50% were deselected as clustered regularly interspaced short palindromic repeat (CRISPR) mutants, while the other pool was further characterized to identify receptor mutants. On the basis of genome sequencing data, phage resistance in putative receptor mutants was attributed to nucleotide changes in genes encoding glycan biosynthetic pathways. Superresolution structured illumination microscopy was used to visualize the interactions between S. thermophilus and its phages. The phages were either regularly distributed along the cells or located at division sites of the cells. The cell wall structures mediating the latter type of phage adherence were further analyzed via phenotypic and biochemical assays. Altogether, our data suggested that phage adsorption to S. thermophilus is mediated by glycans associated with the bacterial cell surface. Specifically, the pac-type phage CHPC951 adsorbed to polysaccharides anchored to peptidoglycan, while the 987-type phage CHPC926 recognized exocellular polysaccharides associated with the cell surface.IMPORTANCEStreptococcus thermophilus is widely used in starter cultures for cheese and yoghurt production. During dairy fermentations, infections of bacteria with bacteriophages result in acidification failures and a lower quality of the final products. An understanding of the molecular factors involved in phage-host interactions, in particular, the phage receptors in dairy bacteria, is a crucial step for developing better strategies to prevent phage infections in dairy plants.

Keywords: Streptococcus thermophilus; adsorption; bacteriophages; cell wall; glycans; polysaccharides; receptors.

FIG 1

Changes in phage binding to S. thermophilus wild-type strains and their phage-resistant mutants. Phage DNA was labeled with SYBR Gold, and the green fluorescence was visualized. (a) Phage CHPC1057 adsorbs to its host STCH_09 (panel 1) and does not adsorb to STCH_09_BIM (panel 2). (b) Phage CHPC926 adsorbs to its host STCH_15 (panel 1), has reduced adsorption with STCH_15_BIM_2 (panel 2), and does not adsorb to STCH_15_BIM_1, STCH_15_BIM_3, STCH_15_BIM_4, or STCH_15_BIM_5 (panels 3 to 6, respectively). Scale bars, 1 μm.

FIG 2

Fluorescence imaging of phage binding to S. thermophilus strains. (a) Adsorption of phages to their hosts was visualized with a conventional fluorescence microscope after labeling phage DNA with SYBR Gold. (b) Superresolution structured illumination microscopy (SR-SIM) images of bacterial cells stained with Nile red (red) and mixed with SYBR Gold DNA-labeled phages (green). Panels with phages and their host strains: 1, CHPC926 and STCH_15; 2, CHPC951 and STCH_12; 3, CHPC1057 and STCH_09; 4, CHPC1014 and STCH_13; 5, CHPC1046 and STCH_14. Two binding patterns are observed: spotty (panel numbers 1, 2, and 3) or diffused (panel numbers 4 and 5). Scale bars, 1 μm.

FIG 3

Superresolution structured illumination microscopy (SR-SIM) images of phage binding to S. thermophilus strains. The pink lines indicate the distance between phage capsids, containing SYBR Gold-labeled DNA (green), and bacterial membranes, stained with Nile red (red). The presented values correspond to the lengths of phage tails and are the averages from 80 measurements. Panels with phages and their host strains: 1, CHPC926 and STCH_15; 2, CHPC951 and STCH_12; 3, CHPC1057 and STCH_09; 4, CHPC1014 and STCH_13; 5, CHPC1046 and STCH_14. Scale bars, 0.5 μm.

FIG 4

Fluorescence imaging of phage binding to cellular fractions of S. thermophilus. Phage DNA was labeled with SYBR Gold and the green fluorescence was visualized. (a) Phage CHPC951 was mixed with cellular fractions of STCH_12. Phage CHPC926 was mixed with cellular fractions of STCH_15 (b), cellular fractions of STCH_15_BIM_1 (c), or cellular fractions of STCH_15_BIM_2 (d). The following samples were used: 1, cells in exponential phase; 2, cells devoid of surface enzymes, membranes, and membrane proteins; 3, purified cell walls; 4, purified peptidoglycan. Scale bars, 1 μm.

FIG 5

Microscopy images of India ink negatively stained S. thermophilus strains. Staining was performed for two wild-type strains and two phage-resistant mutants: 1, STCH_15; 2, STCH_15_BIM_1; 3, STCH_15_BIM_2; 4, STCH_12. Presence of capsular polysaccharide is indicated with arrows. Scale bars, 1 μm.

FIG 6

Profiles of monosaccharide compositions in purified cell walls isolated from S. thermophilus strains. HPAEC-PAD analyses were performed for cell walls purified from two wild-type strains (STCH_12 and STCH_15) and two phage-resistant mutants (STCH_15_BIM_1 and STCH_15_BIM_2). Standards for different saccharides were eluted under the same conditions to enable peak identification. Differences in the heights of glucosamine and glucose peaks are indicated with arrows.