Streptococcus gordonii Type I Lipoteichoic Acid Contributes to Surface Protein Biogenesis

Abstract

Lipoteichoic acid (LTA) is an abundant polymer of the Gram-positive bacterial cell envelope and is essential for many species. Whereas the exact function of LTA has not been elucidated, loss of LTA in some species affects hydrophobicity, biofilm formation, and cell division. Using a viable LTA-deficient strain of the human oral commensal Streptococcus gordonii, we demonstrated that LTA plays an important role in surface protein presentation. Cell wall fractions derived from the wild-type and LTA-deficient strains of S. gordonii were analyzed using label-free mass spectroscopy. Comparisons showed that the abundances of many proteins differed, including (i) SspA, SspB, and S. gordonii 0707 (SGO_0707) (biofilm formation); (ii) FtsE (cell division); (iii) Pbp1a and Pbp2a (cell wall biosynthesis and remodeling); and (iv) DegP (envelope stress response). These changes in cell surface protein presentation appear to explain our observations of altered cell envelope homeostasis, biofilm formation, and adhesion to eukaryotic cells, without affecting binding and coaggregation with other bacterial species, and provide insight into the phenotypes revealed by the loss of LTA in other species of Gram-positive bacteria. We also characterized the chemical structure of the LTA expressed by S. gordonii Similarly to Streptococcus suis, S. gordonii produced a complex type I LTA, decorated with multiple d-alanylations and glycosylations. Hence, the S. gordonii LTA appears to orchestrate expression and presentation of cell surface-associated proteins and functions.IMPORTANCE Discovered over a half-century ago, lipoteichoic acid (LTA) is an abundant polymer found on the surface of Gram-positive bacteria. Although LTA is essential for the survival of many Gram-positive species, knowledge of how LTA contributes to bacterial physiology has remained elusive. Recently, LTA-deficient strains have been generated in some Gram-positive species, including the human oral commensal Streptococcus gordonii The significance of our research is that we utilized an LTA-deficient strain of S. gordonii to address why LTA is physiologically important to Gram-positive bacteria. We demonstrate that in S. gordonii, LTA plays an important role in the presentation of many cell surface-associated proteins, contributing to cell envelope homeostasis, cell-to-cell interactions in biofilms, and adhesion to eukaryotic cells. These data may broadly reflect a physiological role of LTA in Gram-positive bacteria.

Keywords: Gram-positive bacteria; LTA; Streptococcus gordonii; cell wall; lipoteichoic acid; surface proteins.

FIG 1

ltaS deletion leads to loss of LTA. (A) ltaS deletion was confirmed by PCR amplification of the wild-type (WT) and the LTA-deficient (ΔltaS) genomic DNA with the primer pair ltaS up For and ltaS Dn Rev. (B) Mouse anti-LTA antibody was used to detect LTA presence on cell wall fractions from the WT strain, the ΔltaS strain, and the complemented LTA deletion strain (ΔltaS^c) using Western blotting.

FIG 2

LTA structure. (A) Proposed structure of S. gordonii type I LTA with glycerol phosphate (X) and terminal glucose-glycerol phosphate (Y) repeat units. (B) NMR spectrum of purified LTA extracted from S. gordonii containing two distinct anomeric proton signals. (C) Purified S. gordonii LTA was also subjected to electrospray ionization-mass spectrometry after monomerization by hydrofluoric acid. Multiply modified glycerol signals are observed at m/z 326.15, 348.14, 439.14, and 488.20. (D) Carbohydrate composition analysis of monomerized LTA by GC/MS (carbohydrate composition) identified glucose as the sole monosaccharide residue in the S. gordonii LTA, comprising 43.2% (mol%) versus 56.8% of glycerol.

FIG 3

ltaS deletion affects growth and chain length. (A) Growth curve of S. gordonii strains (WT, ΔltaS, and ΔltaS^c) as determined by optical density (λ = 600 nm). Data represent means of results from three independent biological replicates (± standard deviations [SD]). (B) Number of cells per chain of S. gordonii in samples from WT and ΔltaS strains collected after 6 and 12 h of growth visualized by light microscopy. Each data point represents one chain. Bars on top of the graph show statistical significance (P < 0.05) as determined by analysis of variance (ANOVA).

FIG 4

SEM of S. gordonii biofilms. S. gordonii strains (WT, ΔltaS, and ΔltaS^c) were allowed to form biofilm on saliva-coated hydroxyapatite disks for 12 h. Biofilms were visualized at ×2,500 (A [WT strain], B [ΔltaS strain], and C [ΔltaS^c strain]) and ×20,000 (D [WT strain], E [ΔltaS strain], and F [ΔltaS^c strain]).

FIG 5

LTA is involved in biofilm formation and interaction with oral keratinocytes. (A) Quantification of biofilm biomass of S. gordonii WT, ΔltaS, ΔltaS^c, and ΔsrtA strains on saliva-coated polystyrene wells. Bars represent means of results from six biological replicates ± SD. *, P < 0.05. (B) Quantitative coaggregation of S. gordonii WT and ΔltaS strains with F. nucleatum, P. gingivalis, and A. naeslundii with or without 50 mM l-arginine (R). Bars represent means of results from six biological replicates ± SD. *, P < 0.05. (C) Number of S. gordonii cell chains from the WT, ΔltaS, and ΔsrtA strains found associated with immortalized human oral keratinocytes (OKF6/TERT-2). A total of 2,921 cells derived from three separate experiments were visualized. (D) Relative hydrophobicity levels of S. gordonii ΔltaS strain, compared to the WT, as determined by association with hexadecane. The bar represents the mean of results from six biological replicates ± SD.

FIG 6

ltaS deletion affects cell wall-associated protein expression. Proteins (60 μg) from cell wall and cell membrane fractions from two biological replicates of WT and ΔltaS strains were resolved on a 4%-to-20%-gradient SDS-PAGE gel and stained with GelCode Blue Safe protein stain.

FIG 7

ltaS deletion affects SspAB and SGO_0707 expression. (A) Protein (60 μg) isolated from the cell wall fraction of two biological replicates of the WT and ΔltaS strains was resolved on a 4%-to-20%-gradient SDS-PAGE gel and transferred to a nitrocellulose membrane. Levels of SspAB were detected by Western immunoblot analysis performed with the anti-P1 antibody. (B) Cell wall fractions (20 μg) of the WT and ΔltaS strains were separated by isoelectric focusing (pH 4 to 7) in the first dimension and 7% SDS-PAGE in the second dimension. The identity of the major spots is shown. (C) Relative expression levels of SGO_0707 and sspA were determined during the exponential-growth phase in chemically defined medium (FMC) by qPCR analysis of total RNA extracted from the WT, ΔltaS, and ΔltaS^c strains at 37°C. Bars represent means of results from six biological replicates ± SD.