Streptococcus pneumoniae, S. pyogenes and S. agalactiae membrane phospholipid remodelling in response to human serum

Abstract

Streptococcus pneumoniae, S. pyogenes (Group A Streptococcus; GAS) and S. agalactiae (Group B Streptococcus; GBS) are major aetiological agents of diseases in humans. The cellular membrane, a crucial site in host-pathogen interactions, is poorly characterized in streptococci. Moreover, little is known about whether or how environmental conditions influence their lipid compositions. Using normal phase liquid chromatography coupled with electrospray ionization MS, we characterized the phospholipids and glycolipids of S. pneumoniae, GAS and GBS in routine undefined laboratory medium, streptococcal defined medium and, in order to mimic the host environment, defined medium supplemented with human serum. In human serum-supplemented medium, all three streptococcal species synthesize phosphatidylcholine (PC), a zwitterionic phospholipid commonly found in eukaryotes but relatively rare in bacteria. We previously reported that S. pneumoniae utilizes the glycerophosphocholine (GPC) biosynthetic pathway to synthesize PC. Through substrate tracing experiments, we confirm that GAS and GBS scavenge lysoPC, a major metabolite in human serum, thereby using an abbreviated GPC pathway for PC biosynthesis. Furthermore, we found that plasmanyl-PC is uniquely present in the GBS membrane during growth with human serum, suggesting GBS possesses unusual membrane biochemical or biophysical properties. In summary, we report cellular lipid remodelling by the major pathogenic streptococci in response to metabolites present in human serum.

Keywords: Streptococcus; cellular membrane; lipidomics; phosphatidylcholine; phospholipids; plasmalogen.

Fig. 1.

Glycolipid and phospholipid biosynthesis pathways in S. pneumoniae , GAS and GBS. Genes of known or predicted function in each pathway are indicated. Lipids and substrates in black are common to all three species, in green are specific to S. pneumoniae (Spn), in red are specific to GBS, in blue are present in S. pneumoniae and GAS, and in purple are present in both GAS and GBS. DAG, diacylglycerol; Glc₂-DAG, diglucosyl-DAG; Gal-Glc-DAG, galactosyl-glucosyl-DAG; Lys-Glc-DAG, lysyl-glucosyl-DAG; PA, phosphatidic acid; CDP-DAG, cytidine diphosphate-DAG; PGP, PG-3-phosphate; PG, phosphatidylglycerol; Lys-PG, lysyl-phosphatidylglycerol; CL, cardiolipin; GPC, glycerophosphocholine; lysoPC, lyso-phosphatidylcholine; PC, phosphatidylcholine; Lyso-pPC, lyso-plasmanyl-PC; pPC, plasmanyl-PC. ‘*’ denotes genes identified by blastp and ‘?’ denotes unidentified genes. It is currently unknown whether GBS scavenge lyso-pPC or pPC from human serum or if it is de novo synthesized from PC. This figure combines lipids and genes described in the literature [14–19, 21–25, 32, 33] and detected lipids from Table 1.

Fig. 2.

PC and Lys-PG detection when streptococci are cultured in different media. Shown are representative positive ESI mass spectra obtained during the LC retention time of 19–20.5 min indicating the presence or absence of PC, pPC and Lys-PG in the membranes of (a) S. pneumoniae TIGR4, (b) S. pyogenes (GAS) MGAS315 and (c) S. agalactiae (GBS) COH1 when cultured in: panel 1, rich undefined medium; panel 2, defined medium; and panel 3, defined medium supplemented with 5 % (v/v) human serum. All cultures were performed in biological triplicate and representative spectra are shown.

Fig. 3.

LysoPC is scavenged by GAS and GBS to synthesize PC. (a) GAS strain NZ131 cultured in defined medium supplemented with 100 µM GPC synthesizes a very low level of PC. (b) GAS strain NZ131 grown in defined medium supplemented with 100 µM lysoPC (20 : 0) synthesizes PC. (c) No PC is detected for GBS strain COH1 cultured in defined medium supplemented with 100 µM GPC. (d) GBS strain COH1 cultured in defined medium supplemented with 100 µM lysoPC (20 : 0) synthesizes PC. Cultures were performed in biological triplicate and representative data are shown.