Streptococcus mitis and S. oralis Lack a Requirement for CdsA, the Enzyme Required for Synthesis of Major Membrane Phospholipids in Bacteria

Abstract

Synthesis and integrity of the cytoplasmic membrane are fundamental to cellular life. Experimental evolution studies have hinted at unique physiology in the Gram-positive bacteria Streptococcus mitis and S. oralis These organisms commonly cause bacteremia and infectious endocarditis (IE) but are rarely investigated in mechanistic studies of physiology and evolution. Unlike in other Gram-positive pathogens, high-level (MIC ≥ 256 μg/ml) daptomycin resistance rapidly emerges in S. mitis and S. oralis after a single drug exposure. In this study, we found that inactivating mutations in cdsA are associated with high-level daptomycin resistance in S. mitis and S. oralis IE isolates. This is surprising given that cdsA is an essential gene for life in commonly studied model organisms. CdsA is the enzyme responsible for the synthesis of CDP-diacylglycerol, a key intermediate for the biosynthesis of all major phospholipids in prokaryotes and most anionic phospholipids in eukaryotes. Lipidomic analysis by liquid chromatography-mass spectrometry (LC-MS) showed that daptomycin-resistant strains have an accumulation of phosphatidic acid and completely lack phosphatidylglycerol and cardiolipin, two major anionic phospholipids in wild-type strains, confirming the loss of function of CdsA in the daptomycin-resistant strains. To our knowledge, these daptomycin-resistant streptococci represent the first model organisms whose viability is CdsA independent. The distinct membrane compositions resulting from the inactivation of cdsA not only provide novel insights into the mechanisms of daptomycin resistance but also offer unique opportunities to study the physiological functions of major anionic phospholipids in bacteria.

Keywords: Streptococcus; daptomycin; lipidomics.

FIG 1

Biosynthetic pathway of the major phospholipids in bacteria. Abbreviations: PA, phosphatidic acid; CDP-DAG, CDP-diacylglycerol; PS, phosphatidylserine; PE, phosphatidylethanolamine; G-3-P, glycerol-3-phosphate; PG-P, phosphatidylglycerol-3-P; PG, phosphatidylglycerol; CL, cardiolipin.

FIG 2

Normal-phase LC-ESI/MS analysis of the total lipid extracts of DAP-susceptible and DAP-resistant strains of S. mitis. The y-axis numbers represent ion intensities of arbitrary units. PG and CL are major lipids found in S. mitis 1643. PG and CL are absent in the DAP-resistant derivative 1643-HA04. After passage without selection, DAP susceptibility was restored in 1643-HA14, as were PG and CL levels.

FIG 3

Normal-phase LC-ESI/MS analysis of the total lipid extracts of DAP-susceptible and DAP-resistant strains of S. oralis. The y-axis numbers represent ion intensities of arbitrary units. PG and CL are major lipids found in DAP-susceptible S. oralis 1647 and 1648. PG and CL are missing from DAP-resistant derivatives 1647-HA06 and 1648-HA08. Upon restoration of DAP susceptibility in 1647-HA16 and 1648-HA18, PG levels return to normal. CL levels are restored in 1648-HA18 but not 1647-HA16.

FIG 4

Summary of CdsA alterations identified in S. mitis and S. oralis after DAP selection. CdsA is depicted as a black rectangle, with amino acid positions indicated at the bottom. Green stripes represent active sites identified previously in Thermotoga maritima (31). Red stripes represent the original mutations identified in the broth-derived DAP-resistant strains. Asterisks depict premature stop codons, diamonds depict amino acid substitutions, triangles depict a nucleotide coding region deletion event of 1 to 2 bp, and the large lines at the top depict large areas of deletion identified in spontaneous-resistance studies on DAP agar. HA04, S. mitis 1643-HA04; HA06, S. oralis 1647-HA06; HA08, S. oralis 1648-HA08.