The Length of Lipoteichoic Acid Polymers Controls Staphylococcus aureus Cell Size and Envelope Integrity

ABSTRACT The opportunistic pathogen Staphylococcus aureus is protected by a cell envelope that is crucial for viability. In addition to peptidoglycan, lipoteichoic acid (LTA) is an especially important component of the S. aureus cell envelope. LTA is an anionic polymer anchored to a glycolipid in the outer leaflet of the cell membrane. It was known that deleting the gene for UgtP, the enzyme that makes this glycolipid anchor, causes cell growth and division defects. In Bacillus subtilis, growth abnormalities from the loss of ugtP have been attributed to both the absence of the encoded protein and the loss of its products. Here, we show that growth defects in S. aureus ugtP deletion mutants are due to the long, abnormal LTA polymer that is produced when the glycolipid anchor is missing from the outer leaflet of the membrane. Dysregulated cell growth leads to defective cell division, and these phenotypes are corrected by mutations in the LTA polymerase gene, ltaS, that reduce polymer length. We also show that S. aureus mutants with long LTA are sensitized to cell wall hydrolases, beta-lactam antibiotics, and compounds that target other cell envelope pathways. We conclude that control of LTA polymer length is important for S. aureus physiology and promotes survival under stressful conditions, including antibiotic stress. IMPORTANCE Methicillin-resistant Staphylococcus aureus (MRSA) is a common cause of community- and hospital-acquired infections and is responsible for a large fraction of deaths caused by antibiotic-resistant bacteria. S. aureus is surrounded by a complex cell envelope that protects it from antimicrobial compounds and other stresses. Here, we show that controlling the length of an essential cell envelope polymer, lipoteichoic acid, is critical for controlling S. aureus cell size and cell envelope integrity. We also show that genes involved in LTA length regulation are required for resistance to beta-lactam antibiotics in MRSA. The proteins encoded by these genes may be targets for combination therapy with an appropriate beta-lactam.

The length of lipoteichoic acid polymers controls Staphylococcus aureus cell size and envelope integrity

ABSTRACTThe opportunistic pathogen Staphylococcus aureus is protected by a cell envelope that is crucial for viability. In addition to peptidoglycan, lipoteichoic acid (LTA) is an especially important component of the S. aureus cell envelope. LTA is an anionic polymer anchored to a glycolipid in the outer leaflet of the cell membrane. It was known that deleting the gene for UgtP, the enzyme that makes this glycolipid anchor, causes cell growth and division defects. In Bacillus subtilis, growth abnormalities from the loss of ugtP have been attributed to the absence of the encoded protein, not to loss of its enzymatic activity. Here, we show that growth defects in S. aureus ugtP deletion mutants are due to the long, abnormal LTA polymer that is produced when the glycolipid anchor is missing from the outer leaflet of the membrane. Dysregulated cell growth leads to defective cell division, and these phenotypes are corrected by mutations in the LTA polymerase, ltaS, that reduce polymer length. We also show that S. aureus mutants with long LTA are sensitized to cell wall hydrolases, beta-lactam antibiotics, and compounds that target other cell envelope pathways. We conclude that control of LTA polymer length is important for S. aureus physiology and promotes survival under stressful conditions, including antibiotic stress.IMPORTANCEMethicillin-resistant Staphylococcus aureus (MRSA) is a common cause of community- and hospital-acquired infections and is responsible for a large fraction of deaths caused by antibiotic-resistant bacteria. S. aureus is surrounded by a complex cell envelope that protects it from antimicrobial compounds and other stresses. Here we show that controlling the length of an essential cell envelope polymer, lipoteichoic acid, is critical for controlling S. aureus cell size and cell envelope integrity. We also show that genes involved in LTA length regulation are required for resistance to beta-lactam antibiotics in MRSA. The proteins encoded by these genes may be targets for combination therapy with an appropriate beta-lactam.

Alanylated lipoteichoic acid primer in Bacillus subtilis

Lipoteichoic acid is a major lipid-anchored polymer in Gram-positive bacteria such asBacillus subtilis. This polymer typically consists of repeating phosphate-containing units and therefore has a predominant negative charge. The repeating units are attached to a glycolipid anchor which has a diacylglycerol (DAG) moiety attached to a dihexopyranose head group. D-alanylation is known as the major modification of type I and type IV lipoteichoic acids, which partially neutralizes the polymer and plays important roles in bacterial survival and resistance to the host immune system. The biosynthesis pathways of the glycolipid anchor and lipoteichoic acid have been fully characterized. However, the exact mechanism of D-alanyl transfer from the cytosol to cell surface lipoteichoic acid remains unclear. Here I report the use of mass spectrometry in the identification of possible intermediate species in the biosynthesis and D-alanylation of lipoteichoic acid: the glycolipid anchor, nascent lipoteichoic acid primer with one phosphoglycerol unit, as well as mono- and di-alanylated forms of the lipoteichoic acid primer. Monitoring these species as well as the recently reported D-alanyl-phosphatidyl glycerol should aid in shedding light on the mechanism of the D-alanylation pathway of lipoteichoic acid.

Alanylated lipoteichoic acid primer in Bacillus subtilis

Lipoteichoic acid is a major lipid-anchored polymer in Gram-positive bacteria such asBacillus subtilis. This polymer typically consists of repeating phosphoglycerol or phosphoribitol units and therefore has a predominant negative charge. The repeating units are attached to a glycolipid anchor which has a diacylglycerol (DAG) moiety attached to a dihexopyranose head group. D-alanylation is known as the major modification of lipoteichoic acid, which partially neutralizes the polymer and plays important roles in bacterial survival and resistance to the host immune system. The biosynthesis pathways of the glycolipid anchor and lipoteichoic acid have been fully characterized. However, the exact mechanism of D-alanyl transfer from the cytosol to cell surface lipoteichoic acid remains unclear. Here I report the use of mass spectrometry in the identification of intermediate species in the biosynthesis and D-alanylation of lipoteichoic acid: the glycolipid anchor, nascent lipoteichoic acid primer with one phosphoglycerol unit, as well as mono- and di-alanylated forms of the lipoteichoic acid primer. Monitoring these species as well as the recently reported D-alanyl-phosphatidyl glycerol would aid in shedding light on the mechanism of the D-alanylation pathway of lipoteichoic acid.

Localization and expression of the Bacillus subtilis dl-endopeptidase LytF are influenced by mutations in LTA synthases and glycolipid anchor synthetic enzymes

Bacillus subtilis LytF plays a principal role in cell separation through its localization at the septa and poles on the vegetative cell surface. In this study, we found that a mutation in a major lipoteichoic acid (LTA) synthase gene – ltaS – results in a considerable reduction in the σD-dependent transcription of lytF. The lytF transcription was also reduced in mutants that affected glycolipid anchor biosynthesis. Immunofluorescence microscopy revealed that both the numbers of cells expressing LytF and the LytF foci in these mutants were decreased. In addition, the transcriptional activity of lytF was almost abolished in the double (ltaS yfnI), triple (ltaS yfnI yqgS), and quadruple (ltaS yfnI yqgS yvgJ) mutants during vegetative growth. Cell separation defects in these mutants were partially restored with artificial expression of LytF. Interestingly, when lytF transcription was induced in the ltaS single or multiple mutants, LytF was localized not only at the septum, but also along the sidewall. The amounts of LytF bound to cell wall in the single (ltaS) and double (ltaS yfnI) mutants gradually increased as compared with that in the WT strain, and those in the triple (ltaS yfnI yqgS) and quadruple mutants were almost similar to that in the double mutant. Moreover, reduction of the lytF transcription and chained cell morphology in the ltaS mutant were completely restored with artificial induction of the yqgS gene. These results strongly suggest that LTA influences the temporal, σD-dependent transcription of lytF and is an additional inhibitory component to the vegetative cell separation enzyme LytF.

Characterization of a Lactobacillus gasseri JCM 1131TLipoteichoic Acid with a Novel Glycolipid Anchor Structure

ABSTRACTWe determined the chemical structure of lipoteichoic acid (LTA) fromLactobacillus gasseriJCM 1131T. The repeating unit was comprised of glycerolphosphate and 2-alanylglycerolphosphate. The glycolipid anchor was tetrahexosylglycerol with two or three acyl groups. To our knowledge, this is the first demonstration of a tetrahexose structure in an LTA glycolipid anchor.