Molecular analysis of lipoteichoic acid from Streptococcus agalactiae

Abstract

A method for the analysis of lipoteichoic acid (LTA) by polyacrylamide gel electrophoresis (PAGE) is described. Purified LTA from Streptococcus agalactiae tended to smear in the upper two-thirds of a 30 to 40% linear polyacrylamide gel, while the chemically deacylated form (cdLTA) migrated as a ladder of discrete bands, reminiscent of lipopolysaccharides. The deacylated polymer appeared to separate in this system on the basis of size, as evident from results obtained from PAGE analysis of cdLTA subjected to limited acid hydrolysis and LTA that had been fractionated by gel filtration. A survey of cdLTA from other streptococci revealed similarities in molecular weight ranges. The polymer from Enterococcus hirae was of a higher molecular weight. This procedure was used to examine the effect of penicillin and chloroamphenicol on the synthesis, turnover, and heterogeneity of LTA in S. agalactiae. Penicillin appeared to enhance LTA synthesis while causing the release of this polymer into the supernatant fluid. In contrast, chloramphenicol inhibited the synthesis of this molecule and resulted in its depletion from the cell surface. Penicillin did not alter the heterogeneity of this polymer, but chloramphenicol caused an apparent shift to a lower-molecular-weight form of the LTA, as determined by PAGE. This shift in the heterogeneity of LTA did not appear to be due to increased carbohydrate substitution, since chloramphenicol did not alter the electrophoretic migration profile of LTA from E. hirae. From a pulse-chase study, it was determined that LTA was released as a consequence of deacylation.Lipoteichoic acid (LTA) is an amphiphilic molecule found in most gram-positive bacteria (12). This molecule is a glycerol-phosphate (GP) polymer that extends through the peptidoglycan layer of the cell wall and is bound to the cell surface through a hydrophobic interaction between its glycolipid anchor and the cytoplasmic membrane (12). Various functions have been attributed to this polymer; they include scavenging divalent cations (15), regulating autolysin activity (9), and mediating adherence of a number of grampositive bacteria to eucaryotic cells (2,6,8,23,31,33). In this last role, it is believed to play a major role in the pathogenesis of several species of streptococci, including Streptococcus pyogenes, the cause of streptococcal pharyngitis (2), and S. agalactiae, one of the leading causes of neonatal sepsis and meningitis in the United States.A recent study demonstrated differences among clinical isolates of S. agalactiae in terms of the levels of LTA synthesized (30) and the relative binding activity of these strains to human fetal lung epithelial cells (31). The chain length of LTA in S. agalactiae (32) and other gram-positive bacteria has previously been defined as the total number of GP units per terminal GP unit or fatty acids present in the glycolipid (12). In this report, an alternative method for analysis of polymer length by polyacrylamide gel electrophoresis (PAGE) is described which allows visualization of th...

D-Alanyl-lipoteichoic acid in Lactobacillus casei: secretion of vesicles in response to benzylpenicillin

“…5, lane 4), which is also heterogeneous in size, was determined to have an average of 60 glycerol phosphate residues per chain (J. H. Pollack and F. C. Neuhaus, unpublished observation). These results confirm similar reports by Maurer & Mattingly (1991) and Leopold & Fischer (1991) that LTA from a number of bacteria is polydisperse. In L. casei chain lengths between 30 and 65 residues were reported.…”

“…The distribution of polymer lengths in LTA was determined by a modification of the methods described by Min & Cowman (1986), Wolters et al (1990, and Maurer & Mattingly (1991). LTA was extracted with 40% (w/v) phenol/water according to the procedure of Fischer et al (1983) and chemically deacylated with 15% NH40H at 20 "C for 12 h. The samples were taken to dryness at 60 "C, dissolved in water, and analysed by PAGE on a 20% (w/v, acrylamide) gel (1-5 mm thickness) using an electrode buffer of 0-25 M-Tris/borate (pH 8.2).…”

Group B Streptococcus and E. coli LPS‐induced NO‐dependent hyporesponsiveness to noradrenaline in isolated intrapulmonary arteries of neonatal piglets

“…Clinical isolates of ** GSB, including type III, recovered from infants with sepsis, possessed significantly higher levels of LTA in their cell wall ** | than those isolated from asymptomatic carriers (Nealon & Mattingly, 1983). The molecular analysis of GBS LTA demonstrated a close similarity with the Group A Streptococcus LTA (Maurer & Mattingly, 1991). Thus, the LTA component of GBS might be responsible for the effects on vascular reactivity and iNOS induction described in the present paper.…”

Occurrence of Lipoteichoic Acid in Oral Streptococci

“…Immunoblotting of SDS-PAGE-separated phenol extracts of representatives of each species studied revealed a cell component from each strain that had a mobility identical to that of standard PGP-LTA isolated from S. sanguis NCTC 7863T and little evidence of cross-reactions with other extracted compounds (data not shown). The PGP-LTA migrated close to but not coincident with the leading edge of the electrophoresis gel and exhibited some heterogeneity in PGP chain length, which is a feature of this compound (29). Sometimes small amounts of faster-migrating material were detected, which may have been LTA degradation products or extracted cardiolipin.…”