Polymer length of teichuronic acid released from cell walls of Micrococcus luteus

Wolters, Paul J.; Hildebrandt, K M; Dickie, Janet; Anderson, John S.

doi:10.1128/jb.172.9.5154-5159.1990

Cited by 25 publications

(22 citation statements)

References 30 publications

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“…The cultures were incubated for 1 h at 37°C. LTA (39). However, the polymer differed from other polysaccharides (13,34) and macromolecules (22) in that it migrated ahead of the bromophenol blue dye front, which is normally used in other systems to mark the extent of migration.…”

Section: Methodsmentioning

confidence: 99%

Molecular analysis of lipoteichoic acid from Streptococcus agalactiae

Maurer

Mattingly

1991

J Bacteriol

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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...

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Section: Methodsmentioning

confidence: 99%

Molecular analysis of lipoteichoic acid from Streptococcus agalactiae

Maurer

Mattingly

1991

J Bacteriol

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“…Extracts were sodium dodecyl sulfate purified, and the wall teichoic acids (WTA) were isolated with extraction in 5% (wt/vol) trichloroacetic acid as described previously (54). WTA were resolved on a 15% (wt/vol) polyacrylamide TBE gels and then revealed by a modified Alcian blue silver staining as described previously (76).…”

Section: Methodsmentioning

confidence: 99%

Isolation and Characterization of Biofilm Formation-Defective Mutants of Staphylococcus aureus

et al. 2007

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Staphylococcus aureus produces biofilm and this mode of colonization facilitates infections that are often difficult to treat and engender high morbidity and mortality. We have exploited bacteriophage Mu transposition methods to create an insertional mutant library in a highly biofilm-forming S. aureus clinical isolate. Our screen identified 38 insertions in 23 distinct genes together with one intergenic region that significantly reduced biofilm formation. Nineteen insertions were mapped in loci not previously known to affect biofilm in this organism. These include insertions in codY, srrA, mgrA, and fmtA, a putative DEAD-box helicase, two members of the zinc-metallo-␤ lactamase/␤-CASP family, and a hypothetical protein with a GGDEF motif. Fifteen insertions occurred in the icaADBC operon, which produces intercellular adhesion antigen (PIA) and is important for biofilm formation in many strains of S. aureus and Staphylococcus epidermidis. Obtaining a high proportion of independent Em-Mu disruptions in icaADBC demonstrated both the importance of PIA for biofilm formation in this clinical strain and the strong validation of the screening procedure that concomitantly uncovered additional mutants. All non-ica mutants were further analyzed by immunoblotting and biochemical fractionation for perturbation of PIA and wall teichoic acid. PIA levels were diminished in the majority of non-ica insertional mutants. Three mutant strains were chosen and were functionally complemented for restored biofilm formation by transformation with plasmids carrying the cloned wild-type gene under the control of a xylose-inducible promoter. This is a comprehensive collection of biofilm-defective mutants that underscores the multifactorial genetic program underlying the establishment of biofilm in this insidious pathogen.

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“…In the isolation process, care was taken to prevent the introduction of endotoxin by ensuring reagents were prepared in endotoxin-free water and containers. The purified LTA was analyzed on a 7.5% polyacrylamide gel in Tris-borate-EDTA buffer (42) and detected as a single band by the cationic dye Stains-All (Sigma-Aldrich) (22). The purified LTA was then freeze-dried.…”

Section: Methodsmentioning

confidence: 99%

Role ofd-Alanylation ofStreptococcus gordoniiLipoteichoic Acid in Innate and Adaptive Immunity

Chan¹,

Mayer²,

Davis³

et al. 2007

Infect Immun

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In recent years, there has been considerable interest in using the oral commensal gram-positive bacterium Streptococcus gordonii as a live vaccine vector. The present study investigated the role of D-alanylation of lipoteichoic acid (LTA) in the interaction of S. gordonii with the host innate and adaptive immune responses. A mutant strain defective in D-alanylation was generated by inactivation of the dltA gene in a recombinant strain of S. gordonii (PM14) expressing a fragment of the S1 subunit of pertussis toxin. The mutant strain was found to be more susceptible to killing by polymyxin B, nisin, magainin II, and human ␤ defensins than the parent strain. When it was examined for binding to murine bone marrow-derived dendritic cells (DCs), the dltA mutant exhibited 200-to 400-fold less binding than the parent but similar levels of binding were shown for Toll-like receptor 2 (TLR2) knockout DCs and HEp-2 cells. In a mouse oral colonization study, the mutant showed a colonization ability similar to that of the parent and was not able to induce a significant immune response. The mutant induced significantly less interleukin 12p70 (IL-12p70) and IL-10 than the parent from DCs. LTA purified from the bacteria induced tumor necrosis factor-alpha and IL-6 production from wild-type DCs but not from TLR2 knockout DCs, and the mutant LTA induced a significantly smaller amount of these two cytokines. These results show that D-alanylation of LTA in S. gordonii plays a role in the interaction with the host immune system by contributing to the relative resistance to host defense peptides and by modulating cytokine production by DCs.

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Polymer length of teichuronic acid released from cell walls of Micrococcus luteus

Cited by 25 publications

References 30 publications

Molecular analysis of lipoteichoic acid from Streptococcus agalactiae

Molecular analysis of lipoteichoic acid from Streptococcus agalactiae

Isolation and Characterization of Biofilm Formation-Defective Mutants of Staphylococcus aureus

Role ofd-Alanylation ofStreptococcus gordoniiLipoteichoic Acid in Innate and Adaptive Immunity

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