In vitro analysis of the Staphylococcus aureus lipoteichoic acid synthase enzyme using fluorescently labeled lipids

Abstract

Lipoteichoic acid (LTA) is an important cell wall component of Gram-positive bacteria. The key enzyme responsible for polyglycerolphosphate lipoteichoic acid synthesis in the Gram-positive pathogen Staphylococcus aureus is the membrane-embedded lipoteichoic acid synthase enzyme, LtaS. It is presumed that LtaS hydrolyzes the glycerolphosphate head group of the membrane lipid phosphatidylglycerol (PG) and catalyzes the formation of the polyglycerolphosphate LTA backbone chain. Here we describe an in vitro assay for this new class of enzyme using PG with a fluorescently labeled fatty acid chain (NBD-PG) as the substrate and the recombinant soluble C-terminal enzymatic domain of LtaS (eLtaS). Thin-layer chromatography and mass spectrometry analysis of the lipid reaction products revealed that eLtaS is sufficient to cleave the glycerolphosphate head group from NBD-PG, resulting in the formation of NBD-diacylglycerol. An excess of soluble glycerolphosphate could not compete with the hydrolysis of the fluorescently labeled PG lipid substrate, in contrast to the addition of unlabeled PG. This indicates that the enzyme recognizes and binds other parts of the lipid substrate, besides the glycerolphosphate head group. Furthermore, eLtaS activity was Mn(2+) ion dependent; Mg(2+) and Ca(2+) supported only weak enzyme activity. Addition of Zn(2+) or EDTA inhibited enzyme activity even in the presence of Mn(2+). The pH optimum of the enzyme was 6.5, characteristic for an enzyme that functions extracellularly. Lastly, we show that the in vitro assay can be used to study the enzyme activities of other members of the lipoteichoic acid synthase enzyme family.

FIG. 1.

eLtaS in vitro assay. (A) Chemical structures of the fluorescently labeled phosphatidylglycerolphosphate lipids NBD-PG and NBD-DAG. The B. cereus phospholipase C (PLC) cleavage site is indicated on the scheme, as is the mass reduction following the release of the glycerolphosphate lipid head group. (B) TLC analysis of eLtaS in vitro reaction products. NBD-PG lipid suspensions were incubated with either eLtaS, the eLtaS-T300A protein, or no enzyme (negative control). Reaction products were separated by TLC, and plates were scanned using a fluorescence imager. As a positive control, a reaction was set up with B. cereus PLC. Note that only 10% of the PLC reaction mixture was run on the TLC. Arrows on the left indicate the positions of the NBD-PG input lipid, the main hydrolysis product, and a lipid of unknown structure (lipid X). The proteins added to each reaction mixture are shown at the top. (C) NBD-DAG standard curve. Twofold dilutions of PLC control reaction mixtures were separated by TLC (inset); the signal for the hydrolysis product was quantified; and the average values and standard deviations from two plates were plotted.

FIG. 2.

MALDI-TOF mass spectra of eLtaS lipid reaction products. For mass spectrometry analysis, PLC (positive control), eLtaS, eLtaS-T300A, and no-enzyme reactions were set up, and lipids were extracted and dried as described in Materials and Methods. Dried lipids, as well as 12.5 μg of the NBD-PG input lipid, were suspended in a 0.5 M DHB MALDI matrix and were spotted onto a MALDI plate. Spectra were recorded in the reflector positive-ion mode on a MALDI micro MX machine (Waters, United Kingdom), and m/z signals were plotted for the DHB matrix only (A), the NBD-PG input lipid (mass signal, 718.4) (B), the PLC reaction (mass signal, 564.2) (C), no enzyme (mass signal, 718.3) (D), the eLtaS-T300A reaction (mass signal, 718.4) (E), and the eLtaS reaction (mass signals, 564.3 and 718.4) (F). Presumed NBD-lipid-specific signals are shown in red.

FIG. 3.

eLtaS requires Mn²⁺ for activity. (A) eLtaS in vitro reactions were set up in 10 mM sodium succinate (pH 6.0) buffer (ionic strength, 20 mM) in the absence or presence of the indicated metal ion at a final concentration of 10 mM. (B) EDTA inhibits eLtaS activity. eLtaS in vitro reactions were set up in 10 mM sodium succinate (pH 6.0) buffer (ionic strength, 50 mM) containing 10 mM MnCl₂ with or without the addition of 16 mM EDTA. (C) Zn²⁺ inhibits eLtaS activity. eLtaS in vitro reactions were set up in 10 mM sodium succinate (pH 6.0) buffer (ionic strength, 20 mM) in the absence of ions or in the presence of a combination of metal ions at a final concentration of 10 mM each as indicated below each bar. (D) Zn²⁺ inhibits eLtaS activity in a dose-dependent manner. Enzyme reactions were set up in the presence of 10 mM MnCl₂ and of ZnCl₂ at concentrations in the range of 0.01 to 10 mM. (E) eLtaS activity is dependent on the MnCl₂ concentration. Enzyme reactions were set up in the presence of MnCl₂ at concentrations ranging from 1 to 250 mM. Two to four independent experiments were performed, and normalized average values and standard deviations were plotted as described in Materials and Methods. Student's t test was used to determine statistically significant differences between the enzyme activity represented by a dark grey bar in each graph and the activity with each of the other enzyme reaction conditions shown in the same plot. Double asterisks indicate statistically significant differences (P, <0.001).

FIG. 4.

pH requirement, ionic strength profile, and reaction kinetics of eLtaS. (A) pH profile of eLtaS. NBD-PG vesicles were prepared in buffers ranging from pH 4.5 to pH 8 (for the exact buffer composition, see Materials and Methods). Reaction mixtures were incubated for 3 h at RT in the presence of 10 mM MnCl₂, and lipids were extracted and analyzed by TLC. Data were plotted and analyzed as described for Fig. 3. (B) Ionic strength profile of eLtaS. NBD-PG vesicles were prepared in 10 mM sodium succinate (pH 6.0) buffer the ionic strength was adjusted to the indicated values with NaCl. Data were plotted and analyzed as described for Fig. 3. Asterisks indicate statistically significant differences (*, P values between 0.001 and 0.05; **, P values below 0.001). (C) Reaction kinetics of eLtaS. Enzyme reactions were set up in 10 mM sodium succinate (pH 6.0) buffer (ionic strength, 50 mM) containing 10 mM MnCl₂, and reaction mixtures were incubated at 37°C for the indicated times. Three independent experiments were performed with duplicate or triplicate samples, and a representative graph is shown.

FIG. 5.

Unlabeled PG but not glycerolphosphate can compete with NBD-PG as a substrate. (A) Standard eLtaS reactions were set up with or without the addition of 175 μM glycerol-1-phosphate (GroP) (10-fold excess over the NBD-PG lipid substrate). (B) Unlabeled PG competes with NBD-PG. Vesicles containing only NBD-PG (0:1) or a mixture of PG and NBD-PG (5:1) were prepared, and eLtaS reactions were performed as described in Materials and Methods. Reaction products were separated by TLC, and the amount of the hydrolysis product was quantified. Data were plotted and analyzed as described for Fig. 3. Double asterisks indicate statistically significant differences (P, <0.001).

FIG. 6.

The substrate specificity of the S. aureus eLtaS enzyme was tested by setting up enzyme assays with NBD-PG, NBD-PS, NBD-PC, or NBD-PE, as indicated. As a control, no enzyme (no enz.) or the active-site variant eLtaS-T300A was added to reaction mixtures, as indicated above the panels. PLC reactions using NBD-PG as a substrate were run alongside to indicate the mobility of the hydrolysis product. Note that only the portions of the TLC plates that correspond to the location of the hydrolysis product are shown.

FIG. 7.

In vitro activities of L. monocytogenes LtaS-type enzymes. Enzyme assays were set up with eLtaP_Lm, eLtaS_Lm, and S. aureus eLtaS using NBD-PG as a substrate. Six independent experiments were performed with triplicate samples. Data were plotted and analyzed as described for Fig. 3. Double asterisks indicate statistically significant differences (P, <0.001).