pH-dependent association of enolase and glyceraldehyde-3-phosphate dehydrogenase of Lactobacillus crispatus with the cell wall and lipoteichoic acids

Abstract

The plasminogen-binding proteins enolase and glyceraldehyde-3-phosphate dehydrogenase of Lactobacillus crispatus were localized on the cell surface at pH 5 but released into the medium at an alkaline pH. These proteins bound to lipoteichoic acids at a pH below their isoelectric point. The results indicate that lactobacilli rapidly modify their surface properties in response to changes in pH.

FIG. 1.

Association of enolase and GAPDH with the cell wall of Lactobacillus crispatus ST1. (A) Immunofluorescence assay of the cells suspended in 50 mM Tris-HCl at pH 5 or pH 8 detected with anti-enolase, anti-GAPDH, and anti-S-layer protein immunoglobulins (left). Phase-contrast images are shown on the right. (B) Western blotting of enolase and GAPDH on the ST1 cell surface and in the supernatant, obtained after 1 h of incubation of the cells at the indicated pH. For comparison, reactivity with anti-S-layer protein and with anti-RNA polymerase (pol) is shown. (C) Time course of enolase and GAPDH release into the supernatant at pH 5 and pH 8. Anti-RNA polymerase antibody (anti-RNA pol) was used to detect possible cell lysis. The reactivity of lysed cell samples is also shown. (D) Release of enolase and GAPDH at pH values from 4.4 to 7.0. ST1 cells were incubated for 1 h in 100 mM sodium acetate buffer at the indicated pH. The release of enolase and GAPDH was analyzed by Western blotting.

FIG. 2.

Protein synthesis and transcription of eno and gap. (A and B) Release of enolase and GAPDH from ST1 cells at pH 8 in the presence and absence of chloramphenicol, an antibiotic affecting protein synthesis, was detected by Western blotting (A) and by enzyme-linked immunosorbent assay (B) with anti-enolase and anti-GAPDH antibodies. Means with standard deviations for eight samples from a representative assay are shown. (C) Transcription levels of enolase and GAPDH in L. crispatus ST1 grown to logarithmic phase at pH 5 or pH 8. The levels of enolase and GAPDH mRNA were detected by hybridization with digoxigenin-labeled eno and gap probes. Ethidium bromide staining of 16S and 23S RNA are shown as controls.

FIG. 3.

Binding of enolase and GAPDH to LTA. (A) The mobility of the purified enolase and GAPDH proteins alone and with LTA or PG of S. aureus was analyzed by electrophoresis in nondenaturating PAGE at pH 4.0 and pH 5.6 and detected by Western blotting. The negative (−) and positive (+) poles and direction of the current (arrows) are indicated. (B) Binding of enolase-, GAPDH-, or CbsA 288-410-coated fluorescent beads to LTA of S. faecalis and PG of S. aureus and BSA at pH 4.4 and pH 7.0. Means with standard deviations for eight microscopic fields are shown.

FIG. 4.

Reassociation of enolase and GAPDH to the cell wall. The proteins were first released from the cell surface at pH 8, the cells were then recovered by centrifugation, and a fraction of the supernatant was added to the cells at either pH 4.4 or pH 7.0. The mixture was incubated for 30 min, and the proteins were visualized by immunofluorescence with anti-enolase and anti-GAPDH immunoglobulins (left). The inhibition effect of LTA on the reassociation was tested at pH 4.4. Phase-contrast images are shown on the right. Arrows indicate the bacterial cell wall.

FIG. 5.

Binding of plasminogen and enhancement of its activation by tPA. Plasminogen was incubated with ST1 cells at pH 5 and pH 8, and the localization of plasminogen on the bacteria and in the supernatant was assessed. (A) Binding of plasminogen to L. crispatus ST1 cells under both pH conditions assessed by Western blotting with antiplasminogen immunoglobulins. The added amount of plasminogen in both buffers is shown on the left. (B) Enhancement of tPA-mediated plasminogen activation by the cell and the supernatant fractions of L. crispatus ST1 measured after adjustment of all the fractions to pH 8 to allow plasmin activity. Plasminogen and tPA incubated in plain buffer are also shown. Enhancement of plasmin formation by laminin is shown as a positive control. Means with standard deviations are shown for two independent assays with triplicate samples.