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Journal of Bacteriology, June 2007, p. 4539-4543, Vol. 189, No. 12
0021-9193/07/$08.00+0     doi:10.1128/JB.00378-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

pH-Dependent Association of Enolase and Glyceraldehyde-3-Phosphate Dehydrogenase of Lactobacillus crispatus with the Cell Wall and Lipoteichoic Acids

Jenni Antikainen, Veera Kupannen, Kaarina Lähteenmäki, and Timo K. Korhonen^*

General Microbiology, Faculty of Biosciences, P.O. Box 56, FIN-00014 University of Helsinki, Finland

Received 14 March 2007/ Accepted 5 April 2007

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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.

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The glycolytic enzymes enolase and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) are found on the surface of several gram-positive bacteria, where they are involved in pathogen-host interactions (3-6, 17, 18, 20, 21). Commensal Lactobacillus crispatus and several other species of the Acidophilus group of Lactobacillus have enolase and GAPDH as major constituents of their extracellular proteome at neutral pHs (12). The secretion and anchoring mechanisms of enolase and GAPDH on the bacterial surface have not been characterized. Lactobacillus species are strongly fermentative and secrete lactic acid as a primary metabolite, which rapidly reduces the pH of the environment to 4. Here, we assessed the role of pH in the surface localization of enolase and GAPDH in L. crispatus.

Association of enolase and GAPDH with the cell wall depends on pH. L. crispatus ST1 (9, 12) was cultivated overnight in De Man, Rogosa, and Sharpe (MRS) broth (Difco), the cells were collected by centrifugation, suspended without washing at 10¹⁰ bacteria/ml in 50 mM Tris-HCl at either pH 5 or pH 8, and incubated at 37°C for 1 h, during which time the pHs of the suspensions decreased to 4.5 and to 7.5. The presence of enolase and GAPDH, as well as of an unrelated surface layer (S-layer) protein, on the cells was analyzed by use of indirect immunofluorescence. The cells were used to coat glass slides and fixed with 3.5% (wt/vol) paraformaldehyde prior to detection with anti-His₆-GAPDH (12), anti-His₆-enolase (12), or anti-S-layer protein (2) immunoglobulins as primary antibodies and tetramethylrhodamine isothiocyanate-labeled antibodies (Dako) as detailed previously (19). Enolase and GAPDH were present on the surface of the cells from the pH 5 suspension, whereas the cells from the pH 8 suspension showed only weak fluorescence (Fig. 1A). In contrast, no change in cell-bound S-layer protein was detected (Fig. 1A). Next, the cells from an overnight culture were incubated for 1 h at pH 5 or pH 8, the cell and the supernatant fractions were separated, and the supernatant was filtered through a 0.2-µm-pore-size membrane (12). Surface-attached proteins were extracted by boiling the cell pellet in reducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) sample buffer (8) for 1 min. Enolase and GAPDH were detected by Western blotting in the supernatant from the pH 8 suspension, but not from the pH 5 suspension, and more of these proteins were found on the surfaces of cells from the pH 5 suspension than from the pH 8 suspension (Fig. 1B). Small amounts of surface-associated enolase and GAPDH were detectable by Western blotting of samples from the pH 8 suspension. A fraction of enolase and GAPDH are embedded within the cell wall (12) and probably released by being boiled briefly in buffer containing SDS. The surface location of the S-layer protein was not dependent on the pH (Fig. 1B). No reactivity of an antibody against the cytoplasmic marker protein RNA polymerase ß1 subunit (NeoClone) (1) was detected on the cell surface or in the supernatants. When the cells were lysed with mutanolysin (50 U/ml) and lysozyme (20 mg/ml), equal amounts of enolase and GAPDH were detected for both pHs (Fig. 1B), indicating similar protein expression levels. A similar pH dependence with respect to the surface localization of enolase and GAPDH was also detected in ST1 cells grown to logarithmic phase in MRS broth at pH 5 or pH 8 (data not shown). Further, an analysis of the release of enolase and GAPDH at pH 5 with sodium chloride or choline chloride concentrations varying from 0.1 to 2 M revealed that these proteins are detached from the cell surface by salt concentrations above 0.25 M (not shown), indicating the importance of ionic interactions in the cell wall association.

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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.

Next, the time course of the release of enolase and GAPDH from ST1 cells suspended in pH 5 and pH 8 buffers was assessed. At pH 5, no release was detected until 24 h, whereas at pH 8 the proteins were immediately released from the cells (Fig. 1C). No reaction with the RNA polymerase ß1 subunit antibody was detected. A stepwise increase of the pH from 4.4 to 7.0 revealed that the release of enolase and GAPDH becomes detectable at pH 5.2 (Fig. 1D), which is close to the pI values of enolase (4.8) and GAPDH (5.2).

The surface localization of enolase and GAPDH in L. acidophilus E507, L. amylovorus JCM5807, L. gallinarum T-50, L. gasserii JCM1130, and L. johnsonii F133, which all express enolase and GAPDH proteins that cross-react serologically with the ST1 proteins (12), was pH dependent in a way similar to that of L. crispatus ST1 (data not shown). In conclusion, lactobacillar enolase and GAPDH are immediately released into the supernatant at neutral pHs and associate with the cell surface at low pHs.

Protein synthesis and transcription. ST1 cells were incubated at pH 8 with or without chloramphenicol (25 µg/ml), and the supernatant fractions were analyzed by Western blotting. No visible difference in the release of enolase or GAPDH was detected in incubations of 1 (Fig. 2A), 2, or 6 h (data not shown). Similarly, no significant differences were detected by enzyme-linked immunosorbent assay (Fig. 2B), which indicates that de novo protein synthesis is not necessary for the release process. Transcription levels of eno and gap from ST1 cells grown to logarithmic phase at pH 5 or pH 8 were assessed by Northern analysis. Total RNA was extracted with the RNeasy mini kit (QIAGEN) after the cells had been treated with mutanolysin (50 U/ml) and lysozyme (20 mg/ml) at 37°C for 15 min. RNA was resolved by 1.5% (wt/vol) formaldehyde-agarose electrophoresis, transferred to Hybond N+ membranes (Amersham Biosciences), and detected with ST1 eno and gap DNA (12) labeled with digoxigenin (Boehringer Mannheim). No notable differences in the mRNA levels were detected (Fig. 2C). We conclude that the release of enolase and GAPDH from ST1 is not related to changes in expression levels but is due solely to distribution between the cell surface and the extracellular proteome.

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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.

Binding of enolase and GAPDH to LTA. Enolase and GAPDH of L. crispatus are positively charged at lower pH values and thus could bind to negatively charged cell wall components, such as lipoteichoic acids (LTA). We assessed the binding of enolase and GAPDH to LTA from Staphylococcus aureus and Streptococcus faecalis (Sigma Aldrich) and to peptidoglycan (PG) from S. aureus (Sigma Aldrich) at pH values below and above their pIs. Purified recombinant His₆-enolase (0.05 µg) (12) and GAPDH (0.05 µg) purified from the extracellular proteome by NAD affinity chromatography (Cibacron blue agarose; Sigma Aldrich) were incubated with LTA or PG (2.5 µg) for 45 min at room temperature in 10 µl of 50 mM sodium acetate buffer of either pH 4.0 or pH 5.6. Samples were electrophoresed on nondenaturating 10% (wt/vol) PAGE in 50 mM sodium acetate buffer with reversed (pH 4.0) or normal polarity (pH 5.6) at 4°C as described previously (8). At pH 4.0, enolase and GAPDH migrated towards the negative pole. However, when either S. aureus (Fig. 3.) or S. faecalis (not shown) LTA was added, no migration towards the anionic pole was detected. At pH 5.6, no shift in protein mobility was observed after addition of LTA, and no shift in mobility after the addition of PG was observed at either pH (Fig. 3). The binding of enolase and GAPDH to LTA and PG was also analyzed by using fluorescent MX Covasphere beads (FBS; Duke Scientific) coated with 70 µg recombinant His₆-enolase and His₆-GAPDH (12) as described previously (23). LTA of S. faecalis, PG, or bovine serum albumin (BSA; 4 µg) were immobilized on glass slides, and FBS were incubated on the slides for 1 h in 100 mM NaAc buffer at pH 4.4 or pH 7.0. After a washing at the same pH, the numbers of bound FBS per microscopic field (area, 3.6 x 10⁴ µm²) were determined by microscopy (Olympus BX50) using Image-Pro Plus analysis software (Media Cybernetics). Enolase- and GAPDH-coated beads bound more efficiently to LTA than to PG or BSA at pH 4.4, whereas only poor binding was detected at pH 7.0 (Fig. 3B). At pH 4.4, FBS showed a slight increase in background binding to PG and BSA. For comparison, we used beads coated with His₆-CbsA 288-410, which is the LTA-binding peptide of the S-layer protein of L. crispatus (2). This peptide bound more efficiently to LTA than PG or BSA, and the binding was similar at both pHs (Fig. 3B). We concluded that enolase and GAPDH interact with LTA at acidic pHs but not at pH values higher than 5.6.

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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.

Reassociation of enolase and GAPDH to the cell wall. Pneumococcal enolase reassociates to the cell surface at neutral pH (5). To analyze the possible reassociation of the L. crispatus enzymes, ST1 cells were incubated for 1 h in 50 mM Tris-HCl, pH 8, and the cells and the supernatant were separated. The supernatant was diluted 1/10 in 100 mM NaAc, pH 4.4 or pH 7.0, and resuspended with 5 x 10⁸ cells/ml. After incubation for 30 min at 22°C, the surface localization of enolase and GAPDH to the cell wall was detected by immunofluorescence with anti-His₆-enolase and anti-His₆-GAPDH immunoglobulins. A granular binding of enolase and GAPDH to the cells at pH 4.4 was evident, whereas at pH 7.0 only a low level of binding was detected (Fig. 4). The addition of S. aureus LTA (0.5 mg/ml) diminished the reassociation of enolase and GAPDH. This verifies that enolase and GAPDH bind to the cell wall at acidic pH and further supports the role of LTA in their anchoring onto the cell wall. Due to background fluorescence in the samples containing PG, PG could not be tested in this assay.

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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.

Plasminogen binding and activation. Like the enolases and GAPDHs of several gram-positive pathogens (3, 5, 10, 15, 17, 18), the enolase and GAPDH of L. crispatus ST1 bind and enhance activation of human plasminogen (12), which was used here as a functional assay for the pH-dependent surface variation. Bacterial cells (10¹⁰/ml) were incubated with plasminogen (100 µg/ml; American Diagnostica) at room temperature for 1 h at pH 5 or pH 8, after which the cells and the supernatant were separated and analyzed by Western blotting with antiplasminogen immunoglobulins (American Diagnostica). Plasminogen was discovered on the surface of the cells from the pH 5 suspension but in the supernatant fraction from the pH 8 suspension (Fig. 5A). Similarly, only weak binding of plasminogen to ST1 cells was previously detected at neutral pHs (12). Enhancement of tissue-type plasminogen activator (tPA)-mediated conversion of plasminogen to plasmin was measured after the adjustment of each sample to pH 8 (12). The cells from the pH 5 sample, but the supernatant from the pH 8 sample, enhanced plasmin formation (Fig. 5B).

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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.

We demonstrate here that the lactobacillar plasminogen-binding proteins enolase and GAPDH attach to the cell wall at acidic pHs but are released to the medium at higher pH values. Lactobacilli colonize several acidic environmental niches, such as the oral cavity (7), the small intestine (22), and the vaginal epithelia (13), where they reduce the environmental pH by secreting lactic acid. The pH dependency of surface protein attachment or release is likely to affect lactobacillar host interactions, as exemplified here with the binding and activation of plasminogen, a zymogen of human broad-spectrum serine protease plasmin. Lactobacillar adherence to fibronectin and fibrinogen is enhanced at low pHs (11). The basis of this finding was not resolved, but it is in accordance with our observation that pH affects the host interactions of lactobacilli. Further, a similar pH dependency was reported for the GAPDH on the surface of Streptococcus gordonii (16). However, no release of GAPDH from the surface of Streptococcus pyogenes was detected at neutral pHs (16) or after treatment with 2 M NaCl or 2% SDS (18), suggesting a different mechanism for surface anchoring of GAPDH in group A streptococci.

We showed that enolase and GAPDH of L. crispatus ST1 bind to LTA at low pHs, which suggests that the negatively charged LTA may be involved in the surface anchoring of these proteins. Other bacterial surface proteins that bind to LTA have been characterized; these include the S-layer protein (CbsA) of L. crispatus JCM5810 (2) and the glycyl-tryptophan (GW) module proteins, such as InlB of Listeria monocytogenes (14). The ST1 enolase and GAPDH sequences have no GW modules and only low similarity to these proteins. The pIs of CbsA and the GW module are above 9; thus, at pHs below 9 they are positively charged and may associate with LTA molecules. No release of these proteins under varying pH conditions has been reported, and indeed, we showed that the L. crispatus S-layer protein remained on the cell surface and bound LTA at both acidic and alkaline pHs. Enolase and GAPDH are multifunctional proteins with a role in bacterium-host interactions, and their rapid detachment from the cell surface at high pHs may be a mechanism by which lactobacilli respond to changing environments.

 
We thank Raili Lameranta for technical assistance.

This study was supported by the Academy of Finland (the Microbes and Man Programme, grant numbers 80666, 105824, 201967, and 211300), the Alfred Kordelin Foundation, and the Foundation for Nutritional Research, as well as by the University of Helsinki.

 
* Corresponding author. Mailing address: General Microbiology, Faculty of Biosciences, P.O. Box 56, FIN-00014 University of Helsinki, Finland. Phone: 358-9-19159250. Fax: 358-9-19159262. E-mail: timo.korhonen{at}helsinki.fi

Published ahead of print on 20 April 2007.

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Journal of Bacteriology, June 2007, p. 4539-4543, Vol. 189, No. 12
0021-9193/07/$08.00+0     doi:10.1128/JB.00378-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

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This Article Abstract Full Text (PDF) An erratum has been published Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Reprints and Permissions Copyright Information Books from ASM Press MicrobeWorld Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Antikainen, J. Articles by Korhonen, T. K. Search for Related Content PubMed PubMed Citation Articles by Antikainen, J. Articles by Korhonen, T. K.