Journal of Bacteriology, August 2004, p . 5529-5532, Vol . 186, No . 16

Characterization of Lipoteichoic Acids as Lactobacillus delbrueckii Phage Receptor Components

Liisa Räisänen,^1* Karin Schubert,² Tiina Jaakonsaari,³ and Tapani Alatossava⁴

Department of Biology, University of Oulu, FIN-90014 Oulu,¹ Biotechnology Laboratory, REDEC of Kajaani, University of Oulu, FIN-88600 Sotkamo,³ Department of Food Technology, University of Helsinki, FIN-00014 Helsinki, Finland,⁴ Department Biologie I, Bereich Mikrobiologie der Universität München, D-80638 Munich, Germany²

Received 4 September 2003/ Accepted 16 May 2004

 
Lipoteichoic acids (LTAs) were purified from Lactobacillus delbrueckii subsp . lactis ATCC 15808 and its LL-H adsorption-resistant mutant, Ads-5, by hydrophobic interaction chromatography . L . delbrueckii phages (LL-H, the LL-H host range mutant, and JCL1032) were inactivated by these poly(glycerophosphate) type of LTAs in vitro in accordance to their adsorption to intact ATCC 15808 and Ads-5 cells .

 
Cell walls of gram-positive eubacteria consist mainly of a thick peptidoglycan layer and various compositions of proteins and accessory polymers, such as polysaccharides and teichoic acids . Lactococcal bacteriophages have been reported to use cell wall carbohydrates as (primary) receptors for adsorption, and for some a requirement of certain membrane proteins has been demonstrated (18, 28) . Glycosylated teichoic acids are essential at least for the adsorption of some Bacillus subtilis, Staphylococcus aureus, and Lactobacillus plantarum phages (9, 11, 19, 30) . Even a peptidoglycan layer has been reported to serve as a receptor substance in phage adsorption (29) . Although (lipoteichoic acids) LTAs are widely distributed in gram-positive eubacteria, no reports of their role as a receptor substance have been published so far . In Lactococcus lactis subsp . cremoris SK110, modified LTAs have been suggested to prevent phage adsorption by masking the actual receptor site (26, 27) .

The isometric-headed Lactobacillus delbrueckii subsp . lactis phage LL-H is the only L . delbrueckii phage that is completely sequenced (2, 22) . The LL-H genome exhibits a limited homology to the genome of the prolate-headed L . delbrueckii subsp . lactis phage JCL1032 (17) . Both phages have noncontractile tails, and their genetic determinants involved in host recognition have been characterized . Gp71 and its homolog ORF474 determine the adsorption specificities of LL-H and JCL1032, respectively (23) . Ads-5, one of the LL-H-resistant mutants of L . delbrueckii subsp . lactis ATCC 15808, is able to block the adsorption of LL-H but not the adsorption of the LL-H host range mutant LL-H-a21 or JCL1032 (23) . In this study, we investigated if purified LTAs isolated from ATCC 15808 and Ads-5 could serve as a receptor substance in the early stage of L . delbrueckii phage infections .

Bacterial strains and bacteriophages. L . delbrueckii subsp . lactis strains were grown at 37°C in MRS broth (Difco Laboratories), and for phage propagation, MRS broth was supplemented with 10 mM CaCl₂ . Bacterial strains and bacteriophages used in this study are listed in Table 1 . For the isolation of LTAs, strains were grown in 1-liter batch cultures (supplemented with 10 mM CaCl₂) to an optical density at 600 nm of 0.5 to 0.6 .

 
Extraction and purification of LTAs by HIC. LTAs were extracted from lipid-free bacterial cells by hot 80% (wt/vol) aqueous phenol (20) . For purification by hydrophobic interaction chromatography (HIC), LTAs extracted from ATCC 15808 cells were dissolved in 50 mM sodium acetate (pH 4.0) containing 15% propan-1-ol and applied to a column of octyl-Sepharose CL 4B (2 by 22 cm) . The column was eluted with a linear gradient (15 to 60% [vol/vol]) of propan-1-ol in 50 mM sodium acetate (pH 4.0) at a flow rate of 20 ml h^–1 . Every second fraction (5 ml) was analyzed for nucleic acids (A₂₆₀) and for phosphorus (4) . Three pools containing phosphorus, shown in Fig . 1, were detected . According to its UV absorbance spectrum, pool I contained nucleic acids (data not shown) . Pools II and III, which eluted between propan-1-ol concentrations of 28 and 39%, were practically free of nucleic acids and proteins . Based on the general elution profile, pools II and III were considered as possible fractions of LTAs . They were dialyzed against distilled water .

 
In the faster purification procedure, dissolved LTAs were applied to a column (2.6 by 13 cm) of octyl-Sepharose 4 Fast Flow (Pharmacia Biotech AB, Uppsala, Sweden) . The column was eluted with a linear gradient (15 to 70% [vol/vol]) of propan-1-ol in 100 mM sodium acetate (pH 5.0) at a flow rate of 1.8 ml min^–1 . Collected fractions (3 ml) were analyzed for nucleic acids and for phosphorus as previously described . LTAs eluted as a single peak between propan-1-ol concentrations of 30 and 41% (data not shown) .

Chemical composition analysis of LTAs purified from ATCC 15808. Pools II and III were investigated for D-alanine, sugars, and polyols (Table 2) . Amino acids were quantitatively determined as previously described (25) . Glycerol, ribitol, and sugars were analyzed as peracetylated or reduced peracetylated derivatives by gas-liquid chromatography (GLC) after hydrolysis in 60% (wt/vol) hydrofluoric acid (3, 6, 25) . For the analysis of sugars, hydrofluoric acid-hydrolyzed material was further hydrolyzed in 2 M HCl (100°C for 3 h) . Both the pools contained almost equal molar amounts of glycerol and phosphorus (Table 2) . They eluted separately most likely due to the different number of fatty acids (12, 15) . On the basis of our composition analysis and survey of the literature, LTAs from L . delbrueckii subsp . lactis ATCC 15808 probably belong to the most widespread type of LTAs, 1-3-linked poly(glycerophosphate)s (13, 14) .

 
Besides glycerol, a small amount of ribitol was also detected by GLC . The presence of ribitol in LTAs is rarely observed . Only the so-called Forsman antigen from Streptococcus pneumoniae has been described to contain ribitol phosphate and choline phosphate repeating units (8) . More often the occurrence of ribitol has been described in wall teichoic acids of some bacilli, staphylococci, and lactobacilli (5, 7, 10) . However, we do not believe the observed ribitol is due to a contamination with teichoic acids . HIC has been shown to effectively separate LTAs from polyanionic contaminants (12) .

Poly(glycerophosphate)s are often substituted by glucosyl and D-alanine residues (13) . In ATCC 15808, glucose was exclusively found in the glycolipid anchors, since no glycosylglycerol was detected in GLC . Only 10 to 20% of the glycerol residues were substituted with D-alanine, which is considerably less than with LTAs from Lactobacillus casei or Lactobacillus helveticus (13) .

LTAs and phage inactivation assays. LTAs were tested for their ability to inactivate the L . delbrueckii phages listed in Table 1 . The observation that both the LTA pools (II and III) of ATCC 15808 were able to inactivate the phage LL-H allowed us to use the LTAs obtained with the faster purification procedure . Usually more than 90% of phages (10⁶ PFU/ml) were inactivated during the first 5 to 10 min of incubation at 37°C when a concentration of 1 ng of LTA-phosphorus per ml was used (data not shown) . Minimum concentrations of LTAs needed for significant inactivation (50%) of different L . delbrueckii phages were determined by incubating phages (10⁶ PFU/ml) with various concentrations of the LTAs in 20-min incubations (Fig . 2) .

 
Minimum dosages for inactivation of LL-H and JCL1032 were estimated as 100 pg of LTA-phosphorus of ATCC 15808 per ml (Fig . 2A and C) . For LL-H-a21, the minimum dosage was closer to 10 than to 100 pg per ml (Fig . 2B) . Approximately fivefold more LTA-phosphorus of Ads-5 was needed to significantly reduce the plaque formation of LL-H-a21 or JCL1032 (Fig . 2B and C) . The phage LL-H, instead, was not inactivated even by 10³-fold dosages of LTA-phosphorus of Ads-5 (Fig . 2A) . Despite the overall genome homology between LL-H and mv4 (2), mv4 was not inactivated by any of the tested LTAs (data not shown) .

The most prominent feature in these assays was the lack of LL-H inactivation with the LTAs of Ads-5 contrary to efficient inactivation of LL-H-a21 . As far as we know, LL-H-a21 differs from LL-H only by a single amino acid in the receptor binding protein Gp71, thus suggesting that the specificity of inactivation reactions resides in the Gp71-LTA interaction (23) . Like LL-H-a21, JCL1032 reacted with the LTAs examined in this study, although more LTAs were needed for significant inactivation . This could be an indication of JCL1032 interaction with a different kind of structural feature of LTAs . Comparative chemical and physical analyses on the LTA structures are required to reveal the crucial structural feature(s) of LTAs responsible for the specific interactions with L . delbrueckii subsp . lactis phages .

We have previously suggested that there are at least three types of receptors for L . delbrueckii phages: two specific for LL-H and its host range mutant LL-H-a21 and one specific for the phage JCL1032 (23) . In this study, we demonstrate that these L . delbrueckii phages are inactivated by purified LTAs from L . delbrueckii subsp . lactis in a manner that is consistent with their behavior with intact cells .

 
We thank Franz Fiedler (Munich, Germany) for the opportunity to analyze our LTA samples in his laboratory .

This study was supported by the grants from the Academy of Finland (SA 46921), EC Biotech II program (BIO4-CT98-0406), the National Technology Agency in Finland (Tekes 70062/01), and by a personal grant from the University of Oulu to L.R .

 
* Corresponding author . Mailing address: Department of Biology, University of Oulu, P.O . Box 3000, FIN-90014, Oulu, Finland . Phone: 358-8-553 1792 . Fax: 358-8-553 1061 . E-mail: liisa.raisanen@oulu.fi .

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