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Journal of Bacteriology, April 2009, p. 2218-2227, Vol. 191, No. 7
0021-9193/09/$08.00+0     doi:10.1128/JB.01636-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Both Thiamine Uptake and Biosynthesis of Thiamine Precursors Are Required for Intracellular Replication of Listeria monocytogenes

Kristina Schauer,^1,3 Jürgen Stolz,² Siegfried Scherer,^1,3 and Thilo M. Fuchs^1,3*

Zentralinstitut für Ernährungs- und Lebensmittelforschung (ZIEL), Abteilung Mikrobiologie,¹ Abteilung Biochemie,² Lehrstuhl für Mikrobielle Ökologie, Technische Universität München, Weihenstephaner Berg 3, and Am Forum 5, 85354 Freising, Germany³

Received 18 November 2008/ Accepted 20 January 2009

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Thiamine pyrophosphate is an essential cofactor involved in central metabolism and amino acid biosynthesis and is derived from thiamine (vitamin B₁). The extent to which this metabolite is available to bacterial pathogens replicating within host cells is still little understood. Growth studies using modified minimal Welshimer's broth (mMWB) supplemented with thiamine or the thiamine precursor hydroxymethylpyrimidine (HMP) showed that Listeria monocytogenes, in agreement with bioinformatic prediction, is able to synthesize thiamine only in the presence of HMP. This appears to be due to a lack of ThiC, which is involved in HMP synthesis. The knockout of thiD (lmo0317), which probably catalyzes the phosphorylation of HMP, inhibited growth in mMWB supplemented with HMP and reduced the replication rate of L. monocytogenes in epithelial cells. Mutation of a predicted thiamine transporter gene, lmo1429, led to reduced proliferation of L. monocytogenes in mMWB containing thiamine or thiamine phosphates and also within epithelial cells but had no influence on the expression of the virulence factors Hly and ActA. The toxic thiamine analogue pyrithiamine inhibited growth of wild-type strain EGD but not of the transporter mutant EGDthiT. We also demonstrated that ThiT binds thiamine, a finding compatible with ThiT acting as the substrate-binding component of a multimeric thiamine transporter complex. These data provide experimental evidence that Lmo1429 homologs including Bacillus YuaJ are necessary for thiamine transport in gram-positive bacteria and are therefore proposed to be annotated "ThiT." Taken together, these data indicate that concurrent thiamine uptake and biosynthesis of thiamine precursors is a strategy of L. monocytogenes and possibly other facultative intracellular pathogens to enable proliferation within the cytoplasm.

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Offensive and defensive virulence factors of bacterial pathogens have been investigated intensively during the last few years. However, little attention has been devoted to metabolic factors required for bacterial virulence. Extensive metabolic redundancies and access to diverse host nutrients have recently been demonstrated for Salmonella enterica (2). The application of in vivo expression technologies to a variety of pathogenic bacteria frequently resulted in the recovery of so-called "housekeeping" genes as putative virulence factors required for intracellular replication (12, 16). However, there is still a lack of detailed knowledge about the requirement and availability of substrates and cofactors within the host organism and how limitations are overcome by pathogenic bacteria. As suggested recently (22), studies on cellular bacterial metabolism will in turn also provide further insight into the physiological status of the host compartments or tissues in which pathogens persist or proliferate.

Listeria monocytogenes is able to replicate within host cells during the infection process. By disrupting the phagosomal membrane, L. monocytogenes escapes from the vacuole into the cytoplasm. Earlier data on auxotrophic but virulent strains or mutants of L. monocytogenes suggested that the cytoplasm of host cells provides sufficient organic and inorganic compounds to meet the nutritional requirements of the pathogen (21). More recent studies, however, indicate that both the metabolic adaptations of the bacteria and the cytosolic content of the infected cell type are relevant in determining the capability of pathogenic bacteria to replicate within host cells (6, 15, 18, 21, 25).

It is an open question which strategies are used by L. monocytogenes to satisfy the demand for cofactors such as thiamine pyrophosphate (TPP), the biologically active form of thiamine, during intracellular proliferation. Reactions catalyzed by TPP-binding proteins include decarboxylation of -keto acids and cleavage of -hydroxyketones or -diketones. Detailed biochemical analyses and comparative genomics have identified the genes and reactions involved in thiamine synthesis and transport in prokaryotes in the model organisms Escherichia coli, S. enterica serovar Typhimurium (S. Typhimurium), and Bacillus subtilis (3, 29). Thiamine biosynthesis requires the separate formation of a pyrimidine and a thiazole that are then coupled to form thiamine monophosphate (TMP). In E. coli and S. Typhimurium, three kinases, namely ThiM, ThiD, and ThiK, are responsible for the salvage of hydroxyethylthiazole (HET), hydroxymethylpyrimidine (HMP), and thiamine from the medium. In enteric bacteria, thiamine, TMP, and TPP are transported by the ABC transporter system ThiBPQ (35). A member of the YuaJ family has only recently been identified as a thiamine-binding protein in Lactobacillus casei (8), and the thiamine uptake is mediated by novel class of modular transporters in gram-positive bacteria (28).

In L. monocytogenes, the gene encoding ThiC involved in phospho-HMP (HMP-P) synthesis (Fig. 1) has not been identified, indicating an incomplete de novo biosynthesis pathway for thiamine (9). This finding is supported by the fact that L. monocytogenes requires 0.5 to 1.0 mg/liter thiamine for optimal growth in vitro (27). lmo1429 has been predicted as a putative thiamine transporter gene within the listerial genome (29). Its transport activity, however, has not been experimentally demonstrated. The listerial genes thiM, thiD, and thiE are organized in an operon (lmo0316 to lmo0318) and were supposed to be nonfunctional in L. monocytogenes. This assumption was based on the presence of a candidate thiamine transporter (29). As alternative explanation, the same authors proposed the existence of unidentified HMP and HET transporters or the erroneous assignment of thiamine specificity to Lmo1429 homologs. lmo0315 within the same listerial operon encodes thiaminase II (TenA), which is involved in thiamine hydrolysis and/or the regeneration of the thiamine precursor HMP (14, 34).

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FIG. 1. Reconstruction of the thiamine biosynthetic and salvage pathways in L. monocytogenes. thiC appears to be the only gene missing for complete de novo biosynthesis of thiamine in Listeria species. A predicted thiamine kinase (ThiK) or ThiG and ThiH homologues have not yet been identified (3, 29). Genes that have been investigated in this study are underlined. Thiamine is transported by ThiT (Lmo1429), as shown in this study. No HMP- or HET-specific transporters have been identified so far. Dashed line, cytoplasma membrane; dashed arrows, possible substrate diffusion or unspecific transport. (Modified from Rodionov et al. [29] according to Begley et al. [3] and the literature cited in the text.)

It is largely unknown under which conditions thiamine precursors are synthesized by L. monocytogenes and which salvage pathways are used during in vitro and intracellular growth. In the present study, the in vitro and intracellular growth properties of L. monocytogenes mutants defective in thiamine biosynthesis or transport were investigated. We experimentally addressed the function of lmo1429, a homologue of yuaJ from Bacillus that encodes a candidate transporter, ThiT, for thiamine (phosphates). L. monocytogenes knockout mutants of thiT (lmo1429) and thiD (lmo317) were also tested for their growth properties in human epithelial cells, revealing the requirement of the uptake of thiamine (phosphate) and biosynthesis of thiamine precursors for wild-type-like intracellular growth of L. monocytogenes. These data indicate that thiamine acquisition is a critical step for bacteria that proliferate within the cytoplasm of host cells.

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Bacterial strains, plasmids, cell lines, and growth conditions. The bacterial strains, cell lines, and plasmids used in this study are listed in Table 1. E. coli was grown in Luria-Bertani (LB) broth, L. monocytogenes EGD was cultivated in brain heart infusion (BHI) or in modified minimal Welshimer's broth (mMWB) with 0.1 g histidine per liter (27) at 37°C or at 30°C and 43°C. The concentration of thiamine, thiamine phosphate, and TPP was 1 mg/liter. If appropriate, the media were supplemented with erythromycin (300 µg/ml for E. coli or 5 µg/ml for L. monocytogenes) or ampicillin (150 µg/ml). For solid media, 1.5% agar (wt/vol) was added. Human colon epithelial cells (Caco-2 cells) were received from the American Type Culture Collection (ATCC HTB-37) and were cultured at 37°C and 5% CO₂ in RPMI 1640 (Biochrom KG) supplemented with 10% fetal calf serum (Perbio Science, Bonn, Germany). For growth curves, L. monocytogenes cells were grown overnight in the appropriate medium at 37°C, diluted as specified, and shaken at 180 rpm in flasks until reaching the stationary phase. The optical density at 550 nm (OD₅₅₀) was measured at appropriate time intervals as indicated.

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TABLE 1. Strains and plasmids used in this study

Standard procedures. DNA manipulations and isolation of chromosomal DNA were performed according to standard protocols (30) and following the manufacturers' instructions. GeneRuler DNA ladder mix from MBI Fermentas (St. Leon-Rot, Germany) was used as a marker for DNA analysis. Plasmid DNA was transformed via electroporation by using a Bio-Rad Gene pulser II as recommended by the manufacturer. PCRs were carried out with Taq polymerase. As a template for PCR, we used 100 to 400 ng chromosomal DNA or an aliquot of a single colony resuspended in 100 µl H₂O. The oligonucleotides used in this study are listed in Table 2. For listerial gene annotation, the Listeria homepage of the Institut Pasteur (http://genolist.pasteur.fr/ListiList/) was used.

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TABLE 2. Oligonucleotides used in this study

Construction of deletion mutants and of plasmid pDG148-Stu-thiT. In-frame deletions of thiD (lmo0317), pdxK (lm0662), and thiT (lmo1429) were performed in parental strain EGD as described recently (16). Briefly, two flanking fragments of 500 bp were amplified from chromosomal DNA derived from strain EGD using the oligonucleotides indicated in Table 2 and ligated via the introduced BglII sites. Following nested PCR and with the ligation mixture as a template, the resulting fragment was cloned into pLSV101 via BamHI and EcoRI, giving rise to the plasmids described in Table 1. The plasmids were transformed into L. monocytogenes EGD by electroporation. Then erythromycin-resistant bacteria harboring the chromosomally integrated plasmid were selected upon incubation at 43°C. Cointegrates were resolved as described previously (16), and erythromycin-sensitive clones were screened by PCR to identify the mutants EGDthiD, EGDpdxK, and EGDthiT. The gene deletions in all three mutants were confirmed by sequencing. To complement the thiT deletion, gene lmo1429 was amplified with the primers Lmo1429Ue_uni and Lmo1429Ue_rev and then cloned into StuI-digested pDG148 via the ligation-independent cloning system as described recently (1, 17).

Test for pyrithiamine sensitivity and HMP utilization. Five milliliters of mMWB was supplemented with 100 µl LB medium, inoculated with the appropriate L. monocytogenes strain, and incubated overnight under shaking (180 rpm) at 37°C. The culture was then diluted 1:10 in mMWB without thiamine in a total volume of 20 to 50 ml and incubated as described above for 12 h. For thiamine depletion, cells were diluted 1:100 in mMWB without thiamine and further cultivated until they reached the stationary phase. The number of living cells was determined by plating. Then pyrithiamine sensitivity and HMP utilization tests were performed as follows. A total of 10⁶ cells were plated on mMWB agar plates with 0.01 mg/liter thiamine (pyrithiamine test) or without thiamine (HMP test). A filter disc was placed in the middle of the appropriate agar plates and impregnated with 10 µl of 2 mM pyrithiamine or 20 µl of 120 µM HMP. The plates were then incubated at 37°C for 3 days.

Epithelial cell infection assays. Caco-2 cells (2.5 x 10⁵ per well) were seeded in a 24-well culture plate and cultivated for 22 h until infection. Cells were washed twice with Mg²⁺- and Ca²⁺-containing phosphate-buffered saline (PBS/Mg²⁺ Ca²⁺) and covered for 1 h with 500 µl RPMI 1640 containing approximately 2.5 x 10⁶ bacteria (multiplicity of infection [MOI], 10) from a glycerol stock washed twice with PBS. For glycerol stocks, strains were grown in 20 ml BHI medium to mid-log phase (OD₅₅₀, 0.6) and supplemented with glycerol (15% final concentration). Aliquots of 1 ml were frozen at –80°C. Prior to infection, samples were thawed, and the number of viable bacteria was determined as CFU per ml.

The average MOI was calculated immediately after infection and ranged from 8 to 11. After an infection period of 1 h, the Caco-2 cells were washed twice with PBS/Mg²⁺ Ca²⁺. Extracellular bacteria were removed by adding 0.5 ml RPMI 1640 containing 100 µg/ml gentamicin for 1 h, and the medium was then replaced by RPMI 1640 with 10 µg/ml gentamicin. At appropriate time points of incubation in the presence of 10 µg/ml gentamicin, the infected Caco-2 cells were washed again with PBS/Mg²⁺ Ca²⁺ and then lysed in 1 ml cold Triton X-100 (0.01%). The intracellular replication behavior of the mutants and the wild type was quantified by plating dilutions of the lysed cells on BHI agar plates that were incubated at 37°C and 43°C, respectively, for 1 day. If appropriate, the plates contained 5 µg/ml erythromycin. To examine adhesion properties of bacterial strains, the infection time was reduced to 35 min, and before lysis, cells were washed four times with PBS/Mg²⁺ Ca²⁺. The capability of bacterial cells to invade Caco-2 cells was investigated as described above, but lysis of the epithelial cells was performed after 1 h, and a higher gentamicin concentration of 50 µg/ml was used. In all experiments, intact eukaryotic cell monolayers were observed prior to cell lysis.

Immunoblotting. To prepare L. monocytogenes protein extracts from infected Caco-2 cells, approximately 3.3 x 10⁶ epithelial cells were seeded in 25-cm² tissue culture flasks and grown for 22 h. The Caco-2 cells were infected at an MOI of 200 bacteria per cell, and the infection assay was performed as described above for 14 h. Following cell lysis with Triton X-100 and centrifugation, the pellet was resuspended in 100 µl of Tris-HCl (pH 8.0). Fifty milligrams of Zirconia silica beads (Roth, Karlsruhe, Germany) 0.1 mm in diameter was added, and bacterial cells were subjected to homogenization in a Ribolyser cell disrupter (MP Biomedicals, Solon, OH) for 20 s at 6.5 m/s. This step was repeated six times, and the suspension was centrifuged at 13,000 rpm and 4°C. The sedimented cells were resuspended in Tris-HCl (pH 8.0). The protein concentrations were determined by the Bradford method and then equilibrated by the addition of 2x Laemmli buffer (20). To gain bacterial protein extracts from in vitro cultures, overnight cultures of EGD and mutant strains were diluted 1:50 in BHI and incubated again in BHI at 37°C until reaching an OD₅₅₀ of 1.0. Then 10 ml of each culture was pelleted and resuspended in 100 µl of 2x Laemmli buffer. The suspensions were then incubated for 20 min at 100°C. After centrifugation at 4°C, aliquots of the supernatant were subjected to 12.5% sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Separated proteins were then transferred to a polyvinylidene difluoride membrane (Amersham, Freiburg, Germany) with a semidry apparatus. The membrane was blocked overnight at 4°C in Tris-buffered saline with 0.1% Tween 20 containing 10% fetal calf serum. Antibodies against ActA and Hly were diluted 1:1,000. Goat anti-rabbit immunoglobulin G (Dianova, Hamburg, Germany) served as the second antibody in a 1:15,000 dilution. Proteins were detected using the alkaline phosphatase system.

Uptake of [³H]thiamine. Overnight cultures of DH5/pDG148-Stu-thiT and DH5/pDG148-Stu were diluted 1:50 in LB and cultivated to an OD₅₅₀ of 0.5. Then isopropyl-β-D-thiogalactopyranoside (IPTG; Roth, Karlsruhe, Germany) was added to a final concentration of 2 mM. The cultures were centrifuged 3 h later, and the cells were resuspended in LB to an OD₅₅₀ of 10. To perform the thiamine uptake assay, 50 µl of the cell suspension was mixed with 450 µl of phosphate-citric acid buffer (pH 5.0), 12.5 µl of 40% glucose, and 6.25 µl of substrate. The substrate was a mixture of [³H]thiamine (American Radiolabeled Chemicals, St. Louis, MO) and unlabeled thiamine with a specific activity of 0.19 Ci/mmol and was present during the uptake assay at a concentration of 1.20 µM. Aliquots of 50 µl were removed at specified time points, rapidly filtered through a 0.45-µm nitrocellulose filter, and washed with an excess of 50 mM NaCl to remove unspecifically bound thiamine. Filters were added to 3 ml scintillation cocktail and counted for 1 min in a Perkin Elmer TriCarb scintillation counter.

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thiD (lmo0317) encoding a putative bifunctional HMP and HMP-P kinase enables growth of L. monocytogenes in the presence of HMP. Two listerial genes, lmo0317 and lmo0662, are annotated to encode enzymes with HMP and HMP-P kinase activity. Lmo0317 is a homolog of B. subtilis BSU11710/YjbV (BLAST search E value of 10^–64, 50% identity, and 68% similarity over 239 amino acids [aa]) and also of BSU38020/ThiD (E value of 10^–48, 43% identity, and 59% similarity over 243 aa). On the other hand, Lmo0662 is the ThiD homolog of BSU38020 (E value of 10^–86, 58% identity, and 75% similarity over 264 aa) and is less similar to YbjV (E value of 10^–48, 43% identity, and 59% similarity over 243 aa). Lmo0317 and Lmo0662 differ significantly from each other (42% similarity), indicating different enzymatic activities. Since the currently annotated ThiD exhibited pyridoxal kinase (PdxK) activity, Park et al. suggested that YjbV of B. subtilis be reannotated as ThiD (26). PdxK is able to phosphorylate several substrates, including HMP, but the physiological significance of this broad substrate specificity is not clear (3). To investigate the functional status of thiamine synthesis and to provide evidence for the function of Lmo0317 as HMP/HMP-P kinase (ThiD) of L. monocytogenes, we first constructed a nonpolar deletion mutant of lmo0317. No growth deficiencies of mutant EGDthiD were observed in BHI (data not shown). The growth of this mutant was then measured in mMWB with thiamine, resulting in a slighty reduced growth in comparison to the wild-type strain. The lmo0662 deletion mutant EGDpdxK, which served as a control, showed a similar growth phenotype (Fig. 2A). These data show that under in vitro conditions in the presence of 1 mg/ml thiamine, both thiD and pdxK contribute only slightly to listerial growth and that most thiamine is taken up from the extracellular medium. To test the ability of L. monocytogenes strain EGD to utilize HMP as a source of thiamine, cells were depleted of thiamine as described above and grown on thiamine-free mMWB agar plates with HMP-impregnated filter discs (Fig. 2B). Growth of EGD demonstrates that the thiazole moiety can be synthesized de novo in L. monocytogenes, although thiG and thiH homologues involved in HET formation have not been identified (Fig. 1). In contrast, mutant EGDthiD did not grow under these conditions (Fig. 2B), strongly suggesting a role of listerial ThiD in catalyzing the phosphorylation of HMP and/or HMP-P. Diminished growth of mutant EGDpdxK in comparison to the wild type was observed (Fig. 2B), indicating a minor role of PdxK in this metabolic pathway. In agreement with the thiamine requirement in vitro, supplementation of mMWB with HET alone was not sufficient to allow listerial growth (data not shown).

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FIG. 2. (A) Growth properties of EGDthiD and EGDpdxK in the presence of thiamine. Listerial cells were grown in mMWB containing 1 mg/ml thiamine, and growth was monitored for 72 h. Each experiment was reproduced three times. Standard deviations for each time point are indicated as error bars. Error bars were omitted when they were smaller than the symbols. (B) Growth assay with HMP. A total of 106 cells of thiamine-depleted strains EGD, EGDthiD, and EGDpdxK were plated on mMWB agar plates without thiamine, and a filter disc impregnated with 20 µl of a 120 µM solution of HMP was added. Plates were then incubated for 3 days at 37°C.

Intracellular growth attenuation of EGDthiD. While a wild-type-like growth curve was derived when mutant EGDthiD was grown at 37°C in BHI (data not shown), a strong attenuation of its growth was observed in an epithelial cell infection assay (Fig. 3). Caco-2 cells were infected with EGD, EGDthiD, and, as further controls, with EGDlmo1538/glpD::pLSV101 and EGD-eutB::pLSV101. The latter two mutants have defects in the utilization of glycerol and ethanolamine, respectively, and showed attenuated growth in epithelial cells (16). The number of viable intracellular bacteria in the epithelial cells determined at the later time points was reduced, indicating an approximately 3.3-fold attenuation of the thiD deletion mutant relative to the wild-type strain after 7 h of intracellular replication. In contrast, deletion of pdxK has only a slight influence on listerial growth within epithelial cells. The lower replication rate of EGDthiD in this experiment indicates an important role of ThiD and therefore of thiamine de novo biosynthesis in the in vivo metabolism of L. monocytogenes.

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FIG. 3. Deletion of thiD attenuates growth in epithelial cells. Caco-2 cells were infected with listerial cells at an MOI of 10. The MOI was determined immediately before infection (0 min). The number of intracellular bacteria was determined 35 min, 120 min, and 480 min after the infection. Bars represent the number of viable cells of each strain divided by the number of viable cells of the wild-type strain at each time point. Mutants EGD-eutB::pLSV101 and EGDlmo1538/glpD::pLSV101 served as controls. Each experiment was independently performed at least four times. Standard deviations are indicated by error bars.

Lmo1429 is essential for wild-type-like growth in mMWB containing thiamine, TMP, or TPP. lmo1429 contains a thi box in its 5' untranscribed region, indicating that TPP directly regulates its expression via a riboswitch mechanism (29, 36). lmo1429 is predicted to encode an ATP-independent thiamine transporter containing six transmembrane segments (29). In a BLASTp analysis, the protein with a length of 200 aa revealed significant homology to the putative thiamine transporter YuaJ of Bacillus spp. (for example Bacillus weihenstephanensis: 43% identity and 65% similarity over a length of 185 aa and E value of 10^–38) and also those of Streptococcus spp., Lactobacillus spp., or Enterococcus faecalis. The genome of L. monocytogenes does not contain genes with sequence similarities to the presumably thiamine precursor-specific ABC transporter encoded by the ykoFEDC (thiUVWX) operon of B. subtilis (31). To provide experimental evidence for the thiamine transport activity of Lmo1429, we constructed the deletion mutant EGDthiT. No growth deficiencies were observed in comparison to the EGD wild type when the mutant was grown in BHI (data not shown). In thiamine-supplemented mMWB, retarded growth of EGDthiT in comparison to the control strain EGD was observed (Fig. 4A). However, uptake of thiamine by diffusion or unspecific transporters probably allows EGDthiT to reach the stationary phase 12 h later than the wild-type control. We also tested the effect of a thiT knockout on the growth properties of L. monocytogenes in the presence of TMP or TPP. Whereas TMP and TPP were equivalent to thiamine for the growth of wild-type cells, the stationary phase of EGDthiT is reached approximately 24 h later in the presence of these thiamine phosphates (Fig. 4A). Dephosphorylation of TMP or TPP to free thiamine by extracellular phosphatases might explain this observation.

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FIG. 4. (A) Growth behavior of mutant EGDthiT in mMWB supplemented with thiamine, TMP, or TPP. Strains EGD and EGDthiT were incubated overnight in mMWB containing 2.5% LB, washed twice with PBS, resuspended in mMWB without thiamine to an OD550 of 3.0, and then diluted 1:500 into mMWB. Following 1:500 dilution, the strains were grown in mMWB with 3 µM thiamine, TMP, or TPP, respectively. Three independent experiments were used to determine OD values at each time point and to derive the standard deviation. Error bars were omitted when they were smaller than the symbols. (B) Thiamine-depleted EGD and EGDthiT cells were plated on mMWB agar containing 0.01 mg/liter thiamine, and a filter disc with 10 µl of a 2 mM pyrithiamine solution was placed in the middle of the plates. Pictures were taken after 3 days at 37°C.

We then investigated the growth behavior of EGDthiT in the presence of HMP. No growth deficiency was observed in comparison to the wild-type strain (data not shown), indicating that Lmo1429 is not involved in transport of HMP into L. monocytogenes. Taken together, the data presented here demonstrate that Lmo1429 is involved in the uptake of thiamine by L. monocytogenes and that thiamine, but not thiamine precursors or phosphorylated thiamine, is transported by Lmo1429. In agreement with other groups, we propose the name "ThiT" for Lmo1429 and its homologs such as YuaJ of B. subtilis (8, 31).

Growth in the presence of pyrithiamine. Pyrithiamine, a toxic thiamine analogue, was used to find further evidence for the transport function of listerial ThiT (Lmo1429). Thiamine-depleted EGD and EGDthiT were plated on mMWB medium containing a suboptimal concentration of thiamine (0.01 mg/liter) and grown in the presence of a filter disc with 2 mM pyrithiamine. A large growth inhibition zone around the disc was observed for EGD, while growth of EGDthiT was not affected obviously because the thiT deletion results in a lack of pyrithiamine uptake (Fig. 4B). Without thiamine depletion, a toxic effect of pyrithiamine on the wild-type strain was not observed, indicating the repression of thiT in the presence of thiamine, which is likely mediated by its thi box.

The thiT deletion mutant is deficient in intracellular replication. The transporter mutant EGDthiT was also tested for its replication rate within epithelial cells. Caco-2 cells were infected with the wild-type strain, EGDthiT, EGD inlACGHE, and EGDinlAB. The latter two served as controls to exclude adhesion and invasion deficiencies as a reason for the reduced number of intracellular EGDthiT cells. Wild-type-like growth was observed when all mutants were grown at 37°C in BHI (data not shown). Seven hours after infection, EGDthiT exhibited a 2.2-fold attenuation within epithelial cells compared to the wild-type strain (Fig. 5). The number of viable cells following lysis of the Caco-2 cells was slightly higher than that of the double mutant affected in glycerol utilization in Fig. 3.

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FIG. 5. Deletion of thiT attenuates growth in cell culture experiments. Caco-2 cells were infected with EGD, EGDthiT, and the two control strains EGDinlACGHE and EGDinlAB with an MOI of 10. For further experimental details, see the legend to Fig. 2. Each experiment was independently performed at least four times. The standard deviations are indicated.

ThiT binds thiamine. To further investigate the functional role of ThiT in thiamine metabolism of L. monocytogenes, E. coli strain DH5 was transformed with plasmid pDG148-Stu-thiT, which allows inducible expression of thiT. Strain DH5/pDG148-Stu served as a control. After induction of gene expression with IPTG, [³H]thiamine was added to the cells, and the amount of [³H]thiamine associated with the cells was determined. In comparison to strain DH5/pDG148-Stu, a four- to fivefold larger amount of [³H]thiamine was associated with strain DH5/pDG148-Stu-thiT at any time point (Fig. 6). There was a rapid increase of cell-associated thiamine within 30 s, followed by a much slower increase at later time points. This phenomenon was not caused by depletion of thiamine from the medium, since the amount of [³H]thiamine in the supernatant was similar to that added to the culture (data not shown), thus excluding that [³H]thiamine is quantatively bound to the cells overexpressing thiT. When the same experiment was performed without IPTG, an average of 5 pmol thiamine (DH5/pDG148-Stu) and 8 pmol thiamine (DH5/pDG148-Stu-1429) was associated with the cells, and this amount increased to 8 pmol thiamine (DH5/pDG148-Stu) or 10 pmol thiamine (DH5/pDG148-Stu-1429) in a 60-min experiment observed (data not shown). Taken together, these data are compatible with thiamine binding to ThiT.

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FIG. 6. [3H]thiamine binds to ThiT expressed in E. coli. E. coli cells transformed with a thiT expression plasmid or an empty vector control were incubated in a phosphate-citric acid buffer, and [3H]thiamine was added for a final concentration of 1.25 µM. The amount of cell-associated [3H]thiamine was determined by rapid filtration of the cells and liquid scintillation counting of the filter and is depicted in pmol/OD550. Three independent experiments were performed. The graphs were drawn for a computer-generated best fit to the results of one representative assay. In similar experiments that used incubation times up to 40 min or varied the amount of cells present during the assay, we consistently found a four- to fivefold-larger amount of [3H]thiamine associated with E. coli cells carrying pDG148-Stu-thiT compared to the control strain.

Expression of ActA and listeriolysin is not altered. It is possible that the knockout of thiT negatively affected the expression of main virulence factors, resulting in the observed intracellular growth deficiencies of the mutant. To exclude this possibility, Western blot analysis was performed with antibodies against actin (ActA) and listeriolysin (Hly), which play a key role in intracellular survival and replication of L. monocytogenes. EGD, EGDthiT, and EGDpdxK, but not the deletion mutants EGDactA and EGDhly, showed expression of ActA and Hly (Fig. 7A and B). Wild-type-like ActA expression was also found in all these strains when they were recovered from Caco-2 cells 24 h after infection (Fig. 7C). Thus, the expression of the known virulence factors ActA and Hly is not reduced by a lack of ThiT, indicating that the reduced activity of TPP-dependent enzymes requiring thiamine as cofactors is responsible for the growth deficiencies observed.

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FIG. 7. Western blot analysis of virulence factors. Total protein extracts from EGD and mutant strains EGDthiT and EGDpdxK were separated in a 12.5% (wt/vol) polyacrylamide gel. Strains EGDactA and EGDhly served as negative controls. (A) Cells were grown in BHI medium to an OD550 of 1.0. Polyclonal antibodies against ActA were used. (B) The experiment was similar to that shown in panel A, but the blot was probed with a polyclonal antibody against Hly. (C) Blot with ActA antibodies. Listerial cells were recovered from Caco-2 cells after 14 h of intracellular growth. The amount of total protein separated is equal in all lanes. The preparation of noninfected Caco-2 cells served as a further control. The positions of molecular mass standards and of ActA and Hly are indicated.

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
A hallmark of pathogens is their metabolic flexibility during infection. Several metabolic pathways have been shown to enable pathogens to occupy specific niches such as epithelial cells or macrophages within their host (24). Examples are the utilization of sugar phosphates and glycerol as alternative carbon sources for intracellularly replicating L. monocytogenes (7, 16) or the cobalamin-dependent degradation of propanediol and ethanolamine by S. Typhimurium within macrophages (19). For L. monocytogenes, we demonstrate in this study that cytosolic replication is reduced 3.3-fold and 2.2-fold upon deletion of thiD and thiT, which are responsible for HMP salvage and thiamine transport, respectively. Thus, the requirement for TPP can be satisfied by two alternative pathways: thiamine uptake or HMP salvage coupled to HET de novo biosynthesis. In other words, thiamine and its precursors are a limiting factor for intracellular pathogens that lack complete thiamine biosynthesis pathways.

The result of the uptake assay suggests that ThiT predominantly functions as a thiamine-binding protein or that its uptake activity is too low to be measured. It is possible that further, yet unidentified components are required for the full activity of listerial ThiT as a thiamine transporter. A similar finding was recently reported for a YuaJ homolog of L. casei, an integral membrane protein able to bind, but not transport, thiamine (8). The authors suggested that additional proteins are required for thiamine uptake in gram-positive bacteria, an assumption that is also supported by a recent work on biotin transport in Rhodobacter capsulatus (13). Comparative genome approaches led to the discovery of an abundant class of vitamin transporters mostly restricted to gram-positive bacteria with an unprecedented architecture that includes an often duplicated energy-coupling module with ATPase activity (the A component) and a small integral membrane protein termed the T component (28). The respective transport system EcfAA'T of B. subtilis is encoded by the gene cassette ybaDEF, and its products are homologous to Lmo2601, Lmo2600, and Lmo2599, which may represent the listerial proteins that function together with ThiT.

The concentration of thiamine in mMWB, as well as in cell culture medium, is 1.0 mg/liter, corresponding to 3.16 µM. It has been reported that the concentration of free TPP in the cytosol of some mammalian cells is 2 to 3 µM (5, 33), but its concentration in epithelial cells is unknown. The data presented here indicate a lower concentration of and/or a higher demand for these substrates during replication within epithelial cells, thus urging the intracellular pathogen L. monocytogenes to de novo synthesize HET and to salvage thiamine via HMP uptake resulting from thiaminase II activity (14). Alternatively, listerial tenA might function as a thiC analog, allowing thiamine biosynthesis under certain conditions. B. subtilis tenA has been shown to complement an E. coli thiC strain (23). This assumption about the thiamine metabolism of L. monocytogenes replicating in human epithelial cells is partially supported by DNA microarray analysis. Significant changes in expression were demonstrated for thiF, thiD (lmo0317), and thiI, which are 2.64-fold and 3.07-fold upregulated and 3.10-fold downregulated, respectively, 6 h after infection of Caco-2 cells (16).

Thiamine-dependent enzymes of L. monocytogenes are listed in Table 3. TPP is a cofactor of many enzymes such as transketolase, pyruvate dehydrogenase, -ketoglutarate dehydrogenase, glyoxylate carboligase, and acetolacetate synthase (3). In addition to their role in pyruvate metabolism, the majority of TPP-dependent enzymes are involved in the pentose-phosphate cycle and in biosynthesis and degradation of the branched-chain amino acids isoleucine, leucine, and valine. Remarkably, it was demonstrated recently by DNA microarray analysis that the pentose phosphate cycle, not glycolysis, is the predominant pathway of sugar metabolism of L. monocytogenes during growth in epithelial cells (16). In the same study, upregulation of genes involved in the biosynthesis of branched-chain amino acids—amino acids that host cells are not able to produce—was observed. This finding may indicate that bacteria require higher thiamine concentrations during intracellular growth than in vitro. The fact that the expression of virulence factors was not affected by the knockout of thiT or pdxK underlines the important role of an intact metabolism for intracellular replication. Thus, TPP appears to be a key cofactor for the metabolism of L. monocytogenes under nutrient-limited conditions. Therefore, the availability of thiamine, its precursors, and phosphates is expected to have a significant effect on the in vivo replication of L. monocytogenes.

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TABLE 3. Annotated TPP-dependent enzymes in L. monocytogenes

 
We thank Werner Goebel for the gift of ActA and Hly polyclonal antibodies and Tadgh Begley for providing HMP.

This work was supported by the Competence Center PathoGenoMik funded by the Federal Ministry of Education and Research (BMBF), Germany, and by the Deutche Forschungsgemeinschaft.

 
* Corresponding author. Mailing address: Zentralinstitut für Ernährungs- und Lebensmittelforschung (ZIEL), Abteilung Mikrobiologie, Technische Universität München, Weihenstephaner Berg 3, D-85354 Freising, Germany. Phone: 49-8161-713859. Fax: 49-8161-714492. E-mail: thilo.fuchs{at}wzw.tum.de

Published ahead of print on 30 January 2009.

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Journal of Bacteriology, April 2009, p. 2218-2227, Vol. 191, No. 7
0021-9193/09/$08.00+0     doi:10.1128/JB.01636-08
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