2 Muenster University Hospital, Institute of Physiological Chemistry and Pathobiochemistry, Waldeyerstr. 15, D-48129, Münster, Germany and 3 Abteilung für Medizinische Mikrobiologie und Hygiene, Universitätsklinikum, Albert-Einstein-Allee 11, Ulm, Germany
Received on April 20, 2004; revised on June 1, 2004; accepted on June 16, 2004
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Abstract |
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Key words: capsule / extracellular polysaccharide synthesis / protoplast membrane / virulence
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Introduction |
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Hyaluronan is elongated at the reducing end by alternate transfer of UDP-hyaluronan to the substrates UDP-GlcNac and UDP-GlcA liberating the UDP-moiety (Prehm, 1983a,b
). Chain initiation does not require a protein backbone as for proteoglycans. Only the presence of the nucleotide sugar precursors is sufficient to initiate new chains. During elongation the chain is retained on the membrane integrated synthase. This mechanism of synthesis operates in vertebrates and in streptococci. Hyaluronan is synthesized in vertebrates at plasma membranes (Prehm, 1984
) and in streptococci at protoplast membrane (Markovitz and Dorfman, 1962
), and nascent chains are directly extruded into the extracellular matrix. The enzymatic activity can be solubilized by digitonin (Triscott and van de Rijn, 1986
) and purified in active form as a single 42-kDa protein (Prehm et al., 1996
). This study indicates that a single 42-kDa protein is sufficient to direct hyaluronan synthesis, but it does not address the mechanism of hyaluronan export.
Two genes of the has operon encode enzymes that are involved into the synthesis of hyaluronan (DeAngelis et al., 1993; Dougherty and van de Rijn, 1994
): The gene hasA is sufficient for the enzymatic activity; hasB encodes a UDP-glucose dehydrogenase, which synthesizes glucuronic acid from UDP-glucose; a third gene hasC encodes a UDP-glucose phosphorylase, which is not directly required (Weigel et al., 1997
). From the structural arrangement of the hydrophobic domains, it was concluded that the catalytic activity faces the cytoplasm (Heldermon et al., 2001
). This raised the question of hyaluronan export through the protoplast membrane. Irradiation inactivation was employed to calculate the size of the capsule producing proteins (Tlapak-Simmons et al., 1998
). From this study the authors concluded that the size excluded the participation of proteins other than the synthase itself. Although this method only allowed the conclusion that the synthase was not covalently associated with other proteins, the existence of an independent hyaluronan transporter could not be ruled out. Studies on the hyaluronan synthase activity in Escherichia coli transfected with the synthase gene were also performed (DeAngelis et al., 1993
), but production of hyaluronan by intact cells was not demonstrated.
Most bacterial polysaccharides exit cells by specialized transporters. Genes for these transporter proteins are often found in the vicinity of genes for polysaccharide synthesis. The upstream chromosomal region of S. pyogenes flanking the has operon was partially analyzed (Ashbaugh and Wessels, 1995; Ashbaugh et al., 1998
). The authors found a gene cluster containing an ABC transporter. Deletion of two open reading frames (ORFs) next to the has operon showed no effect on capsule formation, and inactivation the ABC transporter itself was not carried out.
Because the available data do not exclude the participation of a transporter in hyaluronan export and because most other extracellular polysaccharides are exported by ABC transporters (Paulsen et al., 1997), we further investigated hyaluronan export. By means of insertion mutagenesis we show that the ABC transporter located upstream of the has gene locus is required for hyaluronan release by streptococci. Genetic complementation experiments also confirm this finding.
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Results |
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Identification of hyaluronan-deficient mutants
Isolation of hyaluronan-deficient mutants was performed by visual inspection of the colonies. Large, glossy colonies produce the hyaluronan capsule, whereas small, opaque colonies lack such capsules (Wilson, 1959). About 10,000 colonies of the CS101pGh9:ISS1 mutant library were screened for loss or reduction of the mucoid capsule on blood agar. Eleven colonies were nonmucoid, and nine colonies were less mucoid. Among the clones with reduced mucoid appearance, we found one with an unaffected synthase activity but with reduced hyaluronan release into the medium (Figure 1) and hyaluronan production (Table I). This mutant was selected for further characterization.
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DNA and protein sequence analysis of the mutated gene locus
SPy2194 belongs to a gene cluster of seven further ORFs located adjacent to the hyaluronan synthase genes and transcribed in opposite direction (Figure 2). The encoded protein has Walker A and B motifs (Walker et al., 1982) at amino acids 3947 (GHTGSGKST) and 146157 (LSGGQMRRVAIA), respectively. The preceding SPy2195 protein also harbors a Walker A motif at amino acids 5765 (GHNGSGKST) and a Walker B motif at amino acids 158169 (LSGGQKQRVAIA) and has 72% sequence similarity to SPy2194. SPy2195 had no transmembrane segment, and SPy2194 had one. SPy2194 was also related to the KpsT transporter for polysialic acid (54% similarity) or the K5 antigen of gram-negative bacteria and to the human multidrug resistance (MDR) transporter (65% similarity) that belong to the ABC-2 subfamily (Paulsen et al., 1997
). Figure 3 shows the sequence alignments of SPy2194 with Kpst transporter for polysialic acid from E. coli and a segment of the human MDR transporter MDR1 ranging from amino acid residue 388 to 678.
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Reconstitution of hyaluronan release by streptococci by genetic complementation
The requirement of a functional ABC transporter for hyaluronan release from streptococci was confirmed by genetic complementation of the entire 3.8-kb chromosomal fragment make up SPy2195, SPy2194, SPy2193, and SPy2191. It was amplified by PCR and subcloned into pAT28 and the construct pAT28hax was obtained. When pAT28hax was transfected into the ABC transporter mutant, colonies displayed the mucoid phenotype on agar plates, whereas bacteria transfected with only pAT28 resembled the original mutant colonies. These results indicated that an ABC transporter gene cluster was involved in hyaluronan production.
Hyaluronan in the media, capsules and cells
The rate of hyaluronan release of the mutant complemented with pAT28hax almost reached the level of the wild-type cells (Figure 1). The amount of hyaluronan in the three compartments of the media, capsules, and cells was determined for the wild-type, mutant, and reconstituted strain (Table I). The data were related to a 1-ml culture at an optical density at 600 nm of 1.0 (A600 = 1.0) that corresponded for all three strains to 1.2 x 109 colony forming units (cfu). The amount of hyaluronan on centrifuged and washed bacteria was defined as the sum of capsular and cellular hyaluronan. Residual hyaluronan that was left on the bacterial sediment after hyaluronidase digestion was defined as cellular hyaluronan. Table I shows that the mutant strain released about 30% of hyaluronan into the medium as compared to the wild-type and reconstituted strain. The capsular hyaluronan was reduced about 40%. After hyaluronidase digestion, the cellular hyaluronan was very low and showed hardly any differences in the wild-type, mutant, and reconstituted strains. The results indicated that an ABC transporter was required for hyaluronan capsule production in intact streptococci.
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Discussion |
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The insertional mutation into an ATP-binding protein of the ABC transporter decreased hyaluronan production of S. pyogenes by about 70% and reduced the capsule by about 40%. A balanced analysis indicated that most hyaluronan was released into the culture medium or resided in the capsule. Only minute amounts of hyaluronan were hyaluronidase-resistant, indicating that hyaluronan was exported immediately after or during ongoing synthesis. Complementation with a chromosomal fragment coding for SPy2195 to SPy2191 restored hyaluronan deposition in the capsule and release into the medium.
The ABC transport system is localized adjacent to the hyaluronan synthase genes with a reading frame in opposite direction. A similar arrangement of the hyaluronan synthesis and export genes is described for Pasteurella multocida A:1 (Chung et al., 1998). Also here the genes are localized in two separate but adjacent clusters. However, the export proteins of Streptococcus and Pasteurella show very little homology to each other, as do the hyaluronan synthases. This supports the notion that these genes have evolved independently.
The gene cluster of the mutated ABC transport system possesses interesting features. SPy2199 encodes an unknown protein with some similarity to RecA protein from Pseudomonas. This protein is made responsible for homologous recombination with incoming DNA that was liberated from autolytic bacteria and picked up by surviving cells (Mortier-Barriere et al., 1998). SPy2198 and SPy2197 encode for proteins with some similarity to zinc proteases; pgsA encodes a CDP-phosphatidylglycerol-glycerophosphate transferase responsible for the biosynthesis of cardiolipin. The enzymatic activity of the hyaluronan synthase is strongly stimulated by cardiolipin, the optimal lipid for its reconstitution (Triscott and van de Rijn, 1986
). SPy2195 and SPy2194 are similar to each other and contain the Walker A and B motifs and the signature motif LSGGQ found in all ATP-binding domains of ABC transporters. The expression of SPy2194 is influenced by the regulatory system covRS that controls virulence factors, such as capsule, cysteine protease, streptokinase, streptolysin S, and streptodornase (Graham et al., 2002
).
The streptococcal hyaluronan transport system has sequence similarity to other bacterial ABC-2 transporters, particularly to the Kps-transport system of E. coli responsible for the export of the K1 and K5 antigens that both resemble hyaluronan in their polyanionic nature. The structure of the K1 antigen is a desulfated analog of heparin consisting of N-acetyl-glucosamine and glucuronic acid dissaccharide units and the K5 antigen is a polysialic acid. The bacterial ABC-2 transport family contains several subunits (Whitfield and Roberts, 1999). In E. coli, KpsM and KpsT are located at the inner cytoplasmic membrane. KpsM is a membrane embedded channel protein and similar to SPy2193. KpsT is an ATP-binding cassette protein in two copies and similar to SPy2195 and SPy2194. Mutations at different positions in the KpsT gene lead to varying degrees of transport failure, but not to complete inactivation (Bliss et al., 1996
). This phenomenon resembles the partial inactivation of hyaluronan production observed in our insertion mutant. Further experiments will be required to determine the roles of the genes for capsule production and the functions of the individual proteins.
Our results do not exclude the possibility that hyaluronan was exported by another mechanism that is dependent on the ABC transport system. However, the sequence similarity of the streptococcal ABC transport proteins with human MDR transporters instigated the analysis of hyaluronan export from eukaryotic cells (Prehm and Schumacher, 2004). We found that hyaluronan export was inhibited by a variety of inhibitors for multidrug resistance transporters. This indicated that hyaluronan was exported by an ABC transporter in eukaryotic cells. Unfortunately, these inhibitors did not block the streptococcal hyaluronan export.
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Materials and methods |
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Bacterial strains and plasmids
Group A S. pyogenes M49 (strain CS101) was from A. Podbielski. E. coli EC101 (TG1 containing a chromosomal copy of the pWV01 repA gene) was provided by K. Leenhout (Duwat et al., 1997). Thermosensitive pGhost9:ISS1 vector containing the ISS1 insertion sequence from Lactococcus lactis and an erythromycin resistance marker was obtained from E. Maguin (Maguin et al., 1996
). pCRII-TOPO vector from Invitrogen (Karlsruhe, Germany) was used to clone and sequence PCR products. pAT28 vector with a spectinomycin resistance marker was from P. Courvalin (Trieu-Cuot et al., 1990
). Streptococci were grown on TH agar supplemented with 3% sheep blood (Oxoid, Wesel, Germany), in TH or in TH supplemented with 0.5% yeast (THY) at 37°C. CS101 mutant strain containing pGhost9:ISS1 vector was subcultured in medium supplemented with erythromycin (5 mg/L) at 37°C.
DNA and protein sequence analysis
The deduced amino acid sequences of these ORFs were compared to those available in the protein sequence database using the blastp algorithm at the NCBI Web site (Altschul et al., 1997). The conserved domain analysis of the deduced amino acid sequence of SPy2195 to SPy2193 and SPy2191 was performed using the program RPS-BLAST and the Conserved Domain Database of Entrez at the NCBI Web site (http://www.genome.ou.edu/strep.html). Topology prediction of transmembrane domains was calculated by the TopRed software (Claros and von Heijne, 1994
).
General DNA techniques
Standard recombinant DNA techniques for nucleic acid preparation and analysis were performed as described (Sambrook et al., 1989). DNA restriction fragments were isolated from agarose gels with the QIAquick Gel Extraction Kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions. Electrotransformation of E. coli was performed by the method of Dower et al. (1988)
with a BioRad Gene Pulser (BioRad Laboratories, Munchen, Germany). Genomic streptococcal DNA was isolated as described previously (O'Connor and Cleary, 1987
). Plasmids were prepared from streptococci by treatment with mutanolysine and lysozyme in 25% sucrose as described (O'Connor and Cleary, 1987
), and protoplasts were separated by centrifugation. The sediment was taken as starting material for the Qiaprep spin miniprep kit. DNA was sequenced on an ABI 310 automated DNA sequencer using the ABI PRISM Big Dye terminator cycle sequencing kit (PE Applied Biosystems, Langen, Germany).
Construction of an S. pyogenes mutant library
The S. pyogenes mutant library was constructed by chromosomal insertion of the thermosensitive pGhost9:ISS1 vector (Maguin et al., 1996). Bacteria were transformed by electroporation with 1 µg purified plasmid DNA. Selection of plasmid-containing strains was performed on blood agar plates supplemented with erythromycin (5 mg/L) at a temperature of 30°C, allowing replication of the vector. To generate chromosomal integration of the plasmid, one of the isolates was grown overnight in TH broth (THB) supplemented with erythromycin (5 mg/L) at 30°C. The saturated culture was diluted 1:100 in fresh THB without antibiotic pressure and incubated for 3 h at 30°C. To reduce the plasmid copy number per bacterial cell, the culture was transferred to a 38°C water bath and incubated for another 2 h. Samples were diluted with fresh THB and plated on erythromycin-containing blood agar plates (1 mg/L). Mutants were selected for further studies after overnight growth at nonpermissive temperature (above 37°C).
Determination of the ISS1 insertion site
To identify the chromosomal insertion site of the ISS1 insertion sequence, 2 µg of total genomic DNA of the pGhost9: ISS1 mutants was digested with EcoRI or HindIII, extracted with phenol/chloroform, and ethanol precipitated. The digested DNA was diluted to a final concentration of 0.5 µg/ml and ligated with the T4 DNA ligase under conditions as described (Ochman et al., 1989). The obtained plasmid mixture was transfected into E. coli EC101 (Duwat et al., 1997
). Erythromycin-resistant E. coli clones containing pGhost9:ISS1 plasmid with genomic streptococcal DNA flanking the insertion site of the plasmid were selected on LB agar with erythromycin (150 mg/L). The genomic DNA contained in the recovered plasmids was sequenced with primers annealing to pGhost9:ISS1 vector sequences. To sequence the plasmids obtained after cloning EcoRI-digested genomic fragments we used primers pGhost5SK and ISpGhost9P8. Primers pGhost5KS and ISpGhost9P7 were used to sequence plasmids obtained after cloning HindIII-digested DNA. The location of the primers is demonstrated in Figure 2, and their sequences are indicated in Table II.
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Measurement of the hyaluronan synthase activity
Bacteria were grown in 50 ml TH media, transferred into an ice bath at the logarithmic phase (A600 = 0.6) for 1 h, harvested by centrifugation for 10 min at 3000 x g and washed with water. The bacteria were suspended in 2 ml 0.1 M Tris-malonate, 1 mM ATP, pH 7.0; sonicated for 5 min at 80 W; centrifuged for 2 min at 10,000 rpm; and incubated in 0.6 ml 80 µM UDP-GlcNac and 4 µM UDP-[14C]GlcA (specific activity 320 mCi/mmol), 1 mM dithiothreitol, 10 mM MgCl2, 0.5 mM ATP, 50 mM Tris-malonate pH 7.0 for 1 h at 37°C. A solution of 10 µl of 10% sodium dodecyl sulfate was added to inactivate the synthase, and the mixture was applied to descending paper chromatography on Whatman MM paper for 18 h in 1 M ammonium acetate, pH 5.5/ethanol (13:7). The origin was cut out and radioactivity determined.
Measurement of hyaluronan release by intact bacteria
The rate of hyaluronan synthesis by intact bacteria was measured by incorporation of [3H]glucosamine into hyaluronan. Overnight cultures of S. pyogenes were diluted at a ratio of 1:10 with fresh media and incubated at 37°C for 3 h to reach exponential growth. Aliquots of 1 ml were sedimented for 1 min at 10,000 x g, suspended in 0.5 ml fresh medium, mixed with 10 ml of a 100 µg/µl solution of UDP-GlcNac and 25 µl of [3H]glucosamine and incubated at 37°C. The high concentration of UDP-GlcNac diluted any UDP-[3H]GlcNac that may have been formed by bacteria from [3H]glucosamine and could distort the results. After different time periods the suspension was again centrifuged for 1 min at 10,000 x g. The supernatant was subjected to the descending paper chromatography as described.
Determination of released, capsular, and cellular hyaluronan
The amount of hyaluronan in the media, capsules, and cells was determined by the method of Schrager et al. (1996) with slight modifications. Streptococci were grown in 3% TH medium supplemented with 0.5% glucose to an A600 = 0.5 and harvested by centrifugation, washed with water, and suspended in 0.5 ml water. Capsular and cellular hyaluronan was released by shaking with 1 ml chloroform. After centrifugation the hyaluronan content in the water phase was determined by addition of 2 ml of a solution of 20 mg Stains-All (1-ethyl-2-[1-ethylnaphto-[1,2-d]thiazolin-2-ylidene)-2-methylpropenol]naphto-[1,2-d]thiazolium bromide) and 60 µl of acetic acid in 100 ml dimethylformamide. The absorbance was read at 650 nm and compared to a standard curve of known hyaluronan concentrations. To determine of hyaluronan in the cellular compartment, the same amount of streptococci was suspended in a solution of 1 ml of testicular hyaluronidase (1 mg/ml) in 0.1 M sodium acetate, pH 4.0, and incubated for 10 min at 37°C. After the digestion of the capsule the cellular hyaluronan was released with 1 ml of chloroform and determined as described. The culture supernatant (1 ml) was treated with 1 ml chloroform and hyaluronan in the water phase of determined as described.
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Acknowledgements |
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Footnotes |
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Abbreviations |
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References |
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