This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowReprints and Permissions
Right arrow Copyright Information
Right arrow Books from ASM Press
Right arrow MicrobeWorld
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Stinson, M. W.
Right arrow Articles by Barnard, J.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Stinson, M. W.
Right arrow Articles by Barnard, J.

 Previous Article  |  Next Article 

Infect Immun, January 1998, p. 259-265, Vol. 66, No. 1
0019-9567/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.

Streptococcal Histone-Like Protein: Primary Structure of hlpA and Protein Binding to Lipoteichoic Acid and Epithelial Cells

Murray W. Stinson,1,2,* Robert McLaughlin,1,dagger Suk Ho Choi,1,Dagger Zaira E. Juarez,1 and John Barnard1

Department of Microbiology, School of Medicine and Biomedical Sciences,1 and Department of Oral Biology, School of Dental Medicine,2 State University of New York at Buffalo, Buffalo, New York 14214

Received 11 August 1997/Returned for modification 25 September 1997/Accepted 15 October 1997

    ABSTRACT
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

In addition to its role in the nucleoid, the histone-like protein (HlpA) of Streptococcus pyogenes is believed to act as a fortuitous virulence factor in delayed sequelae by binding to heparan sulfate-proteoglycans in the extracellular matrix of target organs and acting as a nidus for in situ immune complex formation. To further characterize this protein, the hlpA genes were cloned from S. pyogenes, S. gordonii, S. mutans, and S. sobrinus, using PCR amplification, and sequenced. The encoded HlpA protein of S. pyogenes has 91 amino acids, a predicted molecular mass of 9,647 Da, an isoelectric point of 9.81, and 90% to 95% sequence identity with HlpA of several oral streptococci. The consensus sequence of streptococcal HlpA has 69% identity with the consensus sequence of the histone-like HB protein of Bacillus species. Oral viridans group streptococci, growing in chemically defined medium at pH 6.8, released HlpA into the milieu during stationary phase as a result of limited cell lysis. HlpA was not released by these bacteria when grown at pH 6.0 or below. S. pyogenes did not release HlpA during growth in vitro; however, analyses of sera from 155 pharyngitis patients revealed a strong correlation (P < 0.0017) between the production of antibodies to HlpA and antibodies to streptolysin O, indicating that the histone-like protein is released by group A streptococci growing in vivo. Extracellular HlpA formed soluble complexes with lipoteichoic acid in vitro and bound readily to heparan sulfate on HEp-2 cell surfaces. These results support a potential role for HlpA in the pathogenesis of streptococcus-induced tissue inflammation.

    INTRODUCTION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

Prokaryotes contain several small, basic, heat-stable proteins in association with the nucleoid. These proteins bind to single- and double-stranded DNA without obvious sequence specificity and are termed histone-like proteins; however, they do not have sequence homology with eukaryotic histones (for reviews, see references 13, 19, 33, and 37). The best-studied histone-like proteins are HU of Escherichia coli (4, 15, 29, 35, 38) and HB of Bacillus species (10, 23, 24, 31, 44). HU is a heterodimer of HU1 and HU2 proteins, which contain 90 amino acid residues each and have 70% sequence identity. HB is a protein highly homologous to HU but existing as a homodimer of a 92-amino-acid subunit (10, 23, 24, 31). Although the biological functions of histone-like proteins are not fully understood, they are known to wrap DNA and restrain negative supercoiling (4, 35). The resulting alterations in DNA structure and topology affect several cellular processes, including initiation of DNA replication (11, 51), DNA partitioning and cell division (12, 50), binding of repressors (3, 17, 30, 34), and transposition of bacteriophage Mu (43).

In addition to the physiological functions of bacterial histone-like proteins, HlpA (previously called GAG-BP and HBP) of Streptococcus species may contribute fortuitously to the virulence of these bacteria when the protein is released into the tissues during infection. Purified HlpA binds selectively in vitro to heparan sulfate in proteoglycans of heart and kidney basement membranes (1, 5, 6, 49). The accumulation of intravenously administered HlpA on renal basement membranes of mice and rabbits and the ensuing in situ immune complex formation (7, 20) indicate that it might be an important virulence factor in acute poststreptococcal glomerulonephritis and the glomerulonephritis that is often associated with streptococcal endocarditis in humans (21, 47). Tissue-bound HlpA may serve as a nidus for in situ immune complex formation leading to the inflammation and immunopathology that typify these diseases. The HlpAs of Streptococcus pyogenes, S. mutans, S. gordonii, and S. mitis are immunologically cross-reactive and exhibit identical binding activities for basement membranes in animal tissues (5, 6, 49).

This study was undertaken to clone and sequence hlpA from group A and viridans group streptococci, to compare the primary structure of HlpAs, and to evaluate the ability of these bacteria to release HlpA protein into the culture medium during growth. The hlpA genes of four Streptococcus species encode proteins of 91 amino acids that have at least 90% sequence identities. Members of the viridans group streptococci released more HlpA during stationary phase of growth than did the group A streptococci, and extracellular HlpA was complexed with soluble lipoteichoic acid (LTA). These antigen complexes bind to the surfaces of human epithelial cells in vitro and can lead to immune complex formation in situ.

    MATERIALS AND METHODS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

Bacteria and growth conditions. S. gordonii G9B, S. mutans MT703, and S. sobrinus B13 were grown in chemically defined broth medium (CDM) (45). S. pyogenes M1 strain SF370, M6 strain D471 (obtained from V. Fischetti, Rockefeller University, New York, N.Y.), M12 (ATCC 11434), M24 (ATCC 10782), and M49 strain F301 (J. Zabriski, Rockefeller University) were grown in CDM supplemented with ultrafiltered yeast extract (48). All cultures were grown at 37°C, and where indicated, the pH of the medium was maintained within the designated range by periodic addition of NaOH.

Autolytic activity of streptococci was determined in some experiments by using a radioactivity release assay. Streptococci were grown for 16 h in CDM containing 2 µCi of [2,8-3H]adenine (ICN Biomedicals, Inc., Costa Mesa, Calif.) per ml. At maximum growth, 1,000-fold-excess unlabeled adenine was added to prevent further uptake of radiolabel. At later intervals, 100-µl aliquots of culture were removed and the bacteria were harvested by filtration (0.22-µm pore size; Millipore Corp.). The bacteria were washed with 5 ml of phosphate-buffered saline (PBS) at pH 7.2, and the incorporation of radioisotope was quantitated by scintillation spectrometry.

HlpA extraction and purification. S. pyogenes D471 cells were harvested at the end of exponential growth (optical density at 600 nm of 0.85 to 0.90), washed twice with PBS, and suspended in 4 volumes of PBS at 0 to 4°C. The suspension was adjusted to pH 11.5 with NaOH, stirred for 18 h at 4°C, and then centrifuged at 14,000 × g for 30 min to remove whole cells and cell wall fragments. Supernatant fluids were collected, sterilized by filtration (0.22-µm-pore-size filter), adjusted to pH 7.0, dialyzed (3,500-molecular-weight exclusion) against water, and lyophilized. HlpA was purified to homogeneity by affinity chromatography on a column of heparin-agarose as previously described (5, 49). HlpA was also isolated from spent culture medium after the bacteria were removed by centrifugation and filtration. The spent culture medium was dialyzed against water and lyophilized.

ELISA. An enzyme-linked immunosorbent assay (ELISA) was used to quantitate HlpA. Lyophilized streptococcal components, isolated from spent culture medium, were dissolved (100 µg of protein/ml) in PBS and used to coat the wells of 96-well vinyl assay plates (Costar, Cambridge, Mass.) at 4°C overnight. After being washed with 0.05% Tween 20 in PBS to remove unbound components, the wells were incubated with dilutions of murine monoclonal antibody (MAb) 3C4 to HlpA of S. pyogenes followed by goat anti-mouse polyvalent immunoglobulins conjugated to alkaline phosphatase (Sigma Chemical Co., St. Louis, Mo.). The wells were washed three times with PBS-Tween 20 and incubated with p-nitrophenylphosphate in 9.7% (vol/vol) diethylamine buffer at pH 9.8 for 15 min. The reaction was stopped with 3-N NaOH, and the A405 in each well was determined with a microplate spectrophotometer (model EL310; Bio-Tek Instruments). Wells without spent culture medium components and wells containing material from uninoculated culture medium were used as reagent controls. Purified HlpA served as the quantitative standard.

Antibodies to HlpA in human sera were quantitated by ELISA. Purified HlpA (100 ng in 0.1 ml of PBS) was incubated in microtiter wells at 4°C for 18 h. The HlpA-coated wells were blocked for 1 h at room temperature with PBS-0.3% Triton X-100 (assay buffer) and washed three times with assay buffer. Human serum, diluted in assay buffer, was added to the wells and incubated for 1 h at room temperature. After the wells were washed five times with assay buffer, they were incubated with alkaline phosphatase-conjugated goat antibodies to human immunoglobulin G (IgG), IgA, and IgM (Sigma), and color was developed as described above. Antibodies to streptolysin O were quantified by using a hemolysin-neutralizing microassay (27).

Gel filtration chromatography. The molecular weight of HlpA from spent culture medium was determined, under nondissociating conditions, by using a column (1.5 by 81 cm) of Bio-Gel P-200 (Bio-Rad, Richmond, Calif.) equilibrated with PBS. The flow rate was 4.8 ml/hr, and the fraction size was 4.8 ml. Molecular mass standards included blue dextran (2,000 kDa), aldolase (158 kDa), ovalbumin (45 kDa), chymotrypsin (25 kDa), and bromphenyl blue (670 Da).

Mobility shift assay. The formation of LTA-HlpA complexes was determined by measuring shifts in the electrophoretic mobility of HlpA during polyacrylamide gel electrophoresis (PAGE) under nondenaturing conditions. Mixtures of purified LTA and HlpA, in 50 mM Na2HPO4-NaH2PO4 buffer at pH 7.2, were incubated at 22°C for 15 min and loaded onto a 1-mm-thick, 5% polyacrylamide gel. The separating and running buffer was also 50 mM phosphate buffer, pH 7.2. Electrophoresis was run for 1.5 h at 18 mA per gel, and HplA was subsequently stained with silver nitrate (32).

Cell culture and IIF assay. Indirect immunofluorescence (IIF) assays were conducted on HlpA-treated monolayers of HEp-2 cells (ATCC CCL-23), a human epithelioid cell line. The HEp-2 cells were grown in Dulbecco's modified Eagle medium supplemented with 10% fetal bovine serum (GIBCO, Grand Island, N.Y.), penicillin (100 U/ml), streptomycin (100 µg/ml), and amphotericin B (0.25 µg/ml). Streptococcal components from spent culture medium or purified HlpA were added to a confluent monolayer in a chambered microscope slide (Nunc, Inc., Naperville, Ill.), and the slides were incubated for 30 min at 37°C. After unbound reactants were removed by washing in PBS, the monolayer was incubated with MAb 3C4 for 30 min, washed in PBS for 30 min, and incubated for 30 min with fluorescein isothiocyanate (FITC)-labeled rabbit antibodies to mouse IgA, IgG, and IgM (Sigma). The slides were viewed with a Leitz fluorescence microscope (Orthoplan 2; Leitz, Wetzlar, Germany).

Cloning strategy. To amplify the hlpA gene from S. pyogenes D471, two degenerate PCR primers were designed based on the N-terminal amino acid sequence of HlpA protein (49) and on the highly conserved C-terminal consensus region of the histone-like gene sequences of Bacillus species (31) (Table 1). Both degenerate primers were biased to a S. pyogenes codon usage table generated from S. pyogenes sequences in the GenBank database.

                              
View this table:
[in this window]
[in a new window]
  		TABLE 1.   Oligonucleotides used as PCR primers in the cloning of hlpA or as probes in Southern blot assays

One microgram of chromosomal DNA from S. pyogenes D471 was used as the template for PCR amplification (30 cycles of denaturation at 94°C for 1 min, annealing at 37°C for 2 min, and extension at 72°C for 3 min) of hlpA genes. The reactions were examined by agarose gel electrophoresis for the presence of an amplified DNA fragment of approximately 300 bp (predicted size of the hlpA gene). The product was then cloned directly from the PCR mixture into the T overhang vector pT7Blue (Novagen) and grown in E. coli DH5alpha F'. The nucleotide sequences of the cloned inserts were determined by double-stranded DNA sequencing. To confirm that hlpA genes had been cloned, the predicted translation products of the clones were compared to the amino acid sequences of other histone-like proteins.

The regions upstream and downstream of the hlpA gene were cloned by inverse PCR. Five micrograms of DNA from D471 was digested to completion with ApoI (New England Biolabs), which produced 0.5- to 3-kb fragments but did not cut within hlpA. After the fragments were religated to form circular molecules, they were precipitated with ethanol and used as templates for PCR amplification (30 cycles of denaturation at 94°C for 1 min, annealing at 60°C for 2 min, and extension at 72°C for 3 min) with primers generated to internal regions of the hlpA structural gene (Table 1). The amplification products of these reactions were cloned into pT7Blue.

Two additional primers (Hlp-forward and Hlp-reverse) were generated specifically to the N-terminal and C-terminal sequences of the hlpA gene of S. pyogenes D471 (Table 1), incorporating the first three codons of the 5' end of the coding sequence and the last four codons of the 3' end of the coding sequence, respectively. These primers were used to amplify and clone the hlpA genes of S. sobrinus B13, S. gordonii G9B, and S. mutans MT703, using amplification conditions as described above for S. pyogenes.

Radiolabeled oligonucleotides. Oligonucleotides corresponding to bases 96 through 117 of the hlpA sequences of S. pyogenes, S. gordonii, S. mutans, and S. sobrinus were synthesized and purified by Integrated DNA Technologies (Coralville, Iowa). Each oligonucleotide was radiolabeled for 30 min at 37°C in a 20-µl reaction mixture containing 20 pmol of oligonucleotide, 62.5 µCi of [gamma -32P]ATP (3,000 Ci/mmol; Dupont-NEN), 10 U of T4 polynucleotide kinase (GIBCO BRL, Grand Island, N.Y.), 0.1 mM spermidine, and 1× Kinase Forward reaction buffer (GIBCO BRL). The kinase reaction was stopped by addition of 25 µl of 10 mM Tris-HCl-1 mM EDTA (pH 7.5) (TE buffer) and 5 µl of 200 mM EDTA (pH 7.0) followed by extraction with phenol-chloroform (1:1) and ethanol precipitation. After being dried, the radiolabeled oligonucleotides were resuspended in 50 µl of TE buffer.

Dot blot hybridization. Genomic DNA was isolated and purified from S. gordonii, S. mutans, S. pyogenes, and S. sobrinus by the procedure of Sun et al. (41). Fifteen micrograms of the purified DNA was spotted onto nitrocellulose paper (MSI Separations Inc., Westboro, Mass.), washed twice with TE buffer, and air dried. The DNA on the paper was denatured with 0.5 M NaOH-1.5 M NaCl for 2 min, neutralized with 0.5 M HCl-1.5 M NaCl (pH 7.4) buffer for 5 min, and washed with 300 mM NaCl-20 mM NaH2PO4-2 mM EDTA (2× SSPE) buffer. The blot was air dried and fixed for 120 min at 80°C in vacuo (36). The nitrocellulose paper was cut into four segments, each containing genomic DNA from all four Streptococcus species, and hybridized for 3 h at 48°C with radiolabeled oligonucleotides (36). The prehybridization solution was 6× SSPE-0.25% nonfat milk-5 mM Na4P2O7. After hybridization, the blots were washed sequentially at 48°C with 2× SSPE-0.1% sodium dodecyl sulfate-5 mM Na4P2O7 and 0.5× SSPE-0.1% sodium dodecyl sulfate-5 mM Na4P2O7. The blots were exposed for 18 h and developed with the Bio-Rad 505 Molecular Imager system.

Sequencing and analysis. All DNA sequencing was done by using double-stranded plasmid DNA and a Sequenase II kit as recommended by the manufacturer (United States Biochemical, Cleveland, Ohio). Analysis of the sequences was done with the University of Wisconsin Genetics Computer Group software package (9). Sequence similarity searches were done by using the on-line BLAST server at the National Institutes of Health.

LTA. LTA concentration was measured by a passive hemagglutination assay as previously described (25). One unit of cell sensitizing activity was defined as the minimum amount of LTA required to sensitize a standard suspension of sheep erythrocytes and yield a positive agglutination with a standard antiserum to LTA of S. mutans. One hemagglutinating unit was equal to 100 ng of LTA. Purified LTA of S. pyogenes and S. mutans was purchased from Sigma. Antibodies to LTA were purified from rabbit antiserum by affinity chromatography on a column of deacylated cardiolipin conjugated to divinyl sulfone-agarose as previously described (25).

Statistical analysis. The relationship between the antibody titers to streptolysin O and HlpA (ASO and AHA titers, respectively) were analyzed with the Spearman rank correlation coefficient and t test and with the Wilcoxon rank sum test.

Nucleotide sequence accession number. The nucleotide sequences of the hlpA genes have been deposited in GenBank with accession numbers L38946 (S. pyogenes D471), L40356 (S. gordonii G9B), L38959 (S. sobrinus B13), and L40355 (S. mutans MT703).

    RESULTS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

Cloning and analysis of the hlpA gene. The HlpA of S. pyogenes has been purified, and the amino acid sequence in the amino-terminal one-third of the protein has been determined (49). Comparison of this sequence with those reported for other bacteria showed extensive homology with the HB histone-like protein of Bacillus species. This information was used in the synthesis of two oligonucleotides, Hlp NT-degenerate and Hlp CT-degenerate (Table 1), which were used to amplify the coding region of the hlpA gene of S. pyogenes D471 by PCR. An amplified DNA fragment of 230 bp was obtained and cloned directly into pT7Blue. The insert was verified by sequencing both strands and comparing the translated sequence with that of the N-terminal sequence of HlpA and with the sequences of other bacterial histone proteins. The promoter and termination regions of the hlpA gene were cloned by inverse PCR of ApoI-digested and religated chromosomal DNA from S. pyogenes D471, using primers internal to the hlpA structural gene (Table 1). The amplification product was cloned and verified as described above. Figure 1 shows that there is a putative ribosome-binding site (GGAGGA) and a potential -10 RNA polymerase-binding site (TAATTA) upstream of the ATG start codon. Complete nucleotide sequencing showed that identical hlpA genes were cloned from three independent chromosomal preparations of S. pyogenes D471. In addition, the nucleotide sequence of hlpA in strain D471 is 100% identical to the single-copy hlpA gene in S. pyogenes SF370 determined by genome sequencing by Ferretti et al. (16).


View larger version (35K):
[in this window]
[in a new window]
  		FIG. 1.   DNA sequence of the hlpA gene of S. pyogenes and the deduced amino acid sequence. The hlpA gene is 276 bp in length and encodes a protein of 91 amino acid residues with a predicted pI of 9.81. The numbers on the left coincide with nucleotide positions relative to the adenine in the ATG initiation codon of hlpA; negative numbers denote positions 5' of the adenine. Amino acid residues for the HlpA (numbered to the right) are given below the DNA sequence in the single-letter designation. The positions of the putative Shine-Dalgarno (SD) ribosome-binding site and the -10 promoter sequence are underlined. *, stop codon.

To obtain the sequence of the hlpA gene from the three other species of streptococci, a pair of PCR primers was generated to the N terminus and C terminus of the S. pyogenes sequence (Table 1). The primers overlapped the first three codons and the last four codons, respectively. The amplified DNA fragments from S. gordonii, S. mutans, and S. sobrinus were cloned, and the sequences were compared with that of hlpA gene of S. pyogenes. All gene sequences were cloned and verified from at least two independent amplification reactions, and multiple clones from each reaction were sequenced in both directions. Although several silent changes were detected in the primary sequences of the hlpA genes from different streptococci (data not shown), the deduced amino acid sequences are very similar (Fig. 2). Two regions show heterogeneity; region 1 (amino acids 31 to 39) has numerous variations in amino acid charge and polarity, whereas region 2 (amino acids 69 to 74) has more limited substitutions. As expected, HlpA has a very high degree of similarity with the other bacterial histones, particularly that of Bacillus species (69% identity). Comparison of the Streptococcus and Bacillus consensus sequences (Fig. 2) shows that the C-terminal half of each protein is highly conserved whereas the N-terminal half is more divergent.


View larger version (36K):
[in this window]
[in a new window]
  		FIG. 2.   Comparison of the amino acid sequences of HlpA proteins of the genus Streptococcus with the HB proteins of Bacillus species. Amino acids are shown for each protein only when they differ from the consensus residue. The boxed sequences represent the conserved alpha -helices and the potential DNA-binding arm of HB protein (43).

To confirm that the PCR-amplified hlpA segments were derived from the indicated Streptococcus species, 22-mer oligonucleotides were synthesized by using the base sequences most unique to each gene; the hypervariable region of hlpA was comprised of bases 96 to 117 (Table 1). Each radiolabeled oligonucleotide was hybridized with dot blots of chromosomal DNA from each Streptococcus species (Fig. 3). In each case, the probe annealed with the DNA of the homologous species but weakly or not at all with DNA from the heterologous species.


View larger version (36K):
[in this window]
[in a new window]
  		FIG. 3.   Southern dot blot showing hybridization of radiolabeled, synthetic oligonucleotides with genomic DNA from S. gordonii (G), S. mutans (M), S. pyogenes (P), and S. sobrinus (S). The sequences of the Hlp oligonucleotides are shown in Table 1 and are designated by the initials of the genus and species from which they were derived.

Release of HlpA by streptococci. We showed previously, using a mouse model, that intravenously administered HlpA is carried by the blood to the kidneys, where it binds to heparan sulfate-proteoglycans of the glomerular capillaries and acts as a nidus for the formation of in situ immune complexes (6, 7). Although immune complex deposition can lead to glomerulonephritis, the tendency of streptococci to release HlpA during localized infections has not been documented. Therefore, serum specimens were obtained from 155 pharyngitis patients at the Buffalo Children's Hospital and assayed for antibodies to streptolysin O and to HlpA. Seventy-seven of the patients had significant ASO titers (>= 200), indicating that they had experienced an infection by S. pyogenes within the previous 3 months (26, 27). Ninety-seven patients had AHA titers of between 1,600 and 25,600 (Fig. 4). There was a strong positive correlation between the history of streptococcal infection, as indicated by the ASO titer, and the AHA titers (P < 0.0001 [Spearman rank correlation coefficient and t test] or P = 0.0017 [Wilcoxon rank sum test]). This result indicates that immunogenic quantities of HlpA are released with streptolysin O by streptococci growing in vivo. None of the patients developed symptoms of delayed sequelae.


View larger version (12K):
[in this window]
[in a new window]
  		FIG. 4.   AHA and ASO titers in human sera. The endpoint data are clustered in relation to the serial dilutions (twofold) of the sera, beginning at 1:100. The average AHA titers are indicated by +.

Analyses of spent culture media of several Streptococcus species indicated that HlpA was released only during the stationary growth phase, as exemplified by S. mutans MT703 (Fig. 5). Maximal yields were 320 ng/ml for S. mutans, 294 ng/ml for S. gordonii CH1, and less than 20 ng/ml for S. pyogenes D471, 11434, and F301. The period of HlpA release by S. mutans, in culture medium maintained at pH 6.8, coincided with a 17% decrease in culture turbidity (Fig. 5) and with a release of 28% of previously assimilated [3H]adenine (data not shown), indicating that limited autolysis had taken place. S. mutans growing at pH 6.0 and below did not release either HlpA or radiolabeled nucleic acids, although the bacteria grew at the same rate and to similar turbidity values as streptococci grown at the higher pH (Fig. 5). S. pyogenes cultures showed neither turbidity decreases nor leakage of cytoplasmic radiolabel during stationary growth phase in any of these growth conditions (data not shown), which is consistent with the relatively low amount of HlpA in the spent culture medium.


View larger version (17K):
[in this window]
[in a new window]
  		FIG. 5.   Release of HlpA by S. mutans during growth in CDM. Symbols: open circle , turbidity of a culture at pH 6.8; bullet , turbidity of a culture at pH 6; square , extracellular HlpA at pH 6.8 as determined by enzyme immunoassay; black-square, extracellular HlpA at pH 6.

Gel filtration chromatography of S. mutans components, from spent culture medium, indicated that HlpA was complexed with other cell constituents because it eluted predominately at the void volume of the column (Fig. 6), whereas chromatography of purified HlpA resulted in a single peak (indicated by bar) with an apparent molecular mass of 31 kDa (6). Addition of DNase to the crude preparation prior to chromatography did not change the elution profile of HlpA (data not shown). LTA, a streptococcal surface polyanion and virulence factor (8, 22), was also detected in the spent culture medium, and its elution with HlpA at the void volume during gel filtration chromatography (Fig. 6) indicated that it might complex ionically with the HplA after their release from the bacteria.


View larger version (16K):
[in this window]
[in a new window]
  		FIG. 6.   Gel filtration chromatography of S. mutans cell components in spent medium. Symbols: open circle , absorbance at 280 nm; bullet , absorbance at 405 nm in ELISA for HlpA; square , LTA concentration determined by passive hemagglutination assay. The elution point of purified HlpA is indicated by the bar.

To determine if HlpA has binding affinity for LTA, purified protein from S. mutans was incubated with LTA (S. mutans), and the mixture was resolved by a PAGE mobility shift assay. Purified LTA caused increased anionic mobility of HlpA during electrophoresis in nondissociating conditions (Fig. 7). Similar results were obtained with reagents from S. pyogenes. Mixing of S. mutans LTA and HlpA at a molar ratio of 5:1 prior to gel filtration chromatography caused HlpA to elute at the void volume instead of its normal location (data not shown). Thus, the constituents of these naturally occurring complexes may exhibit altered tissue-binding properties for host cell surfaces.


View larger version (106K):
[in this window]
[in a new window]
  		FIG. 7.   Mobility shift assay of HlpA-LTA complexes in nondenaturing PAGE. Lane 1, 2 µg of HlpA only. For lanes 2 to 8, 2 µg of HlpA was mixed with 63 ng, 125 ng, 250 ng, 500 ng, 1 µg, 2 µg, and 4 µg of LTA, respectively. Lane 9 contained only 4 µg of LTA. The LTA and HlpA were obtained from S. mutans.

Binding to epithelial cells. To evaluate the tissue binding activity of the HlpA-LTA complexes released by streptococci, naturally occurring complexes were obtained from spent culture medium of S. mutans by gel filtration column chromatography and incubated with growing monolayers of HEp-2 cells (Fig. 8). IIF assay using MAb 3C4 showed that HlpA bound to the cells in a granular pattern (Fig. 8A), which was identical to that observed with purified HlpA (Fig. 8B). Staining of the treated HEp-2 cells with affinity-purified antibodies to streptococcal LTA (22) produced more uniform and linear patterns (Fig. 8D) which are consistent with the binding pattern of purified LTA (Fig. 8E), also reported by others (8, 40). The binding of HlpA, but not LTA, to HEp-2 cells was inhibited by preincubation of the bacterial extract with 10 µl of heparin per ml, further indicating that these bacterial antigens were binding to different receptors on the epithelial cell surfaces. Similar binding properties and IIF staining patterns were observed with LTA and HlpA of S. pyogenes.


View larger version (142K):
[in this window]
[in a new window]
  		FIG. 8.   IIF assay showing HlpA and LTA binding to HEp-2 cell monolayers. (A) Cells treated with HlpA-LTA complexes (50 µg of protein) isolated from spent culture medium of S. mutans and stained for HlpA with MAb 3C4 and FITC-conjugated rabbit antibodies to mouse immunoglobulins; (B) cells treated with 20 µg of purified HlpA and stained as described for panel A; (C) cells treated with PBS (control) and stained as described for panel A; (D) cells treated with HlpA-LTA complexes isolated from spent culture medium and stained with affinity-purified rabbit antibodies to LTA and FITC-conjugated goat antibodies to rabbit IgG; (E) cells treated with 50 µg of pure LTA and stained as described for panel D; (F) control cells treated with only the primary and secondary antibodies. Magnification, ×328.

    DISCUSSION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

We have cloned, sequenced, and characterized the hlpA genes of S. pyogenes, S. gordonii, S. mutans, and S. sobrinus. Analysis of the nucleotide sequence predicts that HlpA is comprised of 91 amino acid residues with a molecular mass of 9,647 Da. Most of the variations (22 of 29) in the deduced amino acid sequences occur between residues 31 and 38, which corresponds to the second alpha -helical domain of the HB histone-like protein of Bacillus species (44). A few amino acid substitutions were also found within the antiparallel beta -ribbon comprising the DNA-binding arm (residues 53 to 78), which showed 92 to 100% identity with this region of the Streptococcus consensus sequence. Specifically, S. pyogenes has a glutamic acid-for-lysine substitution at position 71; S. mutans has glutamic acid-for-alanine and lysine-for-alanine substitutions at positions 68 and 73, respectively; and S. gordonii has lysine-for-alanine and threonine-for-isoleucine substitutions at positions 68 and 70, respectively. Based on the crystallographic data for HB (44), these latter amino acid substitutions fall within the return strand of the arm (residues 65 to 78), whereas the amino acid sequences in the outgoing strands (residues 53 to 64) of the four streptococcal HlpAs are identical. Significantly, the outgoing strand contains all four arginines of the protein and is believed to constitute the DNA-binding site of the histone (31, 44). The high percentage of sequence identity between HlpA and HB proteins and gel filtration chromatography (6) also suggests that HlpA forms a homodimeric structure similar to that of HB (44). The amino acids responsible for the hydrophobic core of the dimer (Phe 30, Phe 48, Phe 51, Phe 80, Leu 7, Ilu/Val 33, Leu 37, and Leu 45) are highly conserved among both Streptococcus and Bacillus species. Consistent with histone-like proteins of other bacteria, HlpAs of streptococci are rich in lysine and arginine and devoid of cysteine and tryptophan. The HlpAs of S. mutans and S. gordonii each contain one tyrosine residue, whereas S. pyogenes and S. sobrinus contain none. The high degree of sequence homology among HlpAs is also consistent with their immunological cross-reactivity and similar binding affinities for heparin and heparan sulfate-proteoglycans of animal tissues (6, 49).

In addition to its essential role in the bacterial nucleoid, HlpA may be an important virulence factor in the streptococcal sequelae, acute poststreptococcal glomerulonephritis and rheumatic fever, when it is released by S. pyogenes during infection of the skin or nasopharynx. Previous studies have shown that purified HlpA in the bloodstream readily adsorbs to heparan sulfate-proteoglycans in the basement membranes of animal tissues (1, 5, 6, 49), where it can complex with circulating antibodies (7, 20). In the present study, the coincident release of [3H]adenine-labeled components and a decrease in culture turbidity indicated that the release of cytoplasmic HlpA to the culture medium by viridans group streptococci during stationary growth phase was most likely the result of limited bacterial lysis. HlpA release was maximal when the pH was maintained between 6.5 and 7 but minimal at more acidic pHs, which is consistent with the pH optima of autolysins of S. pneumoniae (14), S. mitis (42), and Enterococcus faecalis (39). Release of HlpA by these streptococci and by viridans group streptococci during infective endocarditis may lead to glomerulonephritis. S. pyogenes did not release significant amounts of HlpA in vitro and is not known to autolyse; however, the amount of HlpA released into tissues by group A streptococci during infection may be enhanced by the interaction of the bacteria with host defense mechanisms (e.g., defensins) that perturb the integrity of bacterial membranes (18). Indeed, the presence of anti-HlpA antibodies in human sera and their close correlation with antibodies to streptolysin O of group A streptococci can be interpreted to indicate that significant amounts of HlpA are released by these streptococci at infection sites. Alternatively, the immune response may result from the processing of cell-associated antigens, after phagocytosis of streptococci, by macrophages. The close correlation of antibody production to HlpA and streptolysin O makes it unlikely that the immune stimulus was HlpA from viridans group streptococci because they lack this hemolysin.

In mice, specific antibodies cross-link and stabilize the HlpA deposits in glomerular capillaries of kidneys (7), whereas in the absence of antibodies, HlpA is removed from the blood and excreted in urine without inducing adverse effects. Extracellular HlpA also binds to heparan sulfate in the extracellular matrix of human epithelial (HEp-2) cells and might contribute to the injury of mucus membranes in some infections. Although the IIF assay showed that HlpA and LTA had different patterns of binding on HEp-2 cells, it is not clear whether HlpA from spent culture medium was bound as free protein or as a complex with streptococcal LTA. Complexing of HlpA with the polyanionic LTA was not expected to preclude its binding to tissue components because previous studies have shown that HlpA has a 100-fold-higher affinity for heparan sulfate than it has for LTA (6). The different IIF staining patterns seen in Fig. 8 probably reflect the selective binding of HlpA to heparan sulfate-proteoglycans (1, 5, 49) and LTA to lipophilic receptors and to membrane lipids (8, 25). Also, the preparation of HlpA-LTA complexes may have contained free LTA; the molar ratio of LTA to HlpA was more than 30:1. Upon entering the blood, HlpA-LTA complexes, as well as free HlpA and LTA, can be carried to the kidneys, where they may bind directly to the glomerular capillary walls. Glomerulonephritis may arise through the activation of complement by in situ immune complexes and from localized production of interleukin-1beta , interleukin-6, and tumor necrosis factor alpha by LTA-stimulated monocytes (2, 46).

The sequence data presented here are important to understanding the pathogenic properties of HlpA during streptococcal infections. Epitope mapping experiments may reveal protein domains that elicit protective antibodies, which will inhibit HlpA binding to heparan sulfate-proteoglycans in tissues, rather than antibodies that stabilize tissue deposits and exacerbate immunopathology.

    ACKNOWLEDGMENTS

This work was supported by Public Health Service grant R01-DE05696 from the National Institute of Dental Research.

We thank Terry Connell for critically reviewing the manuscript and making many helpful suggestions. We thank Susan Alder, Judy Colby, and Frank Watson for excellent technical assistance. We are grateful to Sousan S. Altaie for providing the specimens of human serum.

    FOOTNOTES

* Corresponding author. Mailing address: Department of Microbiology, School of Medicine and Biomedical Sciences, State University of New York at Buffalo, Buffalo, NY 14214. Phone: (716) 829-2178. Fax: (716) 829-2178. E-mail: mstinson{at}ubmedb.buffalo.edu.

dagger Present address: Department of Microbiology and Immunology, University of Oklahoma Health Sciences Center, Oklahoma City, OK 73190.

Dagger Present address: Department of Dairy Science, Sangji University, Woosandong Wonju, Kangwondo 220-702, Seoul, South Korea.

Editor:  V. A. Fischetti

    REFERENCES
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

1. Bergey, E. J., and M. W. Stinson. 1988. Heparin-inhibitable basement membrane-binding protein of Streptococcus pyogenes. Infect. Immun. 56:1715-1721[Abstract/Free Full Text].
2. Bhakdi, S., T. Klonisch, P. Nuber, and W. Fischer. 1991. Stimulation of monokine production by lipoteichoic acids. Infect. Immun. 59:4614-4620[Abstract/Free Full Text].
3. Bonnefoy, E., and J. Rouvière-Yaniv. 1992. HU, the major histone-like protein of E. coli, modulates the binding of IHF to ori C. EMBO J. 11:4489-4496[Medline].
4. Broyles, S., and D. Pettijohn. 1986. Interaction of the Escherichia coli HU protein with DNA. Evidence for formation of nucleosome-like structures with altered helical pitch. J. Mol. Biol. 187:47-60[Medline].
5. Choi, S. H., and M. W. Stinson. 1989. Purification of a Streptococcus mutans protein that binds to heart tissue and glycosaminoglycans. Infect. Immun. 57:3834-3840[Abstract/Free Full Text].
6. Choi, S. H., and M. W. Stinson. 1991. Binding of a Streptococcus mutans cationic protein to kidneys in vitro. Infect. Immun. 59:537-543[Abstract/Free Full Text].
7. Choi, S. H., X. Zhang, and M. W. Stinson. 1995. Dynamics of streptococcal histone retention by mouse kidneys. Clin. Immunol. Immunopathol. 76:68-74[Medline].
8. Courtney, H., I. Ofek, W. A. Simpson, and E. H. Beachey. 1981. Characterization of lipoteichoic acid binding to polymorphonuclear leukocytes of human blood. Infect. Immun. 32:625-631[Abstract/Free Full Text].
9. Devereux, J. R. 1989. . University of Wisconsin Genetics Computer Group Software. University of Wisconsin, Madison, Wis.
10. Dijk, J., S. W. White, K. S. Wilson, and K. Appelt. 1983. On the DNA binding protein II from Bacillus stearothermophilus. I. Purification, studies in solution and crystallization. J. Biol. Chem 258:4003-4006[Abstract/Free Full Text].
11. Dixon, N. E., and A. Kornberg. 1984. Protein HU in the enzymatic replication of the chromosomal origin of Escherichia coli. Proc. Natl. Acad. Sci. USA 81:424-425[Abstract/Free Full Text].
12. Dri, A-M., J. Rouvière-Yaniv, and P. Moreau. 1991. Inhibition of cell division in hupA hupB mutant bacteria lacking HU protein. J. Bacteriol. 173:2852-2863[Abstract/Free Full Text].
13. Drlica, K., and J. Rouvière-Yaniv. 1987. Histone-like proteins of bacteria. Microbiol. Rev. 51:301-319[Free Full Text].
14. Dubos, R. J. 1937. Mechanism of the lysis of pneumococci by freezing and thawing, bile and other agents. J. Exp. Med. 66:101-112[Abstract].
15. Dürrenberger, M., M.-A. Bjornsti, T. Uetz, J. A. Hobot, and E. Kellenberger. 1988. Intracellular location of the histonelike protein HU in Escherichia coli. J. Bacteriol. 170:4757-4768[Abstract/Free Full Text].
16. Ferretti, J. J., S. W. Clifton, B. A. Roe, A. N. Suvorov, and M. W. McShan. 1997. Personal communication.
17. Flashner, Y., and J. D. Gralla. 1988. DNA dynamic flexibility and protein recognition: differential stimulation by bacteria histone-like protein HU. Cell 54:713-721[Medline].
18. Ganz, T., and R. I. Lehrer. 1995. Defensins. Pharmacol. Ther. 66:191-205[Medline].
19. Geider, K., and H. Hoffmann-Berling. 1981. Proteins controlling the helical structure of DNA. Annu. Rev. Biochem. 50:233-260[Medline].
20. Glurich, I., B. Winters, B. Albini, and M. Stinson. 1991. Identification of Streptococcus pyogenes proteins that bind to rabbit kidney in vitro and in vivo. Microb. Pathog. 10:209-220[Medline].
21. Gutman, R. A., G. E. Striker, B. C. Gilliland, and R. E. Cutler. 1972. The immune complex glomerulonephritis of bacterial endocarditis. Medicine 51:1-25[Medline].
22. Hyzy, J., V. Sciotti, B. Albini, and M. W. Stinson. 1992. Deposition of circulating streptococcal lipoteichoic acid in mouse tissues. Microb. Pathog. 13:123-132[Medline].
23. Imber, R., H. Bächinger, and T. A. Bickle. 1982. Purification and characterization of a small DNA-binding protein, HB from Bacillus globigii. Eur. J. Biochem. 122:627-632[Medline].
24. Imber, R., M. Kimura, N. Groch, and V. Heinemann. 1987. DNA-binding properties and primary structure of HB protein from Bacillus globigii. Eur. J. Biochem. 165:547-552[Medline].
25. Josephson, S. L., M. W. Stinson, S. J. Millar, and R. E. Cohen. 1986. Purification of lipoteichoic acid by chromatography in water-organic solvents. Infect. Immun. 51:378-384[Abstract/Free Full Text].
26. Kaplan, E. L., and B. B. Huew. 1980. The sensitivity and specificity of an agglutination test for antibodies to streptococcal extracellular antigens: a quantitative analysis and comparison of the streptozyme test with the anti-streptolysin O and anti-deoxyribonuclease B tests. J. Pediatr. 96:367-373[Medline].
27. Klein, G. C., M. D. Moody, C. N. Baker, and B. V. Addison. 1968. Micro antistreptolysin O test. Appl. Microbiol. 16:184[Medline].
28. Laemmli, U. K. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature (London) 227:680-685[Medline].
29. Laine, B., D. Kmiecik, P. Sautiere, G. Biserte, and M. Cohen-Solal. 1980. Complete amino acid sequences of DNA-binding proteins HU-1 and HU-2 from Escherichia coli. Eur. J. Biochem. 103:447-461[Medline].
30. Mensa-Wilmot, K., K. Carroll, and R. McMacken. 1989. Transcriptional activation of bacteriophage lambda  DNA replication in vitro: regulatory role of histone-like protein HU of Escherichia coli. EMBO J. 8:2393-2404[Medline].
31. Micka, B., N. Groch, U. Heinemann, and M. A. Marahiel. 1991. Molecular cloning, nucleotide sequence, and characterization of the Bacillus subtilis gene encoding the DNA-binding protein HBsu. J. Bacteriol. 173:3191-3198[Abstract/Free Full Text].
32. Morrisey, J. H. 1981. Silver stain for proteins in polyacrylamide gels: a modified procedure with enhanced uniform sensitivity. Anal. Biochem. 117:307-310[Medline].
33. Pettijohn, D. E. 1988. Histone-like proteins and bacterial chromosome structure. J. Biol. Chem. 263:12793-12796[Free Full Text].
34. Preobrajenskaya, O., A. Boullard, F. Boubrik, M. Schnarr, and J. Rouviere-Yaniv. 1994. The protein HU can displace the Lex A repressor from its DNA-binding sites. Mol. Microbiol. 13:459-467[Medline].
35. Rouvière-Yaniv, J., J. Germond, and M. Yaniv. 1979. E. coli DNA binding protein HU forms nucleosome-like structures with circular double-stranded DNA. Cell 117:265-274.
36. Sambrook, J., E. F. Fritch, and T. Maniatis. 1989. . Molecular cloning: a laboratory manual, 2nd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.
37. Schmidt, M. B. 1988. Structure and function of the bacterial chromosome. Trends Biochem. Sci. 13:131-135[Medline].
38. Shellman, V. L., and D. E. Pettijohn. 1991. Introduction of proteins into living bacterial cells: distribution of labeled HU protein in Escherichia coli. J. Bacteriol. 173:3047-3059[Abstract/Free Full Text].
39. Shockman, G. D., J. S. Thompson, and M. J. Conover. 1967. The autolytic enzyme system of Streptococcus faecalis. II. Partial characterization of the autolysin and its substrate. Biochemistry 6:1054-1065[Medline].
40. Simpson, W. A., I. Ofek, C. Sarasohn, J. Morrison, and E. H. Beachey. 1980. Characteristics of the binding of streptococcal lipoteichoic acid to human oral epithelial cells. J. Infect. Dis. 141:457-462[Medline].
41. Sun, J.-W., S.-Y. Wanda, A. Camilli, and R. Curtiss. 1994. Cloning and DNA sequencing of the dextranase inhibitor gene (dei) from Streptococcus sobrinus. J. Bacteriol. 176:7213-7222[Abstract/Free Full Text].
42. Sund, M.-L., and L. Linder. 1976. Autolysis in strains of viridans streptococci. J. Gen. Microbiol. 96:87-94[Abstract/Free Full Text].
43. Surette, M. G., S. J. Buch, and G. Chaconas. 1987. Transposomes: stable protein-DNA complexes involved in the in vitro transposition of Mu DNA. Cell 49:253-262[Medline].
44. Tanaka, I., K. Appelt, J. Dijk, S. W. White, and K. S. Wilson. 1984. 3-Å resolution structure of a protein with histone-like properties in procaryotes. Nature (London) 310:376-381[Medline].
45. Terleckyj, B., N. P. Willett, and G. D. Shockman. 1975. Growth of several cariogenic strains of oral streptococci in a chemically defined medium. Infect. Immun. 11:649-655[Abstract/Free Full Text].
46. Vernier, A., M. Diab, M. Soell, G. Haan-Archipoff, A. Beretz, D. Wachsmann, and J.-P. Klein. 1996. Cytokine production by human epithelial and endothelial cells following exposure to oral viridans streptococci involves lectin interactions between bacteria and cell surface receptors. Infect. Immun. 64:3016-3022[Abstract].
47. Vogt, A., S. Batsford, B. Rodríguez-Iturbe, and R. García. 1983. Cationic antigens in post-streptococcal glomerulonephritis. Clin. Nephrol. 20:271-279[Medline].
48. Wang, J.-R., and M. W. Stinson. 1994. M6 protein mediates streptococcal adhesion to HEp-2 cells. Infect. Immun. 62:442-448[Abstract/Free Full Text].
49. Winters, B. D., N. Ramasubbu, and M. W. Stinson. 1993. Isolation and characterization of a Streptococcus pyogenes protein that binds to basal laminae of human cardiac muscle. Infect. Immun. 61:3259-3264[Abstract/Free Full Text].
50. Yasuzawa, K., N. Hayashi, N. Goshima, K. Kohno, F. Imamoto, and Y. Kano. 1992. Histone-like proteins are required for cell growth and constraint of supercoils in DNA. Gene 122:9-15[Medline].
51. Yung, B. Y., and A. Kornberg. 1989. The dnaA initiator protein binds separate domains in the replication origin of Escherichia coli. J. Biol. Chem. 264:6146-6150[Abstract/Free Full Text].

Infect Immun, January 1998, p. 259-265, Vol. 66, No. 1
0019-9567/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.


This article has been cited by other articles:

  • Nair, G. B., Ramamurthy, T., Bhattacharya, S. K., Dutta, B., Takeda, Y., Sack, D. A. (2007). Global Dissemination of Vibrio parahaemolyticus Serotype O3:K6 and Its Serovariants. Clin. Microbiol. Rev. 20: 39-48 [Abstract] [Full Text]  
  • Terao, Y., Yamaguchi, M., Hamada, S., Kawabata, S. (2006). Multifunctional Glyceraldehyde-3-phosphate Dehydrogenase of Streptococcus pyogenes Is Essential for Evasion from Neutrophils. J. Biol. Chem. 281: 14215-14223 [Abstract] [Full Text]  
  • Yu, H. B., Zhang, Y. L., Lau, Y. L., Yao, F., Vilches, S., Merino, S., Tomas, J. M., Howard, S. P., Leung, K. Y. (2005). Identification and Characterization of Putative Virulence Genes and Gene Clusters in Aeromonas hydrophila PPD134/91. Appl. Environ. Microbiol. 71: 4469-4477 [Abstract] [Full Text]  
  • Okura, M., Osawa, R., Arakawa, E., Terajima, J., Watanabe, H. (2005). Identification of Vibrio parahaemolyticus Pandemic Group-Specific DNA Sequence by Genomic Subtraction. J. Clin. Microbiol. 43: 3533-3536 [Abstract] [Full Text]  
  • Neuhaus, F. C., Baddiley, J. (2003). A Continuum of Anionic Charge: Structures and Functions of D-Alanyl-Teichoic Acids in Gram-Positive Bacteria. Microbiol. Mol. Biol. Rev. 67: 686-723 [Abstract] [Full Text]  
  • Moreno, A. J., Fontes, M., Murillo, F. J. (2001). ihfA Gene of the Bacterium Myxococcus xanthus and Its Role in Activation of Carotenoid Genes by Blue Light. J. Bacteriol. 183: 557-569 [Abstract] [Full Text]  
  • Lei, B., Mackie, S., Lukomski, S., Musser, J. M. (2000). Identification and Immunogenicity of Group A Streptococcus Culture Supernatant Proteins. Infect. Immun. 68: 6807-6818 [Abstract] [Full Text]  
  • Zhang, Y. L., Ong, C. T., Leung, K. Y. (2000). Molecular analysis of genetic differences between virulent and avirulent strains of Aeromonas hydrophila isolated from diseased fish. Microbiology 146: 999-1009 [Abstract] [Full Text]  
  • Zhang, L., Ignatowski, T. A., Spengler, R. N., Noble, B., Stinson, M. W. (1999). Streptococcal Histone Induces Murine Macrophages To Produce Interleukin-1 and Tumor Necrosis Factor Alpha. Infect. Immun. 67: 6473-6477 [Abstract] [Full Text]  
  • Juarez, Z. E., Stinson, M. W. (1999). An Extracellular Protease of Streptococcus gordonii Hydrolyzes Type IV Collagen and Collagen Analogues. Infect. Immun. 67: 271-278 [Abstract] [Full Text]  

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowReprints and Permissions
Right arrow Copyright Information
Right arrow Books from ASM Press
Right arrow MicrobeWorld
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Stinson, M. W.
Right arrow Articles by Barnard, J.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Stinson, M. W.
Right arrow Articles by Barnard, J.

Copyright © 2009 by the American Society for Microbiology.   For an alternate route to Journals.ASM.org, visit: http://intl-journals.asm.org | More Info»