Streptococcal histone-like protein: primary structure of hlpA and protein binding to lipoteichoic acid and epithelial cells

Abstract

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.

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.

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 α-helices and the potential DNA-binding arm of HB protein (43).

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.

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 +.

FIG. 5

Release of HlpA by S. mutans during growth in CDM. Symbols: ○, turbidity of a culture at pH 6.8; •, turbidity of a culture at pH 6; □, extracellular HlpA at pH 6.8 as determined by enzyme immunoassay; ▪, extracellular HlpA at pH 6.

FIG. 6

Gel filtration chromatography of S. mutans cell components in spent medium. Symbols: ○, absorbance at 280 nm; •, absorbance at 405 nm in ELISA for HlpA; □, LTA concentration determined by passive hemagglutination assay. The elution point of purified HlpA is indicated by the bar.

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.

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.