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. 2001 Feb;69(2):949-58.
doi: 10.1128/IAI.69.2.949-958.2001.

Identification and characterization of a novel family of pneumococcal proteins that are protective against sepsis

J E Adamou  1 , J H Heinrichs, A L Erwin, W Walsh, T Gayle, M Dormitzer, R Dagan, Y A Brewah, P Barren, R Lathigra, S Langermann, S Koenig, S Johnson
Affiliations

VSports最新版本 - Identification and characterization of a novel family of pneumococcal proteins that are protective against sepsis

J E Adamou et al. Infect Immun. 2001 Feb.

Abstract

Four pneumococcal genes (phtA, phtB, phtD, and phtE) encoding a novel family of homologous proteins (32 to 87% identity) were identified from the Streptococcus pneumoniae genomic sequence. These open reading frames were selected as potential vaccine candidates based upon their possession of hydrophobic leader sequences which presumably target these proteins to the bacterial cell surface. Analysis of the deduced amino acid sequences of these gene products revealed the presence of a histidine triad motif (HxxHxH), termed Pht (pneumococcal histidine triad) that is conserved and repeated several times in each of the four proteins. The four pht genes (phtA, phtB, phtD, and a truncated version of phtE) were expressed in Escherichia coli. A flow cytometry-based assay confirmed that PhtA, PhtB, PhtD and, to a lesser extent, PhtE were detectable on the surface of intact bacteria. Recombinant PhtA, PhtB, and PhtD elicited protection against certain pneumococcal capsular types in a mouse model of systemic disease. These novel pneumococcal antigens may serve as effective vaccines against the most prevalent pneumococcal serotypes. VSports手机版.

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FIG. 1
FIG. 1
Multiple alignment of the Pht protein sequences from S. pneumoniae strain Norway 4. The Pht protein sequences (PhtA, PhtB, PhtD, and PhtE) were aligned using the CLUSTAL algorithm in the DNAStar computer package. Amino acid residues that are conserved in at least three Pht proteins are shaded, and gaps introduced to maximize alignments are indicated by dashed lines. The predicted signal peptides, histidine triad motifs, α-helical coiled-coil regions, proline-rich regions, and 61-amino-acid repeat regions are indicated.
FIG. 2
FIG. 2
Southern blot analysis of the phtA gene. Genomic DNA prepared from 23 strains represented in current pneumococcal PS vaccines, as well as strain SJ2, were digested with BamHI and PvuII. Southern blots were probed with a PCR-generated fragment encompassing the phtA gene (A), the 5′ region (B), or the 3′ region (C) of the phtA gene. Serotypes are identified at the top of each lane. HindIII-digested λ DNA (M) was included, and the molecular weights are shown in kilobases. (D) Schematic representation of the proposed recombination between phtA (open box) and phtB (hatched box) genes of certain strains of S. pneumoniae. The thin line denotes flanking genomic DNA. The phtA-phtB hybrid gene shown is based on the sequence information of pneumococcal strain, ATCC 10354 (serotype 15B). Restriction fragments expected after BamHI and PvuII digestion are shown.
FIG. 2
FIG. 2
Southern blot analysis of the phtA gene. Genomic DNA prepared from 23 strains represented in current pneumococcal PS vaccines, as well as strain SJ2, were digested with BamHI and PvuII. Southern blots were probed with a PCR-generated fragment encompassing the phtA gene (A), the 5′ region (B), or the 3′ region (C) of the phtA gene. Serotypes are identified at the top of each lane. HindIII-digested λ DNA (M) was included, and the molecular weights are shown in kilobases. (D) Schematic representation of the proposed recombination between phtA (open box) and phtB (hatched box) genes of certain strains of S. pneumoniae. The thin line denotes flanking genomic DNA. The phtA-phtB hybrid gene shown is based on the sequence information of pneumococcal strain, ATCC 10354 (serotype 15B). Restriction fragments expected after BamHI and PvuII digestion are shown.
FIG. 3
FIG. 3
Immunoblot analysis of whole-cell lysates of S. pneumoniae. Whole-cell lysates of S. pneumoniae strains N4 and SJ2 were evaluated by immunoblot analysis with mouse antiserum raised against PhtD (residues 21 to 839) (A), PhtA (residues 21 to 816) (B), the C-terminal half of PhtA (residues 386 to 816) (C), and the N-terminal half of PhtE (residues 21 to 484) (D). The reactive bands are indicated by arrows. The molecular masses (in kilodaltons) of the protein standards are indicated.
FIG. 4
FIG. 4
Bacterial cell surface reactivity of anti-PhtA and anti-PhtD antisera. The indicated rabbit (A) and mouse (B) immune polyclonal antisera were tested for binding to intact S. pneumoniae strain N4. Bacteria were analyzed by fluorescence-activated cell sorter analysis and compared with samples treated with either rabbit preimmune sera or sera from sham-immunized mice.
FIG. 5
FIG. 5
Immunogenicity of PhtA and PhtD during pneumococcal infection in humans. The reactivity of sera collected from patients with culture-proven pneumococcal bacteremia (patients 1 and 4, serotype 5; patients 2, 3, and 5, serotypes 1, 12, and 18C, respectively) with PhtA (residues 21 to 816) (A), the N-terminal half of PhtA (residues 21 to 361) (B), the C-terminal half of PhtA (residues 386 to 816) (C), or PhtD (residues 21 to 839) (D) was determined by Western blot analysis. Lanes labeled A (acute) were probed with serum collected shortly after the diagnosis of pneumococcal infection; lanes C (convalescent) were probed with serum collected either 1 month (patients 1, 2, and 3) or 8 days after the first serum collection (patients 4 and 5). The molecular masses (in kilodaltons) of the protein standards are indicated.
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