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Comparative Study
. 2000 Jan;68(1):294-302.
doi: 10.1128/IAI.68.1.294-302.2000.

"VSports在线直播" M-like proteins of Streptococcus dysgalactiae

J Vasi  1 , L Frykberg, L E Carlsson, M Lindberg, B Guss
Affiliations
Comparative Study

M-like proteins of Streptococcus dysgalactiae

J Vasi et al. Infect Immun. 2000 Jan.

Abstract

Streptococcus dysgalactiae is one of the most important bacterial species isolated from bovine mastitis. To identify potential virulence factors of this species we prepared chromosomal DNA from strain 8215 and constructed a phage display library. By affinity selection of the library against fibrinogen (Fg), we isolated and characterized a gene, called demA, encoding a protein with the molecular mass of approximately 58 kDa, called DemA, displaying both plasma protein binding properties and sequence similarities with the M and M-like proteins of other streptococcal species VSports手机版. Purified recombinant DemA protein was found to completely inhibit Fg-binding to cells of S. dysgalactiae. A continued sequence analysis revealed that the demA gene was preceded by an open reading frame (dmgA) coding for a putative protein, called DmgA, with high similarities to the Mga proteins of Streptococcus pyogenes. By additional cloning, the corresponding dmgA and demA genes from another strain, called Epi9, were isolated and analyzed. These genes, called dmgB and demB, respectively, revealed a high degree of similarity to the corresponding genes in strain 8215. Increased binding of Fg by cells of strain Epi9, grown in an atmosphere with 10% CO(2), was correlated to an enhanced transcription of the demB gene as shown in a Northern blot. Strain 8215 did not respond to CO(2), which could be explained by a nonfunctional dmgA gene due to insertion of an insertion sequence element. Based on sequence similarities of the described proteins to Mga, M, and M-like proteins and the response to elevated level of CO(2), we suggest that the dmg and dem genes are members of a regulon similar to the described mga regulon in S. pyogenes, which encodes several virulence factors in this species. .

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Figures

FIG. 1
FIG. 1
Schematic presentation of deduced gene products encoded by the pDEMA6 clone derived from S. dysgalactiae 8215. pDEMA1 to pDEMA5 are phagemid clones isolated after panning a phage display library of strain 8215 against Fg. pGDEMA7 and pGDEMA8 are expression clones of the pGEX-2T vector with inserts corresponding to the phagemid clone pDEMA5 and a PCR fragment derived from pDEMA1 encoding amino acids 41 to 518 of DemA, respectively, yielding GST fusion proteins. The figures within parentheses after the names of the clones indicate the encoded amino acids of protein DemA. The DemA protein, including the signal peptide, consists of 548 amino acid residues as indicated above the schematic drawing. S, signal peptide, followed by the cell surface exposed part of the protein with repetitive sequences marked by different patterns; W, the cell wall spanning region; M, the membrane spanning region directly followed by five charged amino acids; ΔDmgA, a truncated protein highly homologous to the Mga regulatory protein from S. pyogenes (32); ΔIS, 276 bp homologous to an IS element. +, the phagemid clone can bind Fg, human IgG, and bovine IgG; −, no detectable binding.
FIG. 2
FIG. 2
Schematic presentation of the alignment of the primary structures of the Fg-binding proteins DemA from S. dysgalactiae 8215 and FgBP from S. equi TW (29). The deduced amino acid sequences of the molecules were aligned by the PALIGNE program of PCGENE and are shown as schematic bars. Numbers along the bars indicate positions of amino acids. Numbers between the bars indicate the percentage of similar residues relative to the total number of amino acids within the compared domains, marked by connecting vertical lines. The percentages of identical residues were calculated in a similar way and are shown within parentheses. S, signal peptide; A, B, and C, various repeated sequences in the respective molecules, each marked with different patterns; W, wall spanning regions; M, membrane spanning regions directly followed by five charged amino acids.
FIG. 3
FIG. 3
SDS-PAGE and Western blotting. (a) The affinity-purified GST fusion proteins coded by pGDEMA7 and pGDEMA8 clones derived from S. dysgalactiae strain 8215 were separated on an SDS-PAGE gel (8 to 25% polyacrylamide) (A) and blotted to an NC membrane. Binding of Fg by the recombinant proteins was tested by probing with 125I-Fg and visualized by exposing the strips to x-ray film (B). IgG binding by the recombinant proteins was probed with HRP-labelled human (C) and rabbit (D) IgG while HRP-labelled protein G was used to detect the bound unlabelled horse IgG (E). Lanes: 1, GST; 2, GDEMA7; 3, GDEMA8. (b) Human Fg in reducing sample buffer and Fab and Fc fragments of human IgG in nonreducing sample buffer were separated on a homogeneous SDS–12% PAGE gel (A), transferred to an NC membrane, and probed with 125I-GDEMA8 protein. The bound probe was detected by exposing the membrane to x-ray film (B). Lanes: 1, Fg (from the top: α-, β-, and γ-chains); 2, human IgG Fab fragment; 3, human IgG Fc fragment. Molecular mass markers (kDa) are indicated.
FIG. 4
FIG. 4
Alignment of the amino acid sequences of proteins DmgB and Mry. The amino acid sequence of protein DmgB from S. dysgalactiae Epi9 was aligned with the Mry (later designated Mga) of S. pyogenes D471 (32) with the PALIGNE program of PCGENE. A vertical bar indicates identical residues while a dot indicates similar residues. Gaps introduced to improve the alignment are indicated by a dashed line. The horizontal arrow above the sequence indicates the beginning of the N-terminally truncated DmgA protein of S. dysgalactiae 8215. The positions of the five changed amino acids in DmgA in comparison to those in DmgB are indicated by bold letters above the sequence. The predicted helix-turn-helix motif in Mry (33) is indicated by bold letters within the sequence. The region in Mry containing amino acids 404 to 528 exhibits homology to the receiver domain of response regulators (32). The alignment reveals an overall 61% similarity (45% identity) between the DmgB and Mry proteins.
FIG. 5
FIG. 5
Northern blot analysis of the RNA transcripts of the dmg and the dem genes. Twenty micrograms of total RNA isolated from cultures of S. dysgalactiae strains 8215 and Epi9 grown to mid-log phase in normal (lanes 1) or 10% CO2-enriched (lanes 2) atmosphere were separated in an 1% agarose gel, vacuum blotted to a nylon membrane, and hybridized with specific DNA probes derived from dmgB and demB (as presented in Materials and Methods). The positions of the 23S and 16S rRNA bands are indicated.
FIG. 6
FIG. 6
Inhibition of binding of 125I-Fg to S. dysgalactiae cells by recombinant DemA protein. Overnight cultures of S. dysgalactiae strains 8215 and Epi9 grown in ambient atmosphere were centrifuged and washed in PBS. The pellets were resuspended in PBS–0.1% ovalbumin–0.05% Tween 20, and the OD600s were adjusted to 1.4 and 0.75, respectively. Three hundred microliters of the respective cell suspensions were mixed with 100-μl serial dilutions of the GST-DemA fusion protein GDEMA8 and 100 μl of the iodine-labelled Fg. The mixtures were incubated with end-over-end rotation for 4 h at RT. The cells were pelletized and washed twice in PBS–0.1% ovalbumin–0.05% Tween 20, and the radioactivity associated with the cells was measured in a γ-counter. A similar assay was performed with the GST protein as the inhibitor of the binding of 125I-Fg to S. dysgalactiae Epi9 cells to exclude a possible effect of the GST part of the fusion protein on the binding.
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