Abstract
BackgroundBinding of serum components by surface M-related proteins, encoded by the emm genes, in streptococci constitutes a major virulence factor in this important group of organisms. The present study demonstrates fibrinogen binding by S. iniae, a Lancefield non-typeable pathogen causing devastating fish losses in the aquaculture industry and an opportunistic pathogen of humans, and identifies the proteins involved and their encoding genes.ResultsFibrinogen binding by S. iniae significantly reduced respiratory burst activity of barramundi peritoneal macrophages in primary cultures compared to BSA-treated or untreated controls, indicating a potentially important role for fibrinogen binding cell-surface proteins in avoiding phagocytic attack in fish. We describe a novel emm-like gene, simA, encoding a 57 kDa fibrinogen binding M-like protein in S. iniae. These SiM proteins and their corresponding tetrameric structures from some sequevar types (~230 kDa) bound fibrinogen in Western blots. simA was most closely related (32% identity) to the demA gene of S. dysgalactiae. Genome walking and sequencing determined the genetic organization of the simA region had similarities to the mgrC regulon in GCS and to S. uberis. Moreover, a putative multigene regulator, mgx was orientated in the opposite direction to the simA gene in common with S. uberis, but contrary to findings in GAS and GCS. In GAS, diversity among emm-genes and consequent diversity of their M-related proteins results in substantial antigenic variation. However, an extensive survey of S. iniae isolates from diverse geographic regions and hosts revealed only three variants of the gene, with one sequevar accounting for all but two of the 50 isolates analysed.ConclusionThese proteins play a role in avoiding oxidative attack by phagocytic cells during infection of fish by S. iniae, but genetic diversity amongst these key surface proteins has not yet arisen. This lack of diversity coupled with a functional role in macrophage resistance suggests that these proteins may constitute important targets for future vaccines against S. iniae in fish.
Highlights
Binding of serum components by surface M-related proteins, encoded by the emm genes, in streptococci constitutes a major virulence factor in this important group of organisms
Fibrinogen binding by S. iniae Wild-type Streptococcus iniae QMA0076 and QMA0072 were capable of binding biotin-labelled human fibrinogen in vitro as evidenced by fluorescence microscopy of cells counter-labelled with avidin conjugated fluorescein isothiocyanate (FITC) (Figure 1)
This was corroborated for strain QMA0076 by an independent dot-blot assay in which whole cells incubated with biotin-labelled fibrinogen and extensively washed were spotted onto PVDF membrane that was subsequently probed with alkaline phosphatase-conjugated avidin (Figure 2a)
Summary
Fibrinogen binding by S. iniae Wild-type Streptococcus iniae QMA0076 and QMA0072 were capable of binding biotin-labelled human fibrinogen in vitro as evidenced by fluorescence microscopy of cells counter-labelled with avidin conjugated FITC (Figure 1). The gene sequence for isolate QMA0141 was most divergent having 100% amino acid residue identity for the first 35 residues and 100% similarity for the first 41 residues This confirms the theoretical position of where the signal sequence is cleaved to produce the mature SiM protein (Figure 4). Putative Mgx protein binding motifs were found in the spacer regions upstream of the simA or simB genes in all isolates sequenced (Figure 9). Analysis by the COIL program [20,21] with a window length of 28 predicted that the protein from isolate QMA0076 (with signal sequence) has two coiled-coil segments from residues 79–174 and 181–454 with a probability of 1.00 for each segment This is a similar result to that of Vasi et al (2000)[18] who identified M-like proteins from S. dysgalactiae. GQFieMgnuAert0eic078o6rganization of the mgrX segment in S. iniae Genetic organization of the mgrX segment in S. iniae QMA0076
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