Evolution of the capsular operon of Streptococcus iniae in response to vaccination.

Streptococcus iniae causes severe septicemia and meningitis in farmed fish and is also occasionally zoonotic. Vaccination against S. iniae is problematic, with frequent breakdown of protection in vaccinated fish. The major protective antigens in S. iniae are the polysaccharides of the capsule, which are essential for virulence. Capsular biosynthesis is driven and regulated by a 21-kb operon comprising up to 20 genes. In a long-term study, we have sequenced the capsular operon of strains that have been used in autogenous vaccines across Australia and compared it with the capsular operon sequences of strains subsequently isolated from infected vaccinated fish. Intriguingly, strains isolated from vaccinated fish that subsequently become infected have coding mutations that are confined to a limited number of genes in the cps operon, with the remainder of the genes in the operon remaining stable. Mutations in strains in diseased vaccinated fish occur in key genes in the capsular operon that are associated with polysaccharide configuration (cpsG) and with regulation of biosynthesis (cpsD and cpsE). This, along with high ratios of nonsynonymous to synonymous mutations within the cps genes, suggests that immune response directed predominantly against capsular polysaccharide may be driving evolution in a very specific set of genes in the operon. From these data, it may be possible to design a simple polyvalent vaccine with a greater operational life span than the current monovalent killed bacterins.

Two hepcidin-like antimicrobial peptides in Barramundi Lates calcarifer exhibit differing tissue tropism and are induced in response to lipopolysaccharide.

Genes encoding two hepcidin-like antimicrobial peptides were discovered in Barramundi, Lates calcarifer (barramundi, Giant sea perch). Analysis of the coding regions indicated that genes for each hepcidin comprised 3 exons and 2 introns. The deduced amino acid sequences for each molecule resulted in a protein comprising a signal sequence of 24 aa in each case, coupled to a prepropeptide of 75 aa for hepcidin 1 and 78 aa for hepcidin 2. A cleavage site was identified in each prepropetide at amino acid 64 with the cleavage motif--QKR/QS--resulting in mature peptides of 25 and 28 amino acids respectively. Each mature peptide contained 8 conserved cysteine residues and 3 dimensional modeling predicted a ß-hairpin and ß-sheet structure characteristic of human Liver Expressed Antimicrobial Peptide (LEAP). Analysis of the deduced amino acid sequences by BLAST with phylogenetic supported indicated that hepcidin 1 was a HAMP1-type peptide closely related to hepcidins identified in other Perciformes (Micropterus and Pseudosciaena), whilst hepcidin 2 was a HAMP2-type peptide most similar to a hepcidin previously identified in black rock fish (Sebastes schlegeli). Both hepcidin genes were inducible in barramundi following intraperitoneal injection with lipopolysaccharide, with elevated expression detected in liver and head kidney 3 h post IP injection for hepcidin 1 and in liver only for hepcidin 2. The elevated expression was transient with return to normal levels within 24-48 h. No significant expression of either peptide was detected in spleen, skin or gill following IP injection with LPS.

Towards control of Streptococcus iniae.

Streptococcus iniae is an emerging zoonotic pathogen; such infections generally occur through injuries associated with preparing whole fresh fish for cooking. Those infected to date have been of Asian descent, are usually elderly (average age 68 years), and have had >/=1 underlying conditions that may predispose them to infection. Studies of the foundations of growth characteristics of S. iniae and its interactions with piscine host cells have recently been complemented by molecular studies. Advances in molecular biology have allowed research groups to identify numerous virulence factors and to explore their roles in the progression of S. iniae infection. Many of these virulence factors are homologous to those found in the major human pathogen S. pyogenes. An increased understanding of the properties of these factors and their effect on the success of infection is leading to novel approaches to control S. iniae infection; in particular, vaccination programs at fish farms have reduced the reservoir of infection for additional clinical cases.

Host-directed evolution of a novel lactate oxidase in Streptococcus iniae isolates from barramundi (Lates calcarifer).

In Streptococcus iniae, lactate metabolism is dependent upon two proteins, lactate permease that mediates uptake and lactate oxidase, a flavin mononucleotide-dependent enzyme that catalyzes oxidation of alpha-hydroxyacids. A novel variant of the lactate oxidase gene, lctO, in Australian isolates of S. iniae from diseased barramundi was found during a diagnostic screen using LOX-1 and LOX-2 primers, yielding amplicons of 920 bp instead of the expected 869 bp. Sequencing of the novel gene variant (type 2) revealed a 51-nucleotide insertion in lctO, resulting in a 17-amino-acid repeat in the gene product, and three-dimensional modeling indicated formation of an extra loop in the monomeric protein structure. The activities of the lactate oxidase enzyme variants expressed in Escherichia coli were examined, indicating that the higher-molecular-weight type 2 enzyme exhibited higher activity. Growth rates of S. iniae expressing the novel type 2 enzyme were not reduced at lactate concentrations of 0.3% and 0.5%, whereas a strain expressing the type 1 enzyme exhibited reduced growth rates at these lactate concentrations. During a retrospective screen of 105 isolates of S. iniae from Australia, the United States, Canada, Israel, Réunion Island, and Thailand, the type 2 variant arose only in isolates from a single marine farm with unusually high tidal flow in the Northern Territory, Australia. Elevated plasma lactate levels in the fish, resulting from the effort of swimming in tidal flows of up to 3 knots, may exert sufficient selective pressure to maintain the novel, high-molecular-weight enzyme variant.

Identification and molecular characterisation of a fibrinogen binding protein from Streptococcus iniae.

BACKGROUND: 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. 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. RESULTS: Fibrinogen 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 (approximately 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. CONCLUSION: These 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.