Martyn L. Gilpin

Renibacterium salmoninarum and bacterial kidney disease — the unfinished jigsaw

Abstract Bacterial kidney disease (BKD) is one of the most important diseases of wild and cultured salmonid fish, and has been reported from many different countries. Despite considerable effort, many pieces are still missing from the “jigsaw puzzle” which represents our knowledge of the disease, and its etiological agent Renibacterium salmoninarum . The purpose of this review is to consider current knowledge about this bacterial pathogen and the pathogenesis of BKD. It is our intention to construct a picture of the possible ways in which pathogen and host interact, in particular by high-lighting the areas where our understanding is poor and indicating how recent advances in methodology offer the prospect of improvement. The exact status of R. salmoninarum as an obligate fish pathogen, and the implications for transmission, entry, colonization, and disease progression are considered. We review the considerable progress that has been made recently through work on the molecular determinants of pathogenicity, particularly attachment, cellular invasion mechanisms, intracellular survival of the bacterium, and its interactions with the host defense mechanisms and immune system. Finally, we consider ways in which the future control of BKD through improved diagnosis, chemotherapy, and vaccination may be realized through an integrated approach to the study of BKD.

Host responses to Renibacterium salmoninarum and specific components of the pathogen reveal the mechanisms of immune suppression and activation

During infection, Renibacterium salmoninarum survives within the pronephric macrophages of salmonid fish. Therefore, to study the initial phases of the interaction we infected macrophages with live bacteria and analysed the responses of host and pathogen. It was found that the expression of msa encoding the p57 antigen of R. salmoninarum, was constitutive, while the expression of hly and rsh, encoding haemolysins, and lysB and grp was reduced after infection. Macrophages showed a rapid inflammatory response in which the expression of interleukin‐1β (IL‐1β), major histocompatibility complex class II (MHC II), inducible cyclo‐oxygenase (Cox‐2), and inducible nitric oxide synthase (iNOS) was enhanced, but tumour necrosis factor‐α (TNF‐α) expression was greatly reduced initially and then increased. After 5 days, except for TNF‐α and MHC II, expression returned to levels approaching those of uninfected macrophages. We propose that R. salmoninarum survives initial contact with macrophages by avoiding and/or interfering with TNF‐α‐dependent killing pathways. The effects of specific R. salmoninarum components were studied in vivo by injecting fish with DNA vaccine constructs expressing msa, hly, rsh, lysB, or grp. We found that msa reduced the expression of IL‐1β, Cox‐2, and MHC II but stimulated TNF‐α while hly, rsh and grp stimulated MHC II but down‐regulated TNF‐α. Constructs expressing hly or lysB stimulated iNOS expression and additionally, lysB stimulated TNF‐α. The results show how p57 suppresses the host immune system and suggest that the immune mechanisms for the containment of R. salmoninarum infections rely on MHC II‐ and TNF‐α‐dependent pathways. Moreover, prolonged stimulation of TNF‐α may contribute to the chronic inflammatory pathology of bacterial kidney disease.

Molecular Diversity of Renibacterium salmoninarum Isolates Determined by Randomly Amplified Polymorphic DNA Analysis

ABSTRACT The molecular diversity among 60 isolates of Renibacterium salmoninarum which differ in place and date of isolation was investigated by using randomly amplified polymorphic DNA (RAPD) analysis. Isolates were grouped into 21 banding patterns which did not reflect the biological source. Four 16S-23S rRNA intergenic spacer (ITS1) sequence variations and two alleles of an exact tandem repeat locus, ETR-A, were the bases for formation of distinct groups within the RAPD clusters. This study provides evidence that the most common ITS1 sequence variant, SV1, possesses two copies of a 51-bp repeat unit at ETR-A and has been widely dispersed among countries which are associated with mainstream intensive salmonid culture.

A gene from Renibacterium salmoninarum encoding a product which shows homology to bacterial zinc-metalloproteases

A genomic library constructed from Renibacterium salmoninarum isolate MT444 DNA in the plasmid vector pBR328 was screened using Escherichia coli host strain DH1 for the expression of genes encoding putative virulence factors. A single haemolytic clone was isolated at 22 degrees C and found to contain a 3.1 kb HindIII fragment of inserted DNA. This fragment was present in seven isolates of R. salmoninarum which were examined. Western blots of extracts from clones exhibiting haemolytic activity were performed with antisera raised against either cellular or extracellular components of R. salmoninarum and failed to identify any additional proteins compared to control E. coli containing pBR328. However, minicell analysis revealed that a polypeptide with an apparent molecular mass of 65 kDa was associated with a haemolytic activity distinct from that previously described for R. salmoninarum. The nucleotide sequence of the gene encoding this product was determined and the amino acid sequence deduced. The product was 548 amino acids with a predicted molecular mass of 66757 Da and a pl of 5.57. The deduced amino acid sequence of the gene possessed strong similarities to those of a range of secreted bacterial zinc-metalloproteases and was tentatively designed hly. Neither protease nor lecithinase activities were detectable in E. coli recombinants expressing gene hly. Haemolytic activity was observed from 6 degrees C to 37 degrees C for erythrocytes from a number of mammalian species and also from fish. Gene hly was expressed in E. coli as a fusion protein consisting of maltose-binding protein at the N-terminus linked to all but the first 24 amino acids, largely constituting the putative signal peptide, of the N-terminus of Hly. The soluble fusion protein was produced and purified by affinity chromatography. Antiserum raised against the purified fusion protein was used to probe Western blots of cell lysates and extracellular products from seven isolates of R. salmoninarum cultured under conditions of iron-sufficiency or iron-restriction. The results indicate that the availability of iron modulates the expression of the hly gene.

Renibacterium salmoninarum isolates from different sources possess two highly conserved copies of the rRNA operon .

The nucleotide sequences of the rRNA genes and the 5′ flanking region were determined for R. salmoninarum ATCC 33209T from overlapping products generated by PCR amplification from the genomic DNA. Comparison of the sequences with rRNA genes from a variety of bacteria demonstrated the close relatedness between R. salmoninarum and the high G+C group of the actinobacteria, in particular, Arthrobacter species. A regulatory element within the 5′ leader of the rRNA operon was identical to an element, CL2, described for mycobacteria. PCR, DNA sequence analysis, and DNA hybridisation were performed to examine variation between isolates from diverse sources which represented the four 16S–23S rRNA intergenic spacer sequevars previously described for R. salmoninarum. Two 23S–5S rRNA intergenic spacer sequevars of identical length were found. DNA hybridisation using probes complementary to 23S rDNA and 16S rDNA identified two rRNA operons which were identical or nearly identical amongst 40 isolates sourced from a variety of countries.

Characterization of the mrgRS locus of the opportunistic pathogen Burkholderia pseudomallei: temperature regulates the expression of a two-component signal transduction system

BackgroundBurkholderia pseudomallei is a saprophyte in tropical environments and an opportunistic human pathogen. This versatility requires a sensing mechanism that allows the bacterium to respond rapidly to altered environmental conditions. We characterized a two-component signal transduction locus from B. pseudomallei 204, mrgR and mrgS, encoding products with extensive homology with response regulators and histidine protein kinases of Escherichia coli, Bordetella pertussis, and Vibrio cholerae.ResultsThe locus was present and expressed in a variety of B. pseudomallei human and environmental isolates but was absent from other Burkholderia species, B. cepacia, B. cocovenenans, B. plantarii, B. thailandensis, B. vandii, and B. vietnamiensis. A 2128 bp sequence, including the full response regulator mrgR, but not the sensor kinase mrgS, was present in the B. mallei genome. Restriction fragment length polymorphism downstream from mrgRS showed two distinct groups were present among B. pseudomallei isolates. Our analysis of the open reading frames in this region of the genome revealed that transposase and bacteriophage activity may help explain this variation. MrgR and MrgS proteins were expressed in B. pseudomallei 204 cultured at different pH, salinity and temperatures and the expression was substantially reduced at 25°C compared with 37°C or 42°C but was mostly unaffected by pH or salinity, although at 25°C and 0.15% NaCl a small increase in MrgR expression was observed at pH 5. MrgR was recognized by antibodies in convalescent sera pooled from melioidosis patients.ConclusionThe results suggest that mrgRS regulates an adaptive response to temperature that may be essential for pathogenesis, particularly during the initial phases of infection. B. pseudomallei and B. mallei are very closely related species that differ in their capacity to adapt to changing environmental conditions. Modifications in this region of the genome may assist our understanding of the reasons for this difference.

Variation in the Spacer Regions Separating tRNA Genes in Renibacterium salmoninarum Distinguishes Recent Clinical Isolates from the Same Location

ABSTRACT A means for distinguishing between clinical isolates ofRenibacterium salmoninarum that is based on the PCR amplification of length polymorphisms in the tRNA intergenic spacer regions (tDNA-ILPs) was investigated. The method used primers specific to nucleotide sequences of R. salmoninarum tRNA genes and tRNA intergenic spacer regions that had been generated by using consensus tRNA gene primers. Twenty-one PCR products were sequenced from five isolates of R. salmoninarum from the United States, England, and Scotland, and four complete tRNA genes and spacer regions were identified. Sixteen specific PCR primers were designed and tested singly and in all possible pairwise combinations for their potential to discriminate between isolates from recent clinical outbreaks of bacterial kidney disease (BKD) in the United Kingdom. Fourteen of the isolates were cultured from kidney samples taken from fish displaying clinical signs of BKD on five farms, and some of the isolates came from the same farm and at the same time. The tDNA-ILP profiles separated 22 clinical isolates into nine groups and highlighted that some farms may have had more than one source of infection. The grouping of isolates improved on the discriminatory power of previously reported typing methods based on randomly amplified polymorphic DNA analysis and restriction fragment length profiles developed using insertion sequence IS994. Our method enabled us to make divisions between closely related clinical isolates of R. salmoninarum that have identical exact tandem repeat (ETR-A) loci, rRNA intergenic spacer sequences, and IS994profiles.

Effects of the microbial secondary metabolites pyrrolnitrin, phenazine and patulin on INS-1 rat pancreatic β-cells

The effects on pancreatic β-cell viability and function of three microbial secondary metabolites pyrrolnitrin, phenazine and patulin were investigated, using the rat clonal pancreatic β-cell line, INS-1. Cells were exposed to 10-fold serial dilutions (range 0-10 μg mL(-1)) of the purified compounds for 2, 24 and 72 h. After 2 h exposure, only patulin (10 μg mL(-1)) was cytotoxic. All compounds showed significant cytotoxicity after 24 h. None of the compounds altered insulin secretion with 2 and 20 mM glucose after 2 h. However, after 24 h treatment, phenazine and pyrrolnitrin (10 and 100 ng mL(-1)) potentiated insulin production and glucose-stimulated insulin secretion, whereas patulin had no effect. Exposure (24 h) to either phenazine (100 ng mL(-1)) or pyrrolnitrin (10 ng mL(-1)) caused similar increases in the Ca(2+) content of INS-1 cells. The outward membrane current was inhibited after 24 h exposure to either phenazine (100 ng mL(-1)) or pyrrolnitrin (10 or 100 ng mL(-1)). This study presents novel data suggesting that high concentrations of pyrrolnitrin and phenazine are cytotoxic to pancreatic β-cells and thus possibly diabetogenic, whereas at lower concentrations these agents are nontoxic and may be insulinotropic. The possible role of such agents in the development of cystic fibrosis-related diabetes is discussed.