José A. Guijarro

Purification and Characterization of a Psychrophilic, Calcium-Induced, Growth-Phase-Dependent Metalloprotease from the Fish Pathogen Flavobacterium psychrophilum

ABSTRACT Flavobacterium psychrophilum is a fish pathogen that commonly affects salmonids. This bacterium produced an extracellular protease with an estimated molecular mass of 55 kDa. This enzyme, designated Fpp1 (F.psychrophilumprotease1), was purified to electrophoretic homogeneity from the culture supernatant by using ammonium sulfate precipitation, ion-exchange chromatography, hydrophobic chromatography, and size exclusion chromatography. On the basis of its biochemical characteristics, Fpp1 can be included in the group of metalloproteases that have an optimum pH for activity of 6.5 and are inhibited by 1,10-phenanthroline, EDTA, or EGTA but not by phenylmethylsulfonyl fluoride. Fpp1 activity was dependent on calcium ions not only for its activity but also for its thermal stability. In addition to calcium, strontium and barium can activate the protein. The enzyme showed typical psychrophilic behavior; it had an activation energy of 5.58 kcal/mol and was more active at temperatures between 25 and 40°C, and its activity decreased rapidly at 45°C. Fpp1 cleaved gelatin, laminin, fibronectin, fibrinogen, collagen type IV, and, to a lesser extent, collagen types I and II. Fpp1 also degraded actin and myosin, basic elements of the fish muscular system. The presence of this enzyme in culture media was specifically dependent on the calcium concentration. Fpp1 production started early in the exponential growth phase and reached a maximum during this period. Addition of calcium during the stationary phase did not induce Fpp1 production at all. Besides calcium and the growth phase, temperature also seems to play a role in production of Fpp1. In this study we found that production of Fpp1 depends on factors such as calcium concentration, growth phase of the culture, and temperature. The combination of these parameters corresponds to the combination in the natural host during outbreaks of disease caused by F. psychrophilum. Consequently, we suggest that environmental host factors govern Fpp1 production.

Development of genetic techniques for the psychrotrophic fish pathogen Flavobacterium psychrophilum.

ABSTRACT Flavobacterium psychrophilum, a member of the Cytophaga-Flavobacterium-Bacteroides group, is an important pathogen of salmonid fish. Previous attempts to develop genetic techniques for this fastidious, psychrotrophic bacterium have met with failure. Here we describe the development of techniques for the genetic manipulation of F. psychrophilum and the identification of plasmids, selectable markers, a reporter system, and a transposon that function in several isolates of this fish pathogen. The antibiotic resistance genes ermF, cfxA, and tetQ function in F. psychrophilum. Cloning vectors based on the F. psychrophilum cryptic plasmid pCP1 which carried these selectable markers were introduced by conjugation from E. coli, resulting in antibiotic-resistant colonies of F. psychrophilum. Conjugative transfer of DNA into F. psychrophilum was strain dependent. Efficient transfer was observed for two of the seven strains tested (THC02-90 and THC04-90). E. coli lacZY functioned in F. psychrophilum when expressed from a pCP1 promoter, allowing its development as a reporter for studies of gene expression. Plasmids isolated from F. psychrophilum were efficiently introduced into F. psychrophilum by electroporation, but plasmids isolated from E. coli were not suitable for transfer by this route, suggesting the presence of a restriction barrier. DNA isolated from F. psychrophilum was resistant to digestion by Sau3AI and BamHI, indicating that a Sau3AI-like restriction modification system may constitute part of this barrier. Tn4351 was introduced into F. psychrophilum from E. coli and transposed with apparent randomness, resulting in erythromycin-resistant colonies. The techniques developed in this study allow for genetic manipulation and analysis of this important fish pathogen.

A Mutation in Flavobacterium psychrophilum tlpB Inhibits Gliding Motility and Induces Biofilm Formation

ABSTRACT Flavobacterium psychrophilum is a psychrotrophic, fish-pathogenic bacterium belonging to the Cytophaga-Flavobacterium-Bacteroides group. Tn4351-induced mutants deficient in gliding motility, growth on iron-depleted media, and extracellular proteolytic activity were isolated. Some of these mutants were affected in only one of these characteristics, whereas others had defects in two or more. FP523, a mutant deficient in all of these properties, was studied further. FP523 had a Tn4351 insertion in tlpB (thiol oxidoreductase-like protein gene), which encodes a 41.4-kDa protein whose sequence does not exhibit high levels of similar to the sequences of proteins having known functions. TlpB has two domains; the N-terminal domains has five transmembrane regions, whereas the C-terminal domains has the Cys-X-X-Cys motif and other conserved motifs characteristic of thiol:disulfide oxidoreductases. Quantitative analysis of the thiol groups of periplasmic proteins revealed that TlpB is required for reduction of these groups. The tlpB gene is part of the fpt (F. psychrophilum thiol oxidoreductase) operon that contains two other genes, tlpA and tpiA, which encode a thiol:disulfide oxidoreductase and a triosephosphate isomerase, respectively. FP523 exhibited enhanced biofilm formation and decreased virulence and cytotoxicity. Complementation with the tlpB loci restored the wild-type phenotype. Gliding motility and biofilm formation appear to be antagonistic properties, which are both affected by TlpB.

Identification of specific in vivo-induced (ivi) genes in Yersinia ruckeri and analysis of ruckerbactin, a catecholate siderophore iron acquisition system.

ABSTRACT This work reports the utilization of an in vivo expression technology system to identify in vivo-induced (ivi) genes in Yersinia ruckeri after determination of the conditions needed for its selection in fish. Fourteen clones were selected, and the cloned DNA fragments were analyzed after partial sequencing. In addition to sequences with no significant similarity, homology with genes encoding proteins putatively involved in two-component and type IV secretion systems, adherence, specific metabolic functions, and others were found. Among these sequences, four were involved in iron acquisition through a catechol siderophore (ruckerbactin). Thus, unlike other pathogenic yersiniae producing yersiniabactin, Y. ruckeri might be able to produce and utilize only this phenolate. The genetic organization of the ruckerbactin biosynthetic and uptake loci was similar to that of the Escherichia coli enterobactin gene cluster. Genes rucC and rupG, putative counterparts of E. coli entC and fepG, respectively, involved in the biosynthesis and transport of the iron siderophore complex, respectively, were analyzed further. Thus, regulation of expression by iron and temperature and their presence in other Y. ruckeri siderophore-producing strains were confirmed for these two loci. Moreover, 50% lethal dose values 100-fold higher than those of the wild-type strain were obtained with the rucC isogenic mutant, showing the importance of ruckerbactin in the pathogenesis caused by this microorganism.

A mutant in one of two exbD loci of a TonB system in Flavobacterium psychrophilum shows attenuated virulence and confers protection against cold water disease.

Flavobacterium psychrophilum is a psychrotrophic fish-pathogenic bacterium that causes cold water disease (CWD) in salmonids. By means of Tn4351 mutagenesis a mutant named FP1033, deficient in growth on iron-depleted medium, was previously isolated. FP1033 recovered the parental phenotype in the presence of iron. The gene disrupted by the transposon in this mutant encoded a protein with similarity to ExbD proteins, which are members of the TonB complex system involved in iron uptake mediated by siderophores. Analysis of the DNA surrounding the transposon insertion showed the presence of a tonB cluster of genes composed of exbB, two exbD (exbD1 and exbD2) and tonB loci. RT-PCR analysis and complementation studies indicated that these genes are transcribed as an operon and that the exbD2 : : Tn4351 phenotype was caused by the lack of ExbD2. FP1033 showed decreased virulence and conferred a high level of protection in rainbow trout fry after vaccination. This is believed to be the first report of a F. psychrophilum attenuated strain that induces a protective immune response in rainbow trout against CWD. These results suggest that the exbD2 locus from this particular TonB system is a suitable target to generate a live attenuated vaccine.

Isolation and analysis of a protease gene with an ABC transport system in the fish pathogen Yersinia ruckeri: insertional mutagenesis and involvement in virulence

Yersinia ruckeri is a Gram-negative pathogen that causes enteric redmouth disease in salmonids. A gene from Y. ruckeri encoding an extracellular protease termed yrp1 (Yersinia ruckeri protease 1) was cloned from a Sau3AI library constructed in pUC19 and analysed in gelatin-supplemented medium. The nucleotide sequence of the yrp1 gene indicated an ORF encoding a protein of 477 aa. On the basis of the high degree of homology in the amino acid sequence as well as its conservative motifs, this protein was included within the serralysin metalloendopeptidase subfamily (EC 3.4.24.12). The yrp1 N-terminal sequence showed a 14 aa propeptide followed by a 10 aa sequence identical to the one deduced previously from the 47 kDa purified protease. Additional results demonstrated that the yrp1 gene encodes the 47 kDa protein. In contrast to other Yersinia species, the yrp1 protease is secreted by a type I Gram-negative bacterial ABC exporter protein secretion system composed of three genes termed yrpD, yrpE and yrpF, and a protease inhibitor inh. The development of genetic methods for this species has allowed the exploration of the organization and the putative role of the Yrp1 genetic locus. Thus, site-directed insertion mutations into the yrp1 and the yrpE genes were constructed by the integration of the mobilizable suicide vector pIVET8 containing internal portions of both coding sequences. Complementation studies of those mutants with different loci indicated that they are organized as a single operon. The mutant strains lacked protease activity as well as the Yrp1 protein and, although physiologically similar to the parental strain when growing on nutrient broth medium, they were attenuated in virulence when bacteria were injected intraperitoneally into rainbow trout (Oncorhynchus mykiss). This is the first report of defined mutations in Y. ruckeri to show the implication of a factor such as an extracellular protease in pathogenesis.

Genome sequence of Lactococcus garvieae IPLA 31405, a bacteriocin-producing, tetracycline-resistant strain isolated from a raw-milk cheese

This work describes the draft genome sequence of Lactococcus garvieae IPLA 31405, isolated from a traditional Spanish cheese. The genome contains a lactose-galactose operon, a bacteriocin locus, two integrated phages, a transposon harboring an active tet(M) gene, and two theta-type plasmid replicons. Genes encoding virulence factors were not recorded.

Flavobacterium psychrophilum vaccine development: a difficult task

Bacterial cold water disease (BCWD) is a globally distributed freshwater fish disease caused by the Gram‐negative bacterium Flavobacterium psychrophilum. It is a particularly devastating infection in fry salmonids and may lead to high levels of mortality. In spite of its economic impact on fish farms, neither the biology of the bacterium nor the bacterium–host interactions are well understood. This review provides a synopsis of the major problems related to critical remaining questions about research into the use of vaccines against F. psychrophilum and the development of a commercial vaccine against this disease. Studies using sera from convalescent rainbow trout have shown the antigenic properties of different proteins such as OmpH, OmpA and FspA, as well as low and high molecular mass lipopolysaccharide of F. psychrophilum, which are potential candidates for subunit vaccines. Inactivated F. psychrophilum bacterins have been successfully tested as vaccines under laboratory conditions by both immersion and intraperitoneal routes. However, the efficacy and the practical usefulness of these preparations still have to be proved. The use of attenuated and wild‐type strains to immunize fish showed that these systems offer high levels of protection. Nevertheless, their application clashes with the regulations for environmental protection in many countries. In conclusion, protective vaccines against BCWD are theoretically possible, but substantial efforts still have to be made in order to permit the development of a commercial vaccine.

Genome sequence of Lactococcus garvieae UNIUD074, isolated in Italy from a lactococcosis outbreak.

Lactococcus garvieae is the etiological agent of lactococcosis disease, affecting many cultured fish species worldwide. In addition, this bacterium is currently considered a potential zoonotic microorganism since it is known to cause several opportunistic human infections. Here we present the draft genome sequence of the L. garvieae strain UNIUD074.

In Vitro and In Vivo Studies of the Yrp1 Protease from Yersinia ruckeri and Its Role in Protective Immunity against Enteric Red Mouth Disease of Salmonids

ABSTRACT Yersinia ruckeri, the etiological agent of the enteric red mouth disease (ERM) of salmonids, produces Yrp1, a serralysin metalloprotease involved in pathogenesis. We describe here the hydrolytic and immunogenic properties of Yrp1. The protease was able to hydrolyze different matrix and muscle proteins as laminin, fibrinogen, gelatine, actin, and myosin but not type II and IV collagens. In addition, the Yrp1 protein, when inactivated by heat and used as an immunogen, was able to elicit a strong protection against the development of ERM. The analysis of different Y. ruckeri strains with (Azo+) or without (Azo−) Yrp1 activity showed that all of them contained the yrp1 operon. By using yrp1::lacZ operon fusions, protease production analysis, and complementation studies, it was possible to show that an Azo− strain was blocked at the transcription level. The transcriptional study of the yrp1 operon under different environmental conditions showed that it was regulated by osmolarity and temperature, without pH influence. Finally, when β-galactosidase activity was used as a probe in vivo, the progression of the disease in the fish could be visualized, and the tropism of the bacterium and affected organs could be defined. This system opens a vast field of study not only with regard to fish disease progression but also in pathogen interactions, temporal gene expression, carrier stages, antibiotic resistance selection, etc.