Identification of a Novel Elastin-Degrading Enzyme from the Fish Pathogen Flavobacterium psychrophilum.

Hydrolytic extracellular enzymes degrading host tissues potentially play a role in bacterial pathogenesis. Flavobacterium psychrophilum is an important bacterial pathogen of salmonid fish reared in freshwater throughout the world. Diversity among isolates has been described at the phenotypic, serological, and genomic levels, but the links between these various traits remain poorly understood. Using a genome-wide association study, we identified a gene encoding a novel elastinolytic enzyme in F. psychrophilum To formally demonstrate enzymatic activity, this gene (FP0506 from strain JIP 02/86) was expressed in the elastinolysis-deficient strain OSU THCO2-90, resulting in proficient elastin-degrading cells. The encoded protein is predicted to be a cell-surface-exposed lipoprotein with no homology to previously reported elastases. FP0506 might belong to the zincin tribe and gluzincin clan of metalloproteases, and this new elastase-encoding gene seems to be present only in some members of the family FlavobacteriaceaeIMPORTANCE Elastin is an important proteinaceous component of vertebrate connective tissues (e.g., blood vessels, lung, and skin), to which it confers elasticity. Elastases have been identified in a number of pathogenic bacteria. They are thought to be required for tissue penetration and dissemination, acting as "spreading factors." Flavobacterium psychrophilum is a devastating bacterial pathogen of salmonid fish (salmon and trout) that is responsible for severe economic losses worldwide. This pathogen displays strong proteolytic activities. Using a variety of techniques, including genome comparisons, we identified a gene encoding a novel elastase in F. psychrophilum The encoded protein is predicted to be a cell-surface-exposed lipoprotein with no homology to previously reported elastases. In addition, this elastase likely belongs to a new family of proteases that seems to be present only in some members of this important group of bacteria.

dltA gene mutation in the teichoic acids alanylation system of Lactococcus garvieae results in diminished proliferation in its natural host.

The dltABCD cluster is involved in the d-alanylation of teichoic acids in gram positive bacteria. In order to determine the role of this alanylation in the physiology and virulence of Lactococcus garvieae, a previously isolated dltA Delta Tn917 signature tagged mutagenesis (STM) clone was analyzed. RT-PCR results revealed that dltABCD genes form an operon. No major differences could be established between the parental and mutant strains with respect to growth rate, autolytic properties, and susceptibility to acid conditions, lysozyme treatment, anionic detergents, or oxidant agents. However, the dltA mutant was more susceptible to nisin than the parental strain, with minimum inhibitory concentration (MIC) values of 8 and 16 microg/ml, respectively. Less proliferation of the mutant was observed in in vivo competence index experiments (CI=0.08). Furthermore, the mutant strain had a 50% lethal dose (LD(50)) 3-fold that of the parental strain. These results, together with the fact that the dltA Delta Tn917 mutant was isolated as a STM clone, reveal that the dltA locus of Lactococcus garvieae is required for full growth and pathogenesis on rainbow trout.

A chromosomally located traHIJKCLMN operon encoding a putative type IV secretion system is involved in the virulence of Yersinia ruckeri.

Nucleotide sequence analysis of the region surrounding the pIVET8 insertion site in Yersinia ruckeri 150RiviXII, previously selected by in vivo expression technology (IVET), revealed the presence of eight genes (traHIJKCLMN [hereafter referred to collectively as the tra operon or tra cluster]), which are similar both in sequence and organization to the tra operon cluster found in the virulence-related plasmid pADAP from Serratia entomophila. Interestingly, the tra cluster of Y. ruckeri is chromosomally encoded, and no similar tra cluster has been identified yet in the genomic analysis of human pathogenic yersiniae. A traI insertional mutant was obtained by homologous recombination. Coinfection experiments with the mutant and the parental strain, as well as 50% lethal dose determinations, indicate that this operon is involved in the virulence of this bacterium. All of these results suggest the implication of the tra cluster in a virulence-related type IV secretion/transfer system. Reverse transcriptase PCR studies showed that this cluster is transcribed as an operon from a putative promoter located upstream of traH and that the mutation of traI had a polar effect. A traI::lacZY transcriptional fusion displayed higher expression levels at 18 degrees C, the temperature of occurrence of the disease, and under nutrient-limiting conditions. PCR detection analysis indicated that the tra cluster is present in 15 Y. ruckeri strains from different origins and with different plasmid profiles. The results obtained in the present study support the conclusion, already suggested by different authors, that Y. ruckeri is a very homogeneous species that is quite different from the other members of the genus Yersinia.