In Vitro andIn Vivo Studies of the Yrp1 Protease from Yersinia ruckeri andIts Role in Protective Immunity against Enteric Red MouthDisease ofSalmonids

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 ␤-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.

In vivo monitoring of Yersinia ruckeri in fish tissues: progression and virulence gene expression

“…These results are consistent with previous studies in which induction of Pcds promoter required the presence of cysteine in the culture medium (Méndez et al ., ) and high concentrations of iron inhibited Pyhl promoter (Fernández et al ., ). On the contrary, the promoter of yctCBA operon (Pyct) involved in in vivo citrate uptake (Navais et al ., ) and the protease Yrp1 (Pyrp1) (Fernández et al ., ) were the promoters with the highest bioluminescence level on NA (Fig. A).…”

“…B and C). The colonization of these organs was previously described by Fernández and colleagues () who used an inducible β‐galactosidase expression system to monitor bacterial tissue distribution in rainbow trout. This bacterial distribution pattern is also consistent with former studies, in which Avci and Birincioglu () described hyperaemia and haemorrhages in the swim bladder after intraperitoneal injection with Y. ruckeri .…”

“…To assess the validity of the pCS26‐PacTER in the study of gene expression in Y. ruckeri , promoters of four well‐characterized operons of Y. ruckeri 150R such as yrp1 (Fernández et al ., ), yhlBA (Fernández et al ., ), yctCBA (Navais et al ., ) and cdsAB (Méndez et al ., ), as well as the constitutive promoter PgyrB from the gyrase B gene were PCR amplified and cloned into the pCS26‐PacTER plasmid. After introducing the corresponding constructions into Y. ruckeri 150R, the resulting strains were analysed by bioluminescence quantification both on NA at 18°C and during bacterial infection of fry rainbow trout.…”

Identification of Specific In Vivo-Induced (ivi) Genes inYersinia ruckeriand Analysis of Ruckerbactin, a Catecholate Siderophore Iron Acquisition System

“…Twelve Y. ruckeri strains (146, 147, 148, 149, 150, 3585, 4319, 1386, 955, 956, A100, and A102) (21,47) were positive for the production of siderophores in CAS-agar plates. In addition, PCR assays for the detection of rucC and rupG confirmed the presence of these genes in all the strains tested, even though three different plasmid profiles, represented by strains 955, 956, and the rest of the strains, respectively, were analyzed.…”

“…Until now, the only molecule whose involvement in virulence has been proved is the protease Yrp1 (20,21), but it is only produced by Azo ϩ strains (47). Previously, Romalde et al (42) showed that the extracellular products of Y. ruckeri, which included lipase, protease, and cytotoxic and hemolytic activities, reproduce some characteristic symptoms of this pathology, such as hemorrhage in the mouth and intestine.…”