Abstract
Piscirickettsia salmonis is a facultative intracellular pathogen and etiological agent of the systemic disease salmonid rickettsial septicemia. It has been suggested that P. salmonis is able to survive in host macrophages, localized within a vacuole like-compartment which prevents lysosomal degradation. However, the relevant aspects of the pathogenesis of P. salmonis as the host modulation that allow its intracellular survival have been poorly characterized. In this study, we evaluated the role of lysosomes in the response to P. salmonis infection in macrophage-enriched cell cultures established from Atlantic salmon head kidneys. Bacterial infection was confirmed using confocal microscopy. A gentamicin protection assay was performed to recover intracellular bacteria and the 16S rDNA copy number was quantified through quantitative polymerase chain reaction in order to determine the replication of P. salmonis within macrophages. Lysosomal activity in Atlantic salmon macrophage-enriched cell cultures infected with P. salmonis was evaluated by analyzing the lysosomal pH and proteolytic ability through confocal microscopy. The results showed that P. salmonis can survive ≥120 h in Atlantic salmon macrophage-enriched cell cultures, accompanied by an increase in the detection of the 16S rDNA copy number/cell. The latter finding suggests that P. salmonis also replicates in Atlantic salmon macrophage-enriched cell cultures. Moreover, this bacterial survival and replication appears to be favored by a perturbation of the lysosomal degradation system. We observed a modulation in the total number of lysosomes and lysosomal acidification following infection with P. salmonis. Collectively, the results of this study showed that infection of Atlantic salmon macrophages with P. salmonis induced limited lysosomal response which may be associated with host immune evasion mechanisms of P. salmonis that have not been previously reported.
Highlights
Piscirickettsia salmonis (P. salmonis) is the etiological agent of the systemic disease termed Piscirickettsiosis or salmonid rickettsial septicemia, which mostly affects salmonid species in saltwater [1], it has been identified in freshwater salmonid cultures [2]
Our first aim was to demonstrate that the bacteria invade the immune system, reside, and replicate in macrophageenriched cell cultures obtained from Atlantic salmon head kidney infected with P. salmonis
To detect and visualize P. salmonis in the macrophages, the cells were infected with Fluorescein Isothiocyanate (FITC)-labeled P. salmonis
Summary
Piscirickettsia salmonis (P. salmonis) is the etiological agent of the systemic disease termed Piscirickettsiosis or salmonid rickettsial septicemia, which mostly affects salmonid species in saltwater [1], it has been identified in freshwater salmonid cultures [2]. P. salmonis was first reported in Chile in 1989 as a pathogenic agent in coho salmon (Oncorhynchus kisutch) and characterized as a Gram-negative bacterium, non-motile, unencapsulated, pleomorphic, usually coccoid, with a diameter between 0.2 and 1.5 μm [1, 3, 4]. The National Fisheries Service (SERNAPESCA, Servicio Nacional de Pesca) has identified P. salmonis as the most serious health concern facing the Chilean salmon industry [10]. It has been reported that once P. salmonis has colonized, the host, it survives and replicates in vacuoles of macrophage-like cells that do not merge with lysosomes [11, 13, 14]. Infection of macrophages with P. salmonis induces an anti-inflammatory milieu, probably involved in the development of its bacterial virulence mechanism to ensure replication and survival [15]
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