Abstract
Piscirickettsia salmonis, an aggressive intracellular pathogen, is the etiological agent of salmonid rickettsial septicemia (SRS). This is a chronic multisystemic disease that generates high mortalities and large losses in Chilean salmon farming, threatening the sustainability of the salmon industry. Previous reports suggest that P. salmonis is able to survive and replicate in salmonid macrophages, inducing an anti-inflammatory environment and a limited lysosomal response that may be associated with host immune evasion mechanisms favoring bacterial survival. Current control and prophylaxis strategies against P. salmonis (based on the use of antibiotics and vaccines) have not had the expected success against infection. This makes it urgent to unravel the host-pathogen interaction to develop more effective therapeutic strategies. In this study, we evaluated the effect of treatment with IgM-beads on lysosomal activity in Atlantic salmon macrophage-enriched cell cultures infected with P. salmonis by analyzing the lysosomal pH and proteolytic ability through confocal microscopy. The impact of IgM-beads on cytotoxicity induced by P. salmonis in infected cells was evaluated by quantification of cell lysis through release of Lactate Dehydrogenase (LDH) activity. Bacterial load was determined by quantification of 16S rDNA copy number by qPCR, and counting of colony-forming units (CFU) present in the extracellular and intracellular environment. Our results suggest that stimulation with antibodies promotes lysosomal activity by lowering lysosomal pH and increasing the proteolytic activity within this organelle. Additionally, incubation with IgM-beads elicits a decrease in bacterial-induced cytotoxicity in infected Atlantic salmon macrophages and reduces the bacterial load. Overall, our results suggest that stimulation of cells infected by P. salmonis with IgM-beads reverses the modulation of the lysosomal activity induced by bacterial infection, promoting macrophage survival and bacterial elimination. This work represents a new important evidence to understand the bacterial evasion mechanisms established by P. salmonis and contribute to the development of new effective therapeutic strategies against SRS.
Highlights
Piscirickettsia salmonis is the etiological agent of piscirickettsiosis or salmonid rickettsial septicemia (SRS), which mostly affects farmed salmonid species [1, 2]
In macrophage-enriched cells cultures infected with P. salmonis and incubated with immunoglobulin M (IgM)-beads (Figure 1D), we observed at 1 hpt a punctate pattern of yellow/green fluorescence and at 3 hpt we observed both dots of yellow/green and cyan fluorescence (Figure 1H), in a similar way to the observed in infected cells (Figures 1B, F)
On the other hand, when the macrophage-enriched cell cultures infected with P. salmonis were incubated with IgM-beads, the results show no difference in the number of lysosomes/cell compared to the non-infected cells (Figure 2A and Supplementary Table 2)
Summary
Piscirickettsia salmonis is the etiological agent of piscirickettsiosis or salmonid rickettsial septicemia (SRS), which mostly affects farmed salmonid species [1, 2]. Piscirickettsia salmonis is a Gram-negative, non-motile, unencapsulated, pleomorphic, and usually coccoid bacterium, between 0.2 and 1.5 mm in diameter [1, 3, 4]. Infected salmonids respond poorly to these treatments, likely because of the intracellular characteristics of the infective cycle of P. salmonis and the insufficient concentrations of antibiotics that reach the intracellular niche to eliminate the bacterium [11]. This situation is further complicated by the lack of effective vaccines against P. salmonis [12], because prophylactic vaccines do not provide acceptable levels of protection [11]
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