ORIGINAL RESEARCH published: 19 March 2019 doi: 10.3389/fimmu.2019.00434 Non-lysosomal Activation in Macrophages of Atlantic Salmon (Salmo salar) After Infection With Piscirickettsia salmonis Diego Pérez-Stuardo 1† , Jonathan Morales-Reyes 2† , Sebastián Tapia 1 , Diego E. Ahumada 1 , Allison Espinoza 1 , Valentina Soto-Herrera 3 , Bernardo Brianson 1 , Valentina Ibaceta 1 , Ana M. Sandino 2,3 , Eugenio Spencer 2,3 , Eva Vallejos-Vidal 4 , Felipe E. Reyes-López 4 , Jorge Valdés 1 and Sebastián Reyes-Cerpa 1* 1 Centro de Genómica y Bioinformática, Facultad de Ciencias, Universidad Mayor, Santiago, Chile, 2 Consorcio de Sanidad Edited by: Acuícola, Ictio Biotechnologies S.A., Santiago, Chile, 3 Centro de Biotecnología Acuícola, Universidad de Santiago de Chile, Geert Wiegertjes, Santiago, Chile, 4 Department of Cell Biology, Physiology and Immunology, Universitat Autonoma de Barcelona, Wageningen University & Research, Barcelona, Spain Netherlands Reviewed by: Mark D. Fast, Piscirickettsia salmonis is a facultative intracellular pathogen and etiological agent of the University of Prince Edward Island, systemic disease salmonid rickettsial septicemia. It has been suggested that P. salmonis Canada is able to survive in host macrophages, localized within a vacuole like-compartment which Tomáš Korytář, University of South Bohemia, Czechia prevents lysosomal degradation. However, the relevant aspects of the pathogenesis *Correspondence: of P. salmonis as the host modulation that allow its intracellular survival have been Sebastián Reyes-Cerpa poorly characterized. In this study, we evaluated the role of lysosomes in the response sebastian.reyes@umayor.cl to P. salmonis infection in macrophage-enriched cell cultures established from Atlantic † These authors have contributed salmon head kidneys. Bacterial infection was confirmed using confocal microscopy. A equally to this work gentamicin protection assay was performed to recover intracellular bacteria and the Specialty section: 16S rDNA copy number was quantified through quantitative polymerase chain reaction This article was submitted to in order to determine the replication of P. salmonis within macrophages. Lysosomal Comparative Immunology, a section of the journal activity in Atlantic salmon macrophage-enriched cell cultures infected with P. salmonis Frontiers in Immunology was evaluated by analyzing the lysosomal pH and proteolytic ability through confocal Received: 16 October 2018 microscopy. The results showed that P. salmonis can survive ≥120 h in Atlantic salmon Accepted: 19 February 2019 macrophage-enriched cell cultures, accompanied by an increase in the detection of the Published: 19 March 2019 16S rDNA copy number/cell. The latter finding suggests that P. salmonis also replicates in Citation: Pérez-Stuardo D, Morales-Reyes J, Atlantic salmon macrophage-enriched cell cultures. Moreover, this bacterial survival and Tapia S, Ahumada DE, Espinoza A, replication appears to be favored by a perturbation of the lysosomal degradation system. Soto-Herrera V, Brianson B, Ibaceta V, Sandino AM, Spencer E, We observed a modulation in the total number of lysosomes and lysosomal acidification Vallejos-Vidal E, Reyes-López FE, following infection with P. salmonis. Collectively, the results of this study showed that Valdés J and Reyes-Cerpa S (2019) infection of Atlantic salmon macrophages with P. salmonis induced limited lysosomal Non-lysosomal Activation in Macrophages of Atlantic Salmon response which may be associated with host immune evasion mechanisms of P. salmonis (Salmo salar) After Infection With that have not been previously reported. Piscirickettsia salmonis. Front. Immunol. 10:434. Keywords: Atlantic salmon (Salmo salar), Piscirickettsiosis, immune evasion mechanisms, macrophages, doi: 10.3389/fimmu.2019.00434 lysosome, proteolytic activity Frontiers in Immunology | www.frontiersin.org 1 March 2019 | Volume 10 | Article 434 Pérez-Stuardo et al. Piscirickettsia salmonis Perturb Lysosomal Activity INTRODUCTION Legionella pneumophila (L. pneumophila), and Coxiella burnetii (C. burnetii) (21, 22). Piscirickettsia salmonis (P. salmonis) is the etiological agent of the Gomez et al. (11), described that P. salmonis—along with the systemic disease termed Piscirickettsiosis or salmonid rickettsial phylogenetically-related bacteria L. pneumophila or C. burnetii— septicemia, which mostly affects salmonid species in saltwater possess the deficient in organelle trafficking/intracellular (1), although it has also been identified in freshwater salmonid multiplication (Dot/Icm) secretion system genes classified as cultures (2). P. salmonis was first reported in Chile in 1989 type IVB secretion system (TIVBSS) or an equivalent to achieve as a pathogenic agent in coho salmon (Oncorhynchus kisutch) a productive infection. The Dot/Icm proteins form part of a and characterized as a Gram-negative bacterium, non-motile, specialized secretion system encoded by the dot/icm genes and unencapsulated, pleomorphic, usually coccoid, with a diameter constitute the major virulence mechanism of L. pneumophila between 0.2 and 1.5 µm (1, 3, 4). P. salmonis is an intracellular and C. burnetii responsible for their intracellular survival and pathogen, classified phylogenetically as a Gammaproteobacteria multiplication (23–25). During infection, L. pneumophila evades in the family Piscirickettsiaceae, and is closely related to phagosome-lysosome fusion and phagosome acidification. Legionella, Francisella, and Coxiella (1). Although subtly increased, this process is delayed for ≥16 h (26). Notably, infection with P. salmonis is responsible for high Moreover, L. pneumophila has the ability to alter the maturation mortality rates only in Chile. In other countries (e.g., Norway, of the endocytic vacuole in which it initially resides, allowing the Canada, and Ireland), outbreaks have been reported with limited establishment of an organelle within phagocytic host cells that impact (5, 6). In Chile, the management of Piscirickettsiosis supports replication (27). Conversely, C. burnetii resides in an is a national priority given its highly aggressive nature, acidified lysosome-like compartment, in which it can replicate in recurring outbreaks, and wide dispersion among other cultivated the presence of several lysosomal proteins such as v-ATPase (22). salmonid species (7–9). In fact, the National Fisheries Service To the best of our knowledge, there is limited evidence regarding (SERNAPESCA, Servicio Nacional de Pesca) has identified the lysosomal acidification and functionality of Atlantic salmon P. salmonis as the most serious health concern facing the macrophages infected with P. salmonis. The availability of such Chilean salmon industry (10). The SERNAPESCA has established evidence may assist in unraveling the host-pathogen interaction a monitoring and control program to reduce the impact of mechanisms associated with P. salmonis infection. salmonid rickettsial septicemia (9). Despite the great impact that The aim of this study was to determine whether the lysosomal P. salmonis has had on the aquaculture industry, key aspects of its activity in Atlantic salmon macrophages is modulated by biology, pathogenesis, and virulence remain poorly understood, infection with P. salmonis. significantly hampering the strategies for its control (11, 12). It has been reported that once P. salmonis has colonized, the host, it survives and replicates in vacuoles of macrophage-like cells that MATERIALS AND METHODS do not merge with lysosomes (11, 13, 14). Moreover, infection Experimental Fish of macrophages with P. salmonis induces an anti-inflammatory Atlantic salmon (Salmo salar) with an average weight of 55 ± milieu, probably involved in the development of its bacterial 15 g were obtained from a local farm and maintained in tanks virulence mechanism to ensure replication and survival (15). with freshwater at a biomass of 10–12 Kg m−3 and controlled Macrophages are part of the first-line cell defense against temperature (14–16◦ C) with continuous aeration. Water quality bacterial infection, specializing in phagocytosis, destruction parameters, such as pH, oxygen, and the levels of nitrogen of foreign microorganism, and presentation of antigens (16, compounds (i.e., nitrate, nitrite, and ammonia) were monitored 17). The impact of an infection depends on the balance daily and maintained at constant values. The fish were fed between the capacity of macrophages to recognize and destroy with commercial pellets twice daily (Golden Optima, Biomar, bacterial pathogens and the ability of pathogens to disrupt the Puerto Montt, Chile), and acclimatized for ≥3 weeks prior to functions of these macrophages (18). Infection of macrophages the experiments. The maintenance of fish was performed in using different molecular effectors to undermine macrophage accordance with the ethical standards of the Institutional Ethics signaling is a common strategy adopted by intracellular Committee of Universidad de Santiago de Chile (approved in pathogens to evade the immune system and ensure systemic internal report n◦ 364) and the relevant legislation in force. spread into their hosts. In this way, different bacterial pathogens use diverse strategies to subvert the functions of Isolation of Macrophages and Cell Culture macrophages. One of the most important strategies is the evasion Macrophage-enriched cell cultures were obtained from Atlantic of phagolysosomal degradation (11, 18). Maturation of the salmon head kidneys as described by Braun-Nesje et al. (28) phagosome that fuses with lysosomal compartments—containing with slight modifications. Briefly, the head kidney of fish was an acid and hydrolytic lumen with enzymes responsible for aseptically extracted and disaggregated using a cell strainer (pore the eradication of internalized bacteria—is critically linked to size: 70 µm) (BD Falcon, Seaton Delaval, England) on a 60 × the destruction of pathogenic bacteria (19, 20). Therefore, 15 mm culture plate (BD Falcon) in the presence of Leibovitz interference with the phagosomal maturation is essential medium (L-15; Hyclone, Thermo Scientific, Massachusetts, for bacteria to enable their presence and replication within USA) supplemented with 2-mercaptoethanol 36 µM (2-ME; macrophages. Importantly, this pathogen strategy has already Gibco, Thermo Scientific), fetal bovine serum 3% (v/v) (FBS; been reported in bacteria closely related to P. salmonis, including Hyclone, GE Healthcare Life Sciences, Utah, USA), penicillin Frontiers in Immunology | www.frontiersin.org 2 March 2019 | Volume 10 | Article 434 Pérez-Stuardo et al. Piscirickettsia salmonis Perturb Lysosomal Activity 200 U/mL, streptomycin 200 µg/mL (Corning, New York, NY, was amplified as described by Karatas et al. to detect the presence USA), amphotericin B 2.5 mg/mL (Corning), and sodic heparin of P. salmonis in the infected cell cultures (31). Genomic DNA 24.7 UI/mL (Leo Pharma, Ballerup, Denmark) (supplement 1). was obtained using the WizardTM Genomic DNA Purification mechanical disaggregation was performed until a homogeneous kit according to the instructions provided by the manufacturer. cell suspension was obtained. The leukocyte fraction was isolated The PCR amplification was performed using the PowerUpTM through a discontinuous gradient in densities of 34% and 51% SYBR R Green master Mix (Thermo Scientific) according to the of Percoll (GE Healthcare) diluted in miliQ water and Hank’s instructions provided by the manufacturer. The primers were Balanced Salt Solution (HBSS 10X, Gibco). The cell suspension added to a final concentration of 0.4 µM, and 12 ng of template was deposited on the discontinuous Percoll gradient and the were used. The qPCR was performed on a QuantStudio 3 Real- column was centrifuged for 40 min at 400 g at 16◦ C. The Time PCR system (Thermo Scientific). The quantification of 16S leukocyte interface was recovered and resuspended in L-15 rDNA copies was determined through interpolation from the medium (supplement 1). To eliminate the traces of Percoll, standard curve with the cycle threshold (Ct) value obtained for the cell suspension was centrifuged twice for 7 min at 250 each sample. The results are expressed as 16S rDNA copy/cell. g at 16◦ C. The cell suspension was placed on 12-well plates at 40,000 cells/cm2 in L-15 medium supplemented with 2- Fluorescein Isothiocyanate (FITC) Staining for ME 36 µM, FBS 10% (v/v) (Hyclone), penicillin 200 U/mL, P. salmonis streptomycin 200 µg/mL (Corning), and amphotericin B 2.5 In order to monitor the P. salmonis infection into macrophage- mg/mL (Corning) (supplement 2) at 16◦ C. At day 1, the enriched cell cultures, we stained the bacteria using FITC primary culture was washed with phosphate-buffered saline (Thermo Scientific). P. salmonis was extracted from the infection (PBS) 1X, non-adherent cells were discarded to enrich the supernatant of CHSE-214 cells with evident cytopathic effect monocytic/macrophage adherent population, and cultivated in and centrifuged for 15 min at 7,500 g at 16◦ C. Subsequently, the L-15 medium containing supplement 2. At days 3, 5, and 7, the bacteria were incubated for 30 min with FITC 100 µg/mL diluted cultured cells were washed with PBS 1X, the non-adherent cells in L-15 medium without supplement. Finally, the bacteria were were discarded, and the medium of the cell cultures was replaced centrifuged for 15 min at 7,500 g at 16◦ C and resuspended in L- by L-15 medium supplemented with 2-ME 36 µM and FBS 10% 15 medium without supplement. The viability of P. salmonis was (v/v) (Hyclone) (supplement 3). determined by comparison of bacterial growth in enriched blood agar plates (32) with non-stained P. salmonis (Figure 1). Growth Cultivation and Propagation of P. salmonis of bacterial colonies was analyzed for the specific detection of 16S Culture and propagation of P. salmonis (strain 9734, ETECMA, rDNA using real time PCR (31) and morphology as previously Puerto Montt, Chile) was performed in salmonid cell line CHSE- described by Mauel et al. (32). 214 (ATCC N◦ CRL-1682) as previously described by Fryer et al. (29). The CHSE-214 cell line was maintained in minimal essential P. salmonis Infection medium (MEM; Corning) FBS 10% (v/v) (Hyclone), HEPES Macrophage-enriched cell cultures from Atlantic salmon buffer 10 mM (Corning), and non-essential amino acids 1% (v/v) head kidneys were incubated for 30 min with 10 µM Cell (Corning) (supplement 4) at 16◦ C. The infection was observed Tracker Red (CMPTX; Thermo Scientific) diluted in L-15 through conventional inverted microscopy (Motic AE31E) 4–6 without supplements. Subsequently, the cells were washed days post-infection (dpi) to determine the cytopathic effect on thrice with PBS 1X and incubated with P. salmonis-FITC cells (30). The bacteria were quantified using a Petroff–Hausser stained at a multiplicity of infection (MOI) of 0.5 bacteria/cell chamber (Hausser Scientific, Pennsylvania, USA) according to (previously quantified using a Petroff–Hausser chamber), the instructions provided by the manufacturer. and cultured in L-15 medium containing supplement 3. Cells were incubated for 24 and 120 h post-infection (hpi). Inactivation of P. salmonis Subsequently, the cell culture was fixed with 4% PFA for 10 min P. salmonis was extracted from the infection supernatant of and washed thrice with PBS 1X. The nucleus was stained CHSE-214 cells with evident cytopathic effect. Cellular debris with 4′ ,6-diamidino-2-phenylindole, dihydrochloride (DAPI) was eliminated through centrifugation for 5 min at 500 g. 0.1 µg/mL for 1 min and the cells were washed twice with Subsequently, P. salmonis was centrifuged for 15 min at 7,500 PBS 1X and once with MiliQ water to wash the residuary g at 16◦ C. The pellet was resuspended in 4% paraformaldehyde salts. Subsequently, the cells were mounted on slides using (PFA; Sigma Aldrich, Missouri, USA) diluted in PBS 1X and the mounting solution FluoromountTM (Thermo Scientific). incubated overnight at 4◦ C. Finally, the bacterial suspension Micrographs were obtained using a confocal microscope Zeiss was centrifuged for 15 min at 7,500 g at 16◦ C, the supernatant LSM 710, the images were processed and analyzed using the Fiji was discarded, and the pellet was resuspended in L-15 medium software (33). containing supplement 3. Gentamicin Protection Assay Detection of P. salmonis Using Quantitative In order to recover the intracellular bacterium from the infected Polymerase Chain Reaction (qPCR) macrophage-enriched cell cultures, a gentamicin protection assay The gene encoding for 16S rRNA (Fw: 5′ -AGG-GAG-ACT-GCC- was performed. Briefly, macrophage-enriched cell cultures were GGT-GAT-A-3′ ; Rv: 5′ -ACT-ACG-AGG-CGC-TTT-CTC-A-3′ ) infected with P. salmonis at a MOI of 10 bacteria/cell for Frontiers in Immunology | www.frontiersin.org 3 March 2019 | Volume 10 | Article 434 Pérez-Stuardo et al. Piscirickettsia salmonis Perturb Lysosomal Activity FIGURE 1 | Intracellular detection of P. salmonis. Macrophage-enriched cultures obtained from Atlantic salmon head kidneys were incubated with P. salmonis-FITC at a MOI of 0.5 bacteria/cell for 24 and 120 hpi in L-15 supplemented medium. The cellular nucleus (stained with DAPI; blue), cell cytoplasm (stained with CMPTX; red), and P. salmonis-FITC (green) are represented. The intracellular location of P. salmonis-FITC was determined by confocal microscope orthogonal image analysis from the z-stack obtained. (A,B) Non-infected macrophages (scale bar 50 and 10 µm, respectively). (C,D) Macrophages at 24 hpi (scale bar 50 and 10 µm, respectively). (E,F) Macrophages at 120 hpi (scale bar 50 and 10 µm, respectively). 24 and 120 h. Recovery of the intracellular bacterium was Evaluation of Lysosomal Acidification performed following the protocol described by Rodriguez et al. Evaluation of lysosomal acidification in P. salmonis-infected (34) Imarai et al. (35), and Fast et al. (36), with modifications. macrophage-enriched cell cultures was performed by Macrophage-enriched cell cultures infected with P. salmonis fluorescence analysis using the LysoSensorTM Yellow/Blue were incubated with gentamicin (100 µg/mL) for 60 min to probe (LSYB; Thermo Scientific). This ratiometric probe can be eliminate bacteria from the extracellular environment. After 3– used to measure the pH of acidic organelles. The LysoSensorTM 5 washes with cold PBS 1X, saponin (Sigma) was added (1% dye produces blue and yellow fluorescence in neutral and acidic p/v in PBS 1X) for 15 min at 16◦ C to permeabilize the cells. environments, respectively. Therefore, a fluorescence shift from Finally, the homogenized cells were diluted in PBS 1X and blue (maximum emission at 430 nm) to yellow (maximum centrifuged at 6,000 g for 10 min at 4◦ C. The supernatant was emission at 535 nm) indicates an increase in lysosomal pH (37). discarded and the pellet was resuspended in 100 µL of PBS The macrophage-enriched cell cultures were established in 12- 1X. Genomic DNA from the homogenized cells was extracted well plates with glass coverslips and infected with P. salmonis using the WizardTM Genomic DNA Purification kit according to at a MOI of 10 bacteria/cell for 1 h in L-15 medium containing the instructions provided by the manufacturer. Quantification of supplement 3. After incubation, the cell cultures were washed intracellular P. salmonis recovered from head kidney cell cultures once with PBS 1X and incubated with L-15 containing was performed through specific qPCR absolute quantification supplement 3. In order to determine the short-term effect using the set of primers described by Karatas et al. (31). on lysosomal acidification in P. salmonis-infected macrophage- The supernatant used for the gentamicin treatment as well as enriched cell cultures, a lysosomal acidification analysis was the PBS used for each wash were recovered and subsequently performed at 3, 6, and 24 hpi. A non-infected and an incubation centrifuged at 6,000 g for 10 min at 4◦ C to eliminate any viable with inactivated P. salmonis conditions were simultaneously extracellular bacteria after treatment with gentamicin or the performed as internal experimental controls. At each time point, subsequent washes. This process was performed to prevent an the infected cells were incubated with LSYB 10 µM for 5 min. overestimation of the intracellular bacteria. The obtained pellets Subsequently, the cells were washed thrice with PBS 1X and were resuspended in 100 µL PBS. Part of these suspensions (50 fixed with 4% PFA (p/v) (Sigma Aldrich) for 10 min, followed µL) was plated in CHAB agar and cultivated by 14 days at 20◦ C. by three additional washes with PBS 1x. Finally, the nucleus The remaining 50 µL were used to extract genomic DNA for the was stained with propidium iodide (PI) 1 µg/mL (incubation for qPCR amplification of 16S rDNA with the same specific primers 1 min) and the cells were washed twice with PBS 1X and once mentioned above. with MiliQ water to remove the residuary salts. The samples were Frontiers in Immunology | www.frontiersin.org 4 March 2019 | Volume 10 | Article 434 Pérez-Stuardo et al. Piscirickettsia salmonis Perturb Lysosomal Activity mounted on slides with FluoromountTM (Sigma Aldrich) and the analysis was performed by obtaining four micrographs with a images were obtained using a Leica SP8 confocal microscope z-stack to determine the total amount of hydrolytic events per (Leica, Wetzlar, Germany). Results were obtained by the division cell that contains in average 30 cells on the micrographs, for each of the emission spectrum of the LSYB probe, specifically to experimental condition and counting every positive event to the obtain an acidic indicator channel (acquired between 500 and fluorescence of DQTM Green BSA. The data were normalized 580 nm) and a neutral-basic indicator channel (acquired between to the number of cells on the micrograph and the results are 450 and 495 nm). According to instructions provided by the represented as the number of proteolytic events/cell. manufacturer, the fluorescence intensity obtained in the acidic indicator channel had to be ≥2-fold than that obtained in the neutral-basic indicator channel to consider a lysosome as Statistical Analysis acidic. The analysis of lysosomal acidity was performed using Statistical differences in the quantification of P. salmonis the software LAS X (version 3.3.0). Briefly, the fluorescence recovered from macrophage-enriched cell cultures were histogram intensity for each indicator channel was obtained for determined using the one-tailed Mann–Whitney U test. each lysosome in the samples, and the fluorescence intensity Statistical differences in proteolytic events between the groups average per count value was calculated using the software. analyzed were determined using two-way analysis of variance Subsequently, the ratio between the values obtained from the two (ANOVA) with a Dunnett’s multiple comparison test. We used indicator channels was used as the acidity index. An acidic index the GraphPad Prism v6.0 for Windows software (GraphPad ≥2 denoted an acidic pH. Conversely, an acidity index 0300 bacterial genes encoding different capacity, antigen-presenting function, secretion of cytokines, and proteins (termed effectors) that are secreted from the bacteria efficient antimicrobial activity (42, 43). Considering that the to the host cell (46, 47). One of these mechanisms is associated macrophages are one of the first cell types which pathogens with the prevention of phagosome acidification through the encounter after their entry into the host, many pathogens recruitment of Rab1 to the phagosome (48). Acidification are internalized by endocytosis or phagocytosis—intracellular of the phagosome is necessary for efficient lysosomal fusion pathways that culminate the degradation in the lysosomal and function. L. pneumophila employs several mechanisms system. In this sense, it is not surprising that some pathogens to recruit Rab1 to the Legionella-containing vacuole (LCV). have developed intracellular immune evasion mechanisms Multiple Dot/Icm secreted factors, such as DrrA/SidM, SidD, against macrophages (14, 44, 45). In the present study, we and LepB function together to manipulate the localization observed that P. salmonis evades host response mechanisms by of Rab1 to the LCV, thus cycling the host protein between reducing the lysosomal acidification and its proteolytic activity the active (anchored to the LCV) and inactive states. On in the first hpi. Thus, this pathogen is able to reside in the other hand, L. pneumophila also manipulates Rab1 macrophage-enriched cell cultures for 24 hpi and replicate for independently of recruitment to the LCV through the action ≥120 hpi (Figure 1). of SidC/SdcA, LidA, and AnkX. This redundancy in the Macrophage infection is a common strategy adopted by mechanisms indicates that Rab1 manipulation by L. pneumophila several intracellular pathogens to colonize and spread into their is extremely important for the intracellular survival of this hosts. This is achieved through interference with normal cell pathogen (49). The molecular mechanisms involved in host signaling and disruption of the normal responses triggered response evasion which are modulated by P. salmonis remain to to eliminate foreign invaders, such as the lysosomal system be elucidated. Frontiers in Immunology | www.frontiersin.org 8 March 2019 | Volume 10 | Article 434 Pérez-Stuardo et al. Piscirickettsia salmonis Perturb Lysosomal Activity In fish, relatively few pathogens have been reported to lysosomes presented an acidic pH. Interestingly, this percentage resist degradation and replicate within macrophages. Virulent diminished at 6 and 24 hpi to values <40%. Conversely, Edwarsiella tarda is capable of survival and replication in head incubation with inactivated P. salmonis showed an acidic pH kidney phagocytes of blue gourami (Trichogaster trichopterus) in >75% of lysosomes, reflecting that macrophage-like cells at least by 6.5 hpi, where induces low levels of reactive oxygen respond to the appropriate stimulus. Previous studies have intermediates by infected phagocytes (14, 50). Photobacterium reported early lysosomal acidification in macrophage-enriched damselae subsp. piscicida is an intracellular pathogen that can cell cultures infected by P. salmonis at 2 hpi, suggesting that survive and multiply within macrophages of hybrid striped bass, the acidification may be the signal that induces the intracellular via inhibition of the phagosome-lysosome fusion and replication overexpression of the P. salmonis dot/icm genes (11). This in vacuoles at least by 18 hpi (Morone saxatilis × white bass dot/icm gene expression may be responsible for the observed M. chrysops) (14, 51). Similarly, Mycobacterium spp. have been decrease in the number of lysosomes with an acidic pH, as found both intact and partially degraded inside phagolysosomes previously described in L. pneumophila (48, 49). The functional of rainbow trout macrophages, suggesting that some degree of consequence of the disruption of lysosomal acidification is a inhibition of the phagosome-lysosome fusion occurs to favor poor hydrolytic activity. In the present study, this was observed bacterial survival (14, 52). Renibacterium salmoninarum survives by the low levels of degradation of DQTM Green BSA in within macrophages even at 4 dpi, suggesting that this bacterium macrophage-like cells infected by P. salmonis compared with escapes from the phagosome and resides in a bacteria-containing those observed in cells incubated with inactivated P. salmonis vesicle with a tightly apposed membrane by interfering with the (Figure 4). However, expression or functional analyses are macrophage-killing pathways (14, 53, 54). required to identify which part of the mechanism of assembly or These examples reflect the possibility that in fish, like function is targeted by P. salmonis during its interference with the in mammals, certain pathogens have developed evasion lysosomal response. mechanisms against macrophages as part of their life-cycle, Collectively, the results showed that infection of Atlantic using the cell as a shield against cell-mediated and humoral salmon macrophages with P. salmonis induced a limited immune responses (44). However, the mechanism used by lysosomal response in the first hpi, which may correspond to fish pathogens to resist the macrophage killing-mechanism is a strategy of immune evasion that has not been previously currently not understood and requires investigation. In the reported. Previous studies have described that P. salmonis does present study, we detected viable P. salmonis in macrophage- not reach lysosomal compartments in which TIVBSS plays a enriched cell cultures after 5 dpi and observed a two-fold central role (11, 14). Currently, there is limited knowledge increase in its detection using qPCR. These results suggest available regarding the pathogenetic mechanisms of P. salmonis. bacterial survival and replication (Figure 2). The increase in However, the similarities observed with the phylogenetically bacterial load detected through qPCR at 120 hpi was inconsistent closely related bacteria L. pneumophila (58) suggest that with the FITC-fluorescence intensity observed at the same time P. salmonis may use a similar mechanism of pathogenesis to points (Figure 1). However, this discrepancy is probably due those of L. pneumophila. Both bacteria target macrophages and to a decline in FITC fluorescence, which has been reported to disruption of the lysosomal degradation pathways appears to be decrease during prolonged incubation times (55). However, this a hallmark of their infection. approach allows us to observe P. salmonis in the infected cells. These results are consistent with those previously observed in macrophages obtained from rainbow trout head kidneys—in DATA AVAILABILITY which concurrent destruction and replication were observed at 5 All datasets generated for this study are included in the dpi—implying that P. salmonis is able to survive and reproduce manuscript and/or the supplementary files. in functional macrophages (14). An analysis performed to characterize the function of the lysosome showed that the number of lysosomes in AUTHOR CONTRIBUTIONS macrophage-enriched cell cultures infected with P. salmonis (Figure 3) does not increase in the first 24 hpi. This evidence DP-S and JM-R performed most of the experiments, analyzed suggests a delayed response, which is further supported by the data, prepared the figures, and contributed to the writing evidence showing that incubation with inactivated P. salmonis of this manuscript. ST, DA, AE, VS-H, BB, and VI performed resulted in an increase in the number of lysosomes by more the experiments. JV, EV-V, AS, and ES reviewed this manuscript. than three-fold 6 h post-incubation. An increase in lysosome SR-C conceived and supervised the study. SR-C and FR-L wrote biogenesis/function has been reported to contribute to limiting this manuscript. intracellular macrophage infection in mice (56). Lysosomes must maintain an acidic luminal pH to activate hydrolytic FUNDING enzymes and degrade internalized macromolecules (57). Thus, lysosomal acidification is a key environmental mechanism for This work was financially supported by FONDECYT Iniciación the correct function of this organelle. Analysis of the lysosomal 11150807, Start-Up UMayor 101205, CORFO 13CTI-21527, pH following infection of macrophage-enriched cell cultures UMayor-Ph.D. fellowships to DP-S and CONICYT-BCH with P. salmonis showed that at 3 hpi, approximately 70% of Postdoctoral fellowship to EV-V. Frontiers in Immunology | www.frontiersin.org 9 March 2019 | Volume 10 | Article 434 Pérez-Stuardo et al. Piscirickettsia salmonis Perturb Lysosomal Activity SUPPLEMENTARY MATERIAL blood agar. Confirmation that identity of colonies correspond to P. salmonis was carried out by specific detection of 16S rDNA by real time PCR. (A) The Supplementary Material for this article can be found FITC-stained P. salmonis plated on enriched blood agar. (B) Melt curve of the gene amplification obtained by real time PCR from a single colonies of FITC online at: https://www.frontiersin.org/articles/10.3389/fimmu. stained P. salmonis (red lines) and positive control (gray lines). (C) P. salmonis 2019.00434/full#supplementary-material plated on enriched blood agar. (D) Melt curve of the gene amplification obtained Supplementary Figure 1 | Viability assay of FITC stained P. salmonis. by real time PCR from a single colonies of P. salmonis (red lines) and positive Non-stained P. salmonis and FITC stained P. salmonis were plated on enriched control (gray lines). REFERENCES infection in a salmonid cell line model. PLoS ONE. (2016) 11:e0163943. doi: 10.1371/journal.pone.0163943 1. Fryer JL, Hedrick RP. Piscirickettsia salmonis: a gram-negative 16. Kum C, Sekkin S. The immune system drugs in fish: immune function, intracellular bacterial pathogen of fish. J Fish Dis. (2003) 26:251–62. immunoassay, drugs. In: Dogu FAAZ, editor. Recent Advances In Fish Farms. doi: 10.1046/j.1365-2761.2003.00460.x Rijekap: Intech open science (2011). p. 169–216. doi: 10.5772/26869 2. Gaggero A, Castro H, Sandino AM. First isolation of Piscirickettsia salmonis 17. Aderem A, Underhill DM. 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This is an open-access article distributed under the terms customized intracellular niche: subversion of host cell signaling by Legionella of the Creative Commons Attribution License (CC BY). The use, distribution or type IV secretion system effectors. Can J Microbiol. (2015) 61:617–35. reproduction in other forums is permitted, provided the original author(s) and the doi: 10.1139/cjm-2015-0166 copyright owner(s) are credited and that the original publication in this journal 48. Misch EA. Legionella: virulence factors and host response. Curr Opin Infect is cited, in accordance with accepted academic practice. No use, distribution or Dis. (2016) 29:280–6. doi: 10.1097/QCO.0000000000000268 reproduction is permitted which does not comply with these terms. Frontiers in Immunology | www.frontiersin.org 11 March 2019 | Volume 10 | Article 434