Piscirickettsia salmonis shedding and tissue burden, and hematological responses during cohabitation infections in chum Oncorhynchus keta, pink O. gorbuscha and Atlantic salmon Salmo salar.

BACKGROUND: Salmonid rickettsial septicemia is an emergent and geographically widespread disease of marine-farmed salmonids caused by infection with the water-borne bacterium Piscirickettsia salmonis. Very little is known about the route, timing, or magnitude of bacterial shedding from infected fish. METHODOLOGY/PRINCIPAL FINDINGS: A cohabitation challenge model was used to assess shedding from chum Oncorhynchus keta, pink O. gorbuscha and Atlantic salmon Salmo salar. Infections in donor fish were established by intraperitoneal injection of P. salmonis. Naïve recipients were cohabitated with donor fish after which cumulative percent morbidity and mortality (CMM) was monitored, and bacterial burdens in kidney and in tank water were measured by qPCR. All donor fish died with mean days-to-death (MDD) among species ranging from 17.5 to 23.9. Among recipients, CMM ranged from 42.7% to 77.8% and MDD ranged from 49.7 to 56.4. In each trial, two peaks of bacterial DNA concentrations in tank water closely aligned with the MDD values of donor and recipient fish. Bacterial tissue burden and shedding rate, and plasma physiological parameters were obtained from individual donors and recipients. Statistically significant positive correlations between the shedding rate and P. salmonis kidney burden were measured in donor pink and in donor and recipient chum salmon, but not in donor or recipient Atlantic salmon. In Atlantic salmon, there was a negative correlation between kidney bacterial burden and hematocrit, plasma Ca++ and Mg++ values, whereas in infected chum salmon the correlation was positive for Na+ and Cl- and negative for glucose. CONCLUSIONS: A dependency of bacterial shedding on species-specific patterns of pathogenesis was suggested. The coincidence of bacterial shedding with mortality will inform pathogen transmission models.

Transcriptional analysis of metabolic and virulence genes associated with biofilm formation in Piscirickettsia salmonis strains.

Piscirickettsia salmonis is a facultative intracellular bacterium that generates piscirickettsiosis affecting salmonids in Chile. The bacterium has the adaptability to survive in the marine environment under multiple stressful conditions. In this sense, this work focused on the analysis of a gene battery associated with biofilm formation under different culture conditions and on the adaptability of this biofilm to different media. The results indicated that the strains LF-89, IBM-034 and IBM-040 were strong biofilm producers, evidencing adaptability to the media by increasing the amount of biofilm through successive growths. Transcript levels of six genes described in various bacteria and P. salmonis, considered to have metabolic functions, and playing a relevant role in biofilm formation, were analyzed to evaluate bacterial functionality in the biofilm. The genes mazE-mazF, implicated in biofilm and stress, were markedly overexpressed in the biofilm condition in the three strains. For its part, gene gltA, an indicator of metabolic activity and related to virulence inhibition in Salmonella typhimurium, also seems to restrain the pathogenesis process in P. salmonis by inhibiting the expression of the virulence-associated genes liso and tcf. Finally, the expression of the glnA gene suggests the use of glutamine as an essential element for the growth of the biofilm.

Cellular stress responses of Eleginops maclovinus fish injected with Piscirickettsia salmonis and submitted to thermal stress.

Fluctuations in ambient temperature along with the presence of pathogenic microorganisms can induce important cellular changes that alter the homeostasis of ectothermic fish. The aim of this study was to evaluate how sudden or gradual changes in environmental temperature together with the administration of Piscirickettsia salmonis modulate the transcription of genes involved in cellular stress response in the liver of Eleginops maclovinus. Fish were subjected to the following experimental conditions in duplicate: C- 12 °C: Injection only with culture medium, C+ 12 °C: Injection with P. salmonis, AM 18 °C: Injection only with culture medium under acclimation at 18 °C, AB 18 °C: Injection with P. salmonis under acclimation at 18 °C, SM 18 °C: Injection only with culture medium and thermal shock at 18 °C and SB 18 °C: Injection with P. salmonis and thermal shock at 18 °C and sampling at 4-, 8-, 12-, 16- and 20-day post injection (dpi). The genes implied in the heat shock response (HSP70, HSC70, HSP90, and GRP78), apoptosis pathway (BAX and SMAC/Diablo), ubiquitination (E2, E3, ubiquitin, and CHIP), and 26 proteasome complex (PSMB7, PSMC1, and PSMA2) showed expression profiles dependent on time and type of injection applied. All the genes greatly increased their expression levels at day 16 and showed moderate increases at day 20, except for PSMA2 which showed a higher increase between 4- and 12-day post challenges. Our results suggest that the changes observed at the final days of the experiment are due to temperature more than P. salmonis.

Transcriptomic Changes of Piscirickettsia salmonis During Intracellular Growth in a Salmon Macrophage-Like Cell Line.

Piscirickettsia salmonis is the causative agent of Piscirickettsiosis, a systemic infection of salmonid fish species. P. salmonis infects and survives in its host cell, a process that correlates with the expression of virulence factors including components of the type IVB secretion system. To gain further insights into the cellular and molecular mechanism behind the adaptive response of P. salmonis during host infection, we established an in vitro model of infection using the SHK-1 cell line from Atlantic salmon head kidney. The results indicated that in comparison to uninfected SHK-1 cells, infection significantly decreased cell viability after 10 days along with a significant increment of P. salmonis genome equivalents. At that time, the intracellular bacteria were localized within a spacious cytoplasmic vacuole. By using a whole-genome microarray of P. salmonis LF-89, the transcriptome of this bacterium was examined during intracellular growth in the SHK-1 cell line and exponential growth in broth. Transcriptome analysis revealed a global shutdown of translation during P. salmonis intracellular growth and suggested an induction of the stringent response. Accordingly, key genes of the stringent response pathway were up-regulated during intracellular growth as well as at stationary phase bacteria, suggesting a role of the stringent response on bacterial virulence. Our results also reinforce the participation of the Dot/Icm type IVB secretion system during P. salmonis infection and reveals many unexplored genes with potential roles in the adaptation to intracellular growth. Finally, we proposed that intracellular P. salmonis alternates between a replicative phase and a stationary phase in which the stringent response is activated.

In vitro genomic and proteomic evidence of a type IV pili-like structure in the fish pathogen Piscirickettsia salmonis.

Piscirickettsia salmonis is an intracellular γ-proteobacteria and the etiological agent of piscirickettsiosis, which causes massive economic losses in the Chilean salmon industry. The type IV pili (T4P) play an important role in adherence to host cell surfaces and bacterial pathogenicity. T4P contains a variable number of components, as predicted in P. salmonis genomes. However, no studies have determined if P. salmonis possesses T4P. The aims of this investigation were to identify T4P components in the P. salmonis type strain LF-89T, evaluate respective transcript expressions, and analyze the main putative T4P proteins using bioinformatics and proteomic approaches. Two main clusters of P. salmonis T4P genes were found. Expression of the pilA gene was upregulated at 4 h post-infection (hpi), while pilQ was upregulated 4 days post-infection. At 16 hpi, pilB and pilD were strongly upregulated. The PilA amino acid sequence analysis showed a conserved N-terminal domain and sequence motifs critical for T4P biosynthesis. MudPIT analysis revealed PilA in the P. salmonis LF-89T proteome, and TEM showed pili-like filamentous structures on the P. salmonis surface. These results strongly suggest the presence of a T4P-like structure in P. salmonis.

Selection and validation of reliable housekeeping genes to evaluate Piscirickettsia salmonis gene expression.

Piscirickettsia salmonis is a highly aggressive facultative intracellular bacterium that challenges the sustainability of Chilean salmon production. Due to the limited knowledge of its biology, there is a need to identify key molecular markers that could help define the pathogenic potential of this bacterium. We think a model system should be implemented that efficiently evaluates the expression of putative bacterial markers by using validated, stable, and highly specific housekeeping genes to properly select target genes, which could lead to identifying those responsible for infection and disease induction in naturally infected fish. Here, we selected a set of validated reference or housekeeping genes for RT-qPCR expression analyses of P. salmonis under different growth and stress conditions, including an in vitro infection kinetic. After a thorough screening, we selected sdhA as the most reliable housekeeping gene able to represent stable and highly specific host reference genes for RT-qPCR-driven P. salmonis analysis.

The Proteome of Biologically Active Membrane Vesicles from Piscirickettsia salmonis LF-89 Type Strain Identifies Plasmid-Encoded Putative Toxins.

Piscirickettsia salmonis is the predominant bacterial pathogen affecting the Chilean salmonid industry. This bacterium is the etiological agent of piscirickettsiosis, a significant fish disease. Membrane vesicles (MVs) released by P. salmonis deliver several virulence factors to host cells. To improve on existing knowledge for the pathogenicity-associated functions of P. salmonis MVs, we studied the proteome of purified MVs from the P. salmonis LF-89 type strain using multidimensional protein identification technology. Initially, the cytotoxicity of different MV concentration purified from P. salmonis LF-89 was confirmed in an in vivo adult zebrafish infection model. The cumulative mortality of zebrafish injected with MVs showed a dose-dependent pattern. Analyses identified 452 proteins of different subcellular origins; most of them were associated with the cytoplasmic compartment and were mainly related to key functions for pathogen survival. Interestingly, previously unidentified putative virulence-related proteins were identified in P. salmonis MVs, such as outer membrane porin F and hemolysin. Additionally, five amino acid sequences corresponding to the Bordetella pertussis toxin subunit 1 and two amino acid sequences corresponding to the heat-labile enterotoxin alpha chain of Escherichia coli were located in the P. salmonis MV proteome. Curiously, these putative toxins were located in a plasmid region of P. salmonis LF-89. Based on the identified proteins, we propose that the protein composition of P. salmonis LF-89 MVs could reflect total protein characteristics of this P. salmonis type strain.

Membrane vesicles from Piscirickettsia salmonis induce protective immunity and reduce development of salmonid rickettsial septicemia in an adult zebrafish model.

Infections caused by the facultative intracellular bacterial pathogen Piscirickettsia salmonis remains an unsolved problem for the aquaculture as no efficient treatments have been developed. As a result, substantial amounts of antibiotic have been used to limit salmonid rickettsial septicemia (SRS) disease outbreaks. The antibiotic usage has not reduced the occurrence, but lead to an increase in resistant strains, underlining the need for new treatment strategies. P. salmonis produce membrane vesicles (MVs); small spherical structures know to contain a variety of bacterial components, including proteins, lipopolysaccharides (LPS), DNA and RNA. MVs mimics' in many aspects their mother cell, and has been reported as alternative vaccine candidates. Here, MVs from P. salmonis was isolated and evaluated as a vaccine candidate against SRS in an adult zebrafish infection model. When zebrafish was immunized with MVs they were protected from subsequent challenge with a lethal dose of P. salmonis. Histological analysis showed a reduced bacterial load upon challenge in the MV immunized group, and the mRNA expression levels of several immune related genes altered, including mpeg1.1, tnfα, il1b, il10 and il6. The MVs induced the secretion of IgM upon immunization, indicating an immunogenic effect of the vesicles. Taken together, the data demonstrate a vaccine potential of MVs against P. salmonis.

Transcriptome Analysis of the Intracellular Facultative Pathogen Piscirickettsia salmonis: Expression of Putative Groups of Genes Associated with Virulence and Iron Metabolism.

The intracellular facultative bacteria Piscirickettsia salmonis is one of the most important pathogens of the Chilean aquaculture. However, there is a lack of information regarding the whole genomic transcriptional response according to different extracellular environments. We used next generation sequencing (NGS) of RNA (RNA-seq) to study the whole transcriptome of an isolate of P. salmonis (FAVET-INBIOGEN) using a cell line culture and a modified cell-free liquid medium, with or without iron supplementation. This was done in order to obtain information about the factors there are involved in virulence and iron acquisition. First, the isolate was grown in the Sf21 cell line; then, the bacteria were cultured into a cell-free liquid medium supplemented or not with iron. We identified in the transcriptome, genes associated with type IV secretion systems, genes related to flagellar structure assembly, several proteases and sigma factors, and genes related to the development of drug resistance. Additionally, we identified for the first time several iron-metabolism associated genes including at least two iron uptake pathways (ferrous iron and ferric iron uptake) that are actually expressed in the different conditions analyzed. We further describe putative genes that are related with the use and storage of iron in the bacteria, which have not been previously described. Several sets of genes related to virulence were expressed in both the cell line and cell-free culture media (for example those related to flagellar structure; such as basal body, MS-ring, C-ring, proximal and distal rod, and filament), which may play roles in other basic processes rather than been restricted to virulence.

Comparative Analysis of Membrane Vesicles from Three Piscirickettsia salmonis Isolates Reveals Differences in Vesicle Characteristics.

Membrane vesicles (MVs) are spherical particles naturally released from the membrane of Gram-negative bacteria. Bacterial MV production is associated with a range of phenotypes including biofilm formation, horizontal gene transfer, toxin delivery, modulation of host immune responses and virulence. This study reports comparative profiling of MVs from bacterial strains isolated from three widely disperse geographical areas. Mass spectrometry identified 119, 159 and 142 proteins in MVs from three different strains of Piscirickettsia salmonis isolated from salmonids in Chile (LF-89), Norway (NVI 5692) and Canada (NVI 5892), respectively. MV comparison revealed several strain-specific differences related to higher virulence capability for LF-89 MVs, both in vivo and in vitro, and stronger similarities between the NVI 5692 and NVI 5892 MV proteome. The MVs were similar in size and appearance as analyzed by electron microscopy and dynamic light scattering. The MVs from all three strains were internalized by both commercial and primary immune cell cultures, which suggest a potential role of the MVs in the bacterium's utilization of leukocytes. When MVs were injected into an adult zebrafish infection model, an upregulation of several pro-inflammatory genes were observed in spleen and kidney, indicating a modulating effect on the immune system. The present study is the first comparative analysis of P. salmonis derived MVs, highlighting strain-specific vesicle characteristics. The results further illustrate that the MV proteome from one bacterial strain is not representative of all bacterial strains within one species.

A functional ferric uptake regulator (Fur) protein in the fish pathogen Piscirickettsia salmonis.

Piscirickettsia salmonis, a Gram-negative fastidious facultative intracellular pathogen, is the causative agent of the salmonid rickettsial septicemia (SRS). The P. salmonis iron acquisition mechanisms and its molecular regulation are unknown. Iron is an essential element for bacterial pathogenesis. Typically, genes that encode for the iron acquisition machinery are regulated by the ferric uptake regulator (Fur) protein. P. salmonis fur sequence database reveals a diversity of fur genes without functional verification. Due to the fastidious nature of this bacterium, we evaluated the functionality of P. salmonis fur in the Salmonella Δfur heterologous system. Although P. salmonis fur gene strongly differed from the common Fur sequences, it restored the regulatory mechanisms of iron acquisition in Salmonella. We concluded that P. salmonis LF-89 has a conserved functional Fur protein, which reinforces the importance of iron during fish infection. [Int Microbiol 2016; 49-55].

Characterization and pathogenic role of outer membrane vesicles produced by the fish pathogen Piscirickettsia salmonis under in vitro conditions.

Piscirickettsia salmonis is one of the major fish pathogens affecting Chilean aquaculture. This Gram-negative bacterium is highly infectious and is the etiological agent of Piscirickettsiosis. Little is currently known about how the virulence factors expressed by P. salmonis are delivered to host cells. However, it is known that several Gram-negative microorganisms constitutively release outer membrane vesicles (OMVs), which have been implicated in the delivery of virulence factors to host cells. In this study, OMVs production by P. salmonis was observed during infection in CHSE-214 cells and during normal growth in liquid media. The OMVs were spherical vesicles ranging in size between 25 and 145 nm. SDS-PAGE analysis demonstrated that the protein profile of the OMVs was similar to the outer membrane protein profile of P. salmonis. Importantly, the bacterial chaperonin Hsp60 was found in the OMVs of P. salmonis by Western-blot and LC-MS/MS analyses. Finally, in vitro infection assays showed that purified OMVs generated a cytopathic effect on CHSE-214 cells, suggesting a role in pathogenesis. Therefore, OMVs might be an important vehicle for delivering effector molecules to host cells during P. salmonis infection.

In sílico identification and characterization of putative Dot/Icm secreted virulence effectors in the fish pathogen Piscirickettsia salmonis.

Piscirickettsia salmonis seriously affects the Chilean salmon industry. The bacterium is phylogenetically related to Legionella pneumophila and Coxiella burnetii, sharing a Dot/Icm secretion system with them. Although it is well documented that L. pneumophila and C. burnetii secrete different virulence effectors via this Dot/Icm system in order to attenuate host cell responses, to date there have been no reported virulence effectors secreted by the Dot/Icm system of P. salmonis. Using several annotations of P. salmonis genome, here we report an in silico analyses of 4 putative Dot/Icm effectors. Three of them contain ankyrin repeat domains and the typical conserved 3D structures of this protein family. The fourth one is highly similar to one of the Dot/Icm-dependent effectors of L. pneumophila. Additionally, all the potential P. salmonis effectors contain a classical Dot/Icm secretion signal in their C-terminus, consisting of: an E-Block, a hydrophobic residue in -3 or -4 and an electronegative charge. Finally, qPCR analysis demonstrated that these proteins are overexpressed early in infection, perhaps contributing to the generation of a replicative vacuole, a key step in the neutralizing strategy proposed for the Dot/Icm system. In summary, this report identifies four Dot/Icm-dependent effectors in P. salmonis.

Evidence of the presence of a functional Dot/Icm type IV-B secretion system in the fish bacterial pathogen Piscirickettsia salmonis.

Piscirickettsia salmonis is a fish bacterial pathogen that has severely challenged the sustainability of the Chilean salmon industry since its appearance in 1989. As this Gram-negative bacterium has been poorly characterized, relevant aspects of its life cycle, virulence and pathogenesis must be identified in order to properly design prophylactic procedures. This report provides evidence of the functional presence in P. salmonis of four genes homologous to those described for Dot/Icm Type IV Secretion Systems. The Dot/Icm System, the major virulence mechanism of phylogenetically related pathogens Legionella pneumophila and Coxiella burnetii, is responsible for their intracellular survival and multiplication, conditions that may also apply to P. salmonis. Our results demonstrate that the four P. salmonis dot/icm homologues (dotB, dotA, icmK and icmE) are expressed both during in vitro tissue culture cells infection and growing in cell-free media, suggestive of their putative constitutive expression. Additionally, as it happens in other referential bacterial systems, temporal acidification of cell-free media results in over expression of all four P. salmonis genes, a well-known strategy by which SSTIV-containing bacteria inhibit phagosome-lysosome fusion to survive. These findings are very important to understand the virulence mechanisms of P. salmonis in order to design new prophylactic alternatives to control the disease.