Location: Office of The Director2020 Annual Report
Rainbow trout are an important recreational and food fish species in the U.S., and it is thus important to improve disease resistance and improve methods for combatting outbreaks of disease to increase production and profitability of U.S. aquaculture. This research plan will focus on the following three Objectives and their supporting Sub-Objectives: Objective 1: Identify virulence factors in pathogenic flavobacterium species and develop strategies to control disease. • Sub-Objective 1.A: Isolate and characterize F. columnare mutants and identify virulence factors associated with disease in rainbow trout. • Sub-Objective 1.B: Develop vaccines to control columnaris disease. • Sub-Objective 1.C: Develop improved genetic manipulation techniques for F. psychrophilum, which causes bacterial cold-water disease. Objective 2: Characterize salmonid antimicrobial peptides and evaluate their biocidal effects against pathogens. • Sub-Objective 2.A: Identify new antimicrobial peptides by mining the rainbow trout big data sets and characterize their actions and regulation. • Sub-Objective 2.B: Characterize the immunomodulatory actions of new trout AMPs in vitro. • Sub-Objective 2.C: Assess effects of new rainbow trout AMPs on flavobacterial biofilms. Objective 3: Identify rhabdoviral virulence factors and develop strategies to reduce pathogenesis in salmonids. • Sub-Objective 3.A: Identify and characterize potential viral targets and strategies for vaccine development. • Sub-Objective 3.B: Characterize the involvement of stress granule formation and function in pathogen response and the establishment of protective immunity in fish cell-lines.
Objective 1: For this objective, our scientific aim is to develop genetic techniques to characterize F. psychrophilum and F. columnare virulence mechanisms. Using these genetic techniques, genes encoding specific secreted proteins and components of flavobacterial secretion systems will be mutated and the effects of these modifications on bacterial pathogenesis will be evaluated using in vitro and in vivo systems. Data from these approaches will improve understanding of host-pathogen interactions and generate attenuated bacterial strains for vaccine development. Objective 2: For this objective, our scientific aim is to identify and characterize new antimicrobial peptides (AMPs) in rainbow trout, understand their actions against flavobacterial and rhabdoviral pathogens, and ascertain their physiological control to improve health of rainbow trout. Using established in vitro systems with trout cell-lines, biological actions of AMPs will be assessed and ability of AMPs to kill important flavobacterial and rhabdoviral pathogens, and flavobacterial biofilms, will be characterized. Objective 3: For this objective, our scientific aim is to understand the innate immune response and components of virulence in two important rainbow trout rhabdoviruses (IHNV and VHSV). Work will involve sequential characterization of the effects of critical rhabdoviral proteins, and modifications to these proteins, on the stress response and host immunity using trout cell-lines, in vitro. The goal is to characterize components of rhabodoviral virulence and how these components (viral proteins) influence host response as a means to identify possible viral targets for new vaccine candidates. Developing new vaccine candidates based on improved understanding of rainbow trout antiviral immune functions should advance our abilities to combat these pathogens in rainbow trout.
For Objective 1, our aim was to use genetic techniques to characterize mechanisms of Flavobacterium columnare and Flavobacterium psychrophilum virulence, and to identify potential strategies to control bacterial disease in rainbow trout aquaculture. For work on F. columnare, we used proteomic analyses to identify over 50 secreted proteins as potential targets. These proteins were prioritized, and 33 genes encoding secreted proteases, chondroitinases, other enzymes, adhesins, and motility proteins were deleted. The mutants were examined for virulence in zebrafish and mutations were identified that resulted in decreased virulence in zebrafish. Experiments are ongoing to identify the most important secreted proteins involved in columnaris disease. Additional gene deletion mutants will be constructed and examined for virulence in zebrafish and rainbow trout. Attenuated mutants will be examined to determine if they function as protective vaccines. For work on F. psychrophilum, we completed research for and published the paper “The type IX secretion system is required for virulence of the fish pathogen Flavobacterium psychrophilum” (Appl. Environ. Microbiol. 2020. In Press). In the coming year we will attempt to optimize gene transfer into F. psychrophilum strain CSF-259-93, the preferred model strain for studies of cold water disease in rainbow trout. This will allow genetic experiments similar to those performed above for F. columnare, to identify F. psychrophilum virulence factors, and to construct strains attenuated for virulence as potential vaccines. If we fail to improve gene transfer into strain CSF-259-93, then we will perform these studies with strain THCO2-90. For Objective 2 , our aim is to characterize salmonid antimicrobial peptides (AMPs) and evaluate their biocidal effects against pathogens. We have identified six new trout AMPs. Synthetic peptide cores were commercially synthesized and tested for antimicrobial activity using Flavobacterial columnare cultures to determine inhibitory concentrations, but results varied substantially. Similarly, efforts were undertaken with ARS collaborators to ascertain whether these peptide cores (and other fish AMPs) could disrupt F. columnare biofilms and, again, results were not consistent. Results suggest that: 1) assays require further refinement, and/or 2) peptide cores need to be re-designed. To address this, we are targeting a different region of these peptides for synthesis. Importantly, one area of interest has been on how AMPs affect teleost mucosal surfaces. Here, the teleost gill is known to be an important mucosal barrier that is also involved in innate/adaptive immunity. Consequently, the gill can modulate the host protective immune response to pathogen challenge via regulating cellular and humoral immune factors. We have successfully generated an in vitro system to culture the rainbow trout RTgill-W1 cells as an in vitro model system to study how the host mucosal system responds to hormones, AMPs and pathogen mimetics. As an initial approach, we have exposed cultures of RTgill-W1 cells to moth cecropin B, and the pathogen mimetic lipopolysaccharide (LPS), to determine if the AMP shows any immunomodulatory actions on the gill cells and, therefore, protects the cells against LPS insult. To examine this, we analyze the expression levels of genes of interest (GOI) by RT-qPCR. Using this approach, we observed consistent modulatory activities in regulating the expression levels of immune relevant genes such as pro-inflammatory cytokines, immunoglobulin, docking proteins, cytokine receptors, inflammatory and antimicrobial enzymes, acute phase proteins, and transcriptional factors respond in RTgill-W1 cells. After exposing RTgill-W1 cells to various concentrations (10uM and 30uM) of cecropin B, the dose responses were evaluated, and the higher concentration resulted in a more significant effect on some GOIs. In addition, the time-course analyses indicate significant perturbations of GOIs at the acute phase, with compensation being observed at 24h post-exposure. Results will be repeated and verified. For Objective 3, our aim is to identify rhabdoviral virulence factors and develop strategies to reduce pathogenesis in salmonids. Previous studies have implicated infectious hematopoietic necrosis virus (IHNV) matrix protein (IHNV-M) in downregulating host cell transcription through unknown mechanisms, which allows IHNV to evade the host immune response. We measured host cell transcription by transfecting fish cells with IHNV-M to observe the effects of the wildtype gene compared to the control vector and IHNV-M deletion mutants. Our control vector was used as a baseline for host cell transcription. By comparing our luciferase reporter levels in transfected cells, we identified deletions that attenuated the inhibitory effects of IHNV-M on host cell transcription. This finding shows a crucial role of a particular amino acid sequence on IHNV-M’s inhibitory effect on host cell transcription. Our focus then shifted towards double mutants that included this mutation in order to identify the ideal deletion mutant on which to focus our vaccine efforts. We have also produced a series of deletion mutants in the NV gene to understand the role that NV has in the regulation of host immune responses. All NV deletion mutants showed an attenuation of wild-type NV modulatory activities. However, we identified specific amino acid deletions that showed the most radical activities. In some cases, these mutants suppressed, rather than augmented, luciferase expression as compared to the empty vector control. Concurrent work shows that the viral hemorrhagic septicemia virus (VHSV) IVb NV protein is involved in viral pathogenesis by mediating a crucial immunological signaling pathway to enable viral protein synthesis and inhibit interferon signaling to evade the host cell immune and stress responses. We have observed that formation of canonical stress granule (SG) is conserved in multiple fish cell types (rainbow trout, fathead minnow, bluegill fry). The propensity of the cells to form SG differs, with RTG2 and BF2 forming SG in response to heat shock, but not oxidative stress, while RTgill form SG in response to oxidative stress but not heat shock, and EPC form no SG in response to oxidative stress. The cause of this host-specific difference remains to be determined. VHSV Ia and IVb infection induces the formation of SG-like structures in their respective host lines, but we cannot confirm (at this time) whether they are bona fide avSG or if they are viral replication complexes formed during infection. The kinetics of SG-like formation differs between all cell lines and with differing time-courses and differences could be due to cell types rather than differences in replication kinetics between viral strains. We determined the role of NV in SG induction using WT and recombinant viruses lacking the entire NV coding sequences or with a mutation in the ATG codons preventing NV protein production while not impacting RNA synthesis. Results implicate the role of IVb NV in stress granule formation during VHSV IVb infection. Removal of functional NV during VHSV IVb infection resulted in a significant increase in infected cells forming SG-like structures compared to rWT VHSV infection, suggesting an important role of NV in regulating the formation of VHSV induced SGs. Treatment with a protein inhibitor, prior to infection with NV deficient VHSV, resulted in a decrease in infected cells forming SGs, suggesting the importance of PERK activation as the primary inducer of SG formation during VHSV IVb infection.