Atlantic salmon type I IFN subtypes show differences in antiviral activity and cell-dependent expression: evidence for high IFNb/IFNc-producing cells in fish lymphoid tissues.

This work reveals distinct roles of the two-cysteine-containing type I IFNs, IFNa and IFNd, and the four-cysteine-containing IFNb and IFNc in antiviral immunity of Atlantic salmon. IFNa and IFNc showed similar antiviral activities and ability to induce antiviral genes, IFNb was less active, and IFNd showed no activity. Expression of IFNs was compared by treatment of cells or fish with the dsRNA polyinosinic-polycytidylic acid [poly(I:C)], which induces IFNs via the viral RNA receptors MDA5 and TLR3/TLR22 and with the imidazoquinoline R848, which induces IFNs via TLR7. Poly(I:C) strongly induced IFNa in cell lines, whereas the other IFNs showed little response, indicating that IFNa is the main IFN subtype induced through the RIG-I/MDA5 pathway. In contrast, IFNb and IFNc are the main IFNs induced through the TLR7 pathway because R848 induced high transcript levels of IFNb and IFNc and low transcript levels of IFNa in the head kidney and spleen. IFNd was constitutively expressed in cells and organs but showed no response to poly(I:C) or R848. Fluorescence in situ hybridization studies showed that poly(I:C) induced IFNa and IFNc in a variety of cells in the head kidney, spleen, gills, liver, and heart, whereas R848 induced coexpression of IFNb and IFNc in distinct cells in head kidney and spleen. These cells are likely to be specialized high IFN producers because they were few in numbers despite high IFNb/IFNc transcript levels in the same organs. High IFN expression in response to TLR7 ligation is a feature shared by mammalian plasmacytoid dendritic cells.

Antiviral activity of salmonid gamma interferon against infectious pancreatic necrosis virus and salmonid alphavirus and its dependency on type I interferon.

We investigated the antiviral activity and gene induction properties of interferon gamma (IFN-γ) compared to type I IFN (IFNa1) in Atlantic salmon. IFN-γ protected salmon cells against infectious pancreatic necrosis virus (IPNV)-induced cytopathic effect (CPE), reduced virus titers, and inhibited the synthesis of the viral structural protein VP3. Moreover, IFN-γ showed potent antiviral activity against salmonid alphavirus 3 (SAV3) measured as a reduction in virus nsP1 transcripts. IFN-γ (a type II IFN) had less specific antiviral activity against IPNV than IFNa1, showing a half-maximal effective concentration of 1.6 ng/ml versus 31 pg/ml determined in the CPE reduction assay. Compared to IFNa1, IFN-γ was a more effective inducer of the antiviral protein GBP, several interferon regulatory transcription factors (IRFs), and the chemokine IP-10. The antiviral activity of IFN-γ may also in part be ascribed to upregulation of Mx, ISG15, and viperin. These are typical type I IFN-induced genes in mammals and were also more strongly induced by IFNa1 than by IFN-γ in salmon cells. Fish and mammalian IFN-γ thus show strikingly similar gene induction properties. Interestingly, the antiviral activity of IFN-γ against IPNV and SAV3 and its ability to induce Mx and ISG15 markedly decreased in the presence of neutralizing antiserum against IFNa1. In contrast, antiIFNa1 had no effect on the induction of IRF-1 and IP-10 by IFN-γ. This suggests that the antiviral activity of IFN-γ is partially dependent on IFNa induction. However, because antiIFNa1 could not abolish the IFN-γ-mediated induction of Mx and ISG15 completely, IFN-γ may possibly also induce such genes directly.

Sculpting the Bacterial O-Glycoproteome: Functional Analyses of Orthologous Oligosaccharyltransferases with Diverse Targeting Specificities.

Protein glycosylation systems are widely recognized in bacteria, including members of the genus Neisseria. In most bacterial species, the molecular mechanisms and evolutionary contexts underpinning target protein selection and the glycan repertoire remain poorly understood. Broad-spectrum O-linked protein glycosylation occurs in all human-associated species groups within the genus Neisseria, but knowledge of their individual glycoprotein repertoires is limited. Interestingly, PilE, the pilin subunit of the type IV pilus (Tfp) colonization factor, is glycosylated in Neisseria gonorrhoeae and Neisseria meningitidis but not in the deeply branching species N. elongata subsp. glycolytica. To examine this in more detail, we assessed PilE glycosylation status across the genus and found that PilEs of commensal clade species are not modified by the gonococcal PglO oligosaccharyltransferase. Experiments using PglO oligosaccharyltransferases from across the genus expressed in N. gonorrhoeae showed that although all were capable of broad-spectrum protein glycosylation, those from a deep-branching group of commensals were unable to support resident PilE glycosylation. Further glycoproteomic analyses of these strains using immunoblotting and mass spectrometry revealed other proteins differentially targeted by otherwise remarkably similar oligosaccharyltransferases. Finally, we generated pglO allelic chimeras that begin to localize PglO protein domains associated with unique substrate targeting activities. These findings reveal previously unappreciated differences within the protein glycosylation systems of highly related bacterial species. We propose that the natural diversity manifest in the neisserial protein substrates and oligosaccharyltransferases has significant potential to inform the structure-function relationships operating in these and related bacterial protein glycosylation systems. IMPORTANCE Although general protein glycosylation systems have been well recognized in prokaryotes, the processes governing their distribution, function, and evolution remain poorly understood. Here, we have begun to address these gaps in knowledge by comparative analyses of broad-spectrum O-linked protein glycosylation manifest in species within the genus Neisseria that strictly colonize humans. Using N. gonorrhoeae as a well-defined model organism in conjunction with comparative genomics, intraspecies gene complementation, and glycoprotein phenotyping, we discovered clear differences in both glycosylation susceptibilities and enzymatic targeting activities of otherwise largely conserved proteins. These findings reveal previously unappreciated differences within the protein glycosylation systems of highly related bacterial species. We propose that the natural diversity manifest within Neisseria species has significant potential to elucidate the structure-function relationships operating in these and related systems and to inform novel approaches to applied glycoengineering strategies.

Infectious salmon anemia virus (ISAV) replication is transiently inhibited by Atlantic salmon type I interferon in cell culture.

Infectious salmon anemia virus (ISAV) is a piscine orthomyxovirus, which causes multisystemic disease in farmed Atlantic salmon that may result in large losses. Previous work has suggested that ISAV is able to resist the antiviral state induced in cells by type I interferon (IFN). These studies were, however, mainly based on cytopathic effect (CPE) reduction assays. Here we have investigated the antiviral activity of Atlantic salmon IFNa1, IFNb and IFNc against ISAV using quantitative PCR (qPCR) of segment 6, Western blot analysis of ISAV proteins and viral yield reduction assays, in addition to CPE reduction assays. Antiviral effects of IFNs were tested against the high virulent strain ISAV4 and the low virulent strain ISAV7 both at the optimum growth temperature 15°C and at 20°C. As expected, IFNa1 showed little protection against CPE development in cells after infection with both strains at 15°C. However, the qPCR and Western blot analysis clearly showed strong inhibition of replication of the virus strains by IFNa1 between 24 and 72h after infection. The inhibitory effect declined four to five days post-infection, which explains the low protection against CPE development 7-10 days later. At 20°C, IFNa1 showed strong protection against CPE development, probably due to slower virus growth. IFNc showed similar antiviral activity as IFNa1 against ISAV4 while IFNb showed lower activity. There were observed differences between ISAV4 and ISAV7 both with respect inhibition by IFNa1 and ability to induce the two IFN-inducible antiviral effector proteins, Mx and ISG15, which may be related to differences in virulence properties and/or adaption to growth in cell culture.

An antiserum against Atlantic salmon IFNa1 detects IFN and neutralizes antiviral activity produced by poly I:C stimulated cells.

Type I interferons (IFNs) play a crucial role in innate immune responses against virus infections in vertebrates. Two IFNs (IFNa1 and IFNa2) have previously been cloned from Atlantic salmon. In the present work a polyclonal antiserum, which was generated against salmon IFNa1 was used to study its production in cells by immunoblot detection and neutralization of antiviral activity. The antiserum was first confirmed to detect and neutralize the antiviral activity of recombinant salmon IFNa1 produced in HEK293 cells. The antiserum also detected IFNa1 and neutralized 95-98% of the antiviral activity in supernatants of poly I:C stimulated salmon TO cells. This suggests that IFNa1/IFNa2 are the major IFNs produced by poly I:C stimulated TO cells. The antiserum neutralized most of the IFN activity in poly I:C stimulated head kidney leucocytes from three of five individuals, but in stimulated leucocytes from the other two individuals only 75% of the antiviral activity was neutralized. This shows that although IFNa1/IFNa2 are major IFNs secreted by poly I:C stimulated leucocytes, these cells can also produce additional molecules with IFN-like activity.

Atlantic salmon IPS-1 mediates induction of IFNa1 and activation of NF-kappaB and localizes to mitochondria.

The striking difference in evolution of type I IFN genes of fish and mammals poses the question of whether these genes are induced through similar or different signalling pathways in the two vertebrate groups. Previous work has shown that expression of both Atlantic salmon (Salmo salar) IFNa1 and mammalian IFN-beta genes is dependent on IRF and NF-kappaB elements in their promoters. In mammals, IFN-beta transcription is induced through the RIG-I/MDA5 pathway where the adaptor protein IPS-1 plays a key role in the signal transduction. In this work we show that an Atlantic salmon homologue of IPS-1 (AsIPS-1) mediates activation of the salmon IFNa1 promoter and an NF-kappaB driven promoter. AsIPS-1 shares only 18% identity in amino acid sequence with human IPS-1, but possesses the CARD, proline-rich and transmembrane domains found in mammalian IPS-1. Overexpression of AsIPS-1 resulted in induction of an antiviral state in the cells apparently due to induction of IFN. Deletion of the CARD and transmembrane domains of AsIPS-1 abolished its ability to activate the IFNa1 promoter and the NF-kappaB driven promoter, and thus its ability to induce an antiviral state. AsIPS-1 is located to mitochondria similar to human IPS-1. Taken together, IPS-1 plays a key role in the induction of Atlantic salmon IFNa1, which appears to be the first and major IFN induced in host cells upon recognition of viral dsRNA.

Functional analysis of putative adhesion factors in Lactobacillus acidophilus NCFM.

Lactobacilli are major inhabitants of the normal microflora of the gastrointestinal tract, and some select species have been used extensively as probiotic cultures. One potentially important property of these organisms is their ability to interact with epithelial cells in the intestinal tract, which may promote retention and host-bacterial communication. However, the mechanisms by which they attach to intestinal epithelial cells are unknown. The objective of this study was to investigate cell surface proteins in Lactobacillus acidophilus that may promote attachment to intestinal tissues. Using genome sequence data, predicted open reading frames were searched against known protein and protein motif databases to identify four proteins potentially involved in adhesion to epithelial cells. Homologous recombination was used to construct isogenic mutations in genes encoding a mucin-binding protein, a fibronectin-binding protein, a surface layer protein, and two streptococcal R28 homologs. The abilities of the mutants to adhere to intestinal epithelial cells were then evaluated in vitro. Each strain was screened on Caco-2 cells, which differentiate and express markers characteristic of normal small-intestine cells. A significant decrease in adhesion was observed in the fibronectin-binding protein mutant (76%) and the mucin-binding protein mutant (65%). A surface layer protein mutant also showed reduction in adhesion ability (84%), but the effect of this mutation is likely due to the loss of multiple surface proteins that may be embedded in the S-layer. This study demonstrated that multiple cell surface proteins in L. acidophilus NCFM can individually contribute to the organism's ability to attach to intestinal cells in vitro.