Characterization of the Pichia pastoris protein-O-mannosyltransferase gene family.

The methylotrophic yeast, Pichiapastoris, is an important organism used for the production of therapeutic proteins. However, the presence of fungal-like glycans, either N-linked or O-linked, can elicit an immune response or enable the expressed protein to bind to mannose receptors, thus reducing their efficacy. Previously we have reported the elimination of ß-linked glycans in this organism. In the current report we have focused on reducing the O-linked mannose content of proteins produced in P. pastoris, thereby reducing the potential to bind to mannose receptors. The initial step in the synthesis of O-linked glycans in P. pastoris is the transfer of mannose from dolichol-phosphomannose to a target protein in the yeast secretory pathway by members of the protein-O-mannosyltransferase (PMT) family. In this report we identify and characterize the members of the P. pastoris PMT family. Like Candida albicans, P. pastoris has five PMT genes. Based on sequence homology, these PMTs can be grouped into three sub-families, with both PMT1 and PMT2 sub-families possessing two members each (PMT1 and PMT5, and PMT2 and PMT6, respectively). The remaining sub-family, PMT4, has only one member (PMT4). Through gene knockouts we show that PMT1 and PMT2 each play a significant role in O-glycosylation. Both, by gene knockouts and the use of Pmt inhibitors we were able to significantly reduce not only the degree of O-mannosylation, but also the chain-length of these glycans. Taken together, this reduction of O-glycosylation represents an important step forward in developing the P. pastoris platform as a suitable system for the production of therapeutic glycoproteins.

Functional characterization of full-length TLR3, IRAK-4, and TRAF6 in zebrafish (Danio rerio).

Recently, the zebrafish, Danio rerio, has been recognized as a useful model for infectious disease and immunity. The Toll-like receptor (TLR) family is an evolutionarily conserved component of the innate immune system that responds to specific pathogen-associated molecular patterns (PAMPs) during an infection. This study reports the identification and characterization of a full-length orthologue of mammalian TLR3, and the key TLR pathway signaling molecules IRAK-4 and TRAF6 in the zebrafish. Sequence analysis of zebrafish TLR3 (zfTLR3), IRAK-4 (zfIRAK-4), and TRAF6 (zfTRAF6) revealed conserved domains shared with insect and mammalian genes. Quantitative real-time PCR showed that all three genes are expressed in a variety of adult tissues and during embryonic development. In in situ hybridization, we showed that zfTLR3, zfIRAK-4, and zfTRAF6 are present in distinct regions of the developing brain at 22hpf and that zfTRAF6 was observed in the developing medial neural tube. Overexpression of zfIRAK-4, zfTRAF6, or a mutant zfTLR3 construct was able to stimulate NF-kappaB activation in ZFL cells as measured by a cotransfected NF-kappaB-luciferase reporter plasmid. Messenger RNA expression profiles of each gene in zebrafish embryos and adults were examined by quantitative real-time PCR following infection with snakehead rhabdovirus (SHRV) or Edwardsiella tarda. Following exposure to SHRV, only zfTLR3 and zfTRAF6 mRNA transcripts were upregulated. Interestingly, exposure of fish to E. tarda resulted in an unexpected increase in mRNA expression of zfTLR3, as well as the anticipated upregulation of zfIRAK-4 and zfTRAF6 mRNA transcripts. These results demonstrate that zebrafish possess conserved TLR-signaling pathways, further emphasizing the utility of the zebrafish as a model for vertebrate immunology.

Pathogenesis and inflammatory response to Edwardsiella tarda infection in the zebrafish.

The zebrafish (Danio rerio) is a widely used model for developmental biology, neurobiology, toxicology, and genetic disease. Recently, the zebrafish has been recognized as a valuable model for infectious disease and immunity. In this study the pathogenesis and inflammatory cytokine response of zebrafish to experimental Edwardsiella tarda infection was characterized. In challenge experiments, zebrafish embryos were susceptible to infection by immersion. Adult fish were susceptible to challenge by intraperitoneal (ip) injection but not static immersion unless the epithelial layer was perturbed by scraping prior to exposure. To determine if E. tarda infection induces a typical acute inflammatory response, mRNA expression levels of interleukin-1beta (IL-1beta) and tumor necrosis factor-alpha (TNFalpha) were assessed by quantitative real-time PCR. The expression levels of IL-1beta and TNFalpha were significantly upregulated in infected zebrafish embryos and adults. The methods developed in this study will be particularly valuable for targeted gene disruption studies of host immune components and in zebrafish genetic screens.

Characterization of snakehead rhabdovirus infection in zebrafish (Danio rerio).

The zebrafish, Danio rerio, has become recognized as a valuable model for the study of development, genetics, and toxicology. Recently, the zebrafish has been recognized as a useful model for infectious disease and immunity. In this study, the pathogenesis and antiviral immune response of zebrafish to experimental snakehead rhabdovirus (SHRV) infection was characterized. Zebrafish 24 h postfertilization to 30 days postfertilization were susceptible to infection by immersion in 10(6) 50% tissue culture infective doses (TCID50) of SHRV/ml, and adult zebrafish were susceptible to infection by intraperitoneal (i.p.) injection of 10(5) TCID50 of SHRV/ml. Mortalities exceeded 40% in infected fish, and clinical presentation of infection included petechial hemorrhaging, redness of the abdomen, and erratic swim behavior. Virus reisolation and reverse transcription-PCR analysis of the viral nucleocapsid gene confirmed the presence of SHRV. Histological sections of moribund embryonic and juvenile fish revealed necrosis of the pharyngeal epithelium and liver, in addition to congestion of the swim bladder by cell debris. Histopathology in adult fish injected i.p. was confined to the site of injection. The antiviral response in zebrafish was monitored by quantitative real-time PCR analysis of zebrafish interferon (IFN) and Mx expression. IFN and Mx levels were elevated in zebrafish exposed to SHRV, although expression and intensity differed with age and route of infection. This study is the first to examine the pathogenesis of SHRV infection in zebrafish. Furthermore, this study is the first to describe experimental infection of zebrafish embryos with a viral pathogen, which will be important for future experiments involving targeted gene disruption and forward genetic screens.

Cloning and characterization of an Mx gene and its corresponding promoter from the zebrafish, Danio rerio.

Type I interferons (IFNs) represent a crucial component of the innate immune response to viruses. An important downstream effector of IFN is the Mx gene, which is activated solely through this pathway. Mx proteins are characterized by a tripartite GTP-binding domain, dynamin family signature, and leucine zipper motif. Mx genes are transcribed upon activation of an interferon-stimulated response element (ISRE) located in the Mx promoter region. In this article, we describe the cloning and analysis of an Mx gene and its corresponding promoter from the zebrafish (Danio rerio). The deduced amino acid sequence of zebrafish Mx contains the conserved GTP-binding domain, dynamin family signature, and leucine zipper motif common to Mx proteins, and shows a 50% identity to human MxA and 69% identity both to rainbow trout and to Atlantic salmon. Zebrafish liver cells produced high levels of Mx mRNA in response to induction by the known IFN-inducer polyinosinic-polycytidylic acid (Poly[I:C]). The zebrafish Mx promoter contains two ISREs homologous to those found in the promoter regions of many IFN-inducible genes, and was able to drive transcription of a luciferase reporter gene when induced by either purified zebrafish IFN or Poly[I:C].

Molecular and functional analysis of an interferon gene from the zebrafish, Danio rerio.

The interferon (IFN) family consisting of alpha IFN (IFN-alpha), IFN-beta, IFN-omega, IFN-delta, IFN-kappa, and IFN-tau is a large group of cytokines involved in the innate immune response against various microorganisms. Genes for IFN have been cloned from a variety of mammalian and avian species; however, IFN genes from lower-order vertebrates have not been forthcoming. Here, we report the cloning and characterization of the IFN gene from the zebrafish, Danio rerio. Zebrafish IFN (zfIFN) is 185 amino acids in length, with the first 22 amino acids representing a putative signal peptide. Treatment with the known IFN inducer polyinosinic acid-polycytidylic acid (poly[I]-poly[C]) resulted in an increase in zfIFN mRNA transcripts. zfIFN was also able to activate the IFN-inducible Mx promoter when cotransfected with a plasmid containing the zebrafish Mx promoter upstream of a luciferase reporter gene. To demonstrate antiviral activity, zebrafish cells were transfected with zfIFN and challenged with a fish rhabdovirus. A 36% reduction in plaque number was seen in zfIFN-transfected cells, compared to cells transfected with a control vector. Phylogenetic analysis has shown zfIFN to be approximately equally divergent from avian and mammalian IFN, consistent with its origin from an IFN present in the most recent common ancestor of these divergent lineages. A putative IFN from puffer, Fugu rubripes, was also found when zfIFN was used to search the fugu genome database, demonstrating that zfIFN can be used to find additional fish IFN genes. These results demonstrate that zebrafish can be used as an effective model for studying innate immunity and immune response to infectious disease.

ORGANIZATION OF THE nif GENES OF THE NONHETEROCYSTOUS CYANOBACTERIUM TRICHODESMIUM SP. IMS101.

An approximately 16-kb fragment of the Trichodesmium sp. IMS101 (a nonheterocystous filamentous cyanobacterium) "conventional"nif gene cluster was cloned and sequenced. The gene organization of the Trichodesmium and Anabaena variabilis vegetative (nif 2) nitrogenase gene clusters spanning the region from nif B to nif W are similar except for the absence of two open reading frames (ORF3 and ORF1) in Trichodesmium. The Trichodesmium nif EN genes encode a fused Nif EN polypeptide that does not appear to be processed into individual Nif E and Nif N polypeptides. Fused nif EN genes were previously found in the A. variabilis nif 2 genes, but we have found that fused nif EN genes are widespread in the nonheterocystous cyanobacteria. Although the gene organization of the nonheterocystous filamentous Trichodesmium nif gene cluster is very similar to that of the A. variabilis vegetative nif 2 gene cluster, phylogenetic analysis of nif sequences do not support close relatedness of Trichodesmium and A. variabilis vegetative (nif 2) nitrogenase genes.

Phylogeny of cyanobacterial nifH genes: evolutionary implications and potential applications to natural assemblages.

DNA sequences of a fragment of nifH from diverse cyanobacteria were amplified, cloned and sequenced to determine the evolutionary relationship of nitrogenase within the cyanobacteria as a group, and to provide a basis for the identification of uncultivated strains of cyanobacteria in the environment. Analysis of 30 nitrogenase DNA and deduced amino acid sequences from cyanobacteria representing five major taxonomic subdivisions showed great variation in phylogenetic distances between the sequences. Sequences from heterocystous cyanobacteria formed a coherent cluster, in which branching forms did not form a clade distinct from the non-branching forms. Nitrogenase sequences from the unicellular cyanobacteria Gloeothece and Synechococcus sp. RF-1 formed a cluster, as did sequences from the genera Xenococcus and Myxosarcina. The nifH sequences of filamentous nonheterocystous cyanobacteria were not closely related to each other, forming deep branches with respect to the heterocystous cyanobacterial nifH sequences. The phylogeny of nifH based on amino acid sequences was consistent with taxonomic relationships among the strains; for example, a sequence obtained form a natural assemblage believed to be dominated by 'Lyngbya' clustered with nifH from Lyngbya lagerheimii. Results also indicate that the phylogeny of nifH among the cyanobacteria is largely consistent with the phylogeny of 16S rRNA, and furthermore that the nifH sequence can be used to identify uncultivated strains of nitrogen-fixing cyanobacteria.