Infection-Derived Enterococcus faecalis Strains Are Enriched in esp, a Gene Encoding a Novel Surface Protein

We report the identification of a new cell wall-associated protein of Enterococcus faecalis. Studies on the distribution of the gene encoding this novel surface protein, Esp, reveal a significant (P < 0.001) enrichment in infection-derived E. faecalis isolates. Interestingly, the esp gene was not identified in any of 34 clinical E. faecium isolates or in 4 other less pathogenic enterococcal species tested. Analysis of the structural gene among various E. faecalis isolates reveals the existence of alternate forms of expression of the Esp protein. The deduced primary structure of the Esp protein from strain MMH594, inferred to be 1,873 amino acids (aa) with a predicted mass of ~202 kDa, reveals a core region consisting of repeat units that make up 50% of the protein. Esp bears global organizational similarity to the Rib and C alpha proteins of group B streptococci. Identity among Esp, Rib, and C alpha proteins is strikingly localized to a stretch of 13 aa within repeats of similar length. The high degree of conservation of this 13-residue sequence suggests that it plays an important role in the natural selection for this trait among infection-derived E. faecalis and group B streptococcal isolates.     

ChlorellaVirus PBCV-1 Encodes Functional Glutamine: Fructose-6-Phosphate Amidotransferase and UDP-Glucose Dehydrogenase Enzymes

DNA sequence analysis of the 330-kbChlorellavirus PBCV-1 genome unexpectedly revealed several open reading frames which encode proteins that are homologous to sugar-manipulating enzymes including glutamine:fructose-6-phosphate amidotransferase (GFAT), UDP-glucose dehydrogenase (UDP-GlcDH), and hyaluronan synthase (HAS). PBCV-1 genes encoding the putative GFAT and UDP-GlcDH enzymes were expressed inEscherichia coli,and both recombinant proteins have the predicted enzyme activity in cell free extracts. These same two genes are transcribed early in PBCV-1 infection, and both genes are widespread among theChlorellaviruses. The products of the reactions catalyzed by these two enzymes are precursors in the biosynthesis of hyaluronan polysaccharide. Previous experiments established that, like the GFAT and UDP-GlcDH genes, the HAS gene is transcribed early and encodes a functional enzyme (DeAngelis, P. L., Jing. W., Graves, M. V., Burbank, D. E., and Van Etten, J. L. (1997)Science278, 1800–1803). Interestingly, the predicted amino-acid sequences of the PBCV-1 GFAT and UDP-GlcDH enzymes are more similar to bacterial GFAT and UDP-GlcDH enzymes than to their eukaryotic counterparts. In contrast, the amino-acid sequence of the PBCV-1 HAS enzyme more closely resembles eukaryotic enzymes.     

Structural and functional characteristics of a dominant‐negative isoform of porcine MHC class II transactivator

The MHC class II transactivator, CIITA, is critical for MHC class II gene expression in all species studied to date. We isolated an interferon (IFN)‐γ‐inducible isoform of porcine CIITA (pCIITA′) encoding a protein of 566 amino acids (aa) with significant homology to human CIITA (hCIITA). Analysis indicated that pCIITA′ lacks the entire GTP‐binding domain that is important for nuclear translocation and activation of target genes by hCIITA. In pCIITA′ this region is replaced by a 14‐aa motif with homology to several signalling peptide sequences. Expression of pCIITA′ in porcine (ST‐IOWA) and human (HeLa) cell lines resulted in suppression of IFN‐γ‐stimulated MHC class II gene expression, at the protein and mRNA levels. We also identified two IFN‐γ‐inducible variants of hCIITA, hCIITAlo and hCIITA′ from Hela cells, both exhibiting dominant‐negative suppression of MHC class II gene expression. Interestingly, hCIITA′ encodes a predicted protein of 546 aa with a strikingly similar organization to pCIITA′ including the 14‐aa GTP‐binding domain‐replacement motif in which 10 out of 14 amino acids are identical to the pig sequence. Expression of hCIITA′ and hCIITAlo sequences in Hela cells suppressed IFN‐γ‐induced MHC class II gene expression. hCIITAlo, a predicted 303‐aa protein with deleted GTP‐binding and carboxy‐terminal domain, displayed a more subtle suppression of IFN‐γ‐induced MHC class II expression. These in vitro data indicate that there may be a role in vivo for isoforms of CIITA that can suppress full‐length CIITA‐mediated MHC class II gene expression. Both humans and now, potentially, pigs are candidate donors for organ and tissue allografts and xenografts, respectively. Regulation of MHC class II gene expression by manipulation of CIITA isoform expression in humans and pigs may provide a useful strategy for attenuation of T‐cell‐mediated cellular rejection.     

Limited Inter- and Intra-patient Sequence Diversity of the Genetic Lineage A human metapneumovirus fusion gene

Human metapneumovirus (hMPV) is associated with respiratory tract illness especially in young children. Two hMPV genetic lineages, A and B, and four sublineages A1, A2 and B1, B2 have been defined. Infection with hMPV occurs through membrane fusion mediated by the hMPV fusion (F) protein. In this study, the inter- and intra-patient genetic diversity of the lineage A hMPV F gene was investigated. Ten isolates were collected from 10 hMPV infected children. Viral RNA was isolated and amplified, and approximately 10 clones from each isolate were sequenced. Altogether 108 clones were successfully sequenced. The average interpatient sequence diversity was 1.68% and 1.64% at nucleotide and amino acid levels, respectively. The samples were divisible into two groups on the basis of intrapatient sequence diversity. In group 1 (4 children) the intra-patient sequence diversity was low (nt: 0.26–0.39%, aa: 0.51–0.94%) whereas group 2 (6 children) had a higher intra-patient sequence diversity (nt: 0.85–1.98%, aa: 1.08–2.22%). Phylogenetic analyses showed that the group 1 children harboured sublineage Al only, but interestingly group 2 children harboured both sublineages Al and A2, indicating they had been infected with at least two viruses. Several independent viruses contained premature stop codons in exactly identical positions resulting in truncated fusion proteins. Possibly this is a mechanism for immune system evasion. The F protein is a major antigenic determinant, and the limited sequence diversity observed lay emphasis on the hMPV F gene as a putative target for future vaccine development.     

A recombinant protein disulfide isomerase homologue from Ancylostoma caninum

The objective of this study was to characterize a recombinant antigen of Ancylostoma caninum that had been identified by immunoscreening with selected antisera as described elsewhere. In vitro expression of clone Ac38-1 produced a protein with an apparent molecular mass of approximately 38 kDa, which reacted in Western blots with the antiserum from rabbits experimentally infected with L3 and also with affinity-purified antibodies against hydrophilic proteins of the cephalic glands obtained from the antiserum against the intestine, cephalic glands, and cervical glands of adult worms. It was recognized not by antisera from dogs percutaneously infected with 1,000 L3 of A. caninum but by antiserum from dogs infected with 100,000 L3 of A. caninum. DNA sequencing of clone Ac38-1 showed a cDNA fragment with a coding region of 1,014 bp. Comparison of clone Ac38-1 with the Genbank DNA data base revealed 78% identity with a 244-bp segment of the cm5b5 clone of the free-living nematode Caenorhabditis elegans coding for a protein disulfide isomerase gene. The deduced amino acid sequence of clone Ac38-1 showed 82% identity with a 334-amino-acid (aa) segment of the protein disulfide isomerase of C. elegans and 73% identity with a 334-aa segment of the protein disulfide isomerase aa sequence of Onchocerca volvulus. Received: 12 May 1998 / Accepted: 27 May 1998     

Identification of major antigenic proteins ofPasteurella piscicida

Two different antigenic protein-coding clones (PPA1 and PPA2) were isolated using anti-Pasteurella piscicidarabbit serum from a genomic DNA library ofP. piscicidastrain KP9038. The PPA1 and PPA2 expressed 7 kDa and 45 kDa proteins inEscherichia coli, respectively, and the molecular sizes of these expressed proteins are the same as these of the major antigenic proteins ofP. piscicida. PPA1 encodes a protein of 83 amino acids residues, which is similar to the bacterial lipoprotein. Comparison of the predicted amino acid sequence of the PPA1-encoded 7 kDa protein ofP. piscicidawith previously reported bacterial lipoprotein sequence data revealed that it shares about 40% amino acid sequence identity. PPA2 has two large open reading frame (ORFs). The larger ORF (encoding 452 amino acid residues) encodes a homolog of DegQ protease, and the smaller ORF (371 amino acid residues) encodes a homolog of DegS protease. The antibodies reacted with the larger ORF-encoded 45 kDa DegQ homolog protein. The DegQ and DegS homolog proteins contain an export signal and a serine protease active site. The structural features of the PPA2-coding locus are similar to those of the loci inE. colifor thedegQanddegSserine protease genes. A sequence in the 3′ non-coding region ofVibrio hollisaethermostable hemolysin gene that is highly homologous with a similar located sequence in thePseudomonas putidap-cresol methylhydroxylase gene is also found in the 3′ non-coding region of thedegShomolog gene of the PPA2.     

Isolation and chromosomal assignment of a human gene encoding protein inhibitor of activated STAT3 (PIAS3)

Mouse PIAS3 (protein inhibitor of activated STAT3) is a specific inhibitor of STAT3 that downregulates its signaling pathway. Here we report the isolation and chromosome mapping of the human PIAS3 gene. Human PIAS3 cDNA encoded a predicted protein of 619 aa which has 83% overall amino acid identity to the mouse counterpart. Based on polymerase chain reaction assisted analysis of a human/rodent mono-chromosomal hybrid cell panel and a radiation hybrid mapping panel, the human PIAS3 gene was mapped to the chromosome 1q21 region. Mapping of a crucial gene in modulating the STAT3 signaling pathway may provide new clues to the understanding of malignancies or genetic disorders caused by this chromosome region. Received: January 4, 1999 / Accepted: February 13, 1999     

Repetitive sequence-mediated rearragements in Chlorella ellipsoidea chloroplast DNA: completion of nucleotide sequence of the large inverted repeat

Summary A 3454 base pair (bp) sequence of the large inverted repeat (IR) of chloroplast DNA (cpDNA) from the unicellular green alga Chlorella ellipsoidea has been determined. The sequence includes: (1) the boundaries between the IR and the large single copy (LSC) and the small single copy (SSC) regions, (2) the gene for psbA and (3) an approximately 1.0 kbp region between psbA and the rRNA genes which contains a variety of short dispersed repeats. The total size of the Chlorella IR was determined to be 15243 bp. The junction between the IR and the small single copy region is located close to the putative promoter of the rRNA operon (906 bp upstream of the-35 sequence on each IR). The junction between the IR and the large single copy region is also just upstream of the putative psbA promoter, 218 bp upstream from the ATG initiation codon. A few sets of unique sequences were found repeatedly around both junctions. Some of the sequences flanking the IR-LSC junction suggest a unidirectional and serial expansion of the IR within the genome. The psbA gene is located close to the LSC-side junction and codes for a protein of 352 amino acid residues. A highly conserved C-terminal Gly is absent. Unlike the psbA of Chlamydomonas species, which contains 2–4 large introns, the gene of Chlorella has no introns. The overall gene organization of the Chlorella IR is very different from that of higher plants, but a similar gene cluster of rrn-psbA is also found in the IR of Chlamydomonas species and in a single copy region of some chlorophyll a/c-containing algae, indicating a common evolutionary lineage of these cpDNAs. The origin and evolution of the IR structure are discussed in the light of these observations.     

Hamster polyomavirus-derived virus-like particles are able to transfer in vitro encapsidated plasmid DNA to mammalian cells

The authentic major capsid protein 1 (VP1) of hamster polyomavirus (HaPyV) consists of 384 amino acid (aa) residues (42 kDa). Expression from an additional in-frame initiation codon located upstream from the authentic VP1 open reading frame (at position −4) might result in the synthesis of a 388 aa-long, amino-terminally extended VP1 (aa −4 to aa 384; VP1^ext). In a plasmid-mediated Drosophila Schneider (S2) cell expression system, both VP1 derivatives as well as a VP1^ext variant with an amino acid exchange of the authentic Met1Gly (VP1^ext-M1) were expressed to a similar high level. Although all three proteins were detected in nuclear as well as cytoplasmic fractions, formation of virus-like particles (VLPs) was observed exclusively in the nucleus as confirmed by negative staining electron microscopy. The use of a tryptophan promoter-driven Escherichia coli expression system resulted in the efficient synthesis of VP1 and VP1^ext and formation of VLPs. In addition, establishment of an in vitro disassembly/reassembly system allowed the encapsidation of plasmid DNA into VLPs. Encapsidated DNA was found to be protected against the action of DNase I. Mammalian COS-7 and CHO cells were transfected with HaPyV-VP1-VLPs carrying a plasmid encoding enhanced green fluorescent protein (eGFP). In both cell lines eGFP expression was detected indicating successful transfer of the plasmid into the cells, though at a still low level. Cesium chloride gradient centrifugation allowed the separation of VLPs with encapsidated DNA from “empty” VLPs, which might be useful for further optimization of transfection. Therefore, heterologously expressed HaPyV-VP1 may represent a promising alternative carrier for foreign DNA in gene transfer applications.     

A putative DNA helicase and novel oligoribonuclease in the Diachasmimorpha longicaudata entomopoxvirus (DlEPV)

Summary.  Diachasmimorpha longicaudata entomopoxvirus (DlEPV) is a symbiotic entomopoxvirus (EPV) of the parasitic wasp Diachasmimorpha longicaudata. It has a double-stranded DNA genome of 250–300 kb and is >60% A-T rich. We describe ten ORFs (RI-35-1 to -10) contained within a 5.64 kb clone, RI-35, from a DlEPV EcoRI genomic library. Our goal was to identify unique motifs and compare them with others in the database, particularly those of poxviruses. Two ORFs (RI-35-1 and RI-35-7, respectively) encode putative proteins (113 aa and 219 aa) that are probably involved in regulating gene expression based on their predicted nuclear localization and the presence of SPxx motifs, leucine-zipper like sequences (113 aa), and a basic domain (219 aa). The largest gene (RI-35-3) is under the control of an intermediate/late promoter and is presumed to encode a cytoplasmic 480 aa DNA-dependent DNA helicase with conserved motifs that are characteristic of DExH helicases. Amino acid analysis of the DNA helicase sequence showed that DlEPV is close to but distinct from the Genus B EPVs. The DlEPV helicase is also distinct from that of the Diadromus pulchellus ascovirus 1a from the D. pulchellus parasitic wasp, with less than 10% amino acid identity. DlEPV encodes a 207 aa oligoribonuclease (RI-35-8) of the DEDDh family of exoribonucleases. The second largest ORF (RI-35-9) is under the control of a poxvirus early promoter and encodes a protein of 329 aa that is likely DlEPV-specific. Three ORFs (RI-35-4, -5, and -6) overlap (in the anti-sense strand) with ORFs encoding putatively important virus replication proteins (which were also under the control of intermediate promoters) and are presumably not expressed in DlEPV. These results support earlier reports that DlEPV is a member of the sub-family Entomopoxvirinae, most likely in Group C, and is the first symbiotic EPV described to date from a parasitic wasp. Received August 2, 2002; accepted December 18, 2002 Published online May 5, 2003     

Knock down of chitosanase expression in phytopathogenic fungus Fusarium solani and its effect on pathogenicity

Chitosanases are lytic enzymes involved in the degradation of chitosan, a component of fungal cell walls. The phytopathogenic fungus Fusarium solani produces an extracellular chitosanase, CSN1, the role of which in the physiology and virulence of the fungus remains to be expounded. Here, we studied the expression of the CSN1 gene through gene silencing and examined its effect on fungal pathogenicity. A vector construct encoding a hairpin RNA (hpRNA) of CSN1 was constructed and introduced into the F. solani 0114 strain. The results revealed that majority of the transformants exhibited a significant reduction in chitosanase activity compared with the wild-type strain. Further, transformants with silenced CSN1 exhibited no change in mycelial growth and spore formation. However, pea pod and seedling bioassays indicated that transformants with silenced CSN1 were more virulent compared with the wild-type strain, and in sharp contrast to strains in which overexpression of the CSN1 gene resulted in virulence reduction. Although the mechanism remains unclear, our findings did suggest that F. solani chitosanase has a negative effect on fungal pathogenicity.     

High-level Expression of the C-terminal Hydrophobic Region of HCV E2 Protein Ectodomain in E. coli

High-level expression of hepatitis C virus (HCV) E2 protein fragments encompassing its C-terminal hydrophobic region (downstream of aa 661) in Escherichia coli has been proven difficult. The extreme hydrophobicity of this region has been suspected to be detrimental to the host. In this work, we found that the C-terminal region (downstream of aa 565) of E2 ectodomain interfered with full-length expression of E2 fragments in E. coli. Nonetheless, when the central region (aa 484–622) of E2 was deleted, full-length protein was efficiently produced. C-terminal region aa 567–700 could not be efficiently expressed individually or as mouse dihydrofolate reductase (DHFR) fusion protein. However, a mutant that emerged in the cloning process was able to express full-length DHFR fusion protein. Sequencing analysis reveals the mutation to be a short frame-shift around the fusion junction, altering aa 568–571 of E2. C-terminal region of E2 ectodomain (aa 567–730) carrying this mutation was successfully expressed as hexa-histidine-tagged protein to a high level. The protein was highly insoluble and was purified under denaturing conditions. The purified protein displayed HCV E2-specific antigenicity in Western blot and specific rabbit antiserum was raised against it. These results demonstrate that hydrophobicity of the C-terminal region of E2 ectodomain is not harmful to E. coli host and has no dominative adverse effect on its bacterial expression. Other nucleotide and/or amino acid sequence properties seem to play a more important role. This finding opens up new possibilities for the development of novel bacterially-derived E2 proteins for research and clinical applications.     

Hibiscus virus S is a new subgroup II tobamovirus: evidence from its unique coat protein and movement protein sequences

Summary.  The coat protein (CP) and movement protein (MP) sequences of a new tobamovirus infecting Hibiscus rosa-sinensis L were determined. The CP gene encodes 163 amino acid (aa) residues and with a theoretical molecular weight of 18.19 kDa. The MP gene encodes 282 amino acids and its theoretical molecular weight is 30.36 kDa. The nucleotide (nt) and aa sequences of the CP were 46.88 % to 51.63 % and 45.34 % to 57.06 % identical to other tobamoviruses, respectively. The nt and aa sequence identities of MP ranged from 38.81 % to 43.90 % and 30.85 % to 37.88 %, respectively. The predicted virion origin of assembly (OAS) was located in the CP gene. Phylogenetic trees generated based on the nt and aa sequences of both CP and MP genes indicate that this new virus clusters with members of subgroup II of tobamoviruses. Although this hibiscus virus shared a high nt and aa sequence identity with Sunn-hemp mosaic virus (SHMV), Western analysis showed that it is serologically unrelated to SHMV. We propose the name Hibiscus virus S (HVS) for this Singapore isolate. This is the first report on partial nt sequence of a tobamovirus that infects hibiscus. Received November 22, 2001; accepted February 28, 2002 Published online June 21, 2002     

Characterization and gene expression analysis of the two main Th17 cytokines (IL-17A/F and IL-22) in turbot,Scophthalmus maximus

This report describes the cloning, characterization and gene expression pattern of two Th17 cytokines, interleukin (IL)-17A/F and -22, in turbot Scophthalmus maximus. The turbot IL-17A/F cDNA contains a 516 bp open reading frame encoding a deduced IL-17A/F protein of 171 amino acid (aa) residues, containing a predicted signal peptide of 31 aa. Turbot IL-22 had a 564 bp ORF coding for a 187 aa protein with a 33 aa signal peptide. The turbot IL-22 protein contained a typical IL-10 family signature. Both cytokines had highest expression levels in the intestine followed by head kidney and gills. Stimulation with the Gram negative bacterium Aeromonas salmonicida was able to modulate IL-17A/F and IL-22 expression in head kidney, spleen and liver but not the intestine. PMA and PHA were also able to induce the expression of both cytokines, suggesting that, as expected, T-cells are likely the main producers of these molecules in turbot as in mammals.     

Nucleotide sequence analysis of an infectious laryngotracheitis virus gene corresponding to the US3 of HSV-1 and a unique gene encoding a 67 kDa protein

Summary The DNA sequence of 4005 nucleotides from the Kpnl O and part of Kpnl K fragments in the short unique region of infectious laryngotracheitis virus (ILTV) was determined. The sequence contained two complete and one partial open reading frames (ORFs). The partial ORF was open at the 5 end of the sequence and represented the NH₂-terminal 118 amino acids (aa) of a polypeptide. Its partial predicted protein product exhibited significant homology to the US2 gene product of HSV-1 (herpes simplex virus type 1) and its homologs in other herpesviruses. ORF 2 is 471 aa long and could encode a protein of 53.8 kDa which shared aa homology with the protein kinases encoded by HSV-1 US3 and its gene homologs. Analysis of the ORF 2 aa sequence revealed domains characteristic of protein-serine/threonine (S/T) kinases of cellular and viral origin. The ORF 3 encoded a predicted protein of 601 aa (Mr 67.5 kDa) which exhibited limited homology (18% overall identity) with the UL47 protein (major tegument protein) of HSV-1. Northern (RNA) blot hybridization and metabolic inhibitors were used to characterize the ILTV protein kinase and the 67K mRNAs. The data revealed that protein kinase is a gamma-1 gene encoding a 1.6 kb mRNa, while the 67K ORF is a gamma-2 gene encoding a 2 kb mRNA.