Hitherto-Unnoticed Self-Transmissible Plasmids Widely Distributed among Different Environments in Japan.

Various conjugative plasmids were obtained by exogenous plasmid capture, biparental mating, and/or triparental mating methods from different environmental samples in Japan. Based on phylogenetic analyses of their whole-nucleotide sequences, new IncP/P-1 plasmids that could be classified into novel subgroups were obtained. Mini-replicons of the plasmids were constructed, and each of them was incompatible with at least one of the IncP/P-1 plasmids, although they showed diverse iteron sequences in their oriV regions. There were two large clades of IncP/P-1 plasmids, clade I and II. Plasmids in clade I and II included antibiotic resistance genes. Notably, nucleotide compositions of newly found plasmids exhibited different tendencies compared with those of the previously well-studied IncP/P-1 plasmids. Indeed, the host range of plasmids of clade II was different from that of clade I. Although few PromA plasmids have been reported, the number of plasmids belonging to PromAß, and -γ subgroups detected in this study was close to that of IncP/P-1 plasmids. The host ranges of PromAγ and PromAδ plasmids were broad and transferred to different and distinct classes of Proteobacteria. Interestingly, PromA plasmids and many IncP/P-1 plasmids do not carry any accessory genes. These findings indicate the presence of "hitherto-unnoticed" conjugative plasmids, including IncP/P-1 or PromA derivative ones in nature. These plasmids would have important roles in the exchange of various genes, including antibiotic resistance genes, among different bacteria in nature. IMPORTANCE Plasmids are known to spread among different bacteria. However, which plasmids spread among environmental samples and in which environments they are present is still poorly understood. This study showed that unidentified conjugative plasmids were present in various environments. Different novel IncP/P-1 plasmids were found, whose host ranges were different from those of known plasmids, showing wide diversity of IncP/P-1 plasmids. PromA plasmids, exhibiting a broad host range, were diversified into several subgroups and widely distributed in varied environments. These findings are important for understanding how bacteria naturally exchange their genes, including antibiotic resistance genes, a growing threat to human health worldwide.

Identification and Structure of an MHC Class I-Encoded Protein with the Potential to Present N-Myristoylated 4-mer Peptides to T Cells.

Similar to host proteins, N-myristoylation occurs for viral proteins to dictate their pathological function. However, this lipid-modifying reaction creates a novel class of "lipopeptide" Ags targeted by host CTLs. The primate MHC class I-encoded protein, Mamu-B*098, was previously shown to bind N-myristoylated 5-mer peptides. Nevertheless, T cells exist that recognize even shorter lipopeptides, and much remains to be elucidated concerning the molecular mechanisms of lipopeptide presentation. We, in this study, demonstrate that the MHC class I allele, Mamu-B*05104, binds the N-myristoylated 4-mer peptide (C14-Gly-Gly-Ala-Ile) derived from the viral Nef protein for its presentation to CTLs. A phylogenetic tree analysis indicates that these classical MHC class I alleles are not closely associated; however, the high-resolution x-ray crystallographic analyses indicate that both molecules share lipid-binding structures defined by the exceptionally large, hydrophobic B pocket to accommodate the acylated glycine (G1) as an anchor. The C-terminal isoleucine (I4) of C14-Gly-Gly-Ala-Ile anchors at the F pocket, which is distinct from that of Mamu-B*098 and is virtually identical to that of the peptide-presenting MHC class I molecule, HLA-B51. The two central amino acid residues (G2 and A3) are only exposed externally for recognition by T cells, and the methyl side chain on A3 constitutes a major T cell epitope, underscoring that the epitopic diversity is highly limited for lipopeptides as compared with that for MHC class I-presented long peptides. These structural features suggest that lipopeptide-presenting MHC class I alleles comprise a distinct MHC class I subset that mediates an alternative pathway for CTL activation.

Promoting notification and linkage of HBs antigen and anti-HCV antibody-positive patients through hospital alert system.

BACKGROUND: In Japan, approximately 0.9% and 1% of the whole population are infected with HBV and HCV, respectively. Doctors from departments other than gastroenterology often order viral hepatitis tests before an invasive examination or an operation. However, the notification of positive results to the patients and linkage to care is not appropriately performed. The in-hospital alert system was constructed to promote the notification and referral to gastroenterologists for patients with positive viral hepatitis tests, and its efficacy was evaluated. METHODS: The patients who tested HBsAg and anti-HCV antibody by chemiluminescent enzyme immunoassays and chemiluminescent immunoassays were investigated for whether they were notified of the positive results and if they were referred to gastroenterologists at our hospital. The notification and referral rate was compared before (from January to December 2014) and after the introduction of the alert system (from February to September 2016). RESULTS: HBsAg-positive rate was 1.1% (69/6543) before the introduction of the alert system and 0.8% (41/5403) after it. The notification rate has significantly improved from 46% to 73% (p = 0.0061) and the referral rate has improved from 16% to 27%, while not significant. Positive rate of anti-HCV antibody was 2.1% (139/6481) before the introduction of the alert system and 2.4% (128/5322) after it. The rate of notification and referral has significantly improved from 35% to 62% (p < 0.0001) and from 6% to 23% (p < 0.0001), respectively. CONCLUSIONS: The in-hospital alert system increased the rates of notification and referral of the patients with positive viral hepatitis tests. Enlightenment of doctors other than gastroenterologists on viral hepatitis and cooperation of medical staffs would be helpful to improve the notification and referral rates.

Development of an active tritium sampler for discriminating chemical forms without the use of combustion gases in a fusion test facility.

A new type of active tritium sampler that can discriminate between chemical forms in a fusion test facility without the use of combustion gases was developed. The proposed tritium sampler was operated using water vapour instead of combustion gases. To test the operation and performance of the device when water vapour is used, we evaluated the catalytic oxidation properties, and the evaporation and collection of water vapour under actual sampling conditions. The properties of the added water mass and the operation temperature of catalysts in the proposed sampling system were then determined. Thereafter, we carried out air sampling for tritium monitoring. The levels of tritium concentration measured by the proposed tritium sampling system were similar to the values measured by the conventional sampling system. Our findings show that the proposed tritium sampling system without combustion gases is a good replacement for the conventional tritium sampling system in a fusion test facility.

Crystal structure of the N-myristoylated lipopeptide-bound MHC class I complex.

The covalent conjugation of a 14-carbon saturated fatty acid (myristic acid) to the amino-terminal glycine residue is critical for some viral proteins to function. This protein lipidation modification, termed N-myristoylation, is targeted by host cytotoxic T lymphocytes (CTLs) that specifically recognize N-myristoylated short peptides; however, the molecular mechanisms underlying lipopeptide antigen (Ag) presentation remain elusive. Here we show that a primate major histocompatibility complex (MHC) class I-encoded protein is capable of binding N-myristoylated 5-mer peptides and presenting them to specific CTLs. A high-resolution X-ray crystallographic analysis of the MHC class I:lipopeptide complex reveals an Ag-binding groove that is elaborately constructed to bind N-myristoylated short peptides rather than prototypic 9-mer peptides. The identification of lipopeptide-specific, MHC class I-restricted CTLs indicates that the widely accepted concept of MHC class I-mediated presentation of long peptides to CTLs may need some modifications to incorporate a novel MHC class I function of lipopeptide Ag presentation.

Molecular requirements for T cell recognition of N-myristoylated peptides derived from the simian immunodeficiency virus Nef protein.

We have recently isolated a rhesus macaque cytotoxic T cell line, 2N5.1, that specifically recognizes an N-myristoylated 5-mer peptide (C(14)-Gly-Gly-Ala-Ile-Ser [C14nef5]) derived from the simian immunodeficiency virus (SIV) Nef protein. Such C14nef5-specific T cells expand in the circulation of SIV-infected monkeys, underscoring the capacity of T cells to recognize viral lipopeptides; however, the molecular basis for the lipopeptide antigen presentation remains to be elucidated. Here, functional studies indicated that the putative antigen-presenting molecule for 2N5.1 was likely to have two separate antigen-binding sites, one for interaction with a C(14)-saturated acyl chain and the other for anchorage of the C-terminal serine residue. Mutants with alanine substitutions for the second glycine residue and the fourth isoleucine residue were not recognized by 2N5.1 but interfered with the presentation of C14nef5 to 2N5.1, indicating that these structural analogues retained the ability to interact with the antigen-presenting molecules. In contrast to the highly specific recognition of C14nef5 by 2N5.1, an additional cytotoxic T cell line, SN45, established independently from a C14nef5-stimulated T cell culture, showed superb reactivity to both C14nef5 and an N-myristoylated Nef 4-mer peptide, and therefore, the C-terminal serine residue was dispensable for the recognition of lipopeptides by the SN45 T cells. Furthermore, the mutants with alanine substitutions were indeed recognized by the SN45 T cells. Given that N-myristoylation of the Nef protein occurs in the conserved motifs and is critical for viral pathogenesis, these observations predict that the lipopeptide-specific T cell response is difficult for viruses to avoid by simply introducing amino acid mutations.