Identification of Linked Legionella pneumophila Genes Essential for Intracellular Growth and Evasion of the Endocytic Pathway

Legionella pneumophila replicates within a specialized phagosome in cultured cells, a function necessary for its pathogenicity. The replicative phagosome lacks membrane marker proteins, such as the glycoprotein LAMP-1, that are indicators of the normal endocytic pathway. We describe the isolation of several Legionella genes essential for intracellular growth and evasion of the endocytic pathway, using a genetic and cell biological approach. We screened 4,960 ethyl methanesulfonate-mutagenized colonies for defects in intracellular growth and trafficking to the replicative phagosome. Six mutant strains of L. pneumophila that had severe intracellular growth defects in mouse bone marrow-derived macrophages were identified. All six mutants were found in phagosomes that colocalized with LAMP-1, indicating defects in intracellular trafficking. The growth defects of two of these strains were complemented by molecular clones from a bank constructed from a wild-type L. pneumophila strain. The inserts from these clones are located in a region of the chromosome contiguous with several other genes essential for intracellular growth. Three mutants could be complemented by single open reading frames placed in trans, one mutant by a gene termed dotH and two additional mutants by a gene termed dotO. A deletion mutation was created in a third gene, dotI, which is located directly upstream of dotH. The ΔdotI strain was also defective for intracellular growth in macrophages, and this defect was complemented by a single open reading frame in trans. Based on sequence analysis and structural predictions, possible roles of dotH, dotI, and dotO in intracellular growth are discussed.     

Identification of Genes Encoding Amino Acid Permeases by Inactivation of Selected ORFs from the Synechocystis Genomic Sequence

Genes encoding elements of four amino acid permeases were identified by insertional inactivation of ORFs from the genomic sequence of the cyanobacterium Synechocystis sp. strain PCC 6803 whose putative products are homologous to amino acid permease proteins from other bacteria. A transport system for neutral amino acids and histidine and a transport system for basic amino acids and glutamine were identified as ABC-type transporters, whereas Na(+)-dependent transport of glutamate was found to be mediated by at least two systems, the secondary permease GltS and a TRAP-type transporter. Except for GltS, substrate specificities of the identified permeases do not match those of previously characterized systems homologous to these permeases.     

Application of Subtype-Specific Monoclonal Antibodies for Rapid Detection and Identification of Influenza A and B Viruses

We established a rapid method for the identification of influenza A and B virus strains: the peroxidase-antiperoxidase (PAP) staining method with two subtype-specific murine monoclonal antibodies, C179 (H1 and H2 specific) and F49 (H3 specific), and an anti-influenza B virus rabbit polyclonal serum. The types and subtypes of 160 strains were examined, and 158 strains were identified to be the same by the hemagglutination-inhibition (HI) test and the PAP method. In contrast to the results by the HI test, two strains were revealed to be a mixture of two subtypes (H1 and H3) by the PAP method, which was confirmed by plaque cloning. We further analyzed clinical specimens by the PAP method by directly inoculating specimens into Madin-Darby canine kidney cells in microplates. After 40 h of incubation, the types and subtypes of viruses in 52 of 152 specimens were clearly identified. Since the reactivities of the two monoclonal antibodies are not influenced by the antigenic drift of influenza virus, the newly developed method should be applicable not only for rapid diagnosis but also for the epidemiological study of influenza.     

Identification of Two Novel Genes Encoding 97- to 99-Kilodalton Outer Membrane Proteins of Chlamydia pneumoniae

Two genes encoding 97- to 99-kDa Chlamydia pneumoniae VR1310 outer membrane proteins (Omp4 and Omp5) with mutual similarity were cloned and sequenced. The proteins were shown to be constituents of the C. pneumoniae outer membrane complex, and the deduced amino acid sequences were similar to those of putative outer membrane proteins encoded by the Chlamydia psittaci and Chlamydia trachomatis gene families. By use of a monospecific polyclonal antibody against purified recombinant Omp4, it was shown that without heating, the protein migrated at 65 to 75 kDa in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Immunoelectron microscopy showed that epitopes of Omp4 were exposed on the surface of C. pneumoniae elementary bodies, reticulate bodies, and outer membrane complex. Proteins encoded by the C. pneumoniae gene family seem to be dominant antigens in experimentally infected mice.     

The Glycoprotein B Genes of Marek's Disease Virus Serotypes 2 and 3: Identification and Expression by Recombinant Fowlpox Viruses

The nucleotide sequences of the glycoprotein B (gB) genes of Marek's disease virus (MDV) serotypes 2 and 3 were determined (gB-2 and gB-3, respectively). The genomic locations of these genes coincide with that of the gB gene of serotype 1 MDV (gB-1). Alignment with gB-1 (Ross et al., 1989, J. Gen. Virol. 70, 1789-1804) revealed predicted amino acid identities of 83 and 82% for gB-2 and gB-3, respectively. Excluding the predicted N-terminal signal sequences, 8 of 9 potential N-linked glycosylation sites and all 10 cysteine residues in gB-1 are conserved in both gB-2 and gB-3. In addition, the putative proteolytic cleavage sites for processing of precursors (gp100s) to gp60s and gp49s are conserved among the three gB homologs. Fowlpox virus (FPV) recombinants expressing either the gB-2 or the gB-3 gene were constructed. We detected expression of authentic gB-2 and gB-3 complexes in cells infected with these FPV recombinants. Digestion of immunoprecipitated gB-1 and gB-3 with endoglycosidases revealed that both gp60s are modified by the additions of O -glycans and complex carbohydrates after cleavage of gp100s, while gp100s and gp49s contain only high-mannose carbohydrates. We confirm that the size differences between gB-1 and gB-3 complexes are due to different carbohydrate modifications.     

Identification of Receptors for Animal Viruses

Present knowledge of cell surface receptors for animal viruses is reviewed. The methods used for enumeration and identification of receptors are critically examined with respect to particular advantages and disadvantages. Specific controls and alternative interpretations are suggested in connection with the reactions of lectins which block viral attachment to cells. Currently available information for each group of animal viruses is summarized in order to define the extent to which the corresponding receptors have been identified. It is concluded that the full range of virus-receptor interactions has not yet been explored even for those viruses of which there is the most detailed knowledge. For some groups, moreover, the receptor is totally uncharacterized. Six areas in which future investigative effort might be productive are identified, including the isolation of membrane components and the immunochemical definition of viral receptors.     

The Neutralization Epitope of Lactate Dehydrogenase-Elevating Virus Is Located on the Short Ectodomain of the Primary Envelope Glycoprotein

We have measured by indirect ELISA the binding of neutralizing and non-neutralizing anti-lactate dehydrogenase-elevating virus (LDV) polyclonal and monoclonal antibodies to synthetic peptides representing unmodified hydrophilic segments of LDV proteins. Using this method a single neutralization epitope has been shown to be located in the very short (about 30 amino acid long) ectodomain of the primary envelope glycoprotein, VP-3P, encoded by ORF 5. Although the neutralization epitopes of neuropathogenic and non-neuropathogenic LDVs differ slightly in amino acid sequences, the neutralizing antibodies bind strongly to the epitopes of both groups of viruses. However, the neutralization epitopes of neuropathogenic and non-neuropathogenic LDVs are associated with different numbers of polylactosaminoglycan chains (1 and 3, respectively) which may affect the binding of neutralizing antibodies to the virions of these LDVs. The ELISA using synthetic peptides containing the neutralization epitope provides a novel, rapid, sensitive, and inexpensive method for quantitating LDV neutralizing antibodies in infected mice.     

Clinical Accuracy of a PLEX-ID Flu Device for Simultaneous Detection and Identification of Influenza Viruses A and B

Respiratory tract infections caused by influenza A and B viruses often present nonspecifically, and a rapid, high-throughput laboratory technique that can identify influenza viruses is clinically and epidemiologically desirable. The PLEX-ID Flu assay (Abbott Molecular Inc., Des Plaines, IL) incorporates multilocus PCR and electrospray ionization-mass spectrometry to detect and differentiate influenza A 2009 H1N1 (H1N1-p), seasonal H1N1 (H1N1-s), influenza A H3N2, and influenza B viruses in nasopharyngeal swab (NPS) specimens. The clinical performance characteristics of the PLEX-ID Flu assay in symptomatic patients were determined in this multicenter trial. A total of 2,617 prospectively and retrospectively collected NPS specimens from patients with influenza-like illness between February 2008 and 28 May 2010 were eligible for inclusion in the study. Each specimen was tested in parallel by the PLEX-ID Flu assay and by the Prodesse ProFLU+ assay (Prodesse Inc., Madison, WI), to detect influenza A and B viruses. Specimens testing positive for influenza A virus by ProFLU+ were subtyped as H1N1-p, H1N1-s, or H3N2 by using the ProFAST+ assay (Gen-Probe Prodesse Inc.). The reproducibility of the PLEX-ID Flu assay ranged from 98.3 to 100.0%, as determined by testing a nine-specimen panel at three clinical sites on each of 5 days. Positive percent agreements (PPAs) and negative percent agreements (NPAs) of the PLEX-ID Flu assay were 94.5% and 99.0% for influenza A virus and 96.0% and 99.9% for influenza B virus, respectively. For the influenza A virus subtyping characterization, the PLEX-ID Flu assay had PPAs and NPAs of 98.3% and 97.5% for H1N1-p, 88.6% and 100.0% for H1N1-s, and 98.0% and 99.9% for H3N2, respectively. The overall agreements between the PLEX-ID and Prodesse ProFLU+/ProFAST+ assays were 97.1 to 100.0%. Bidirectional Sanger sequencing analysis revealed that 87.5% of 96 discrepant results between the PLEX-ID Flu and ProFLU+/ProFAST+ assays were found upon influenza A virus detection and H1N1-p subtyping. The PLEX-ID Flu assay demonstrated a high level of accuracy for the simultaneous detection and identification of influenza A and B viruses in patient specimens, providing a new laboratory tool for the rapid diagnosis and management of influenza A and B virus infections.     

Peptidergic Regulation of Expression of Genes Encoding Antioxidant and Anti-Inflammatory Proteins

Geroprotective peptide T-34 regulates the expression of mRNA for various genes. The development of gastric ulcer is associated with morphological and molecular changes resulting from modulation of the synthesis of antioxidant and anti-inflammatory proteins. Peptide T-34 normalizes the synthesis of these proteins by regulating the expression of the corresponding genes.     

Clinical Relevance and Diversity of Two Homologous Genes Encoding Glycosyltransferases in Helicobacter pylori

Helicobacter pylori is known to be a major cause of peptic ulceration. The jhp0562 gene, encoding a glycosyltransferase involved in the synthesis of the lipopolysaccharide, was associated with peptic ulcer disease (PUD) in children. The β-(1,3)-galactosyltransferase [β-(1,3)GalT] gene (jhp0563), involved in Lewis (Le) antigen expression, is highly similar to jhp0562. The clinical significance and diversity of both genes were examined by PCR and sequencing of clinical strains (n = 117) isolated from children with PUD (n = 57) and nonulcer dyspepsia (NUD; n = 60). The prevalence of the jhp0562 gene was significantly higher in strains with a more-virulent profile (strains positive for the cag pathogenicity island [PAI], vacA sl allele, babA, homB, phase-variable gene oipA “on” [i.e., functional], and hopQ I allele). The distribution of genotypes according to clinical outcome showed that the presence of jhp0562 represented one of the greatest risks for the development of PUD. Moreover, the triple-positive genotype for the cag PAI, jhp0562, and homB provided the best discriminatory model for distinguishing PUD and NUD outcomes in children. Sequence and in vitro expression analyses of jhp0562 showed the presence of a complete open reading frame, while the β-(1,3)GalT gene was shown to be a phase-variable gene. The regular presence of jhp0562 in strains with a truncated β-(1,3)GalT gene suggests that jhp0562 may also be implicated in the regulation of Le antigen expression. Overall, the results of this study suggest that the jhp0562 gene is of great clinical relevance, being a useful comarker for severe H. pylori-related disease and contributing to host adaptation.Helicobacter pylori colonizes the human gastric mucus layer and is the major causative agent of chronic active gastritis, peptic ulcer disease (PUD), gastric carcinoma, and gastric mucosa-associated lymphoid tissue (MALT) lymphoma (8, 16, 19, 28). Progression to diseases of greater severity occurs only in certain infected individuals and seems to depend on a number of factors, including host genetic susceptibility, environmental factors, and differences in H. pylori strain virulence.Comparison of H. pylori strains isolated from patients with symptomatic gastric disease, such as gastric cancer or PUD, with strains isolated from patients with no such history is useful in identifying bacterial genetic markers associated with gastric disease. Recently, the jhp0562 gene was isolated from a duodenal ulcer (DU)-associated strain, by means of PCR-based subtractive hybridization, in which the genomic content of two H. pylori strains isolated from two young children, one presenting with DU and the other with recurrent abdominal pain and gastritis, was analyzed (27). The jhp0562 gene was found to be highly associated with PUD in children (15 with PUD and 30 with nonulcer dyspepsia [NUD] with gastritis) (27). This gene encodes a glycosyltransferase involved in the synthesis of the chemical structure of the lipopolysaccharide (LPS). It is present in the genome of the J99 strain but absent in the 26695 strain and located immediately upstream of the β-(1,3)-galactosyltransferase [β-(1,3)GalT] gene (jhp0563 or HP0619 according to the annotation of these strains), which in turn encodes a β-(1,3)GalT involved in type I Lewis (Le) antigen (Le^a and Le^b) synthesis of the LPS (3, 31). jhp0562 and the β-(1,3)GalT gene are highly similar (>80%), especially at their 5′ and 3′ ends (2, 9).The present study aimed to evaluate (i) the clinical relevance of the jhp0562 gene by examining its association with H. pylori virulence-associated genes and with clinical outcome as well as (ii) the jhp0562 gene polymorphisms among clinical isolates, including in vitro gene expression. Due to the high-level similarity between jhp0562 and the β-(1,3)GalT gene, the genetic polymorphism of the latter was also studied. H. pylori strains from a pediatric population were chosen, since children are by definition recently colonized, therefore constituting a privileged natural model with less interference of environmental and age-related factors.     

Identification and Molecular Analysis of the Gene Encoding Rickettsia typhi Hemolysin

Rickettsia typhi, the causative agent of murine typhus, grows directly within the host cell cytoplasm, accumulating a large number of progeny, and eventually lyses the cells. Typhus group rickettsiae (R. typhi and R. prowazekii) adhere to and lyse human and sheep erythrocytes. However, the molecular mechanism underlying erythrocyte lysis by R. typhi has not been defined. Here we describe the cloning and nucleotide sequence analysis of the gene (tlyC) encoding a hemolysin from R. typhi. DNA sequence analysis of R. typhi tlyC revealed an open reading frame of 912 bp, which encodes a protein of 304 amino acids with a predicted molecular mass of 38 kDa. To associate the R. typhi tlyC gene product with hemolytic activity, we performed complementation studies with hemolysin-negative Proteus mirabilis WPM111 (a HpmA(-) mutant of BA6163) transformed with R. typhi tlyC or R. typhi GFPuv-tlyC constructs. We demonstrated that the cloned tlyC gene conferred a hemolytic phenotype on an otherwise nonhemolytic mutant of P. mirabilis. The availability of the cloned R. typhi tlyC will permit further characterization and definition of its role in rickettsial virulence.     

HIV-Neutralizing Antibodies: Epitope Identification and Significance for Future Vaccine

Recent studies of the TCR α and β chains expressed by normal human IELs suggest that these intestinal lymphocytes are directed at a limited set of antigens, presumably on intestinal epithelial cells in view of their anatomic location. The direct sequence analysis of these cells has indicated that they are oligoclonal and cannot, therefore, be responding to the complex mixture of antigens which are present in the lumen. The abundant expression of the CD8 accessory molecule by the IELs, in addition, indicates that these putative intestinal epithelial cell antigens are presented by MHC class I or I-like molecules. The expression of CD8 also suggests that these cells function biologically in part as cytolytic T lymphocytes which is consistent with a variety of functional studies. Taken together with their expression of the CD45RO isoform, these phenotypic and functional observations suggest that iIELs are cytolytic, memory cells which are responsive to an extremely limited number of antigens bound to major histocompatibility complex (MHC) class I or class I-like molecules. Several non-polymorphic MHC class I-like molecules such as Qa, the thymus leukemia antigen (TL) and CD1 in the mouse and CD1 in humans represent important candidate ligands for these oligoclonal iIELs. TL and CD1 are expressed specifically by murine intestinal epithelial cells. In humans, CDld is constitutively expressed by intestinal epithelial cells. In addition, we have isolated iIEL T cell clones which specifically recognize members of the CD1 gene family when expressed on a transfected B cell line that lacks HLA-A and B and have shown that the proliferation of peripheral blood T cells to intestinal epithelial cells is CDld dependent. Thus, the evidence to date strongly implicate the nonpolymorphic, class lb molecules as novel restriction elements for unique populations of lymphocytes within the intestinal epithelium.     

小儿急性感染性胃肠炎的病毒病原研究

对全国１７省市收集的１７７４例腹泻患儿，９８例非腹泻儿和１３５例正常新生儿，以及３６例腹泻与３７例无腹泻成人粪标本，用不同方法研究其病毒病原。行病毒分离的２１６份标本中，肠道病毒检出率为６．９％，已鉴定的病毒为ＥＣＨＯ病毒。     

Identification of a Promiscuous T-Cell Epitope in Mycobacterium tuberculosis Mce Proteins

The characterization of Mycobacterium tuberculosis antigens inducing CD4(+) T-cell responses could critically contribute to the development of subunit vaccines for M. tuberculosis. Here we performed computational analysis by using T-cell epitope prediction software (known as TEPITOPE) to predict promiscuous HLA-DR ligands in the products of the mce genes of M. tuberculosis. The analysis of the proliferative responses of CD4(+) T cells from patients with pulmonary tuberculosis to selected peptides displaying promiscuous binding to HLA-DR in vitro led us to the identification of a peptide that induced proliferation of CD4(+) cells from 50% of the tested subjects. This study demonstrates that a systematic computational approach can be used to identify T-cell epitopes in proteins expressed by an intracellular pathogen.     

双表位多肽法区分胰腺癌与免疫性胰腺炎

目的 将免疫性胰腺炎1-7肽段(SKDERRF)与幽门螺旋杆菌纤溶酶原结合蛋白肽段(AKEERRY)通过连接臂制成双表位多肽,接着偶联载体蛋白,以之作为抗原包被,优化并建立酶免体系,探索是否可用于快速鉴别胰腺癌与免疫性胰腺炎.方法 募集免疫性胰腺炎患者、胰腺癌患者和健康人各24例,分离病人血清,制备双表位试剂盒进行检测.结果 以OD值0.269为阳性判定标准,在免疫性胰腺炎患者中,阳性患者17例,阳性率为71％;在胰腺癌患者中,阳性患者0例;在匹配的健康人对照组中同样未检测出阳性.结论 研究中设计采用的双表位多肽所对应的自身抗体,可以在大部分免疫性胰腺炎患者中检出,而胰腺癌患者与健康人中未显示阳性反应.经过进一步优化完善,该方法可望对快速鉴别免疫性胰腺炎和胰腺癌具有一定的辅助临床参考价值.     

Identification of the Cilium Binding Epitope of the Mycoplasma hyopneumoniae P97 Adhesin

Mycoplasma hyopneumoniae colonizes the swine respiratory tract at the level of ciliated cells by attaching specifically to the cilium membrane. This interaction involves an adhesin called P97; the cilium binding activity of this protein was localized to the carboxy terminus, which included two repeat regions, R1 and R2 (T. Hsu, S. Artiushin, and F. C. Minion, J. Bacteriol. 179:1317–1323, 1997). To further delineate the molecular mechanisms of M. hyopneumoniae interactions with ciliated epithelium, we used a bank of transposon inserts in the cloned P97 gene to identify the site for cilium binding by testing the truncated gene products in an in vitro microtiter plate adherence assay. These studies showed that the cilium binding site was located in the AAKPV(E) repeat sequence of P97, referred to as the R1 repeat. For functional binding, at least seven AAKPV(E) repeats were required. The adherence-blocking monoclonal antibody F1B6 also recognized this region but required fewer AAKPV(E) repeats for recognition. We then constructed R1 region-lacZ gene fusions and used the resulting R1 repeat–β-galactosidase fusion proteins in an in vitro assay to confirm the role of R1 in cilium binding. A comparison of the R1 regions of M. hyopneumoniae strains displaying variation in cilium adherence failed to identify changes that could account for the differences in adherence shown by the strains. Thus, we concluded that other proteins, in addition to P97, must be involved in cilium adherence, possibly in combination with P97.     

Functional Analysis of vif Genes Derived from Various Primate Immunodeficiency Viruses

Replication property in cells of human and simian immunodeficiency viruses (HIVs and SIVs) lacking intact vif gene was evaluated. Of 10 vif mutants constructed in vitro of the major four HIV/SIV groups, only those derived from HIV-1 and HIV-2/SIVmac displayed replication defect. The cell lines non-permissive for the vif mutants of HIV-1 and SIVmac were found to be different. To determine whether Vif is exchangeable between HIV-1 and SIVmac, chimeric virus clones with respect to the vif gene were constructed and virus replication in the cells non-permissive for the vif mutant viruses was monitored. Productive infection in these cells of chimeric viruses clearly indicated that Vif is functionally exchangeable, and that Vifs of different virus origin act through a similar mechanism. This revised version was published online in July 2006 with corrections to the Cover Date.