Role of Campylobacter jejuni flagella as colonization factors for three-day-old chicks: analysis with flagellar mutants.

Campylobacter jejuni, an important cause of human gastrointestinal infection, is a major food-borne pathogen in the United States and worldwide. Since poultry becomes colonized and/or contaminated during the early stages of production and is a major food-borne source for this organism, we studied the role of C. jejuni flagella on the ability of the bacterium to colonize the chicken gastrointestinal tract. Three-day-old chicks were orally challenged with a motile wild-type strain of C. jejuni IN9 or with flagellar mutants created from IN9 by disrupting the flagellin genes with a kanamycin resistance cassette by using shuttle mutagenesis (A. Labigne-Roussel, P. Courcoux, and L. Tompkins, J. Bacteriol. 170:1704-1708, 1988). One mutant, IN9-N3, lacked flagella and was nonmotile. The other, IN9-N7, produced a truncated flagellum and was partially motile. Three-day-old chicks were orally challenged with different doses of the wild-type strain and the two mutants. At challenge doses ranging from 3.0 x 10(4) to 6.6 x 10(8) CFU per chick, only the fully motile, wild-type strain colonized the chick ceca. Our results show that intact and motile flagella are important colonization factors for C. jejuni in chicks.     

Coronavirus proteins: structure and function of the oligosaccharides of the avian infectious bronchitis virus glycoproteins.

The recent finding that the E1 glycoproteins of murine coronaviruses contain only O-linked oligosaccharides suggested that this unusual modification might be a distinguishing feature of coronaviruses and might play an essential role in the life cycle of this family of viruses. To examine these possibilities, we analyzed the oligosaccharide moieties of the membrane proteins of the avian coronavirus infectious bronchitis virus. In addition, we determined the effect of inhibiting the glycosylation of these proteins on viral maturation and infectivity. Infectious bronchitis virus virions contain nine proteins. Four of these proteins, GP36, GP31, GP28, and P23, are closely related structurally and appear to be homologous to the E1 proteins of murine coronaviruses. We found that the oligosaccharides of GP31 and GP28 could be removed with endoglycosidase H and that neither of these glycoproteins was detectable in tunicamycin-treated cells. These two results indicated that GP31 and GP28 contain N-linked oligosaccharides. Therefore, O-linked oligosaccharides are not a universal feature of the small coronavirus membrane glycoproteins. Tunicamycin inhibited glycosylation of all of the viral glycoproteins but did not inhibit production of virions by infectious bronchitis virus-infected cells. The virions released by these cells contained only the three non-glycosylated viral proteins P51, P23, and P14. These particles were not infectious. Therefore, it appears that glycosylated infectious bronchitis virus polypeptides are not required for particle formation. However, the viral glycoproteins are apparently indispensible for viral infectivity.     

Structure of human dipeptidyl peptidase I (cathepsin C): exclusion domain added to an endopeptidase framework creates the machine for activation of granular serine proteases.

Dipeptidyl peptidase I (DPPI) or cathepsin C is the physiological activator of groups of serine proteases from immune and inflammatory cells vital for defense of an organism. The structure presented shows how an additional domain transforms the framework of a papain-like endopeptidase into a robust oligomeric protease-processing enzyme. The tetrahedral arrangement of the active sites exposed to solvent allows approach of proteins in their native state; the massive body of the exclusion domain fastened within the tetrahedral framework excludes approach of a polypeptide chain apart from its termini; and the carboxylic group of Asp1 positions the N-terminal amino group of the substrate. Based on a structural comparison and interactions within the active site cleft, it is suggested that the exclusion domain originates from a metallo-protease inhibitor. The location of missense mutations, characterized in people suffering from Haim-Munk and Papillon-Lefevre syndromes, suggests how they disrupt the fold and function of the enzyme.     

Albumin 3′untranslated region facilitates increased recombinant protein production from Chinese hamster ovary cells

Chinese hamster ovary (CHO) cells are used for recombinant protein production in the pharmaceutical industry but there is a need to improve expression levels. In the present work experiments were carried out to test the effectiveness of different 3′untranslated regions (3′UTRs) in promoting production of a naturally secreted luciferase. Seamless cloning was used to produce expression vectors in which Gaussia princeps luciferase coding sequences were linked to the human albumin, immunoglobulin or chymotrypsinogen 3′UTR. Stably transfected CHO cells expressing these constructs were selected. Luciferase activity in the culture medium was increased 2–3‐fold by replacing the endogenous 3′UTR with the albumin 3′UTR and decreased by replacement with immunoglobulin or chymotrypsinogen 3′UTR. Replacement of the native 3′UTR with the albumin 3′UTR led to a 10‐fold increase in luciferase mRNA levels. Deletion analysis of the albumin 3′UTR showed that loss of nucleotides 1–50, which removed an AU‐rich complex stem loop region, caused significant reductions in both luciferase protein expression and luciferase mRNA levels. The results suggest that recombinant protein expression and yield could be improved by the careful selection of appropriate 3′UTR sequences.     

Murine Borrelia arthritis is highly dependent on ASC and caspase-1, but independent of NLRP3

Introduction

The protein platform called the NOD-like-receptor -family member (NLRP)-3 inflammasome needs to be activated to process intracellular caspase-1. Active caspase-1 is able to cleave pro-Interleukin (IL)-1β, resulting in bioactive IL-1β. IL-1β is a potent proinflammatory cytokine, and thought to play a key role in the pathogenesis of Lyme arthritis, a common manifestation of Borrelia burgdorferi infection. The precise pathways through which B. burgdorferi recognition leads to inflammasome activation and processing of IL-1β in Lyme arthritis has not been elucidated. In the present study, we investigated the contribution of several pattern recognition receptors and inflammasome components in a novel murine model of Lyme arthritis.

Methods

Lyme arthritis was elicited by live B. burgdorferi, injected intra-articularly in knee joints of mice. To identify the relevant pathway components, the model was applied to wild-type, NLRP3-/-, ASC-/-, caspase-1-/-, NOD1-/-, NOD2-/-, and RICK-/- mice. As a control, TLR2-/-, Myd88-/- and IL-1R-/- mice were used. Peritoneal macrophages and bone marrow-derived macrophages were used for in vitro cytokine production and inflammasome activation studies. Joint inflammation was analyzed in synovial specimens and whole knee joints. Mann-Whitney U tests were used to detect statistical differences.

Results

We demonstrate that ASC/caspase-1-driven IL-1β is crucial for induction of B. burgdorferi-induced murine Lyme arthritis. In addition, we show that B. burgdorferi-induced murine Lyme arthritis is less dependent on NOD1/NOD2/RICK pathways while the TLR2-MyD88 pathway is crucial.

Conclusions

Murine Lyme arthritis is strongly dependent on IL-1 production, and B. burgdorferi induces inflammasome-mediated caspase-1 activation. Next to that, murine Lyme arthritis is ASC- and caspase-1-dependent, but NLRP3, NOD1, NOD2, and RICK independent. Also, caspase-1 activation by B. burgdorferi is dependent on TLR2 and MyD88. Based on present results indicating that IL-1 is one of the major mediators in Lyme arthritis, there is a rationale to propose that neutralizing IL-1 activity may also have beneficial effects in chronic Lyme arthritis.     

Identification and characterization of specific sequences encoding pathogenicity associated proteins in the genome of commensal Neisseria species

Abstract The distribution of distinct sequences in pathogenic and commensal Neisseria species was investigated systematically by dot blot analysis. Probes representing the genes of Rmp, pilin and IgA1 protease were found to hybridize exclusively to the chromosomal DNA of the pathogenic species, Neisseria gonorrhoeae and/or Neisseria meningitidis . In contrast, specific sequences for the genes of the porin protein Por and the opacity protein (Opa) were also detected in a panel of commensal Neisseria species such as N. lactamica, N. subflava, N, flava, N. mucosa and N. sicca . Using opa -specific oligonucleotides as probes in chromosomal blots, the genomes of the commensal Neisseria species show a totally reduced repertoire of cross-hybridizing loci compared to the complex opa gene family of N. gonorrhoeae . DNA sequence analysis of one opa -related gene derived from N. flava and N. sicca , respectively, revealed a large degree of homology with previously described gonococcal and meningococcal genes e.g., a typical repetitive sequence in the leader peptide and the distribution of the hypervariable and conserved regions. This observation, together with the finding, that the gene is constitutively transcribed, leads to the assumption that some of the commensal Neisseria species may have the potential for the expression of a protein harboring similar functions as the Opa proteins in pathogenic Neisseriae .     

Molecular identification, tissue distribution and subcellular localization of mST3GalV/GM3 synthase

A molecular screen for a mouse homologue of a Drosophila carbohydrate binding protein, called Gliolectin, yielded a cDNA encoding mST3GalV/GM3 synthase (CMP-NeuAc: lactosylceramide alpha2, 3-sialyltransferase). By in situ hybridization and immunohistochemistry, mST3GalV exhibits differential expression in neural and non-neural tissues. Although expressed by all neurons in the central nervous system, neuronal populations that contribute their axons to myelinated efferent projections, such as cerebellar Purkinje cells and spinal motorneurons, demonstrate the highest ST3GalV expression. When stained with anti-mST3GalV antiserum (designated CS2), subpopulations of neurons display an elaborate Golgi apparatus, frequently extending into one or more dendritic processes. The extended spatial distribution of the neuronal Golgi apparatus, particularly in spinal motorneurons, allowed the confocal immunohistochemical colocalization of mST3GalV with markers for medial/trans-Golgi but not the cis-Golgi or trans-Golgi network, consistent with previous observations suggesting that ganglioside glycosyltransferases are enriched in late Golgi compartments. Among non-neural tissues, liver and testes demonstrate cell-type specific CS2 staining. In liver, endothelial cells lining a ring of sinusoids, concentric with the central vein, express mST3GalV. Kupffer cells are also stained with CS2 antiserum but hepatocyte expression is undetectable. In the seminiferous tubules of the testes, ST3GalV is found in somatic (Leydig, Sertoli) and early germline cells (spermatogonia and primary spermatocytes); the epididymal epithelium exhibits intense ST3GalV expression. Since GM3 is a precursor for the synthesis of a- and b-series gangliosides, the range of mST3GalV/GM3 synthase expression among various cell populations indicates that certain cell types possess greater reliance on ganglioside function than others.