Transcriptomes of the major human pancreatic cell types.

AIMS/HYPOTHESIS: We sought to determine the mRNA transcriptome of all major human pancreatic endocrine and exocrine cell subtypes, including human alpha, beta, duct and acinar cells. In addition, we identified the cell type-specific distribution of transcription factors, signalling ligands and their receptors. METHODS: Islet samples from healthy human donors were enzymatically dispersed to single cells and labelled with cell type-specific surface-reactive antibodies. Live endocrine and exocrine cell subpopulations were isolated by FACS and gene expression analyses were performed using microarray analysis and quantitative RT-PCR. Computational tools were used to evaluate receptor-ligand representation in these populations. RESULTS: Analysis of the transcriptomes of alpha, beta, large duct, small duct and acinar cells revealed previously unrecognised gene expression patterns in these cell types, including transcriptional regulators HOPX and HDAC9 in the human beta cell population. The abundance of some regulatory proteins was different from that reported in mouse tissue. For example, v-maf musculoaponeurotic fibrosarcoma oncogene homologue B (avian) (MAFB) was detected at equal levels in adult human alpha and beta cells, but is absent from adult mouse beta cells. Analysis of ligand-receptor interactions suggested that EPH receptor-ephrin communication between exocrine and endocrine cells contributes to pancreatic function. CONCLUSIONS/INTERPRETATION: This is the first comprehensive analysis of the transcriptomes of human exocrine and endocrine pancreatic cell types-including beta cells-and provides a useful resource for diabetes research. In addition, paracrine signalling pathways within the pancreas are shown. These results will help guide efforts to specify human beta cell fate by embryonic stem cell or induced pluripotent stem cell differentiation or genetic reprogramming.

Bacterial lactoferrin receptors.

Lactoferrin is thought to play a pivotal role in prevention of infection in the host and its ability to sequester iron from potential pathogens has been considered an important component of its antimicrobial function. A number of bacterial species in the Neisseriaceae have developed a mechanism for acquiring iron directly from this host glycoprotein which involves surface receptors capable of specifically binding lactoferrin. Initial attempts at identifying the receptor proteins in Neisseria and Moraxella species using affinity isolation with immobilized lactoferrin under high stringency conditions presumptively identified a single 100 kDa receptor protein, LbpA (formerly Lbp1). Under modified affinity isolation conditions a second 84 kDa lactoferrin binding protein was isolated and had been presumptively identified as LbpB. This protein was not isolated from a CopB-ve isogenic mutant of Moraxella catarrhalis, indicating that it was in fact CopB. However, another lactoferrin binding protein isolated under high stringency conditions, that comigrated with LbpA in most, but not all, M. catarrhalis strains, was identified by convalescent antisera. Its biochemical properties suggested that it indeed was LbpB. The identity of these proteins was confirmed by preparing isogenic mutants with the lbpA and lbpB genes. Growth studies with isogenic mutants deficient in LbpB, LbpA, CopB or FbpA were performed to evaluate their role in iron acquisition from lactoferrin. LbpA and FbpA were essential for this process, supporting prior models of the iron acquisition pathway. LbpB was not essential which is remniscent of studies with the bacterial transferrin receptors. The isogenic CopB-ve isogenic mutants were deficient in iron acquisition from both transferrin and lactoferrin, suggesting that it is a key component in both pathways. A model providing an alternate explanation of the data is presented. The role and surface accessibility of the lactoferrin receptor proteins suggests that they might be useful vaccine antigens and the preferentially reactivity of convalescent antisera with LbpB suggests that it may be the prime candidate.

Preparation and characterization of Neisseria meningitidis mutants deficient in production of the human lactoferrin-binding proteins LbpA and LbpB.

Pathogenic members of the family Neisseriaceae produce specific receptors facilitating iron acquisition from transferrin (Tf) and lactoferrin (Lf) of their mammalian host. Tf receptors are composed of two outer membrane proteins, Tf-binding proteins A and B (TbpA and TbpB; formerly designated Tbp1 and Tbp2, respectively). Although only a single Lf-binding protein, LbpA (formerly designated Lbp1), had previously been recognized, we recently identified additional bacterial Lf-binding proteins in the human pathogens Neisseria meningitidis and Moraxella catarrhalis and the bovine pathogen Moraxella bovis by a modified affinity isolation technique (R. A. Bonnah, R.-H. Yu, and A. B. Schryvers, Microb. Pathog. 19:285-297, 1995). In this report, we characterize an open reading frame (ORF) located immediately upstream of the N. meningitidis B16B6 lbpA gene. Amino acid sequence comparisons of various TbpBs with the product of the translated DNA sequence from the upstream ORF suggests that the region encodes the Lf-binding protein B homolog (LbpB). The LbpB from strain B16B6 has two large stretches of negatively charged amino acids that are not present in the various transferrin receptor homologs (TbpBs). Expression of the recombinant LbpB protein as a fusion with maltose binding protein demonstrated functional Lf-binding activity. Studies with N. meningitidis isogenic mutants in which the lbpA gene and the ORF immediately upstream of lbpA (putative lbpB gene) were insertionally inactivated demonstrated that LbpA, but not LbpB, is essential for iron acquisition from Lf in vitro.

Biochemical analysis of lactoferrin receptors in the Neisseriaceae: identification of a second bacterial lactoferrin receptor protein.

Bacterial transferrin receptors that have been described in the families Pasteurellaceae and Neisseriaceae are composed of two receptor proteins, transferrin binding proteins 1 and 2 (Tbp1 and Tbp2). In contrast, bacterial lactoferrin receptors have only been described for human pathogens in the family Neisseriaceae, and were believed to consist of a single protein, Lbp1, which is highly homologous to Tbp1. We describe a modified affinity isolation procedure that facilities isolation of a second lactoferrin receptor protein Lbp2 (a presumptive Tbp2 homologue) from Neisseria meningitidis, Moraxella catarrhalis and Moraxella bovis using immobilized lactoferrin. Antiserum specific for either the M. catarrhalis Tbp1+2 molecules, the M. catarrhalis Lbp1 molecule, or for a commercial preparation of human lactoferrin did not react on western blots with the same organisms' affinity purified Lbp2. In addition, the M. catarrhalis Lbp2 could be isolated in a functional form without contaminating Lbp1 or Tbp1+2. We also demonstrate that the bovine pathogen, M. bovis, produces functional transferrin and lactoferrin receptors specific for the bovine forms of these glycoproteins. A putative lbpB gene, recently speculated to reside immediately upstream of the N. meningitidis Lbp1 structural gene, lbpA, likely encodes the newly isolated Lbp2 protein from this bacterial species.

Analysis of the immunological responses to transferrin and lactoferrin receptor proteins from Moraxella catarrhalis.

Moraxella catarrhalis expresses surface receptor proteins that specifically bind host transferrin (Tf) and lactoferrin (Lf) in the first step of the iron acquisition pathway. Acute- and convalescent-phase antisera from a series of patients with M. catarrhalis pulmonary infections were tested against Tf and Lf receptor proteins purified from the corresponding isolates. After the purified proteins had been separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting, we observed strong reactivity against Tf-binding protein B (TbpB; also called OMP1) and Lf-binding protein B (LbpB) but little or no reactivity against Tf-binding protein A (TbpA) or Lf-binding protein A (LbpA), using the convalescent-phase antisera. Considerable antigenic heterogeneity was observed when TbpBs and LbpBs isolated from different strains were tested with the convalescent-phase antisera. Comparison to the reactivity against electroblotted total cellular proteins revealed that the immune response against LbpB and TbpB constitutes a significant portion of the total detectable immune response to M. catarrhalis proteins. Preparations of affinity-isolated TbpA and LbpA reacted with convalescent-phase antisera in a solid-phase binding assay, but blocking with soluble TbpB, soluble LbpB, or extracts from an LbpA(-) mutant demonstrated that this reactivity was attributed to contaminants in the TbpA and LbpA preparations. These studies demonstrate the immunogenicity of M. catarrhalis TbpB and LbpB in humans and support their potential as vaccine candidates.

Characterization of Moraxella (Branhamella) catarrhalis lbpB, lbpA, and lactoferrin receptor orf3 isogenic mutants.

Pathogenic members of the family Neisseriaceae produce specific receptors to acquire iron from their host's lactoferrin and transferrin. Recently, putative Moraxella catarrhalis lactoferrin receptor genes and a third open reading frame (lbpB, lbpA, and orf3) were cloned and sequenced. We describe the preliminary characterization of isogenic mutants deficient in LbpB, LbpA, or Orf3 protein.

Alteration of epithelial cell transferrin-iron homeostasis by Neisseria meningitidis and Neisseria gonorrhoeae.

Iron is an essential element for nearly all organisms. In mammals, iron is transported to body tissues by the serum glycoprotein transferrin. Transferrin-iron is internalized by binding to specific receptors followed by endocytosis. In vitro, Neisseria meningitidis and Neisseria gonorrhoeae can use iron from a variety of iron-containing compounds, including human transferrin. In vivo, transferrin is an important source of iron for N. gonorrhoeae: a mutant that is unable to bind and use transferrin-iron is unable to colonize the urethra of men or initiate disease at this site. As pathogenic Neisseria and its human host derive much of their iron from transferrin, we reasoned that a competition may exist between microbe and host epithelial cells for transferrin-iron at certain stages of infection. We therefore tested the hypothesis that N. meningitidis and N. gonorrhoeae may actively interfere with host transferrin-iron metabolism. We report that Neisseria-infected human epithelial cells have reduced levels of transferrin receptor messenger RNA and cycling transferrin receptors. The ability of infected cells to internalize transferrin receptor is also reduced. Finally, the relative distribution of surface and cycling transferrin receptors is altered in an infected cell. We conclude that Neisseria infection alters epithelial cell transferrin-iron homeostasis at multiple levels.

Biochemical and immunological properties of lactoferrin binding proteins from Moraxella (Branhamella) catarrhalis.

The Neisseriaceae can acquire iron (Fe) from lactoferrin (Lf) using host-Lf receptors on the bacterial surface. The binding proteins that are proposed to constitute the receptor have been identified by isolation with immobilized Lf. Using CopB-specific monoclonal antibodies and isogenic CopB mutants, we demonstrate that the 84 kDa protein isolated with immobilized human Lf from Moraxella catarrhalis using low stringency conditions is CopB, an 84 kDa membrane-spanning protein with similarities to other TonB-dependent outer membrane proteins. Affinity isolation of Lf receptors from a variety of M. catarrhalis strains using high stringency conditions revealed a 95 kDa protein migrating slightly faster than LbpA on SDS-PAGE in some strains. Convalescent human antisera from patients infected with M. catarrhalis reacted specifically with this protein, but not LbpA. Proteolysis experiments demonstrated that, unlike LbpA, it was rapidly degraded. The 95 kDa protein, but not LbpA, binds labelled Lf after SDS-PAGE and electroblotting, suggesting the 95 kDa protein is LbpB, the homologue of TbpB. This protein comigrates with LbpA in most strains, which may explain why it had not been previously identified.

Lectinlike properties of pertussis toxin.

We have examined the lectinlike properties of pertussis toxin by binding-inhibition assays and affinity chromatography of goose erythrocyte membranes. Although pertussis toxin and wheat germ agglutinin apparently recognize similar sugar sequences on glycoproteins, the binding activities of the two lectins are not identical.