The physicochemical characterisation of pepsin degraded pig gastric mucin

Mucins are the main macromolecular components of the mucus secretions that cover the oral cavity, gastrointestinal and urogenital tracts of animals. The properties of the mucus secretions are therefore directly correlated with the physicochemical properties of mucin glycoproteins. In this study, mucins were obtained from pig gastric mucous after digestion with pepsin at 37 ⁰C for 4 hours, these mucins were characterised in terms of compositional and hydrodynamic properties. Compositional analysis showed that this mucin contains protein (15%), carbohydrates (55%) of which the constituents are: fucose (4%), galactose (9%), glucosamine (55%), glucosamine (33%) and sialic acid (2%). The latter component gives the mucin polymer a pH-dependant negative charge, with a -potential of -3 mV at pH 1.2 up to -11 mV at pH 7.4. The weight average molar mass was ~1 x 106 g/mol and intrinsic viscosity was ~0.42 dL/g although there was a small pH dependency due to the polyelectrolyte behaviour of the polymer. The measurements of viscosity versus shear rate showed shear thinning behaviour and the critical overlap concentration was determined to be 10-11% w/v indicating a compact structure. Knowledge of these properties is fundamental to the understanding interactions of mucins, with for example, novel drug delivery systems

Surface Co-Expression of Two Different PfEMP1 Antigens on Single Plasmodium falciparum-Infected Erythrocytes Facilitates Binding to ICAM1 and PECAM1

The Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) antigens play a major role in cytoadhesion of infected erythrocytes (IE), antigenic variation, and immunity to malaria. The current consensus on control of variant surface antigen expression is that only one PfEMP1 encoded by one var gene is expressed per cell at a time. We measured var mRNA transcript levels by real-time Q-PCR, analysed var gene transcripts by single-cell FISH and directly compared these with PfEMP1 antigen surface expression and cytoadhesion in three different antibody-selected P. falciparum 3D7 sub-lines using live confocal microscopy, flow cytometry and in vitro adhesion assays. We found that one selected parasite sub-line simultaneously expressed two different var genes as surface antigens, on single IE. Importantly, and of physiological relevance to adhesion and malaria pathogenesis, this parasite sub-line was found to bind both CD31/PECAM1 and CD54/ICAM1 and to adhere twice as efficiently to human endothelial cells, compared to infected cells having only one PfEMP1 variant on the surface. These new results on PfEMP1 antigen expression indicate that a re-evaluation of the molecular mechanisms involved in P. falciparum adhesion and of the accepted paradigm of absolutely mutually exclusive var gene transcription is required

Haemophilus influenzae adhesins and the innate immune response

Haemophilus influenzae is a human specific Gram-negative respiratory tract pathogen. Encapsulated H. influenzae strains exist in six different serotypes a-f, of which type b (Hib) is the most virulent. Hib is protected by the polysaccharide capsule when invading the blood circulaton and may cause diseases such as meningitis and epiglottitis. Non-encapsulated H. influenzae referred to as nontypeable H. influenzae (NTHi), are frequently associated with acute otitis media in children and chronic obstructive pulmonary disease (COPD) among the elderly. H. influenzae expresses several outer membrane proteins of which the adhesins play an important role in the initial interactions with the airway epithelium. In this thesis, we have studied two Haemophilus influenzae adhesins and their interactions with innate defense mechanisms. Vitronectin is a glycoprotein that regulates the terminal pathway of the complement system by inhibiting the membrane attack complex. We demonstrate that Haemophilus influenzae surface fibrils (Hsf) may increase the bacterial survival by interacting with vitronectin in serum. We also describe the isolation and characterization of a novel H. influenzae adhesin designated protein E (PE). PE was isolated by using an IgD myeloma serum which specifically recognized the protein on the bacterial surface. Furthermore, PE was found to be a 16 kDa protein and classified as a lipoprotein. PE displays adhesive activity to three respiratory epithelial cell lines and erythrocytes, both when expressed on the surface of H. influenzae and as a recombinant protein in E. coli. The active adhesive binding domain has been determined to be located within the central part of the molecule, PE amino acids 84-108. PE shows stimulatory effects when incubated with respiratory epithelial cells by inducing high levels of IL-8 and ICAM-1. These inflammatory mediators play an important role for the innate immune response and the recruitment of neutrophils. Finally, PE promotes bacterial invasion into epithelial cells. In summary, the work presented in this thesis shows that Hsf may prolong the bacterial survival by interacting with the complement regulator vitronectin. The novel protein PE has been isolated, characterized and shown to be important for interactions of H. influenzae with the innate immune system

Identification of a novel Haemophilus influenzae protein important for adhesion to epithelial cells.

Non-typable Haemophilus influenzae (NTHi) is an important human-specific respiratory pathogen colonizing the mucosa of the upper respiratory tract. The bacterium is a common cause of acute otitis media in children and exacerbations in patients with chronic obstructive pulmonary disease (COPD). An immunoglobulin (Ig) D-lambda myeloma protein was found to detect a 16kDa surface protein that we designated protein E (PE). The pe gene was cloned using an NTHi genomic DNA library, and a truncated PE-derived protein lacking the endogenous signal peptide (PE22-160) was synthesized and produced in large amounts in Escherichia coli. Interestingly, PE was expressed at the bacterial surface of NTHi as revealed by flow cytometry using the IgD-lambda myeloma protein or PE-specific polyclonal antibodies. A PE-deficient NTHi mutant was produced and lost 50% of its adhesive capacity as compared to the wild-type counterpart when analysed for adhesion to type II lung alveolar epithelial cells. In parallel, E. coli expressing full-length PE1-160 adhered significantly more efficiently to epithelial cells as compared to wild-type E. coli. Recombinant IgD that recognized the chemical dansyl-chloride did not interact with PE indicating that the IgD-lambda myeloma protein most likely was an antibody directed against the H. influenzae surface epitope. In conclusion, we have discovered a novel NTHi outer membrane protein with adhesive properties using an IgD-myeloma protein

Analysis of Single-cell Gene Transcription by RNA Fluorescent In Situ Hybridization (FISH)

Adhesion of Plasmodium falciparum infected erythrocytes (IE) to human endothelial receptors during malaria infections is mediated by expression of PfEMP1 protein variants encoded by the var genes. The haploid P. falciparum genome harbors approximately 60 different var genes of which only one has been believed to be transcribed per cell at a time during the blood stage of the infection. How such mutually exclusive regulation of var gene transcription is achieved is unclear, as is the identification of individual var genes or sub-groups of var genes associated with different receptors and the consequence of differential binding on the clinical outcome of P. falciparum infections. Recently, the mutually exclusive transcription paradigm has been called into doubt by transcription assays based on individual P. falciparum transcript identification in single infected erythrocytic cells using RNA fluorescent in situ hybridization (FISH) analysis of var gene transcription by the parasite in individual nuclei of P. falciparum IE(1). Here, we present a detailed protocol for carrying out the RNA-FISH methodology for analysis of var gene transcription in single-nuclei of P. falciparum infected human erythrocytes. The method is based on the use of digoxigenin- and biotin- labeled antisense RNA probes using the TSA Plus Fluorescence Palette System(2) (Perkin Elmer), microscopic analyses and freshly selected P. falciparum IE. The in situ hybridization method can be used to monitor transcription and regulation of a variety of genes expressed during the different stages of the P. falciparum life cycle and is adaptable to other malaria parasite species and other organisms and cell types

Nontypeable Haemophilus influenzae Adhesin Protein E: Characterization and Biological Activity.

The adhesin protein E (PE) of the human respiratory pathogen nontypeable Haemophilus influenzae (NTHi) exists in all clinical isolates. In the present study, NTHi adherence to epithelial cells of various origins was further analyzed. The number of intraepithelial PE-deficient NTHi was decreased compared with PE-expressing NTHi. Interestingly, PE-expressing NTHi or Escherichia coli transformants, in addition to soluble recombinant PE22-160 without a lipid moiety, induced a proinflammatory cell response. The adhesive PE domain was defined within PE84-108, and preincubation of epithelial cells with this peptide blocked adhesion of several clinical NTHi isolates. Mice immunized with PE84-108 cleared NTHi up to 8-fold more efficiently on pulmonary challenge than did mice immunized with a control peptide. Finally, anti-PE mouse antibodies from vaccinated mice prevented NTHi adhesion. Our data suggest that the ubiquitous adhesin PE plays an important role in the pathogenesis of NTHi infection