WP1 Virulence proteins of enteric bacteria in the gut lumen and epithelium

Anaerobiosis and gene regulation
The Shigella lipoprotein export protein, Lol, is down-regulated under anaerobic conditions. Lol is involved in transport of lipoproteins required for the Type Three Secretion System (TTSS), an essential virulence factor. Using a range of molecular genetic approaches, it was shown that the lipoprotein transport activity of LolA is necessary for efficient transport of the lipoprotein MxiM to the surface of the bacteria. MxiM in turn is necessary for the transportation of a fundamental component of the TTSS outer ring structure called MxiD (Francesco Berlanda Scorza, seconded from Novartis to Imperial College London). RNA-sequencing (RNA-seq) experiments in the presence and absence of oxygen have identifed further genes regulated by oxygen. In particular, MxiA, another protein involved in TTSS function was found to be regulated by oxygen, thus confirming the role of oxygen in regulating the TTSS (Marta Vergara Irigaray, recruited by Imperial College London). In addition, an improved purification protocol for the native TTSS has been developed that has allowed further structural and functional characterization of the TTSS (Anastasia Gazi, recruited by Institut Pasteur).

Adhesion in mucosal pathogens
Interactions between adhesive pilin proteins PilM, PilN, PilO and PilP of meningococcus have been characterized after immunoprecipitation. The 4 proteins form a complex in the inner membrane in the absence of other pilins. The pilE, pilF, pilD (or pilDG) were then introduced into E. coli. Expression of each Pil- component, although expression levels were variable, was confirmed using a battery of antibodies. It was therefore possible to express simultaneously seven or eight proteins of Neisseria in E. coli (Marta Castagnini, seconded from Novartis to Imperial College London).

Manipulation of the host adaptive immune response by Shigella
A manuscript entitled “The Shigella flexneri Type Three Secretion System effector IpgD inhibits T cell migration by manipulating host phosphoinositide metabolism” has been published. In addition, it was found out that the “in vitro” infection of Jurkat cells by Shigella impairs the formation of the immune synapse (Elisabetta Frigimelica, seconded from Novartis to Institut Pasteur).

Mechanisms of regulation of GNA2132 in Neisseria meningitdis
Using transcriptome analysis, progress has been made in understanding the role of temperature and carbon source (glucose and lactose) in regulation of components of an experimental vaccine against meningococcal meningitis. The mechanisms behind the regulation have, in part, been elucidated In addition, progress has also been made in understanding the role of the contact regulatory element of Neisseria, CREN, in regulation of expression of GNA2132 and other virulence factors (Ana Sofia Maceira Antunes, seconded from Institut Pasteur to Novartis).

The function of vaccine candidates in Staphylococcus aureus
Two candidate antigens for a novel vaccine against S. aureus infection (EsxA and EsxB) were shown to be necessary for efficient intracellular survival of the bacteria. The data suggest that EsxA delays host cell death by interfering with the autophagic and apoptotic pathways, and together with EsxB, mediates the release of S. aureus from the host cell (Charalampia Korea, recruited by Novartis).

WP2 Creation of a pilus or pilus subunits based vaccine against S. pneumoniae

The interaction of the S. pneumoniae pilus adhesin RrgA with macrophages has been characterized using fluorescent microspheres conjugated with recombinant RrgA. The data indicate that the RrgA adhesin directly promotes phagocytosis due to a direct interaction of RrgA with CR3. Specific monoclonal antibodies against murine and human CD11b were used demonstrate that RrgA binds CD11b and its CD11b-I domain. These data were confirmed by Far Western experiments showing binding of RrgA to CD11b (Tiziana Spadafina, seconded from Novartis to Karolinska Institutet). S. pneumoniae produces high levels of hydrogen peroxide by converting pyruvate to acetyl phosphate and hydrogen peroxide via a pyruvate oxidase encoded by the spxB gene. Natural and recombinant spxB mutants showed decreased binding ability and were less internalized by phagocytic cells compared to wild type strains. The results suggest that spxB mutants are selected in vivo because they are more resistant to clearance by host macrophages (Marilena Gallotta, seconded from Novartis to Karolinska Institutet). Further experiments in this work package addressed new studies on S. pneumoniae choline binding proteins most of which are actively secreted to the cell surface, where they bind by affinity to the phosphoryl-choline residues on the lipoteichoic and wall teichoic acids of the bacteria (Albino T et al. 1970). In this work a protocol was developed to visualize LytA by high-resolution microscopy using the STED (Stimulated Emission Depletion) approach. It could be shown that the binding of LytA by its choline-binding domain (CBD) attached to teichoic acids in contrast to its substrate-binding profile mediated via its amidase domain. These two localization patterns differ, indicating that LytA can in principle bind all over the cell surface but that the substrate for the amidase domain is only present close to the equatorial site, where cell division occurs (Alice Eberhardt, recruited by Karolinska Institutet).

WP3 WP3 Vaccination against Helicobacter pylori and exploration of determinants of immune protection

H. pylori permanently modifies gene expression in host cells during infection. Bacterial produced γ-glutamyltransferase (GGT) was identified as the main virulence factor that induces transcription of SIRT1, a histone deacetylase which also has important non-histone targets, including P53, and this induction is dependent on GGT enzymatic activity. This mechanism was confirmed by infecting epithelial cells with a GGT KO mutant or treatment with purified recombinant wild type or mutant GGT protein. Interestingly SIRT1 has been recently implicated in cancer progression as well as in suppression of inflammatory response through repression of both NFkB and P53 activity (Matteo Metruccio, seconded from Novartis to Max Planck Institute for Infection Biology). Next generation sequencing has been used to analyze the regulation of host gene expression after H. pylori infection. A pipeline that allows to perform robust analyses of Next Generation Sequencing Data has been established (publicly available at: http://qualimap.bioinfo.cipf.es). This pipeline covers all the steps from mapping of the reads to obtaining the differentially methylated regions (DMRs). The database generated will be invaluable for future studies (Fernando Garcia Alcalde, recruited at Max Planck Institute for Infection Biology). A second approach is studying the induction of anti-microbial peptides in human H. pylori infected dendritic and T-cells. Preliminary data show robust anti-H.pylori responses. In particular, human Beta Defensin 3 was shown to bind H. pylori and shows antimicrobial activity (Paolo Montanari, seconded from Novartis to Max Planck Institute for Infection Biology). Experiments to compare infected with non-infected individuals in order to identify biomarkers in the gastric mucosa that are regulated upon H. pylori infection were performed. A meta-analysis of internal and published microarray data sets was used to build up a detailed picture of activated transcription factors and signalling pathways.in order to pinpoint the most important biomarkers. The meta-analysis was further evaluated using RankProd software to identify the rank of the gene in whole datasets. Further analysis of these data are likely to give clearer insights into the interaction of H. pylori with the host (Elif Dagdan, recruited at Max Planck Institute for Infection Biology).