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Capture Compound Mass Spectrometry - A Powerful Tool to Identify Novel c-di-GMP Effector Proteins

Considerable progress has been made during the last decade towards the identification and characterization of enzymes involved in the synthesis ...

Biozentrum of the University of Basel

For the efficient surface presentation and release of virulence factors especially pathogenic Gram-negative bacteria have developed several distinct secretion mechanisms. An increasing number of pathogens in various species employs a mechanism denoted the 'autotransporter' pathway. This pathway is characterised by an outer membrane translocator module representing the C-terminal domain of the transported protein itself. An intriguing potential application of such systems involves the transport and surface expression of recombinant proteins or peptides, like e.g. the presentation of antigens for the generation of live oral vectors as vaccine carriers. Here we report on the incorporation of heterologous (poly-) peptides in permissive sites of the translocator module of the adhesin-involved-in-diffuse-adherence (AIDA) autotransporter system. We demonstrate the presentation of the B subunit of the heat labile enterotoxin of Escherichia coli (LTB) as well as of functional T-cell epitopes of Yersinia enterocolitica heat-shock protein 60 (Y-hsp60) on the surface of E. coli.

Cell surface presentation of recombinant (poly-) peptides including functional T-cell epitopes by the AIDA autotransporter system.

The species Escherichia coli comprises not only non-pathogenic or commensal variants that belong to the normal intestinal flora of most mammals, but also various pathogenic strains causing diverse intestinal and extraintestinal infections in man and animals. Virulence factors and mechanisms involved in pathogenesis have been successfully analyzed for many years resulting in a wealth of knowledge about many E. coli pathotypes. However, our knowledge on the genome content, diversity and variability between pathogenic and also non-pathogenic subtypes is only slowly accumulating. Pathotypes have been largely defined by the presence or absence of particular DNA segments that in most cases appear to have been acquired via horizontal gene transfer events. As these regions are frequently subjected to excisions, rearrangements, and transfers they contribute to the previously unexpected and underestimated rapid evolution of E. coli variants resulting in the development of novel strains and even pathotypes. In these studies various novel aspects of genome diversity and plasticity in extraintestinal and intestinal pathogenic E. coli pathotypes have been addressed and the results have been directly applied for the improvement of diagnostic methods.

Aspects of genome plasticity in pathogenic Escherichia coli.

Horizontal gene transfer plays an important role in bacterial evolution. DNA acquired by horizontal gene transfer has to be incorporated into existing regulatory networks. The histone-like nucleoid structuring protein H-NS acts as a silencer of horizontally acquired genes to avoid potential damage. However, specific regulators can overcome H-NS repression, resulting in the integration of newly acquired genes into existing regulatory networks. Here, we analyzed the influence of H-NS on the transcription of the Yersinia enterocolitica hreP gene and its regulators pypA, pypB, and pypC by establishing a dominant-negative H-NS version. Using transcriptional fusions and electrophoretic mobility shift assays, we show that H-NS silences hreP, pypA, pypB, and pypC by direct interactions. While the H-NS antagonist RovA activates pypC, it has no effect on pypA and pypB. Furthermore, H-NS affects biofilm formation in Y. enterocolitica.

The H-NS protein silences the pyp regulatory network of Yersinia enterocolitica and is involved in controlling biofilm formation.

Extracellular Gram-negative pathogenic bacteria target essential cytoplasmic processes of eukaryotic cells by using effector protein delivery systems such as the type III secretion system (T3SS). These secretion systems directly inject effector proteins into the host cell cytoplasm. Among the T3SS-dependent Yop proteins of pathogenic Yersinia, the function of the effector protein YopM remains enigmatic. In a recent study, we demonstrated that recombinant YopM from Yersinia enterocolitica enters host cells autonomously without the presence of bacteria and thus identified YopM as a novel bacterial cell-penetrating protein. Following entry YopM down-regulates expression of pro-inflammatory cytokines such as tumor necrosis factor α. These properties earmark YopM for further development as a novel anti-inflammatory therapeutic. To elucidate the uptake and intracellular targeting mechanisms of this bacterial cell-penetrating protein, we analyzed possible routes of internalization employing ultra-cryo electron microscopy. Our results reveal that under physiological conditions, YopM enters cells predominantly by exploiting endocytic pathways. Interestingly, YopM was detected free in the cytosol and inside the nucleus. We could not observe any colocalization of YopM with secretory membranes, which excludes retrograde transport as the mechanism for cytosolic release. However, our findings indicate that direct membrane penetration and/or an endosomal escape of YopM contribute to the cytosolic and nuclear localization of the protein. Surprisingly, even when endocytosis is blocked, YopM was found to be associated with endosomes. This suggests an intracellular endosome-associated transport of YopM.

Alexander Schmidt

Proteomics Core Facility

Biozentrum of the University of Basel

Capture Compound Mass Spectrometry - A Powerful Tool to Identify Novel c-di-GMP Effector Proteins

Alexander Schmidt

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Proteomics Core Facility

Biozentrum of the University of Basel

Capture Compound Mass Spectrometry - A Powerful Tool to Identify Novel c-di-GMP Effector Proteins

Proteomics Core Facility