Direct binding of gangliosides to Helicobacter pylori vacuolating cytotoxin (VacA) neutralizes its toxin activity.

Gangliosides are target receptors for bacterial entry, yet those present in human milk exhibit a protective role against bacterial infection. Here, we show that treatment with ganglioside mixture at a concentration of 100 microg/mL resulted in significant inhibition of the vacuole formation activity of Helicobacter pylori vacuolating cytotoxin (VacA) in gastric epithelial cancer AZ-521 cells. All gangliosides (GM1, GM2, GM3, GD1a, GD1b, GD3 and GT1b) examined showed good neutralizing capacity against VacA. A pull-down assay was performed using lyso-GM1 coupled to Sepharose as the tagged polysaccharide polymer to capture VacA from H. pylori culture supernatant. GM1-VacA complexes were successfully precipitated, suggesting that GM1 binds directly to VacA. The hydrodynamic binding of lyso-GM1 and VacA measured by fluorescence correlation spectroscopy had a K(d) value of 190 nM. VacA also bound to lyso-GM1 at pH 2 corresponding to the physiological pH of human stomach. Collectively, these results showed that direct binding of H. pylori VacA to free gangliosides neutralizes the toxin activity of VacA. These findings offer an alternative insight into the role of gangliosides in VacA toxicity and the pathogenesis of H. pylori

Virulence factors of Actinobacillus pleuropneumoniae involved in colonization, persistence and induction of lesions in its porcine host

Actinobacillus pleuropneumoniae is the causative agent of porcine pleuropneumonia. The virulence factors of this microorganism involved in colonization and the induction of lung lesions have been thoroughly studied and some have been well characterized. A. pleuropneumoniae binds preferentially to cells of the lower respiratory tract in a process involving different adhesins and probably biofilm formation. Apx toxins and lipopolysaccharides exert pathogenic effects on several host cells, resulting in typical lung lesions. Lysis of host cells is essential for the bacterium to obtain nutrients from the environment and A. pleuropneumoniae has developed several uptake mechanisms for these nutrients. In addition to persistence in lung lesions, colonization of the upper respiratory tract – and of the tonsils in particular – may also be important for long-term persistent asymptomatic infection. Information on virulence factors involved in tonsillar and nasal cavity colonization and persistence is scarce, but it can be speculated that similar features as demonstrated for the lung may play a role

Differential Modulation of TNF-α–Induced Apoptosis by Neisseria meningitidis

Infections by Neisseria meningitidis show duality between frequent asymptomatic carriage and occasional life-threatening disease. Bacterial and host factors involved in this balance are not fully understood. Cytopathic effects and cell damage may prelude to pathogenesis of isolates belonging to hyper-invasive lineages. We aimed to analyze cell–bacteria interactions using both pathogenic and carriage meningococcal isolates. Several pathogenic isolates of the ST-11 clonal complex and carriage isolates were used to infect human epithelial cells. Cytopathic effect was determined and apoptosis was scored using several methods (FITC-Annexin V staining followed by FACS analysis, caspase assays and DNA fragmentation). Only pathogenic isolates were able to induce apoptosis in human epithelial cells, mainly by lipooligosaccharide (endotoxin). Bioactive TNF-α is only detected when cells were infected by pathogenic isolates. At the opposite, carriage isolates seem to provoke shedding of the TNF-α receptor I (TNF-RI) from the surface that protect cells from apoptosis by chelating TNF-α. Ability to induce apoptosis and inflammation may represent major traits in the pathogenesis of N. meningitidis. However, our data strongly suggest that carriage isolates of meningococci reduce inflammatory response and apoptosis induction, resulting in the protection of their ecological niche at the human nasopharynx

Gene expression during normal and FSHD myogenesis

<p>Abstract</p> <p>Background</p> <p>Facioscapulohumeral muscular dystrophy (FSHD) is a dominant disease linked to contraction of an array of tandem 3.3-kb repeats (D4Z4) at 4q35. Within each repeat unit is a gene, <it>DUX4</it>, that can encode a protein containing two homeodomains. A <it>DUX4 </it>transcript derived from the last repeat unit in a contracted array is associated with pathogenesis but it is unclear how.</p> <p>Methods</p> <p>Using exon-based microarrays, the expression profiles of myogenic precursor cells were determined. Both undifferentiated myoblasts and myoblasts differentiated to myotubes derived from FSHD patients and controls were studied after immunocytochemical verification of the quality of the cultures. To further our understanding of FSHD and normal myogenesis, the expression profiles obtained were compared to those of 19 non-muscle cell types analyzed by identical methods.</p> <p>Results</p> <p>Many of the ~17,000 examined genes were differentially expressed (> 2-fold, <it>p </it>< 0.01) in control myoblasts or myotubes vs. non-muscle cells (2185 and 3006, respectively) or in FSHD vs. control myoblasts or myotubes (295 and 797, respectively). Surprisingly, despite the morphologically normal differentiation of FSHD myoblasts to myotubes, most of the disease-related dysregulation was seen as dampening of normal myogenesis-specific expression changes, including in genes for muscle structure, mitochondrial function, stress responses, and signal transduction. Other classes of genes, including those encoding extracellular matrix or pro-inflammatory proteins, were upregulated in FSHD myogenic cells independent of an inverse myogenesis association. Importantly, the disease-linked <it>DUX4 </it>RNA isoform was detected by RT-PCR in FSHD myoblast and myotube preparations only at extremely low levels. Unique insights into myogenesis-specific gene expression were also obtained. For example, all four Argonaute genes involved in RNA-silencing were significantly upregulated during normal (but not FSHD) myogenesis relative to non-muscle cell types.</p> <p>Conclusions</p> <p><it>DUX4</it>'s pathogenic effect in FSHD may occur transiently at or before the stage of myoblast formation to establish a cascade of gene dysregulation. This contrasts with the current emphasis on toxic effects of experimentally upregulated <it>DUX4 </it>expression at the myoblast or myotube stages. Our model could explain why <it>DUX4</it>'s inappropriate expression was barely detectable in myoblasts and myotubes but nonetheless linked to FSHD.</p

Global Effects of Catecholamines on Actinobacillus pleuropneumoniae Gene Expression

Bacteria can use mammalian hormones to modulate pathogenic processes that play essential roles in disease development. Actinobacillus pleuropneumoniae is an important porcine respiratory pathogen causing great economic losses in the pig industry globally. Stress is known to contribute to the outcome of A. pleuropneumoniae infection. To test whether A. pleuropneumoniae could respond to stress hormone catecholamines, gene expression profiles after epinephrine (Epi) and norepinephrine (NE) treatment were compared with those from untreated bacteria. The microarray results showed that 158 and 105 genes were differentially expressed in the presence of Epi and NE, respectively. These genes were assigned to various functional categories including many virulence factors. Only 18 genes were regulated by both hormones. These genes included apxIA (the ApxI toxin structural gene), pgaB (involved in biofilm formation), APL_0443 (an autotransporter adhesin) and genes encoding potential hormone receptors such as tyrP2, the ygiY-ygiX (qseC-qseB) operon and narQ-narP (involved in nitrate metabolism). Further investigations demonstrated that cytotoxic activity was enhanced by Epi but repressed by NE in accordance with apxIA gene expression changes. Biofilm formation was not affected by either of the two hormones despite pgaB expression being affected. Adhesion to host cells was induced by NE but not by Epi, suggesting that the hormones affect other putative adhesins in addition to APL_0443. This study revealed that A. pleuropneumoniae gene expression, including those encoding virulence factors, was altered in response to both catecholamines. The differential regulation of A. pleuropneumoniae gene expression by the two hormones suggests that this pathogen may have multiple responsive systems for the two catecholamines