Toll-Like Receptor 2 and Lipoprotein-Like Lipoproteins Enhance Staphylococcus aureus Invasion in Epithelial Cells.

Staphylococcus aureus contains a certain subclass of lipoproteins, the so-called lipoprotein-like lipoproteins (Lpl's), that not only represent Toll-like receptor 2 (TLR2) ligands but are also involved in host cell invasion. Here we addressed the question of which factors contribute to Lpl-mediated invasion of epithelial cells and keratinocytes. For this purpose, we compared the invasiveness of USA300 and its Δlpl mutant under different conditions. In the presence of the matrix proteins IgG, fibrinogen (Fg), and fibronectin (Fn), and of fetal bovine serum (FBS), the invasion ratio was increased in both strains, and always more in USA300 than in its Δlpl mutant. Interestingly, when we compared the invasion of HEK-0 and HEK-TLR2 cells, the cells expressing TLR2 showed a 9-times-higher invasion frequency. When HEK-TLR2 cells were additionally stimulated with a synthetic lipopeptide, Pam3CSK4 (P3C), the invasion frequency was further increased. A potential reason for the positive effect of TLR2 on invasion could be that TLR2 activation by P3C also activates F-actin formation. Here we show that S. aureus invasion depends on a number of factors, on the host side as well as on the bacterial side.

Proteome and phosphoproteome analysis of commensally induced dendritic cell maturation states.

Dendritic cells (DCs) can shape the immune system towards an inflammatory or tolerant state depending on the bacterial antigens and the environment they encounter. In this study we provide a proteomic catalogue of differentially expressed proteins between distinct DC maturation states, brought about by bacteria that differ in their endotoxicity. To achieve this, we have performed proteomics and phosphoproteomics on murine DC cultures. Symbiont and pathobiont bacteria were used to direct dendritic cells into a semi-mature and fully-mature state, respectively. The comparison of semi-mature and fully-mature DCs revealed differential expression in 103 proteins and differential phosphorylation in 118 phosphosites, including major regulatory factors of central immune processes. Our analyses predict that these differences are mediated by upstream elements such as SOCS1, IRF3, ABCA1, TLR4, and PTGER4. Our analyses indicate that the symbiont bacterial strain affects DC proteome in a distinct way, by downregulating inflammatory proteins and activating anti-inflammatory upstream regulators. Biological significance In this study we have investigated the responses of immune cells to distinct bacterial stimuli. We have used the symbiont bacterial strain B. vulgatus and the pathobiont E. coli strain to stimulate cultured primary dendritic cells and performed a shotgun proteome analysis to investigate the protein expression and phosphorylation level differences on a genome level. We have observed expression and phosphorylation level differences in key immune regulators, transcription factors and signal transducers. Moreover, our subsequent bioinformatics analysis indicated regulation at several signaling pathways such as PPAR signaling, LXR/RXR activation and glucocorticoid signaling pathways, which are not studied in detail in an inflammation and DC maturation context. Our phosphoproteome analysis showed differential phosphorylation in 118 phosphosites including those belonging to epigenetic regulators, transcription factors and major cell cycle regulators. We anticipate that our study will facilitate further investigation of immune cell proteomes under different inflammatory and non-inflammatory conditions.

Staphylococcus aureus Lpl Lipoproteins Delay G2/M Phase Transition in HeLa Cells.

The cell cycle is an ordered set of events, leading to cell growth and division into two daughter cells. The eukaryotic cell cycle consists of interphase (G1, S, and G2 phases), followed by the mitotic phase and G0 phase. Many bacterial pathogens secrete cyclomodulins that interfere with the host cell cycle. In Staphylococcus aureus four cyclomodulins have been described so far that all represent toxins and are secreted into the culture supernatant. Here we show that the membrane-anchored lipoprotein-like proteins (Lpl), encoded on a genomic island called νSaα, interact with the cell cycle of HeLa cells. By comparing wild type and lpl deletion mutant it turned out that the lpl cluster is causative for the G2/M phase transition delay and also contributes to increased invasion frequency. The lipoprotein Lpl1, a representative of the lpl cluster, also caused G2/M phase transition delay. Interestingly, the lipid modification, which is essential for TLR2 signaling and activation of the immune system, is not necessary for cyclomodulin activity. Unlike the other staphylococcal cyclomodulins Lpl1 shows no cytotoxicity even at high concentrations. As all Lpl proteins are highly conserved there might be a common function that is accentuated by their multiplicity in a tandem gene cluster. The cell surface localized Lpls' suggests a correlation between G2/M phase transition delay and host cell invasion.