Although considered to be an extracellular pathogen, Staphylococcus aureus is able to invade a variety of mammalian, non-professional phagocytes and can also survive engulfment by professional phagocytes such as neutrophils and monocytes. In both of these cell types S. aureus promptly escapes from the endosomes/phagosomes and proliferates within the cytoplasm, which quickly leads to host cell death. In this report we show that S. aureus interacted with human monocyte-derived macrophages in a very different way to those of other mammalian cells. Upon phagocytosis by macrophages, S. aureus persisted intracellularly in vacuoles for 3–4 days before escaping into the cytoplasm and causing host cell lysis. Until the point of host cell lysis the infected macrophages showed no signs of apoptosis or necrosis and were functional. They were able to eliminate intracellular staphylococci if prestimulated with interferon-γ at concentrations equivalent to human therapeutic doses. S. aureus survival was dependent on the alternative sigma factor B as well as the global regulator agr, but not SarA. Furthermore, isogenic mutants deficient in α-toxin, the metalloprotease aureolysin, protein A, and sortase A were efficiently killed by macrophages upon phagocytosis, although with different kinetics. In particular α-toxin was a key effector molecule that was essential for S. aureus intracellular survival in macrophages. Together, our data indicate that the ability of S. aureus to survive phagocytosis by macrophages is determined by multiple virulence factors in a way that differs considerably from its interactions with other cell types. S. aureus persists inside macrophages for several days without affecting the viability of these mobile cells which may serve as vehicles for the dissemination of infection.

Cucurbit[n]urils (CB[n]) are potential stabilizing, solubilizing, activating, and delivering agents for drugs. The toxicity of the macrocyclic host molecules cucurbit[7]uril (CB[7]), the most water-soluble homologue, as well as cucurbit[8]uril (CB[8]) has been evaluated. In vitro studies on cell cultures revealed an IC₅₀ value of 0.53 ± 0.02 mM for CB[7], corresponding to around 620 mg of CB[7] per kg of cell material. Live-cell imaging studies performed on cells treated with subtoxic amounts of CB[7] showed no detrimental effects on the cellular integrity as assessed by mitochondrial activity. For CB[8], no significant cytotoxicity was observed within its solubility range. The bioadaptability of the compounds was further examined through in vivo studies on mice, where intravenous administration of CB[7] showed a maximum tolerated dosage of 250 mg kg⁻¹, while oral administration of a CB[7]/CB[8] mixture showed a tolerance of up to 600 mg kg⁻¹. The combined results indicate a sufficiently low toxicity to encourage further exploration of CB[n] as additives for medicinal and pharmaceutical use.

Background: Cysteine cathepsins are known to primarily cleave their substrates at reducing and acidic conditions within endo-lysosomes. Nevertheless, they have also been linked to extracellular proteolysis, that is, in oxidizing and neutral environments. Although the impact of reducing or oxidizing conditions on proteolytic activity is a key to understand physiological protease functions, redox conditions have only rarely been considered in routine enzyme activity assays. Therefore we developed an assay to test for proteolytic processing of a natural substrate by cysteine cathepsins which accounts for redox potentials and pH values corresponding to the conditions in the extracellular space in comparison to those within endo-lysosomes of mammalian cells. Results: The proteolytic potencies of cysteine cathepsins B, K, L and S towards thyroglobulin were analyzed under conditions simulating oxidizing versus reducing environments with neutral to acidic pH values. Thyroglobulin, the precursor molecule of thyroid hormones, was chosen as substrate, because it represents a natural target of cysteine cathepsins. Thyroglobulin processing involves thyroid hormone liberation which, under physiological circumstances, starts in the extracellular follicle lumen before being continued within endo-lysosomes. Our study shows that all cathepsins tested were capable of processing thyroglobulin at neutral and oxidizing conditions, although these are reportedly non-favorable for cysteine proteases. All analyzed cathepsins generated distinct fragments of thyroglobulin at extracellular versus endo-lysosomal conditions as demonstrated by SDS-PAGE followed by immunoblotting or N-terminal sequencing. Moreover, the thyroid hormone thyroxine was liberated by the action of cathepsin S at extracellular conditions, while cathepsins B, K and L worked most efficiently in this respect at endo-lysosomal conditions. Conclusion: The results revealed distinct cleavage patterns at all conditions analyzed, indicating compartment-specific processing of thyroglobulin by cysteine cathepsins. In particular, proteolytic activity of cathepsin S towards the substrate thyroglobulin can now be understood as instrumental for extracellular thyroid hormone liberation. Our study emphasizes that the proteolytic functions of cysteine cathepsins in the thyroid are not restricted to endo-lysosomes but include pivotal roles in extracellular substrate utilization. We conclude that understanding of the interplay and fine adjustment of protease networks in vivo is better approachable by simulating physiological conditions in protease activity assays.

The histone‐like protein HU is a highly abundant DNA architectural protein that is involved in compacting the DNA of the bacterial nucleoid and in regulating the main DNA transactions, including gene transcription. However, the coordination of the genomic structure and function by HU is poorly understood. Here, we address this question by comparing transcript patterns and spatial distributions of RNA polymerase in Escherichia coli wild‐type and hupA/B mutant cells. We demonstrate that, in mutant cells, upregulated genes are preferentially clustered in a large chromosomal domain comprising the ribosomal RNA operons organized on both sides of OriC. Furthermore, we show that, in parallel to this transcription asymmetry, mutant cells are also impaired in forming the transcription foci—spatially confined aggregations of RNA polymerase molecules transcribing strong ribosomal RNA operons. Our data thus implicate HU in coordinating the global genomic structure and function by regulating the spatial distribution of RNA polymerase in the nucleoid.

While mast cells and basophils constitutively express the high-affinity IgE receptor (FcεRI), it is absent or weakly expressed on APCs from normal donors. FcεRI is strongly upregulated on APCs from atopic donors and involved in the pathophysiology of atopic diseases. Despite its clinical relevance, data about FcεRI regulation on APCs are scarce. We show that in all donors intracellular α chain of the FcεRI (FcεRIα) accumulates during DC differentiation from monocytes. However, expression of γ chains of the FcεRI (FcεRIγ), mandatory for surface expression, is downregulated. It is low or negative in DCs from normal donors lacking surface FcεRI (FcεRI^neg DCs). In contrast, DCs from atopics express surface FcεRI (FcεRI^pos DCs) and show significant FcεRIγ expression, which can be coprecipitated with FcεRIα. In FcεRI^neg DCs lacking FcεRIγ, immature and core glycosylated FcεRIα accumulates in the endoplasmic reticulum. In FcεRI^pos DCs expressing FcεRIγ, an additional mature form of FcεRIα exhibiting complex glycosylation colocalizes with FcεRIγ in the Golgi compartment. IgE binding sustains surface-expressed FcεRI on DCs from atopic donors dependent on baseline protein synthesis and transport and enhances their IgE-dependent APC function. We propose that enhanced FcεRI on DCs from atopic donors is driven by enhanced expression of otherwise limiting amounts of FcεRIγ and is preserved by increased IgE levels.