Biology subjects

Junne, T.

Publications and source records attributed to Junne, T..

3 recordsLinked to original sources

bioRxiv2023

Transwell-based microphysiological platform for high-resolution imaging of airway tissues

Transwell-based airway models have become increasingly important to study the effects of respiratory diseases and drug treatment at the air-liquid interface of the lung epithelial barrier. However, the underlying mechanisms at tissue and cell level often remain unclear, as transwell inserts feature limited live-cell imaging compatibility. Here, we report on a novel microphysiological platform for the cultivation of transwell-based lung tissues providing the possibility to alternate between air-liquid and liquid-liquid interfaces. While the air-liquid interface recapitulates physiological conditions for the lung model, the liquid-liquid interface enables live-imaging of the tissue at high spatiotemporal resolution. The plastics-based microfluidic platform enables insertion and recuperation of the transwell inserts, which allows for tissue cultivation and analysis under standardized well plate conditions. We used the device to monitor infections of Pseudomonas aeruginosa in human stem-cell-derived bronchial epithelial tissue. We continuously imaged the progression of a P. aeruginosa infection in real time at high resolution, which provided insights into bacterial spreading and invasion on the apical tissue surface, as well as insights into tissue breaching and destruction over time. The airway tissue culture system is a powerful tool to visualize and elucidate key processes of developing respiratory diseases and to facilitate drug testing and development.

bioengineering↗

bioRxiv2023

Goblet cell invasion promotes breaching of respiratory epithelia by an opportunistic human pathogen

While commensal bacteria generally respect natural barriers of the human body, pathogens are able to breach epithelia, invade deeper tissue layers and cause life-threatening infections. Pseudomonas aeruginosa, an opportunistic human pathogen, is a leading cause of severe hospital-acquired pneumonia, with mortality rates as high as 50% in mechanically ventilated patients1-3. Effective colonization and breaching of lung mucosa are hallmarks of P. aeruginosa pathogenesis4. Although virulence factors and behavioral strategies of P. aeruginosa have been described5,6, it has remained unclear how this pathogen disseminates on functional mucosal surfaces, how it avoids mucociliary clearance and how it invades the tissue barrier. Using fully differentiated human lung epithelia, we demonstrate that P. aeruginosa efficiently spreads on the apical tissue surface before it breaches epithelia by specifically invading mucus secreting goblet cells. Internalization leads to host cell death and expulsion and the formation of ruptures of the epithelial barrier. Rupture sites are rapidly colonized by extracellular bacteria through active chemotaxis, leading to increasing tissue damage and successful pathogen translocation to the unprotected basolateral side of the epithelium. We show that cell invasion is promoted by two Type-6 toxin secretion systems (T6SS), while Type-3 (T3SS) mediates cell death of infected goblet cells. T3SS mutants invade goblet cells normally, but internalized bacteria fail to trigger goblet cell expulsion and instead show unrestrained intracellular replication. While the effective shedding of infected host cells reveals potent tissue protection mechanisms, the discovery of an intracellular lifestyle of P. aeruginosa in human lung epithelia provides new entry points into investigating the intersection of antibiotic and immune mechanisms during lung infections. By demonstrating that P. aeruginosa uses a combination of specific virulence factors and collective behavior to invade goblet cells and breach the lung tissue barrier from within, these studies reveal novel mechanisms underlying lung infection dynamics under physiological conditions.

microbiology↗

bioRxiv2022

A common mechanism of Sec61 translocon inhibition by small molecules

The Sec61 complex forms a protein-conducting channel in the endoplasmic reticulum (ER) membrane that is required for secretion of soluble proteins and production of many membrane proteins. Several natural and synthetic small molecules specifically inhibit the Sec61 channel, generating cellular effects that are potentially useful for therapeutic purposes, but their inhibitory mechanisms remain unclear. Here we present near-atomic-resolution structures of the human Sec61 channel inhibited by a comprehensive panel of structurally distinct small molecules-- cotransin, decatransin, apratoxin F, ipomoeassin F, mycolactone, cyclotriazadisulfonamide (CADA) and eeyarestatin I (ESI). Remarkably, all inhibitors bind to a common lipid-exposed pocket formed by the partially open lateral gate and plug domain of the channel. Mutations conferring resistance to the inhibitors are clustered at this binding pocket. The structures indicate that Sec61 inhibitors stabilize the plug domain of Sec61 in a closed state, thereby preventing the protein-translocation pore from opening. Our study reveals molecular interactions between Sec61 and its inhibitors in atomic detail and offers the structural framework for further pharmacological studies and drug design.

biochemistry↗