Search

The opportunistic pathogen Pseudomonas aeruginosa has gained precedence over the years due to its ability to develop resistance to existing antibiotics, thereby necessitating alternative strategies to understand and combat the bacterium. Our previous work identified the interaction between the bacterial lectin LecA and its host cell glycosphingolipid receptor globotriaosylceramide (Gb3) as a crucial step for the engulfment of P. aeruginosa via the lipid zipper mechanism. In this study, we define the LecA-associated host cell membrane domain by pull-down and mass spectrometry analysis. We unraveled a predilection of LecA for binding to saturated, long fatty acyl chain-containing Gb3 species in the extracellular membrane leaflet and an induction of dynamic phosphatidylinositol (3,4,5)-trisphosphate (PIP 3 ) clusters at the intracellular leaflet co-localizing with sites of LecA binding. We found flotillins and the GPI-anchored protein CD59 not only to be an integral part of the LecA-interacting membrane domain, but also majorly influencing bacterial invasion as depletion of either of these host cell proteins resulted in about 50% reduced invasiveness of the P. aeruginosa strain PAO1. In summary, we report that the LecA-Gb3 interaction at the extracellular leaflet induces the formation of a plasma membrane domain enriched in saturated Gb3 species, CD59, PIP 3 and flotillin thereby facilitating efficient uptake of PAO1.

Introduction: Nephronophthisis (NPH) is an inherited form of cystic kidney disease with various extrarenal manifestations accounting for the largest amount of endstage renal disease in childhood. Patient mutations of Anks6 have also been found to cause NPH like phenotypes in animal models. However, little is known about functionality of Anks6., Objectives/methods: We investigated the impact of Anks6 depletion on cellular metabolism of inner medullary collecting duct cells by GC-MS profiling and targeted LC-MS/MS analysis using two different shRNA cell lines for tetracycline-inducible Anks6 downregulation, namely mIMCD3 krab shANKS6 i5 2 and mIMCD3 krab shANKS6 i1 2 ., Results: In combination, we could successfully identify 158 metabolites of which 20 compounds showed similar alterations in both knockdown systems. Especially, large neutral amino acids, such as phenylalanine, where found to be significantly downregulated indicating disturbances in amino acid metabolism. Arginine, lysine and spermidine, which play an important role in cell survival and proliferation, were found to be downregulated. Accordingly, cell growth was diminished in tet treated mIMCD3 krab shANKS6 i5 2 knockdown cells. Deoxynucleosides were significantly downregulated in both knockdown systems. Hence, PARP1 levels were increased in tet treated mIMCD3 krab shANKS6 i5 2 cells, but not in tet treated mIMCD3 krab shANKS6 i1 2 cells. However, yH2AX was found to be increased in the latter., Conclusion: In combination, we hypothesise that Anks6 affects DNA damage responses and proliferation and plays a crucial role in physiological amino acid and purine/pyrimidine metabolism.

Endolysosomal cysteine cathepsins functionally cooperate. Cathepsin B (Ctsb) and L (Ctsl) double-knockout mice die 4 weeks after birth accompanied by (autophago-) lysosomal accumulations within neurons. Such accumulations are also observed in mouse embryonic fibroblasts (MEFs) deficient for Ctsb and Ctsl. Previous studies showed a strong impact of Ctsl on the MEF secretome. Here we show that Ctsb alone has only a mild influence on extracellular proteome composition. Protease cleavage sites dependent on Ctsb were identified by terminal amine isotopic labeling of substrates (TAILS), revealing a prominent yet mostly indirect impact on the extracellular proteolytic cleavages. To investigate the cooperation of Ctsb and Ctsl, we performed a quantitative secretome comparison of wild-type MEFs and Ctsb (-/-) Ctsl (-/-) MEFs. Deletion of both cathepsins led to drastic alterations in secretome composition, highlighting cooperative functionality. While many protein levels were decreased, immunodetection corroborated increased levels of matrix metalloproteinase (MMP)-2. Re-expression of Ctsl rescues MMP-2 abundance. Ctsl and to a much lesser extent Ctsb are able to degrade MMP-2 at acidic and neutral pH. Addition of active MMP-2 to the MEF secretome degrades proteins whose levels were also decreased by Ctsb and Ctsl double deficiency. These results suggest a degradative Ctsl-MMP-2 axis, resulting in increased MMP-2 levels upon cathepsin deficiency with subsequent degradation of secreted proteins such as collagen α-1 (I).