Gutsmann, T. | Search bioRxiv

bioRxiv2024

Cytoadhesion of Plasmodium falciparum-infected red blood cells changes the expression of cytokine-, histone- and antiviral protein-encoding genes in brain endothelial cells

Malaria remains a significant global health problem, mainly due to Plasmodium falciparum, which is responsible for most fatal infections. Infected red blood cells (iRBCs) evade spleen clearance by adhering to endothelial cells (ECs), triggering capillary blockage, inflammation, endothelial dysfunction and altered vascular permeability, prompting an endothelial transcriptional response. The iRBCIT4var04/HBEC-5i model, where iRBCs present IT4var04 (VAR2CSA) on their surface, was used to analyse the effects of iRBC binding on ECs at different temperature (37{degrees}C vs. 40{degrees}C). Binding of non-infected RBCs (niRBCs) and fever alone altered the expression of hundreds of genes in ECs. Comparing the expression profile of HBEC-5i cells cultured either in the presence of iRBCs or in the presence of niRBCs revealed significant upregulation of genes linked to immune response, nucleosome assembly, NF-kappa B signaling, angiogenesis, and antiviral immune response/interferon-alpha/beta signaling. Raising the temperature to 40{degrees}C, simulating fever, led to further upregulation of many genes, particularly those involved in cytokine production and angiogenesis. In summary, the presence of iRBCs stimulates ECs, activating several immunological pathways and affecting antiviral (- parasitic) mechanisms and angiogenesis. Our data uncovered the induction of the interferon-alpha/beta signaling pathway in ECs in response to iRBCs.

microbiology↗

bioRxiv2023

Structure-function relationships underpin disulfide loop cleavage-dependent activation of Legionella pneumophila lysophosholipase A PlaA

Legionella pneumophila, the causative agent of a life-threatening pneumonia, intracellularly replicates in a specialized compartment in lung macrophages, the Legionella-containing vacuole (LCV). Secreted proteins of the pathogen govern important steps in the intracellular life cycle including bacterial egress. Among these is the type II secreted PlaA which, together with PlaC and PlaD, belongs to the GDSL phospholipase family found in L. pneumophila. PlaA shows lysophospholipase A (LPLA) activity which increases after secretion and subsequent processing by the zinc metalloproteinase ProA at residue E266/L267 located within a disulfide loop. Activity of PlaA contributes to the destabilization of the LCV in the absence of the type IVB-secreted effector SdhA. We here present the 3D structure of PlaA which shows a typical /{beta} hydrolase fold and reveals that the uncleaved disulfide loop forms a lid structure covering the catalytic triad S30/D278/H282. This leads to reduction of both substrate access and membrane interaction before activation; however, the catalytic and membrane interaction site gets more accessible when the disulfide loop is processed. After structural modelling, a similar activation process is suggested for the GDSL hydrolase PlaC, but not for PlaD. Furthermore, the size of the PlaA substrate binding site indicated preference towards phospholipids comprising ~16 carbon fatty acid residues which was verified by lipid hydrolysis, suggesting a molecular ruler mechanism. Indeed, mutational analysis changed the substrate profile with respect to fatty acid chain length. In conclusion, our analysis revealed the structural basis for the regulated activation and substrate preference of PlaA.

biochemistry↗