Pseudomonas aeruginosa exoenzymes U and Y induce a transmissible endothelial proteinopathy

Abstract

We tested the hypothesis that Pseudomonas aeruginosa type 3 secretion system effectors exoenzymes Y and U (ExoY and ExoU) induce release of a highmolecular-weight endothelial tau, causing transmissible cell injury characteristic of an infectious proteinopathy. Both the bacterial delivery of ExoY and ExoU and the conditional expression of an activity-attenuated ExoU induced time-dependent pulmonary microvascular endothelial cell gap formation that was paralleled by the loss of intracellular tau and the concomitant appearance of high-molecular-weight extracellular tau. Transfer of the highmolecular-weight tau in filtered supernatant to naïve endothelial cells resulted in intracellular accumulation of tau clusters, which was accompanied by cell injury, interendothelial gap formation, decreased endothelial network stability in Matrigel, and increased lung permeability. Tau oligomer monoclonal antibodies captured monomeric tau from filtered supernatant but did not retrieve higher-molecular-weight endothelial tau and did not rescue the injurious effects of tau. Enrichment and transfer of high-molecularweight tau to naïve cells was sufficient to cause injury. Thus we provide the first evidence for a pathophysiological stimulus that induces release and transmissibility of high-molecular-weight endothelial tau characteristic of an endothelial proteinopathy.

Pseudomonas aeruginosa infection liberates transmissible, cytotoxic prion amyloids

“…Immunoblot studies were performed by using standard procedures (21, 47). Finally, in some experiments, cytotoxic supernatant was treated with a 10‐fold excess of 1,1,1,3,3,3‐hexafluor‐2‐propanol (HFIP; 003409; Oakwood Chemical, Estill, SC, USA) for either 5, 15, or 30 mins.…”

“…Cytotoxicity was then determined by direct observation as previously detailed. BAL fluids were then assayed by immunoblot analysis using previously detailed procedures (21, 47). The protein concentration in each sample was determined by using previously described methods (47), and equal amounts of protein were loaded for each BAL fluid sample.…”

The Pseudomonas aeruginosa Exoenzyme Y: A Promiscuous Nucleotidyl Cyclase Edema Factor and Virulence Determinant

“…Interestingly, ExoU activity is also sufficient to induce oligomeric tau production and release. ExoU visibly damages endothelial cells within 3–4 h, whereas ExoY disrupts cell morphology within 6–8 h (Morrow et al 2016) (Fig. 4).…”

“…Tau accumulates in the endothelial cytosol for several hours following infection, and then is released in a high molecular weight, injurious form (Morrow et al 2016) (see below). Interestingly, tau oligomerization and release closely parallels the increase in cUMP levels.…”

“…We have seen that ExoY functions not only as an adenylyl cyclase, but rather, as a promiscuous purine and pyrimidine nucleotidyl cyclase in vascular endothelium, generating cGMP, cAMP, cUMP and, to a lesser extent, cCMP (Morrow et al 2015). The elevation of these cyclic nucleotide monophosphates causes endothelial tau hyperphosphorylation, which leads to microtubule breakdown and endothelial hyperpermeability (Balczon et al 2013; Morrow et al 2016; Ochoa et al 2012; Prasain et al 2009; Sayner et al 2011). Hyperphosphorylation appears to trigger tau oligomerization and release, contributing to a transmissible proteinopathy that perpetuates cytotoxicity during infection (Morrow et al 2016).…”