Pseudomonas aeruginosa exoenzyme Y directly bundles actin filaments

Abstract

Pseudomonas aeruginosa uses a type III secretion system (T3SS) to inject cytotoxic effector proteins into host cells. The promiscuous nucleotidyl cyclase, exoenzyme Y (ExoY), is one of the most common effectors found in clinical P. aeruginosa isolates. Recent studies have revealed that the nucleotidyl cyclase activity of ExoY is stimulated by actin filaments (F-actin) and that ExoY alters actin cytoskeleton dynamics in vitro, via an unknown mechanism. The actin cytoskeleton plays an important role in numerous key biological processes and is targeted by many pathogens to gain competitive advantages. We utilized total internal reflection fluorescence microscopy, bulk actin assays, and EM to investigate how ExoY impacts actin dynamics. We found that ExoY can directly bundle actin filaments with high affinity, comparable with eukaryotic F-actin–bundling proteins, such as fimbrin. Of note, ExoY enzymatic activity was not required for F-actin bundling. Bundling is known to require multiple actin-binding sites, yet small-angle X-ray scattering experiments revealed that ExoY is a monomer in solution, and previous data suggested that ExoY possesses only one actin-binding site. We therefore hypothesized that ExoY oligomerizes in response to F-actin binding and have used the ExoY structure to construct a dimer-based structural model for the ExoY–F-actin complex. Subsequent mutational analyses suggested that the ExoY oligomerization interface plays a crucial role in mediating F-actin bundling. Our results indicate that ExoY represents a new class of actin-binding proteins that modulate the actin cytoskeleton both directly, via F-actin bundling, and indirectly, via actin-activated nucleotidyl cyclase activity.

Pseudomonas aeruginosa: An Audacious Pathogen with an Adaptable Arsenal of Virulence Factors

“…Consequently, it causes irreversible actin microtubule disassembly, cell necrosis, and alteration of endothelial barrier integrity, following lung injury and end-organ dysfunction [ 120 , 122 ]. Recently, it was shown that ExoY possesses one actin-binding site that directly bundles actin filaments in the host cell [ 123 ]. Strikingly, ExoY downregulates the activation of transforming growth factor β-activated kinase 1 (TAK1), thereby inhibiting the production of proinflammatory cytokines by both macrophages and epithelial cells [ 124 ].…”

Bacterial Nucleotidyl Cyclases Activated by Calmodulin or Actin in Host Cells: Enzyme Specificities and Cytotoxicity Mechanisms Identified to Date

“…Pa -ExoY can indeed alter F-actin turnover independently of its NC activity in Pa -ExoY-transfected epithelial cells [ 17 ]. Consistent with effects observed in cells, an inactive variant of Pa -ExoY inhibits the acceleration of F-actin disassembly induced by the regulatory ABP ADF (actin-depolymerizing factor)/cofilin in vitro [ 17 ] or can induce F-actin bundling in vitro [ 99 ]. Our recent biochemical studies of Vn -ExoY have also shown that its interaction with free or profilin-bound actin affects actin self-assembly dynamics in vitro independently of the NC activity of this toxin [ 101 ].…”

“…RID-mediated inactivation of Rac1 leads to actin cytoskeleton disruption and inhibits, together with ABH, pro-inflammatory response [ 98 ]. ExoY may also impact actin self-assembly [ 17 , 99 ]. ACD induces the formation of toxic actin monomers [ 23 , 100 ] that will lead, together with RID, to a disruption of tight junctions (TJ).…”

Functional and structural insights into the multi-step activation and catalytic mechanism of bacterial ExoY nucleotidyl cyclase toxins bound to actin-profilin

“…Further investigations are needed to see whether the formation of such a Vn-ExoY:G-actin:profilin abortive ternary complex for barbed-end elongation, which can be recruited by PRMs of elongation factors or other modular ABPs, may also contribute to Vn-ExoY cytotoxicity in host cells independently of its NC activity. Several observations suggest that Pa-ExoY may also affect eukaryotic target cells independently of its enzymatic activity (5,41,42). The interaction of Vn-ExoY with actin:profilin may be especially important for its subcellular localisation and activation at regions of active actin cytoskeleton remodelling, where G-actin:profilin and elongation-promoting factors such as formin or VASP together play important roles (40,(43)(44)(45).…”