Photorhabdus luminescens is an entomopathogenic bacterium that kills insect larvae by secreting toxic proteins. One of the major toxins produced by bacteria is TccC3, which ADP-ribosylates Thr148 residue of actin – an essential cytoskeletal protein. This modification causes excessive clustering of filamentous-actin (F-actin) and leads to inhibition of phagocytosis in insect immune cells. Thr148 is located near a major binding site for many actin-binding proteins (ABPs). Accordingly, Thr148-ADP-ribosylation weakens the interaction of actin with thymosin β4 (TMSB4), cofilin, and gelsolin, compromising their functional activities. For actin-specific toxins, large-scale toxicity can be expected when only a small fraction of actin is modified. Yet, the effects of partially ADP-ribosylated-actin were not studied. We re-evaluated the effects of partially ADP-ribosylated-actin by quantitative methods while also extending the panel of tested ABPs. We found that the modification of G-actin is inhibited by profilin and TMSB4, while that of F-actin is inhibited by tropomyosin (TM). These constraints leave only the dynamic, TM-free fraction of F-actin as TccC3’s physiological substrate. We confirmed previous findings that Thr148-ADP-ribosylation potently inhibited cofilin-facilitated actin depolymerization. However, the inhibition was weak when a small fraction of F-actin was ADP-ribosylated as expected at the early stages of infection. While spontaneous polymerization of actin was mildly weakened by Thr148-ADP-ribosylation, it was faster in the presence of profilin and TMSB4. Furthermore, the modification promoted the Arp2/3 complex-mediated actin polymerization. The predicted net effect of these changes on actin dynamics are i) accelerated spontaneous nucleation (normally minimal or non-existing) as well as ii) potentiated Arp2/3-controlled nucleation of actin filaments at undesirable cellular locations. In F-actin, Thr148 is located by the D-loop of an adjacent actin subunit. In agreement with D-loop being a part of a tandem calponin homology (t-CH) domain binding site, we found that Thr148-ADP-ribosylation potently inhibited actin-bundling ability of t-CH proteins plastins and α-actinin, but not filamin. Since α-actinin is a component of stress fibers and plastins are involved in the stabilization of the cell cortex, lamellipodia, and microvilli, the observed effects are expected to contribute to the disorganization of actin superstructures.

Functional consequences of actin ADP-ribosylation by Photorhabdus luminescens TccC3 toxin

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