Discussion
In this study, we identified HERC5 as a host substrate of ADP-riboxanation catalyzed by S. flexneri effector OspC1, which promoted its E3 ISG15 ligase activity. Considering that HERC5 itself showed quite strong enzymatic activity in the overexpression system (Figure 4b), it was not surprising to observe that the ISGylation was subtly enhanced by ADP-riboxanated HERC5, comparing to unmodified HERC5 (Figure 4c). Besides, ADP-riboxanated HERC5 specifically upregulates the ISGylation of certain substrates with varying degrees (Figure 4d-e), so that the global change of ISGylation on the whole cell level were neutralized by those unchanged or changed slightly.
In addition to ADP-riboxanated peptide, ADP-ribosylated peptide of HERC5 was also detected in our LC-MS/MS analysis (Figure 3a). Coincidentally, ADP-ribosylated peptide of caspase-3 had also been detected when studying ADP-riboxanation (also designated as ADPR-deacylization) of caspasa-3 catalyzed by C. violaceum CopC (Peng et al., 2022). The trace amount of ADP-ribosylated peptide of caspase-3 detected led them to interpret it as the intermediate of ADP-riboxanated caspase-3. However, the stoichiometric ratio of ADP-ribosylated HERC5 to ADP-riboxanated HERC5 observed in our system was 100 times higher than that of ADP-ribosylated caspase-3 to ADP-riboxanated caspase-3 (Figure 3b). Despite no additional experimental evidence currently, we tend to believe ADP-ribosylated HERC5 is the reversible product of ADP-riboxanated HERC5 (Figure 3c).
Traditionally, cytosolic nucleic acid sensors such as RIG-I, MDA5, and STING, and downstream transcription factor IRF3 were believed to only respond to viral RNA or DNA. However, a growing body of studies suggests that they also sense bacterial DNA and RNA and induce innate immune response defensing against bacterial infection by promoting the expression of type I interferons. Upon bacterial infection and type I interferon stimulation, the ISG15-conjugating system are also highly induced to modify a huge subset of host substrates, especially those participating anti-bacterial defence, by ISGylation. As mentioned above, RIG-I and STING activation were blocked by the C. burnetiieffector EmcB and S. flexneri effector IpaJ respectively (Duncan-Lowey et al., 2023; Dobbs et al., 2015), indicating that RIG-I and STING signaling play indispensable roles in anti-bacterial defence. Additionally, bacteria also evolved strategies to counteract host ISG15- and ISGylation-mediated anti-bacterial defence (Radoshevich et al., 2015; Swaim et al., 2017). As shown in Figure 5, we found that S. flexneri effector OspC1 could target host E3 ISG15 ligase HERC5 for ADP-riboxanation and then specifically upregulate the ISGylation of RIG-I, thereby inhibiting its activation. It provided novel insights into the roles of ADP-riboxanation employed by S. flexnerito manipulate host anti-bacterial defence.