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.