Introduction
Shigella flexneri is a gram-negative bacterial pathogen that
causes communicable human bacillary dysentery (Trofa et al., 1999).
During infection, S. flexneri manipulate various host processes
by delivering effector proteins into host cells via its type III
secretion system (T3SS) to promote development of its intracellular
replicative niche and intercellular transmission (Mattock et al., 2017).
Bacterial effectors are always found to be enzymes that often target
host substrates for post-translational modifications (PTM) (Chambers et
al., 2020). Among S. flexneri T3SS effectors, OspC family
proteins, including OspC1, OspC2, and OspC3, have recently been
demonstrated to possess ADP-riboxanase activity and catalyze
ADP-riboxanation (541.06 Da), a derivative of ADP-ribosylation (524.03
Da) (Li et al., 2021). In the same study, human caspase-4 and mouse
caspase-11 were identified to be the host targets of OspC3, which blocks
the host pyroptosis-mediated defence. However, the host targets of its
paralogues, OspC1 and OspC2, are still unknown and remain to be further
investigated.
Accumulating evidence indicates that secreted bacterial nucleic acids
could also be recognized by the traditionally believed cytosolic viral
RNA and DNA sensors RIG-I, MDA5, and STING thereby triggering
IFN-β production and anti-bacterial defence (Abdullah et al.,
2012; Dobbs et al., 2015). To establish a vacuolar niche inside host
cells, bacterial pathogens have evolved effectors to hijack these innate
immune pathways. For instance, the Coxiella burnetii effector
EmcB acts as a deubiquitinase and thwarts host RIG-I signaling by
preferentially cleaving its K63-linked ubiquitin chains (Duncan-Lowey et
al., 2023), whereas the S. flexneri effector IpaJ blocks STING
translocation from endoplasmic reticulum (ER) to ER-Golgi intermediate
compartments (ERGIC) thereby inhibiting its activation (Dobbs et al.,
2015).
In the present study, we identified HERC5 as a host substrate of
ADP-riboxanation catalyzed by S. flexneri effector OspC1 using a
previously described strategy involving eAf1521 enrichment and mass
spectrometry (MS)-based proteomics (Jin et al., 2023). HERC5 is highly
inducible upon type I interferon stimulation and bacterial infection
(Han et al., 2018), and functions as an E3 ISG15 ligase catalyzing the
ISGylation of proteins (i.e., RIG-I, MDA5, and IRF3) implicated in
innate immunity (Kim et al., 2008; Liu et al., 2021; Shi et al., 2010).
Given that ISG15 and ISGylation themselves counteract Listeriamonocytogenes and mycobacteria infection via different mechanisms
(Radoshevich et al., 2015; Swaim et al., 2017), we proposed thatS. flexneri effector OspC1 may subvert host anti-bacterial
defence by targeting HERC5 for ADP-riboxanation and thus inhibiting
HERC5-catalyzed ISGylation. However, to our surprise, OspC1-catalyzed
HERC5 ADP-riboxanation promotes its E3 ISG15 ligase activity and the
overall ISGylation levels in the whole cell lysates slightly. To
understand this confusing experimental result, we extensively reviewed
the related literatures in detail, and found that not all ISGylation
positively regulate host innate immune response. Indeed, HERC5-catalyzed
ISGylation of MDA5 and IRF3 promote the host antiviral immunity (Liu et
al., 2021; Shi et al., 2010), whereas HERC5-catalyzed RIG-I ISGylation
induced by Type I interferon negatively regulates RIG-I-mediated
antiviral signaling as a feedback mechanism (Kim et al., 2008). It is
reasonable that HERC5 targets different proteins for ISGylaton to
positively and negatively regulate the host immunity precisely and thus
avoid excessive inflammatory damage. Inspired by this finding, we
determined two documented HERC5-catalyzed ISGylation substrates (i.e.,
HERC5 itself and RIG-I) with ISG15-conjugating system in the presence of
wild-type or catalytically mutated OspC1 (Wong et al., 2006; Kim et al.,
2008; Zhao et al., 2005), and found that OspC1-mediated HERC5
ADP-riboxanation upregulates the ISGylation of HERC5 and RIG-I,
resulting in the downregulation of host anti-Shigella defence.