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\begin{document}

\title{The ever increasing necessity of mass spectrometry in dissecting protein
post-translational modifications catalyzed by bacterial effectors}



\author[1]{Xiaoyun Liu}%
\author[1]{Jie Jin}%
\author[1]{Yi Yuan}%
\author[1]{Wei Xian}%
\author[1]{Zhiheng Tang}%
\author[2]{Jiaqi Fu}%
\affil[1]{Peking University School of Basic Medical Sciences}%
\affil[2]{The First Hospital of Jilin University}%


\vspace{-1em}



  
  \date{May 25, 2024}


\begingroup
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\selectlanguage{english}
\begin{abstract}
Protein post-translational modifications (PTMs), such as
ADP-ribosylation and phosphorylation, regulate multiple fundamental
biological processes in cells. During bacterial infection, effector
proteins are delivered into host cells through dedicated bacterial
secretion systems and can modulate important cellular pathways by
covalently modifying their host targets. These strategies enable
intruding bacteria to subvert various host processes, thereby promoting
their own survival and proliferation. Despite rapid expansion of our
understanding of effector-mediated PTMs in host cells, analytical
measurements of these molecular events still pose significant challenges
in the study of host-pathogen interactions. Nevertheless, with major
technical breakthroughs in the last two decades, mass spectrometry (MS)
has evolved to be a valuable tool for detecting protein PTMs and mapping
modification sites. Additionally, large-scale PTM profiling, facilitated
by different enrichment strategies prior to MS analysis, allows
high-throughput screening of host enzymatic substrates of bacterial
effectors. In this review, we summarize the advances in the studies of
two representative PTMs (i.e., ADP-ribosylation and phosphorylation)
catalyzed by bacterial effectors during infection. Importantly, we will
discuss the ever increasing role of MS in understanding these molecular
events and how the latest MS-based tools can aid in future studies of
this booming area of pathogenic bacteria-host interactions.%
\end{abstract}%



\sloppy


The ever increasing necessity of mass spectrometry in dissecting protein
post-translational modifications catalyzed by bacterial effectors

Jie Jin\textsuperscript{1}, Yi Yuan\textsuperscript{1}, Wei
Xian\textsuperscript{1}, Zhiheng Tang\textsuperscript{1}, Jiaqi
Fu\textsuperscript{3}, Xiaoyun Liu\textsuperscript{1, 2*}

\textsuperscript{1}Department of Microbiology and Infectious Disease
Center, School of Basic Medical Sciences, Peking University Health
Science Center, Beijing 100191, China

\textsuperscript{2}NHC Key Laboratory of Medical Immunology, Peking
University, Beijing 100191, China

\textsuperscript{3}Department of Respiratory Medicine, Infectious
Diseases and Pathogen Biology Center, Key Laboratory of Organ
Regeneration and Transplantation of the Ministry of Education, State Key
Laboratory for Zoonotic Diseases, The First Hospital of Jilin
University, Changchun 130021, China

\textbf{Key words} :

Mass spectrometry, Bacterial effectors, Post-translational
modifications, ADP-ribosylation, Phosphorylation

*Correspondence

Xiaoyun Liu, Department of Microbiology, School of Basic Medical
Sciences, Peking University Health Science Center, Beijing, China.

Email:\emph{xiaoyun.liu@bjmu.edu.cn}

\textbf{Abstract}

Protein post-translational modifications (PTMs), such as
ADP-ribosylation and phosphorylation, regulate multiple fundamental
biological processes in cells. During bacterial infection, effector
proteins are delivered into host cells through dedicated bacterial
secretion systems and can modulate important cellular pathways by
covalently modifying their host targets. These strategies enable
intruding bacteria to subvert various host processes, thereby promoting
their own survival and proliferation. Despite rapid expansion of our
understanding of effector-mediated PTMs in host cells, analytical
measurements of these molecular events still pose significant challenges
in the study of host-pathogen interactions. Nevertheless, with major
technical breakthroughs in the last two decades, mass spectrometry (MS)
has evolved to be a valuable tool for detecting protein PTMs and mapping
modification sites. Additionally, large-scale PTM profiling, facilitated
by different enrichment strategies prior to MS analysis, allows
high-throughput screening of host enzymatic substrates of bacterial
effectors. In this review, we summarize the advances in the studies of
two representative PTMs (i.e., ADP-ribosylation and phosphorylation)
catalyzed by bacterial effectors during infection. Importantly, we will
discuss the ever increasing role of MS in understanding these molecular
events and how the latest MS-based tools can aid in future studies of
this booming area of pathogenic bacteria-host interactions.



\textbf{INTRODUCTION}

Gram-negative bacterial pathogens such as \emph{Shigella flexneri}
,\emph{Salmonella enterica} , \emph{Legionella pneumophila} , etc.,
cause a broad range of communicable human diseases (Jennison \& Verma,
2004; Knodler \& Elfenbein, 2019; Gon\selectlanguage{ngerman}çalves et al., 2021). Their
virulence is often mediated by dedicated protein secretion systems
(i.e., type III for \emph{S. flexneri} and \emph{S. enterica} or type IV
for \emph{L. pneumophila} ), which can deliver effector proteins
directly into host cells to modulate diverse cellular pathways
(Muthuramalingam et al., 2021; Jennings et al., 2017; Hubber \& Roy,
2010; Isberg et al., 2009). In the last decade, a growing body of
studies has shown that many secreted bacterial effectors harbor
enzymatic activities that often lead to post-translational modifications
(PTMs) of their host targets (Jennings et al., 2017; Mattock \& Blocker,
2017; Qiu \& Luo, 2017; Macek et al., 2019; Chambers \& Scheck, 2020).
From the perspective of bacterial pathogens, covalently modifying key
host proteins represents a more economic and rapid means (relative to
mechanisms altering protein abundance) to regulate host biological
processes including anti-bacterial defense system, thereby promoting
bacterial survival and/or proliferation (Ashida et al., 2014). Indeed,
accumulating evidence suggests that bacteria-mediated modifications of
host targets are much more prevalent than we initially thought.

Rapid growth of this emerging field (i.e., pathogen-mediated host
modifications) is, at least in part, spurred by technical innovations
that facilitate accurate and precise measurements of these molecular
events such as protein PTMs. Among those tools probing covalent
modifications, mass spectrometry (MS) is arguably the most powerful and
versatile analytical approach thanks to major technological
breakthroughs (e.g., in terms of instrument sensitivity, mass resolution
and throughput) that were witnessed in the last two decades.
Nevertheless, the application of this analytical tool in dissecting
effector-mediated PTMs is, at least in our opinion, still far below
where it is supposed to be. This review will focus on the application of
MS-based approaches in the study of host protein modifications catalyzed
by bacterial effectors. Specifically, we will highlight some recent work
on protein ADP-ribosylation (and related ADP-riboxanation) and classical
phosphorylation as examples to illustrate our current understanding of
this fascinating area in host-pathogen interactions (Figure 1).
Furthermore, we will critically discuss the increasing role and
capability of MS in dissolving some looming challenges for further
advancements in the field.

\textbf{ADP-ribosylation and ADP-riboxanation}

ADP-ribosylation involves the attachment of ADP-ribose (ADPr) groups
from nicotinamide adenine dinucleotide (NAD\textsuperscript{+}) to
substrates by enzymes called ADP-ribosyltransferases (ARTs), together
with the release of nicotinamide (NA) (Palazzo et al., 2017). Given
bacterial toxins (i.e., Cholera and Diphtheria toxins) as the first
reported enzymes of this group (Honjo et al., 1968; Cassel \& Pfeuffer,
1978), the other ARTs identified afterwards are often classified as
either Cholera toxin-like ART (ARTC) or Diphtheria toxin-like ART (ARTD)
superfamily (Hottiger et al., 2010). Recently, there seems to be a spike
in the number of reports on bacterial virulence factors possessing ART
activities. For instance, \emph{Salmonella} Typhimurium effector SopF
targets Gln124 of ATP6V0C in host V-ATPase for ADP-ribosylation, thereby
blocking antibacterial autophagy (xenophagy) via disrupting
V-APTase-ATG16L1 association (Xu et al., 2019). In a follow-up study,
the authors found SopF stably associates with ADP-ribosylation factor
(ARF) GTPases and requires these host factors for its activation (Xu et
al., 2022). Another type III effector CetC from \emph{Chromobacterium
violaceum} catalyzes ADP-ribosylation of ubiquitin to disrupt host
ubiquitin signaling, representing the first of its kind on the threonine
residue (Yan et al., 2020). In the above studies, tandem mass
spectrometry (MS/MS) involving dissociation of modified protein
fragments/peptides plays an indispensable role in ascertaining the
modification in the first place as well as pinpointing the exact
modified sites.

Recently, a novel PTM dubbed as ADP-riboxanation, a derivative of
ADP-ribosylation, was uncovered and catalyzed by bacterial effectors
OspC3 and CopC from \emph{S. flexneri} and \emph{C. violaceum} ,
respectively (Li et al., 2021; Peng et al., 2022). Distinct from
ADP-ribosylation, ADP-riboxanation involves an additional deamination
upon the classical modification. In other words, this novel modification
couples two enzymatic reactions through the activity of a single
effector. This unique reaction mechanism was supported by the effector
variants (OspC3\textsuperscript{D177A} and CopC\textsuperscript{D172E})
capable of catalyzing conventional ADP-ribosylation without the
subsequent deamination. The initial assignment/characterization of this
unconventional modification was greatly facilitated by a suite of MS
tools including multi-stage MS (MS/MS and MS/MS/MS), high-resolution
measurements and stable isotope labeling MS. Unlike canonical
arginine\selectlanguage{greek}\emph{N\textsuperscript{ω}} -\selectlanguage{english}ADP-ribosylation (ANT1 by Ceg3 or
Rab4a and Ras by ExoS) (Fu et al., 2022; Bette-Bobillo et al., 1998;
Vareechon et al., 2017), OspC3-catalyzed ADP-riboxanation proceeds
with\selectlanguage{greek}\emph{N\textsuperscript{δ}} -\selectlanguage{english}ADP-ribosylation and subsequent
deamination with the ribosyl-2'-OH of ADPR to remove
one\selectlanguage{greek}\emph{N\textsuperscript{ω}} , \selectlanguage{english}resulting in an oxazolidine ring. Thus
far, both \emph{S. flexneri} OspC3 and \emph{C. violaceum} CopC were
found to modify host caspases via ADP-riboxanation. While OspC3 targets
inflammatory caspases (-4 and -11) to block host pyroptosis (Li et al.,
2021), CopC inactivates a broader spectrum of caspases (-3, -7, -8, and
-9) to interfere with host cell death pathways including apoptosis,
necroptosis and pyroptosis (Peng et al., 2022).

Of note, upon elucidation of enzymatic activities of bacterial
effectors, a rate-limiting step of most studies can often be associated
with the discovery of their modified targets. As discussed above,
enzymatic substrates are mostly identified by inference from perturbed
cellular pathways or through conventional genetic and biochemical
assays. Nevertheless, in many cases one could encounter a scenario where
there are no observable phenotypes associated with the effector of
interest. Then we would argue that cellular ADP-ribosylome profiling
(i.e., global analysis of ADP-ribosylated proteome) can be a generic and
unbiased strategy for high-throughput screening of ART substrates. Such
work is facilitated by the development of an engineered macro
domain-containing protein (eAf1521) from \emph{Archaeoglobus fulgidus} .
This variant of Af1521 exhibits high affinity towards ADP-ribosylated
proteins, thereby allowing efficient enrichment of cellular
ADP-ribosylome prior to MS analyses (Hendriks et al., 2019; Nowak et
al., 2020). Indeed, such a strategy has been applied in the study
of\emph{S.} Typhimurium SopF (Xu et al., 2019) and \emph{L.
pneumophila}effector Ceg3 (Fu et al., 2022; Kubori et al., 2022), though
in a more targeted manner as only those Af1521-enriched proteins with
the size of interest were analyzed by LC-MS. Notably, Ceg3 and Larg1
were found to target ADP/ATP translocases by reversible ADP-ribosylation
in host mitochondria to temporally regulate their ADP/ATP exchange
activity during \emph{L. pneumophila} infection. Alternatively, the use
of an NAD derivative, biotin-17-NAD, in \emph{in vitro} ADP-ribosylation
assays permits pull-down of modified ART substrates prior to MS
identification. By using this strategy, \emph{L. pneumophila} effector
Lart1 was found to ADP-ribosylate yeast glutamate dehydrogenase 2 (Black
et al., 2021).

\textbf{Phosphorylation}

Phosphorylation often occurs on serine, threonine, and tyrosine residues
of target proteins with the addition of phosphate by kinases and its
removal by phosphatases. As a highly efficient modification,
phosphorylation modulates protein functions by altering conformation,
promoting ubiquitination and degradation, influencing protein-protein
interactions, or regulating enzyme activities (Huttlin et al., 2010).
Due to long-standing co-evolution with mammalian host cells, many
bacterial pathogens have evolved effector proteins harboring kinase or
phosphatase activities to directly engage with host phosphorylation
signaling (Grishin et al., 2015). \emph{Yersinia pseudotuberculosis}YpkA
is arguably the first reported prokaryotic virulence factor possessing
Ser/Thr protein kinase activity (Galyov et al., 1993) and its activation
is dependent on a host factor, actin (Barz et~al., 2000, Dukuzumuremyi
et~al., 2000, Juris et~al., 2000). YpkA phosphorylates the
heterotrimeric G protein, G\selectlanguage{greek}α\selectlanguage{english}q, on a critical serine residue, thereby
inhibiting its GTP binding (Navarro et al., 2007). In addition to its
kinase activity, YpkA also possesses the guanidine nucleotide
dissociation inhibitor (GDI) domain, and together they work
synergistically to disrupt host actin cytoskeleton (Prehna et al.,
2006). Later, vasodilator-stimulated phosphoprotein (VASP) was reported
to be another enzymatic substrate of YpkA and its modification led to
the disruption of host actin dynamics (Ke et al., 2015).
\emph{S.}Typhimurium effector SteC harbors kinase activity that is
required for the formation of the F-actin meshwork associated
with\emph{Salmonella} -containing vacuoles (SCVs). Several enzymatic
substrates of SteC have been identified by various approaches, including
Hsp27 (Imaimi et al., 2013), formin family FMNL proteins (Walch et al.,
2021; Poh et al., 2008) and the MAP kinase MEK (Odendall et al., 2012).
Together, SteC-mediated phosphorylation of these target proteins
coordinately modulates the host F-actin cytoskeleton (Heggie et al.,
2021).

\emph{L. pneumophila} exploits phosphorylation signaling pathways by
delivering an array of kinase effectors into host cells including
LegK1-K4, LegK7 and Lem28/Lpg2603 (Hervet et al., 2011; Lee et al.,
2018; Sreelatha et al., 2020). LegK1 was first identified in a screen
of\emph{L. pneumophila} effectors that activate the NF-\selectlanguage{greek}κ\selectlanguage{english}B pathway (Ge et
al., 2009). Mechanistically, LegK1 directly phosphorylates the I\selectlanguage{greek}κ\selectlanguage{english}B
family of inhibitors. LegK2 targets the ARP2/3 complex whose
phosphorylation triggers actin cytoskeleton remodeling in host cells
(Michard et al., 2015). LegK7 hijacks the host Hippo pathway by
phosphorylating the scaffolding protein MOB1, leading to the degradation
of the transcriptional regulators TAZ and YAP1 (Lee et al., 2018). The
host targets of LegK4 were identified to be the Hsp70 chaperone family
by using a chemical genetic screen (Moss et al., 2019). LegK4-dependent
phosphorylation of cytosolic Hsp70 inhibits its ATPase activity and
hence protein folding capacity, resulting in global translation arrest
in host cells. Thus far, host targets of other \emph{L.
pneumophila}kinase effectors are still elusive and their functions in
bacterial infection remain to be investigated.

Other than kinases, bacterial pathogens also encode and secret effector
proteins harboring phosphatase activities. \emph{Y.
pseudotuberculosis}YopH is perhaps the first reported bacterial effector
possessing potent tyrosine phosphatase activity (Bliska et al., 1991).
Later, two independent reports found that YopH targets focal adhesions
in host cells by dephosphorylating p130Cas and FAK, thereby disrupting
peripheral focal complexes and inhibiting bacterial uptake into HeLa
cells (Persson et al., 1997; Black et al., 1997). In T cells, YopH
targets the tyrosine kinases Lck and ZAP-70 together with the adaptors
SLP-76 and LAT, and their dephosphorylation alters T cell-mediated
immune responses (Alonso et al., 2004; Gerke et al., 2005). Overlapping
with the previously reported targets in different host cells, the
PRAM-1/SKAP-HOM and SLP-76 signaling pathways are also dephosphorylated
in a YopH-dependent manner to impair neutrophil responses in an animal
infection model (Rol\selectlanguage{ngerman}án et al., 2013). In many of these studies, a
catalytic mutant of YopH was often employed to precipitate the
interacting proteins for identifying potential phosphatase substrates.
Furthermore, \emph{Salmonella} effector SptP has a C-terminal protein
tyrosine phosphatase domain fused to its N-terminal GTPase activating
protein (GAP) domain (Stebbins \& Galán, 2000). It dephosphorylates the
host AAA+ ATPase VCP to regulate the biogenesis of\emph{Salmonella}
-containing vacuoles (SCVs) (Humphreys et al., 2009). Again, the
substrate VCP was identified from the pull-down samples by using a
phosphatase inactive mutant of SptP. Furthermore, SptP targets a
tyrosine kinase Syk and a vesicle fusion protein,
N-ethylmalemide-sensitive factor to suppress immune responses in mast
cells through its phosphatase activity (Choi et al., 2013). Additional
phosphatase effectors include WipA, WipB, Ceg4 and Lem4 from \emph{L.
pneumophila} (Pinotsis \& Waksman, 2017; Prevost et al., 2017; Quaile et
al., 2018; Beyrakhova et al., 2018) and \emph{Coxiella burnetii}effector
CinF that dephosphorylates I\selectlanguage{greek}κ\selectlanguage{english}B\selectlanguage{greek}α \selectlanguage{english}to inhibit host NF-\selectlanguage{greek}κ\selectlanguage{english}B signaling (Zhang
et al., 2022).

Nevertheless, our understanding of bacterial manipulation of host
phosphorylation signaling pathways remains rather limited, largely
because of technical challenges in sensitively and reliably measuring
phosphorylation events of proteins (especially on a global level).
Therefore, many studies on bacterial kinases or phosphatases, in
particular some of the early work as we discussed above, were carried
out without the assistance of the latest MS tools on more definitive
measurements of protein modifications as well as precise determination
of phosphorylation sites. In addition, the potential impact(s) of a
bacterial kinase/phosphatase was not probed on systems-level during
bacterial infection. Such holistic views are particularly desired when a
single effector (e.g., YpkA, SteC, YopH and SptP) may target multiple
host pathways. Fortunately, the arrival and maturation of MS-based
phosphoproteomics in the last two decades renders large-scale analyses
of cellular phosphorylation network possible.

Indeed, phosphopeptides can be efficiently enriched (e.g., by titanium
dioxide (TiO\textsubscript{2})) from digested total cell lysates at
relatively low cost, allowing high-throughput phosphoproteome profiling
by LC-MS (Olsen et al., 2010; Vill\selectlanguage{ngerman}én et al., 2007). Global
phosphoproteomic analyses of host cells infected by bacterial pathogens
were also reported (Rogers et al., 2011; Imami et al., 2013; Schmutz et
al., 2013). Such phosphoproteome measurements, if designed with proper
controls, can be exploited as a universal and powerful approach to
identify host enzymatic substrates of a given effector protein with
either kinase or phosphatase activities. For example,
enteropathogenic\emph{E. coli} (EPEC) effectors NleH1/2 are Ser/Thr
protein kinases and they have been shown by a number of studies to
attenuate NF-\selectlanguage{greek}κ\selectlanguage{english}B activation (Gao et al., 2009; Royan et al., 2010; Wan et
al., 2011) and inhibit host apoptotic pathway during infection
(Hemrajani et al., 2010). Nevertheless, no phosphorylated substrates of
NleH1/2 were reported until a recent study in which a large-scale
phosphoproteomic screen successfully identified host microvillus protein
Eps8 as the kinase target (Pollock et al., 2022). Phosphorylation of
Eps8 on Ser775 inhibits its bundling activity, leading to its dispersal
from the attaching and effacing (AE) lesion during EPEC infection. This
work exemplifies the utility of phosphoproteomics in identifying
enzymatic substrates of bacterial kinases.

\textbf{CONCLUSION}

In general, host target discovery is a demanding task in the study of
bacterial effector proteins. In particular, it often becomes more
challenging when the effector of interest is an enzyme that may catalyze
a potential PTM of host targets. In many cases, one would rely on
biochemical isolation of protein complexes comprising effectors and host
targets. The success of such strategies often requires the stable
interaction/association of host substrates with bacterial factors. There
are at least two scenarios where these approaches (e.g.,
immunoprecipitation of protein complexes) would be less effective in
identifying target proteins. First, the transient or weak
enzyme-substrate affinities prevent successful isolation of protein
complexes unless additional techniques such as crosslinking methods are
employed to preserve such labile interactions before
immunoprecipitation. For instance, in addition to its N-terminal
ADP-riboxanase domain \emph{S. flexneri} effector OspC3 has a C-terminal
ARD domain that is thought to recognize specific caspases for
ADP-riboxanation. That being said, the binding constant
(K\textsubscript{D}) between OspC3 and caspases were measured at
\textasciitilde{}5 \selectlanguage{greek}µ\selectlanguage{english}M (Hou et al., 2023). In contrast, efficient
immunoprecipitation often requires binding affinities in the range of nM
at least. Additionally, another complication may arise from the scenario
where effectors do have stably interacting partners and yet such
interactors are not enzymatic substrates. Rather, stably associated
factors can be host factors required for activation of bacterial
effectors. For example, both \emph{S. flexneri} OspC3 and \emph{C.
violaceum} CopC rely on the interaction with an abundant host factor,
calmodulin, for their activation (Hou et al., 2023; Peng et al., 2022;
Liu et al., 2022). The stable association of ARF GTPases
with\emph{Salmonella} effector SopF also falls into this category (Xu et
al., 2022). Therefore, alternative approaches without the necessity of
stable protein-protein interactions (i.e., effector-substrate
association) would be highly desired for identification of enzymatic
targets.

As we discussed above, recent advancements in MS-based proteomic
approaches make it possible to comprehensively identify host proteins
modified by bacterial effectors. Successful application of such
strategies has been seen at least in ADP-ribosylation and
phosphorylation mediated by bacterial effectors. As the macro
domain-containing protein Af1521 is able to recognize the newly
discovered ADP-riboxanation as well, we would anticipate similar
utilization of PTM profiling to identify modified substrates for the
class of effectors possessing ADP-riboxanase activity. Furthermore, as
effectors may have multiple substrates by targeting different host
processes, such PTM profiling strategies can also be utilized to uncover
any additional host targets of some characterized effectors (e.g.,
potential non-caspase substrates of OspC3 and CopC). Last but not the
least, large-scale analyses of additional PTMs (e.g., ubiquitination and
acetylation) are also feasible with the development of specific
antibodies recognizing these modifications (Xu et al., 2010; Choudhary
et al., 2009).

In summary, we believe that mass spectrometry will play a more prevalent
role in studying host protein modifications catalyzed by bacterial
effectors. It will not only aid in the characterization of novel
modifications (e.g., ADP-riboxanation), but also greatly facilitate the
global, unbiased discovery of enzymatic substrates of bacterial
effectors. We certainly envisage a flourishing field of
bacteria-mediated PTMs and further expansion of our knowledge on
host-pathogen interactions in the near future.

CONFLICT OF INTEREST

The authors declare no competing interests.

DATA AVAILABILITY STATEMENT

Data sharing is not applicable to this article as no new data were
created or analyzed in this study.

\textbf{Acknowledgement}

We thank the members of the Liu laboratory for critical review of this
manuscript. We also thank Dr. Jiazhang Qiu from Jilin University for the
useful discussion on \emph{Legionella} kinase effectors. Due to time
constraint as well as space limitation, we apologize to those colleagues
whose work was not discussed or cited in this paper. The work in our
group was financially supported by grants from National Key Research and
Development Program of China (2022YFA1304500), the Natural Science
Foundation of China (22174003 and 21974002), Peking University Medicine
Seed Fund for Interdisciplinary Research and the Fundamental Research
Funds for the Central Universities.

ORCID

Jie Jin https://orcid.org/0000-0001-8641-8843

Jiaqi Fu https://orcid.org/0000-0003-0081-6133

Xiaoyun Liu https://orcid.org/0000-0001-7083-5263

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\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image1/image1}

\end{center}
\end{figure}

FIGURE 1 Schematic representation of exploitation of host cell signaling
by bacterial pathogens via effector-mediated post-translational
modifications. Effector proteins secreted by invading pathogenic
bacteria have been shown to harbor diverse enzymatic activities,
subverting host cellular pathways. For instance, \emph{Shigella}effector
OspC3 targets host caspase-4/11 for ADP-riboxanation, which blocks the
cleavage of GSDMD by caspase-4/11 (Li et al., 2021).\emph{Salmonella}
kinase SteC remodels the host F-actin cytoskeleton by phosphorylating
FMNL1 (Poh et al., 2008), whereas enteropathogenic\emph{Escherichia
coli} effector kinases NleH1 and NleH2 target Ser775 of host microvillus
protein Eps8 for phosphorylation, thus inhibiting its bundling activity
and driving its dispersal from the attaching and effacing (AE) lesion
during infection (Pollock et al., 2022). In addition, Lpg0080 (also
named as Ceg3) and Lpg0081 from \emph{Legionella pneumophila}
coordinately modulate host mitochondrial ADP/ATP exchange via reversible
ADP-ribosylation of ANTs (Kubori et al., 2022; Fu et al., 2022). Due to
space limitation, only a few representative effectors and their
associated pathways are shown in the figure.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/Figure-1-20230214/Figure-1-20230214}

\end{center}
\end{figure}

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