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). Yersinia pseudotuberculosisYpkA 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, Gαq, 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). S.Typhimurium effector SteC harbors kinase activity that is required for
the formation of the F-actin meshwork associated withSalmonella -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).
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 ofL. pneumophila effectors that activate the NF-κB pathway (Ge et
al., 2009). Mechanistically, LegK1 directly phosphorylates the IκB
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 L. pneumophilakinase 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. Y. pseudotuberculosisYopH 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 (Rolán 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, 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 ofSalmonella -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 L.
pneumophila (Pinotsis & Waksman, 2017; Prevost et al., 2017; Quaile et
al., 2018; Beyrakhova et al., 2018) and Coxiella burnetiieffector CinF that dephosphorylates IκBα to inhibit host NF-κB 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 (TiO2)) from digested total cell lysates at
relatively low cost, allowing high-throughput phosphoproteome profiling
by LC-MS (Olsen et al., 2010; Villén 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, enteropathogenicE. coli (EPEC) effectors NleH1/2 are Ser/Thr protein kinases and
they have been shown by a number of studies to attenuate NF-κB
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.