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 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
(KD) between OspC3 and caspases were measured at
~5 µM (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 S. flexneri OspC3 and 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 withSalmonella 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.