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.