Molecular Cell: Uncovering new strategies for pathogenic infections

Professor Li Shan's research group, College of Life Science and Technology, School of Biomedicine and Health, Huazhong Agricultural University, and Fu Xuan's research group of Southern University of Science and Technology, published a report entitled "Structural insights into caspase ADPR-deacylization catalyzed by a bacterial effector and host calmodulin" online in Molecular Cell of papers
.
This study comprehensively uses multidisciplinary methods such as biological mass spectrometry, structural biology, biochemistry, and cell biology to reveal the molecular basis of pathogenic bacteria catalyzing the post-translational modification of proteins with a new backbone ADP-ribose deacylization to modify caspase and then regulate host programmed cell death, which provides a new target and theoretical basis
for the development of related bacterial infectious disease drugs.

Pathogenic bacteria use the Type III Secretion System (T3SS) to "inject" effector proteins into the host, simulate or manipulate the host's signal transduction pathway, and promote infection and colonization, which is the core infection strategy
of many gram-negative pathogenic bacteria.
Chromobacter purple is an emerging human pathogenic bacterium with multi-drug resistance and high fatality rate of infection, but its pathogenic mechanism is still unclear
.
Professor Li Shan's laboratory has conducted research on the molecular mechanism of "pathogen-host" interaction, and has made a series of progress
in the pathogenesis of Chromobacter purple.

Mechanism of action of CopC effector protein Macrobacterium purple

Earlier, Professor Li Shan's laboratory found that the T3SS effector protein CopC of Chromobacter purple can catalyze a new backbone protein post-translational modification targeting multiple caspase in host cells, thereby regulating a variety of programmed cell death signaling pathways and promoting self-infection and reproduction
。 The research group named the new protein post-translational modification catalyzed by CopC as ADPR-deacylization, and found that effector proteins with the same enzymatic activity as CopC also existed in other pathogenic bacteria, indicating that CopC family proteins modify caspase protein through ADP-ribose dehumidification cyclization to interfere with host cell death signaling pathway is a pathogenic strategy
commonly used by pathogenic bacteria 。 It is worth noting that this novel protein post-translational modification needs to be completed with the help of calmodulin (CaM), which is specifically present in the human host, ensuring that pathogenic bacterial virulence proteins only function
after entering the host.

In order to further study how CopC catalyzes the post-translational modification of ADP-ribose deamination cyclization of a novel protein and how the cofactor CaM regulates the enzymatic activity of CopC, Professor Li Shan's group has cooperated with the research group of Fu Xuan of Southern University of Science and Technology to carry out further research
.
The researchers first used tandem mass spectrometry to identify that CopC performed multisite modification on caspase-7/-8/-9, and only single-site modification on caspase-3, so caspase-3 was selected as a suitable substrate protein for subsequent structural studies
。 The researchers successfully captured the high-resolution structure of CaM-CopC-caspase-3 ternary complex and ligand NAD+ in three states before, during and after the reaction by single-particle cryo-EM technology, combined with biochemical experimental analysis and functional verification, comprehensively revealed the mechanism of CopC recognition of ligands/substrates/cofactors, and elucidated the molecular basis of CopC's unique enzymatically catalyzed reaction and the molecular mechanism of substrate departure after the reaction was completed through structural comparison of different reaction states
。

Mechanism of CopC regulation of Chromobacterium violaeus effector protein

The calmodulin CaM, which is specifically present in eukaryotic hosts, is the switch that controls CopC activity, so how does CaM help CopC function? The researchers compared the structure of the CopC-caspase-3 binary complex and the CaM-CopC-caspase-3 ternary complex and found that the NCD catalytic domain of CopC can be properly folded and assembled
in an orderly manner in the presence of CaM 。 In vitro biochemical analysis showed that CaM in the form of Ca2+-free can significantly promote the catalytic activity of CopC and enhance the binding ability to the substrate caspase-3, while Ca2+-bound form of CaM makes CopC in a state without catalytic activity, which is consistent
with the resting state of host cells in a low calcium environment.
The researchers upregulated or downregulated the expression of calmodulin in cells, which has a corresponding regulatory effect
on the activity of CopC when pathogenic bacteria are infected.
The ability of CopC to bind to CaM in mouse models of Chrobacillus purple infection has also been shown to be critical
for the colonization of pathogenic bacteria in the mouse liver and the lethality of infection in mice.
The choice of CaM as a cofactor of its own virulence factor is a clever choice, which cleverly uses the change of Ca2+ concentration in the host cell to achieve the regulation of virulence protein activity
.

This research system revealed the molecular mechanism of Hysmogella purple using the effector protein CopC to catalyze the protein post-translational modification of the new backbone to target the host cell death signaling pathway, and for the first time comprehensively elaborated the mechanism of host-specific cofactor CaM regulating CopC activity in vitro and in vitro, which provided a new perspective for in-depth understanding of the pathogenic mechanism of pathogenic bacteria, and provided a potential target and theoretical basis
for the development of specific antibacterial drugs based on the pathogen-host interaction process.

Professor Li Shan and Professor Fu Huan are co-corresponding authors
of the paper.
Dr.
Zhang Kuo and Dr.
Tian Miao of Fu Xuan's research group, Peng Ting and Tao Xinyuan, doctoral students of Li Shan's research group, are co-first authors, and a number of master's and doctoral students in Li Shan's research group participated in the study
.