Pathogen hijacks programmed cell death signaling by arginine ADPR-deacylization of caspases.

Caspases are evolutionarily conserved cysteine proteases that are essential for regulating cell death and are involved in multiple development and disease processes, including immunity. Here, we show that the bacterial type III secretion system (T3SS) effector CopC (Chromobacterium outer protein C) from the environmental pathogen Chromobacterium violaceum attacks caspase-3/-7/-8/-9 by ADPR-deacylization to dysregulate programmed cell death, including apoptosis, necroptosis, and pyroptosis. This modification involves ADP-ribosylation- and deamination-mediated cyclization on Arg207 of caspase-3 by a mechanism that requires the eukaryote-specific protein calmodulin (CaM), leading to inhibition of caspase activity. The manipulation of cell death signaling by CopC is essential for the virulence of C. violaceum in a mouse infection model. CopC represents a family of enzymes existing in taxonomically diverse bacteria associated with a wide spectrum of eukaryotes ranging from humans to plants. The unique activity of CopC establishes a mechanism by which bacteria counteract host defenses through a previously unrecognized post-translational modification.

Structural insights into caspase ADPR deacylization catalyzed by a bacterial effector and host calmodulin.

Programmed cell death and caspase proteins play a pivotal role in host innate immune response combating pathogen infections. Blocking cell death is employed by many bacterial pathogens as a universal virulence strategy. CopC family type III effectors, including CopC from an environmental pathogen Chromobacterium violaceum, utilize calmodulin (CaM) as a co-factor to inactivate caspases by arginine ADPR deacylization. However, the molecular basis of the catalytic and substrate/co-factor binding mechanism is unknown. Here, we determine successive cryo-EM structures of CaM-CopC-caspase-3 ternary complex in pre-reaction, transition, and post-reaction states, which elucidate a multistep enzymatic mechanism of CopC-catalyzed ADPR deacylization. Moreover, we capture a snapshot of the detachment of modified caspase-3 from CopC. These structural insights are validated by mutagenesis analyses of CopC-mediated ADPR deacylization in vitro and animal infection in vivo. Our study offers a structural framework for understanding the molecular basis of arginine ADPR deacylization catalyzed by the CopC family.

Interbacterial Chemical Communication-Triggered Nascent Proteomics.

Metabolic labeling with clickable noncanonical amino acids has enabled nascent proteome profiling, which can be performed in a cell-type-specific manner. However, nascent proteomics in an intercellular communication-dependent manner remains challenging. Here we develop communication-activated profiling of protein expression (CAPPEX), which integrates the LuxI/LuxR quorum sensing circuit with the cell-type-specific nascent proteomics method to enable selective click-labeling of newly synthesized proteins in a specific bacterium upon receiving chemical signals from another reporter bacterium. CAPPEX reveals that E. coli competes with Salmonella for tryptophan as the precursor for indole, and the resulting indole suppressed the expression of virulence factors in Salmonella. This tryptophan-indole axis confers attenuation of Salmonella invasion in host cells and living mice. The CAPPEX strategy should be widely applicable for investigating various interbacterial communication processes.

Clutter Elimination and Random-Noise Denoising of GPR Signals Using an SVD Method Based on the Hankel Matrix in the Local Frequency Domain.

Ground-penetrating radar (GPR) is a kind of high-frequency electromagnetic detection technology. It is mainly used to locate targets and interfaces in underground structures. In addition to the effective signals reflected from the subsurface objects or interfaces, the GPR signals in field work also include noise and different clutters, such as antenna-coupled waves, ground clutters, and radio-frequency interference, which have similar wavelet spectral characteristics with the target signals. Clutter and noise seriously interfere with the target's response signal. The singular value decomposition (SVD) filtering method can select appropriate singular values and characteristic components corresponding to the effective signals for signal reconstruction to filter the GPR data. However, the conventional time-domain SVD method introduces fake signals when eliminating direct waves, and does not have good suppression of random noise around non-horizontal phase axes. Here, an SVD method based on the Hankel matrix in the local frequency domain of GPR data is proposed. Different numerical models and real field GPR data were handled using the proposed method. Based on the power of fake signals introduced via different processes, qualitative and quantitative analyses were carried out. The comparison shows that the newly proposed method could improve efforts to suppress random noise around non-horizontal phase reflection events and weaken the horizontal fake signals introduced by eliminating clutter such as ground waves.

Arg-GlcNAcylation on TRADD by NleB and SseK1 Is Crucial for Bacterial Pathogenesis.

Death receptor signaling is critical for cell death, inflammation, and immune homeostasis. Hijacking death receptors and their corresponding adaptors through type III secretion system (T3SS) effectors has been evolved to be a bacterial evasion strategy. NleB from enteropathogenic Escherichia coli (EPEC) and SseK1/2/3 from Salmonella enterica serovar Typhimurium (S. Typhimurium) can modify some death domain (DD) proteins through arginine-GlcNAcylation. Here, we performed a substrate screen on 12 host DD proteins with conserved arginine during EPEC and Salmonella infection. NleB from EPEC hijacked death receptor signaling through tumor necrosis factor receptor 1 (TNFR1)-associated death domain protein (TRADD), FAS-associated death domain protein (FADD), and receptor-interacting serine/threonine-protein kinase 1 (RIPK1), whereas SseK1 and SseK3 disturbed TNF signaling through the modification of TRADD Arg235/Arg245 and TNFR1 Arg376, respectively. Furthermore, mouse infection studies showed that SseK1 but not SseK3 rescued the bacterial colonization deficiency contributed by the deletion of NleBc (Citrobacter NleB), indicating that TRADD was the in vivo substrate. The result provides an insight into the mechanism by which attaching and effacing (A/E) pathogen manipulate TRADD-mediated signaling and evade host immune defense through T3SS effectors.

Arginine GlcNAcylation of Rab small GTPases by the pathogen Salmonella Typhimurium.

Salmonella enterica serovar Typhimurium, an intracellular Gram-negative bacterial pathogen, employs two type III secretion systems to deliver virulence effector proteins to host cells. One such effector, SseK3, is a Golgi-targeting arginine GlcNAc transferase. Here, we show that SseK3 colocalizes with cis-Golgi via lipid binding. Arg-GlcNAc-omics profiling reveals that SseK3 modifies Rab1 and some phylogenetically related Rab GTPases. These modifications are dependent on C-termini of Rabs but independent of the GTP- or GDP-bound forms. Arginine GlcNAcylation occurs in the switch II region and the third α-helix and severely disturbs the function of Rab1. The arginine GlcNAc transferase activity of SseK3 is required for the replication of Salmonella in RAW264.7 macrophages and bacterial virulence in the mouse model of Salmonella infection. Therefore, this SseK3 mechanism of action represents a new understanding of the strategy adopted by Salmonella to target host trafficking systems.