Makes caterpillars floppy-like effector-containing MARTX toxins require host ADP-ribosylation factor (ARF) proteins for systemic pathogenicity

Abstract

Upon invading target cells, multifunctional autoprocessing repeats-in-toxin (MARTX) toxins secreted by bacterial pathogens release their disease-related modularly structured effector domains. However, it is unclear how a diverse repertoire of effector domains within these toxins are processed and activated. Here, we report that Makes caterpillars floppy-like effector (MCF)-containing MARTX toxins require ubiquitous ADP-ribosylation factor (ARF) proteins for processing and activation of intermediate effector modules, which localize in different subcellular compartments following limited processing of holo effector modules by the internal cysteine protease. Effector domains structured tandemly with MCF in intermediate modules become disengaged and fully activated by MCF, which aggressively interacts with ARF proteins present at the same location as intermediate modules and is converted allosterically into a catalytically competent protease. MCF-mediated effector processing leads ultimately to severe virulence in mice via an MCF-mediated ARF switching mechanism across subcellular compartments. This work provides insight into how bacteria take advantage of host systems to induce systemic pathogenicity.

Keywords: ADP-ribosylation factor protein; MARTX toxin; effector.

Fig. 1.

MARTX toxins undergo a bilateral procedure to process effector modules. (A−C) Effector modules of V. vulnificus MARTX toxins are not completely processed by CPD. Processed products confirmed by Edman sequencing are shown at the bottom of the gels. aCPD, autoprocessed CPD. (D) The ABH–MCF pair was processed in cells in a CPD-independent manner. The indicated constructs were coexpressed in HEK293T cells and subjected to Western blotting. (E) Pull-down assays showing that MCF interactions are specific for ARF proteins, but not ARF-like proteins. ARL4C, ARF-like protein 4C; SAR1A, secretion-associated Ras-related GTPase 1A. (F) In vitro pull-down assay showing that MCF interacts with the active form ARF3_Q71L, but not with the inactive form ARF3_T31N. Data are representative of at least 3 independent experiments, each with similar results (A−F).

Fig. 2.

Host ADP ribosylation factors are essential for processing MCF-containing MARTX toxins from a broad range of pathogenic bacteria. (A) Active form ARF is the cellular activator of MCF-mediated processing of the ABH/MCF pair. (B and C) ARF protein-specific MCF activation and effector pair processing. Processing of the purified ABH/MCF pair was examined using active forms of ARF (ARF1_Q71L and ARF6_Q67L) (B) and ARF-like protein SAR1A_H79G (C). (D) ARF-activated MCF processing, not the CPD-mediated processing, is responsible for the ABH/MCF pair processing. (E) More than 34% of MARTX toxins contain at least 1 MCF (or its homolog, DmX) domain. Asterisks indicate nonspecific products generated by in vitro cleavage. Data are representative of at least 3 independent experiments, each with similar results (A−D).

Fig. 3.

Crystal structures of MCF_C/S either in tandem arrangement with ABH or in complex with ARF3_Q71L. (A) ITC analysis showing a high-affinity interaction between MCF and ARF. (B) Schematic diagram of the ABH/MCF pair (residues 2902−3586), MCF (residues 3219−3586), and ARF3 (residues 14−176) used for the structural studies. L, linker connecting ABH and MCF. (C and D) Overall structures of ABH/MCF_C/S (C) and the MCF_C/S–ARF3_Q71L complex (D). GTP bound to ARF3 is shown as a yellow stick representation (D). (E) Catalytic site within the MCF_C/S–ARF3_Q71L complex. (F) Essential roles of catalytic residues during N-terminal autoprocessing of MCF. The indicated uncleaved MCF constructs (residues 3206−3586) were transiently expressed in HEK293T cells and subjected to Western blotting. EV, empty vector. Data are representative of at least 2 (A) or 3 (F) independent experiments, each with similar results.

Fig. 4.

Molecular basis of ARF-mediated MCF activation. (A−C) The MCF mutant (MCF_4MT, mutated at the positions of E3310L, R3317L, Y3381E, and E3397G) does not interact with ARF. ARF-induced effector processing both in vitro (B) and in vivo (C). (D−F) Molecular mechanism underlying ARF-induced allosteric activation of MCF. Inactive MCF_A in ABH/MCF (D) is converted into a catalytically competent cysteine protease, MCF_B, upon ARF binding (E). Expanded view of the catalytic and oxyanion residues within each structure (F). Data are representative of 3 independent experiments, each with similar results (A−C).

Fig. 5.

ARF binding to MCF is essential for full activation of tandemly paired effectors. (A) Schematic representation of modified MARTX toxins in the engineered strains. Mutated positions in MCF are indicated. (B and C) Actin in HeLa cells infected with the indicated strains was analyzed by Western blotting (B). Band intensities were quantified, and levels of actin cross-linking relative to those in ACD/MCF_WT were calculated and presented (C). Data are expressed as the mean ± SD of biological triplicates (****P < 0.0001; ***P < 0.001; **P < 0.01; *P < 0.05; NS, not significant; ND, not detected).

Fig. 6.

MCF switches interacting ARF proteins across subcellular compartments. (A) Overall structure of free MCF_C/S. (B) Structural significance of inactive MCF_C/S released from ARF, showing a large conformational change in the SD (red). The conformation of the SD in active MCF_B complexed with ARF3 is shown by black lines. (C) MCF switches interacting ARF proteins in HEK293T cells. (Scale bars, 10 μm.) (D) Interaction between MCF and ARF proteins results in Golgi dispersion and cell shrinkage. Before confocal microscopy analysis, the cis-Golgi marker GM130 and nuclei were stained. Margins of transfected cells in the GM130 panel are indicated by yellow dashed lines. (Scale bars, 20 μm.) Data are representative of 3 independent experiments, each with similar results (C and D).

Fig. 7.

Significance of processing and activation of MCF-containing MARTX toxins. (A) Schematic representation of modified MARTX toxins in the engineered strains. Mutated positions are indicated. (B) Survival of mice (n = 10 per group; pooled data from 2 experiments) challenged subcutaneously with the engineered strains, illustrating the significance of MCF interactions with ARF proteins. (C) Proposed model showing processing and activation of MCF-containing MARTX toxins. After translocation, MARTX effector domains are first processed by CPD in the cytoplasm, resulting in tandemly arranged effector-MCF intermediate modules as well as free single effector(s). The effector intermediate (ABH/MCF) relocalizes to the plasma membrane, where MCF interacts with and is activated by ARF6, followed by a second round of processing. ARFGAP-mediated inactivation of ARF6 may release MCF into the cytoplasm, which subsequently sequesters other ARF proteins and/or modifies unknown cellular substrate(s). Other intermediate effector modules tandemly paired with MCF may undergo similar processing events. As a result, MCF causes Golgi fragmentation and cell shrinkage, while other effectors have cytopathic/cytotoxic consequences.