Abstract
Studies have successfully elucidated the mechanism of action of several effector domains that comprise the multifunctional-autoprocessing repeats-in-toxins (MARTX) toxins of Vibrio vulnificus. However, the biochemical linkage between the cysteine proteolytic activity of Makes Caterpillars Floppy (MCF)-like effector and its cellular effects remains unknown. In this study, we identify the host cell factors that activate in vivo and in vitro MCF autoprocessing as adenosine diphosphate (ADP)-Ribosylation Factor 1 (ARF1) and ADP-Ribosylation Factor 3 (ARF3). Autoprocessing activity is enhanced when ARF1 is in its active [guanosine triphosphate (GTP)-bound] form compared to the inactive [guanosine diphosphate (GDP)-bound] form. Subsequent to auto-cleavage, MCF is acetylated on its exposed N-terminal glycine residue. Acetylation apparently does not dictate subcellular localization as MCF is found localized throughout the cell. However, the cleaved form of MCF gains the ability to bind to the specialized lipid phosphatidylinositol 5-phosphate enriched in Golgi and other membranes necessary for endocytic trafficking, suggesting that a fraction of MCF may be subcellularly localized. Traditional thin-section electron microscopy, high-resolution cryoAPEX localization, and fluorescent microscopy show that MCF causes Golgi dispersal resulting in extensive vesiculation. In addition, host mitochondria are disrupted and fragmented. Mass spectrometry analysis found no reproducible modifications of ARF1 suggesting that ARF1 is not post-translationally modified by MCF. Further, catalytically active MCF does not stably associate with ARF1. Our data indicate not only that ARF1 is a cross-kingdom activator of MCF, but also that MCF may mediate cytotoxicity by directly targeting another yet to be identified protein. This study begins to elucidate the biochemical activity of this important domain and gives insight into how it may promote disease progression.
| Original language | English (US) |
|---|---|
| Article number | e13133 |
| Journal | Cellular Microbiology |
| Volume | 22 |
| Issue number | 2 |
| DOIs | |
| State | Published - Feb 1 2020 |
Funding
The authors thank Heike Folsch for sharing ARF expression plasmids. We thank the Purdue CryoEM Facility at Hockmeyer Hall for access to the high‐pressure freezer, freeze substitution unit, microtome and chemical hood. We thank Dr. Christopher Gilpin for access to the T12 electron microscope at the Purdue Life Sciences EM Core Facility where thin sectioning was carried out. We also thank Dr. Arvanitis and the Center for Advanced Microscopy and Nikon Imaging Center at Northwestern University for their invaluable help and resources. This work was supported by National Institutes of Health grants AI092825 (to K.J.F.S), R01GM10092 (to S.M.), as well as Indiana Clinical and Translational Sciences Institute grant CTSI‐106564 (to S.M.), and Purdue University Institute for Inflammation, Immunology and Infectious Diseases grant PI4D‐209263 (to S.M.), GM104610 (to R.R.O.L.), and GM103479 (to J.A.L.). A.H. was supported by a Ruth L. Kirschstein Institutional National Research Service Award (5T32AI007476). J.M. was supported by a UCLA Molecular Biology Institute Whitcome Fellowship. Immunofluorescence imaging work was performed at the Northwestern University Center for Advanced Microscopy generously supported by NCI CCSG P30 CA060553 awarded to the Robert H Lurie Comprehensive Cancer Center. The authors thank Heike Folsch for sharing ARF expression plasmids. We thank the Purdue CryoEM Facility at Hockmeyer Hall for access to the high-pressure freezer, freeze substitution unit, microtome and chemical hood. We thank Dr. Christopher Gilpin for access to the T12 electron microscope at the Purdue Life Sciences EM Core Facility where thin sectioning was carried out. We also thank Dr. Arvanitis and the Center for Advanced Microscopy and Nikon Imaging Center at Northwestern University for their invaluable help and resources. This work was supported by National Institutes of Health grants AI092825 (to K.J.F.S), R01GM10092 (to S.M.), as well as Indiana Clinical and Translational Sciences Institute grant CTSI-106564 (to S.M.), and Purdue University Institute for Inflammation, Immunology and Infectious Diseases grant PI4D-209263 (to S.M.), GM104610 (to R.R.O.L.), and GM103479 (to J.A.L.). A.H. was supported by a Ruth L. Kirschstein Institutional National Research Service Award (5T32AI007476). J.M. was supported by a UCLA Molecular Biology Institute Whitcome Fellowship. Immunofluorescence imaging work was performed at the Northwestern University Center for Advanced Microscopy generously supported by NCI CCSG P30 CA060553 awarded to the Robert H Lurie Comprehensive Cancer Center.
ASJC Scopus subject areas
- Microbiology
- Immunology
- Virology