Biology subjects

Satchell, K. J. F.

Publications and source records attributed to Satchell, K. J. F..

6 recordsLinked to original sources

bioRxiv2026

Cryo-EM structure of detergent-bound Vibrio cholerae MakA captures a candidate intermediate between the soluble form and mature assembly of an α-pore-forming toxin

The tripartite toxin MakA/MakB/MakE from Vibrio cholerae belongs to a family of -pore-forming toxins that undergo extensive conformational rearrangements upon interacting with membrane. While soluble structures of MakA, MakB and MakE have been determined, the structural basis of membrane association and pore assembly remains poorly understood. Here, we report a cryo-electron microscopy structure of a detergent-associated form of MakA at a global resolution of 3.13 Angstrom, with focused refinement of a flexible region improving the local resolution to 2.68 Angstrom. This structure consists of a trimer of MakA asymmetric homodimers, in which each subunit adopts a transmembrane -helical conformation distinct from the previously reported soluble structure. The asymmetric homodimer closely resembles the building block observed in the previously reported helical MakA assembly and is structurally similar to the homodimeric building blocks of the Aeromonas hydrophila AhlB and Serratia marcescens SmhA pores, and the heterodimeric building blocks of the Xenorhabdus nematophila XaxAB and Yersinia enterocolitica YaxAB pores, suggesting a conserved assembly principle among distantly related -pore-forming toxins. Complementary hydropathy analysis, AlphaFold2 modeling, and liposome-based assays reveal distinct roles for the three components, with MakA acting as the primary membrane-inserting subunit, MakE exhibiting low but consistent membrane-association, and MakB alone remaining largely soluble. Together, these results support a model in which the MakA asymmetric homodimer represents a plausible early membrane-insertion unit and building block for pore assembly, while the trimer of dimers represents an early assembly intermediate.

microbiology↗

bioRxiv2023

Vibrio MARTX toxin processing and degradation of cellular Rab GTPases by the cytotoxic effector Makes Caterpillars Floppy

Vibrio vulnificus causes life threatening infections dependent upon the effectors released from the Multifunctional-Autoprocessing Repeats-In-Toxin (MARTX) toxin. The Makes Caterpillars Floppy-like (MCF) cysteine protease effector is activated by host ADP ribosylation factors (ARFs), although the targets of processing activity were unknown. In this study we show MCF binds Ras-related proteins in brain (Rab) GTPases at the same interface occupied by ARFs and then cleaves and/or degrades 24 distinct members of the Rab GTPases family. The cleavage occurs in the C-terminal tails of Rabs. We determine the crystal structure of MCF as a swapped dimer revealing the open, activated state of MCF and then use structure prediction algorithms to show that structural composition, rather than sequence or localization, determine Rabs selected as MCF proteolytic targets. Once cleaved, Rabs become dispersed in cells to drive organelle damage and cell death to promote pathogenesis of these rapidly fatal infections.

microbiology↗

bioRxiv2021

Functional and Structural Characterization of OXA-935, a Novel OXA-10-family β-lactamase from Pseudomonas aeruginosa

Resistance to antipseudomonal penicillins and cephalosporins is often driven by the overproduction of the intrinsic {beta}-lactamase AmpC. However, OXA-10-family {beta}-lactamases are a rich source of resistance in Pseudomonas aeruginosa. OXA {beta}-lactamases have a propensity for mutation leading to extended spectrum cephalosporinase and carbapenemase activity. In this study, we identified isolates from a subclade of the multidrug-resistant (MDR) high risk clonal complex CC446 with resistance to ceftazidime. Genomic analysis revealed that these isolates harbored a plasmid containing a novel allele of blaOXA-10, named blaOXA-935, which was predicted to produce an OXA-10 variant with two amino acid substitutions: an aspartic acid instead of glycine at position 157 and a serine instead of phenylalanine at position 153. The G157D mutation, present in OXA-14, is associated with resistance to ceftazidime. Deletion of blaOXA-935 restored sensitivity to ceftazidime and susceptibility profiling of P. aeruginosa laboratory strains expressing blaOXA-935 revealed that OXA-935 conferred ceftazidime resistance. To better understand the impact of the variant amino acids, we determined the crystal structures of OXA-14 and OXA-935. In OXA-14, one of two monomers contained the canonical carbamylated lysine-70 (K70). In contrast, both monomers of OXA-935 were decarbamylated at K70, and the F153S mutation conferred increased flexibility to the omega ({Omega}) loop. Compared to OXA-14, the catalytic efficiency of OXA-935 for nitrocefin was significantly reduced. Amino acid changes that confer extended spectrum cephalosporinase activity to OXA-10-family {beta}-lactamases are concerning given rising reliance on novel {beta}-lactam/{beta}-lactamase inhibitor combinations such as ceftolozane-tazobactam and ceftazidime-avibactam to treat MDR P. aeruginosa infections.

microbiology↗

bioRxiv2020

Cell cycle arrest following pan-RAS inhibition can occur through upregulation of p27 in both KRAS-dependent and KRAS-independent colon cancer cell lines

Ras-specific proteases to degrade RAS within cancer cells are under active development as an innovative strategy to treat tumorigenesis. The naturally occurring biological toxin effector called RAS/RAP1-specific endopeptidase (RRSP) is known to cleave all RAS within a cell, including HRAS, KRAS, NRAS and mutant KRAS G13D. In the course of studies developing RRSP as an anti-cancer therapeutic, it was shown that cleavage of total RAS by RRSP results in a range of cell fates from cytotoxicity to moderate growth inhibition. Despite the considerable amount of evidence demonstrating RRSP anti-tumor effects in vivo, our understanding of the mechanisms involved are unknown. Here, we first demonstrate, using isogenic mouse fibroblasts expressing a single isoform of RAS or mutant KRAS, that RRSP equally inactivates all isoforms of RAS as well as the major oncogenic KRAS mutants. The cleavage of RAS inhibited phosphorylation of ERK and cell proliferation regardless of the RAS isoform. To investigate further how RAS processing might lead to varying outcomes in cell fate within cancer cells, we tested RRSP against four colorectal cancer cell lines with a range of cell fates. While cell lines highly susceptible to RRSP (HCT116 and SW1463) undergo cytotoxic death, RRSP treatment of GP5d cells induces G1 cell cycle arrest, and SW620 cells instead induces growth inhibition through cell senescence. In three of four cell lines tested, growth effects were dictated by rescued expression of the tumor suppressor protein p27 (Kip1). The ability of RRSP to inactivate all RAS and inhibit cancer cell growth through a variety of mechanisms highlights the antitumor potential of RRSP, and further warrants investigation as a potential anti-tumor therapeutic.

cancer biology↗

bioRxiv2019

N-terminal autoprocessing and acetylation of MARTX Makes-Caterpillars Floppy-like effector is stimulated by ADP-Ribosylation Factor 1 in advance of Golgi fragmentation

Studies have successfully elucidated the mechanism of action of several effector domains that comprise the MARTX toxins of Vibrio vulnificus. However, the biochemical linkage between the cysteine proteolytic activity of Makes Caterpillars Floppy-like (MCF) 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 ADP-Ribosylation Factor 1 (ARF1) and ADP-ribosylation Factor 3 (ARF3). Autoprocessing activity is enhanced when ARF1 is in its active (GTP-bound) form compared to the inactive (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 a fraction of MCF may be subcellular 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. Surprisingly, ARF1 is not itself processed or post-translationally modified by MCF. Further, only catalytically inactive MCF stably associates with ARF1, thus serving as a substrate trap. Our data indicate that ARF1 is a cross-kingdom activator of MCF, but reveal that MCF mediates cytotoxicity likely 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.

microbiology↗

bioRxiv2019

Vibrio cholerae MARTX toxin multifunctionality silences inflammatory response to toxin mediated cytoskeletal collapse

AbstractMultifunctional autoprocessing repeats-in-toxin (MARTX) toxins are pore-forming toxins that translocate multiple functionally independent effector domains into a target eukaryotic cell. Vibrio cholerae colonizes intestinal epithelial cells (IECs) and utilizes a MARTX toxin with three effector domains -- an actin cross-linking domain (ACD), a Rho inactivation domain (RID), and an /{beta} hydrolase domain (ABH) -- to suppress innate immunity and enhance colonization. We investigated whether these multiple catalytic enzymes delivered from a single toxin function in a coordinated manner to regulate intestinal innate immunity. Using cultured IECs, we demonstrate that ACD-induced cytoskeletal collapse activated ERK, p38, and JNK mitogen-activated protein kinase (MAPK) signaling to elicit a robust proinflammatory response characterized by production of interleukin-8 (IL-8) and expression of CXCL8, TNF, and other proinflammatory genes. However, RID and ABH, which are naturally delivered along with ACD, blocked MAPK activation via Rac1 and thus prevented the ACD-induced inflammation. RID also abolished IL-8 secretion induced by heat-killed bacteria, tumor necrosis factor, and latrunculin A. Thus, MARTX toxins utilize enzymatic multifunctionality to silence the host response to bacterial factors and to the damage it causes. Further, these data show how V. cholerae MARTX toxin suppresses intestinal inflammation and contributes to cholera being classically defined as non-inflammatory diarrheal disease.

microbiology↗