Biology subjects

Keyel, P. A.

Publications and source records attributed to Keyel, P. A..

9 recordsLinked to original sources

bioRxiv2026

Leishmanial GP63 acts as a protease for the small pore forming toxin aerolysin

The eukaryotic pathogen Leishmania major causes disfiguring cutaneous lesions, whose resolution can be complicated by secondary bacterial infections. Bacteria, including Aeromonas spp., also interact with L. major promastigotes in the sandfly midgut. The mechanisms by which L. major competes with bacteria and resists their toxins are poorly defined. Prior work proposed that L. major resists the Aeromonas-produced pore-forming toxin aerolysin using an altered GPI-anchor. However, we found that L. major is sensitive to aerolysin. Here, we determined the mechanism by which L. major promastigotes are sensitive to aerolysin, using flow cytometry and biochemical approaches to analyze promastigotes genetically deficient in enzymes that produce key membrane components. The virulence factor lipophosphoglycan protected L. major from aerolysin cytotoxicity. The metalloproteinase GP63 exerted the necessary furin-like protease activity to activate aerolysin. Leishmanial GPI-anchored proteins were necessary for aerolysin heptamerization and killing of L. major promastigotes. Finally, mutation of the GPI-anchor binding domain of aerolysin crippled its cytotoxicity, consistent with its reliance on the GPI-anchor binding site to engage GPI anchors on the surface of L. major promastigotes. Taken together, we propose the L. major virulence factor lipophosphoglycan defends against pore-forming toxins made by bacterial competitors, while the GP63 metalloproteinase activates pro-aerolysin like furin. Overall, this study highlights approaches microbes use to compete with each other. Graphical AbstractAerolysin cytotoxicity depends on gp63 and LPG in Leishmania major promastigotes. (A) Wild type Leishmania major promastigotes are sensitive to aerolysin, which forms lethal heptameric pore complexes in the plasma membrane (B) L. major lpg1-- promastigotes are highly sensitive to aerolysin challenge because they lack LPG. (C) L. major gp63-- promastigotes have wild type sensitivity to aerolysin challenge but resist pro-aerolysin. (D) L. major gpi8-- knockout promastigotes are resistant to aerolysin and show no heptameric pore complexes in the plasma membrane. Created in BioRender.

cell biology↗

bioRxiv2025

Functional dissection of Leishmania major membrane components in resistance to cholesterol-dependent cytolysins

Bacteria use cholesterol-dependent cytolysins (CDCs) to damage eukaryotes. While well-studied in mammals, the mechanisms by which CDCs bind to and kill protozoans remains unclear. CDCs bind to the human pathogen Leishmania major, but only kill in the absence of sphingolipids. The contribution of other leishmanial membrane components to CDC binding and cytotoxicity remains unknown. Here, we used genetic knockouts and inhibitors to determine the contribution of key membrane components to CDC binding and killing in L. major. We analyzed toxin binding and killing using flow cytometry and western blotting. Loss of the virulence factor GP63 enhanced toxicity of perfringolysin O, but not streptolysin O. Plasmenylethanolamine and lipophosphoglycan had minimal contributions to CDC binding and cytotoxicity. Removal of sterols protected cells from CDCs, yet failed to reduce binding. We used CDCs defective in engaging glycans or cholesterol to confirm that CDCs deficient in sterol binding, but not glycan binding, could bound to L. major. Thus, in non-mammalian systems, CDCs may rely on glycans for binding, while using sterols for pore-formation. This suggests that CDCs may not be sterol-specific probes in some non-mammalian systems. We conclude that early-branching eukaryotes use distinct mechanisms from mammals to limit CDC pore-formation and killing.

cell biology↗

bioRxiv2025

Vav2 is a master regulator of repair against bacterial pore-forming toxins.

Despite antibiotic therapy, 25-35% of patients with necrotizing soft tissue infections (NSTIs) die. The etiologic agents for NSTIs include Streptococcus pyogenes and Clostridium perfringens, which secrete the cholesterol-dependent cytolysins (CDCs) streptolysin O and perfringolysin O to disrupt cell membranes. While cells resist this damage by activating Ca2+-dependent repair pathways, including MEK-dependent microvesicle shedding, dysferlin-mediated patch repair, and annexin-mediated membrane clogging, the upstream regulators of these responses have remained elusive. Here, we demonstrated that the Rac GEF Vav2 accounts for almost all of Ca2+-dependent repair against CDCs. Inhibiting or knocking down Vav2 sensitized multiple cell types to CDCs, whereas blocking other Rac GEFs did not. Mechanistically, Vav2 triggered the critical MLK3-MEK-dependent repair pathway. MEK activation rescued repair in Vav2-inhibited cells. Blocking dysferlin or annexins failed to increase damage beyond Vav2 inhibition, suggesting Vav2 coordinates multiple repair pathways. Thus, Vav2 controls multiple Ca2+-activated repair pathways that protect cells from CDCs produced during NSTIs. TeaserThis one protein accounts for almost all Ca2+-dependent repair by activating at least 3 distinct pathways.

cell biology↗

bioRxiv2025

The fungal peptide toxin candidalysin induces distinct membrane repair mechanisms compared to bacterial pore-forming toxins

The common fungal pathogen, Candida albicans, relies on the pore-forming toxin candidalysin to damage host cells. Cells counteract pore-forming toxins by Ca2+-dependent mechanisms, such as microvesicle shedding and annexin recruitment to resist cholesterol-dependent cytolysins like streptolysin O (SLO), or annexin involvement and patch repair in the case of aerolysin. However, the specific Ca2+-dependent repair pathways engaged in response to candidalysin remain poorly understood. Here, we determined the involvement of different Ca2+-dependent repair mechanisms to candidalysin and compared responses to SLO and aerolysin using flow cytometry and high-resolution microscopy. We report that candidalysin triggered Ca2+-dependent repair, but patch repair and ceramide failed to provide significant protection. MEK-dependent repair and annexins A1, A2 and A6 contributed partially to repairing damage caused by candidalysin. However, annexin translocation after candidalysin challenge was delayed compared to SLO or aerolysin challenge. Surprisingly, extracellular Cl- improved cell survival after candidalysin challenge, but not after challenge with SLO or aerolysin. Finally, we found that candidalysin is removed via extracellular vesicle shedding. These findings reveal that Ca2+-dependent microvesicle shedding protects cells from candidalysin and can be engaged by multiple molecular mechanisms during membrane repair. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=142 SRC="FIGDIR/small/653080v2_figa1.gif" ALT="Figure 1"> View larger version (43K): org.highwire.dtl.DTLVardef@433b3aorg.highwire.dtl.DTLVardef@1e583f3org.highwire.dtl.DTLVardef@139f086org.highwire.dtl.DTLVardef@de07b0_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOGraphical Abstract.C_FLOATNO Candidalysin is resisted by distinct repair mechanisms compared to bacterial PFTs.After pore formation and membrane damage by each toxin, multiple repair pathways are triggered downstream of Ca{superscript 2} flux. Candidalysin induces a protective Cl- influx and activates MEK-dependent repair, which contributes to cell protection. Annexin translocation occurs slowly and provides minor protection, while patch repair is ineffective. In contrast, aerolysin does not benefit from Cl- influx or MEK protection. Aerolysin triggers moderate annexin translocation and relies primarily on patch repair as the main protective mechanism. Streptolysin O elicits rapid annexin translocation and activates MEK signaling, both of which contribute to robust protection. Patch repair plays only a minor protective role against SLO. The figure was created using BioRender. C_FIG

cell biology↗

bioRxiv2023

Pathogenic Mutations in the C2A Domain of Dysferlin form Amyloid that Activates the Inflammasome.

Limb-Girdle Muscular Dystrophy Type-2B/2R is caused by mutations in the dysferlin gene (DYSF). This disease has two known pathogenic missense mutations that occur within dysferlins C2A domain, namely C2AW52R and C2AV67D. Yet, the etiological rationale to explain the disease linkage for these two mutations is still unclear. In this study, we have presented evidence from biophysical, computational, and immunological experiments which suggest that these missense mutations interfere with dysferlins ability to repair cells. The failure of C2AW52R and C2AV67D to initiate membrane repair arises from their propensity to form stable amyloid. The misfolding of the C2A domain caused by either mutation exposes {beta}-strands, which are predicted to nucleate classical amyloid structures. When dysferlin C2A amyloid is formed, it triggers the NLRP3 inflammasome, leading to the secretion of inflammatory cytokines, including IL-1{beta}. The present study suggests that the muscle dysfunction and inflammation evident in Limb-Girdle Muscular Dystrophy types-2B/2R, specifically in cases involving C2AW52R and C2AV67D, as well as other C2 domain mutations with considerable hydrophobic core involvement, may be attributed to this mechanism.

biophysics↗

bioRxiv2023

Deficiency of macrophage-derived Dnase1L3 causes lupus-like phenotypes in mice

Systemic Lupus Erythematosus (SLE) is a chronic autoimmune disease caused by environmental factors and loss of key proteins. One such protein is a serum endonuclease secreted by macrophages and dendritic cells, Dnase1L3. Loss of Dnase1L3 causes pediatric-onset lupus in humans is Dnase1L3. Reduction in Dnase1L3 activity occurs in adult-onset human SLE. However, the amount of Dnase1L3 necessary to prevent lupus onset, if the impact is continuous or requires a threshold, and which phenotypes are most impacted by Dnase1L3 remain unknown. To reduce Dnase1L3 protein levels, we developed a genetic mouse model with reduced Dnase1L3 activity by deleting Dnase1L3 from macrophages (cKO). Serum Dnase1L3 levels were reduced 67%, though Dnase1 activity remained constant. Sera were collected weekly from cKO and littermate controls until 50 weeks of age. Homogeneous and peripheral anti-nuclear antibodies were detected by immunofluorescence, consistent with anti-dsDNA antibodies. Total IgM, total IgG, and anti-dsDNA antibody levels increased in cKO mice with increasing age. In contrast to global Dnase1L3-/- mice, anti-dsDNA antibodies were not elevated until 30 weeks of age. The cKO mice had minimal kidney pathology, except for deposition of immune complexes and C3. Based on these findings, we conclude that an intermediate reduction in serum Dnase1L3 causes mild lupus phenotypes. This suggest that macrophage-derived DnaselL3 is critical to limiting lupus.

immunology↗

bioRxiv2022

Patch repair protects cells from the small pore-forming toxin aerolysin

Small pore-forming toxins in the aerolysin family lyse cells by damaging the membrane, but membrane repair responses used to resist them, if any, remain controversial. Four membrane repair mechanisms have been proposed: toxin removal by caveolar endocytosis, clogging by annexins, microvesicle shedding catalyzed by MEK, and patch repair. Which of these repair mechanisms aerolysin triggers is unknown. Furthermore, Ca2+ flux triggered by aerolysin is controversial, yet membrane repair responses require Ca2+. Here, we determined Ca2+ influx and repair mechanisms activated by aerolysin. In contrast to cholesterol-dependent cytolysins (CDCs), removal of extracellular Ca2+ protected cells from aerolysin. Aerolysin triggered sustained Ca2+ influx. Since aerolysin triggered Ca2+ flux, we investigated Ca2+-dependent repair pathways. Caveolar endocytosis failed to protect cells from aerolysin or CDCs. MEK-dependent repair did not protect against aerolysin. Aerolysin triggered slower annexin A6 membrane recruitment compared to CDCs. In contrast to CDCs, expression of the patch repair protein dysferlin potently protected cells from aerolysin. We propose that aerolysin triggers a Ca2+-dependent death mechanism that obscures repair responses, and the primary repair mechanism used to resist aerolysin is patch repair. We conclude that different classes of bacterial toxins trigger distinct repair mechanisms.

cell biology↗

bioRxiv2022

Sphingolipids protect ergosterol in the Leishmania major membrane from sterol-binding toxins

Susceptibility of Leishmania to the first line treatment amphotericin B remains poorly understood. Amphotericin B targets ergosterol, so one approach to improving drug efficacy and reducing side effects could be improving access to ergosterol. While the surface exposure of ergosterol in Leishmania is unknown, sterols in mammalian cells can be sheltered from sterol-binding agents by membrane components, including sphingolipids. Here, we tested the ability of the Leishmania major sphingolipids inositol phosphorylceramide (IPC), and ceramide to shelter ergosterol by preventing binding and cytotoxicity of the sterol-specific toxins streptolysin O and perfringolysin O using flow cytometry. In contrast to mammalian systems, Leishmania sphingolipids did not preclude toxin binding to sterols in the membrane. However, IPC interfered with cytotoxicity. Ceramide reduced perfringolysin O, but not streptolysin O, cytotoxicity in cells. Ceramide sensing was controlled by the toxin L3 loop. Ceramide was sufficient to protect L. major promastigotes from amphotericin B. We propose a mechanism whereby pore-forming toxins engage additional lipids like ceramide to determine the optimal environment to sustain pore formation. Thus, L. major offers a genetically tractable model organism for understanding toxin-membrane interactions. Furthermore, our findings suggest targeting ceramide may enhance the efficacy of ergosterol-targeting anti-leishmanial drugs. Abstract ImportanceLeishmaniasis is a neglected tropical disease with [~]1.5-2 million new cases and [~]70,000 deaths annually. One first-line treatment for leishmaniasis is liposomal amphotericin B, which is expensive and damages the kidneys. Cost and side effects can be minimized by improving efficacy. To improve efficacy, we must learn how amphotericins target--ergosterol--is protected by other components of Leishmania. The human ergosterol equivalent is protected by components called sphingolipids. We tested the ability of sphingolipids to protect ergosterol using pore-forming toxins. Pore-forming toxins use ergosterol to bind and kill Leishmania. Unlike human cells, toxins bound to ergosterol--indicating that they had access--when sphingolipids were present. However, sphingolipids protected Leishmania from toxins and amphotericin. Thus, Leishmania organizes sterol-protective components differently from humans. Further, toxins and Leishmania serve as a system to understand fundamental rules governing sterol-protecting component membrane organization. We can use this information to help improve drugs targeting sterols.

microbiology↗

bioRxiv2022

Structural features of Dnase1L3 responsible for serum antigen clearance

Autoimmunity develops when extracellular DNA released from dying cells is not cleared from serum. While serum DNA is primarily digested by Dnase1 and Dnase1L3, Dnase1 does not rescue autoimmunity arising from Dnase1L3 deficiencies. Dnase1L3 uniquely degrades antigenic forms of cell-free DNA, including DNA complexed with lipids and proteins. The distinct activity of Dnase1L3 relies on its unique C-terminal Domain (CTD), but the mechanism is unknown. We used multiple biophysical techniques and functional assays to study the interplay between the core catalytic domain and the CTD. While the core domain resembles Dnase1, there are several key differences between the two enzymes. Dnase1L3 is not inhibited by actin due to multiple differences in the actin recognition site. The CTD augments the ability of the core to bind DNA, thereby facilitating the degradation of complexed DNA to prevent autoimmune pathology. Together, these structural insights will inform the development of Dnase1L3-based therapies for autoimmunity.

biochemistry↗