Ebselen Not Only Inhibits Clostridioides difficile Toxins but Displays Redox-Associated Cellular Killing.

Ebselen, a reactive organoselenium compound, was shown to inhibit toxins TcdA and TcdB by covalently binding to their cysteine protease domains. It was suggested that ebselen lacked antimicrobial activity against Clostridioides difficile. However, this perception conflicts with C. difficile having essential cysteine-containing enzymes that could be potential targets and the reported antimicrobial activity of ebselen against other species. Hence, we reevaluated the anti-C. difficile properties of ebselen. Susceptibility testing revealed that its activity was either slightly reduced by pyruvate found in Wilkins-Chalgren agar or obliterated by blood in brucella agar. In brain heart infusion (BHI) agar, ebselen inhibited most C. difficile strains (MICs of 2 to 8 µg/ml), except for ribotype 078 that was intrinsically resistant (MIC = 32 to 128 µg/ml). Against C. difficile R20291, at concentrations below its minimal bactericidal concentration (MBC), 16 µg/ml, ebselen inhibited production of toxins and spores. Transcriptome analysis revealed that ebselen altered redox-associated processes and cysteine metabolism and enhanced expression of Stickland proline metabolism, likely to regenerate NAD+ from NADH. In cellular assays, ebselen induced uptake of cysteine, depleted nonprotein thiols, and disrupted the NAD+/NADH ratio. Taken together, killing of C. difficile cells by ebselen occurs by a multitarget action that includes disrupting intracellular redox, which is consistent with ebselen being a reactive molecule. However, the physiological relevance of these antimicrobial actions in treating acute C. difficile infection (CDI) is likely to be undermined by host factors, such as blood, which protect C. difficile from killing by ebselen. IMPORTANCE We show that ebselen kills pathogenic C. difficile by disrupting its redox homeostasis, changing the normal concentrations of NAD+ and NADH, which are critical for various metabolic functions in cells. However, this antimicrobial action is hampered by host components, namely, blood. Future discovery of ebselen analogues, or mechanistically similar compounds, that remain active in blood could be drug leads for CDI or probes to study C. difficile redox biology in vivo.

Chronic whipworm infection exacerbates Schistosoma mansoni egg-induced hepatopathology in non-human primates.

BACKGROUND: Schistosomiasis continues to inflict significant morbidity and mortality in the tropical and subtropical regions of the world. The disease endemicity overlaps with the transmission of other parasitic diseases. Despite the ubiquity of polyparasitism in tropical regions, particularly in rural communities, little is known about the impact of multiple helminth infections on disease progression. In this pilot study, we describe the influence of chronic Trichuris trichiura infection on Schistosoma mansoni egg-induced hepatopathology in infected baboons. METHODS: Baboons with or without underlying whipworm infection were challenged with S. mansoni cercariae to establish schistosomiasis. Adult S. mansoni worms were recovered by perfusion and enumerated, hepatic granulomas were quantified via light microscopy, and transcriptional profiling of tissues were completed using RNA sequencing technologies. RESULTS: Co-infection with both S. mansoni and T. trichiura resulted in higher female schistosome worm burden and significantly larger liver granuloma sizes. Systems biology analyses of peripheral blood mononuclear cells (PBMC) revealed pathways associated with increased liver damage in co-infected baboons. CONCLUSIONS: Underlying chronic whipworm infection intensified schistosome egg-induced liver pathology in infected baboons. RNA-Seq analysis provided insight into pathways associated with increased liver damage, corroborating histological findings.

Alpha-Tocotrienol Prevents Oxidative Stress-Mediated Post-Translational Cleavage of Bcl-xL in Primary Hippocampal Neurons.

B-cell lymphoma-extra large (Bcl-xL) is an anti-apoptotic member of the Bcl2 family of proteins, which supports neurite outgrowth and neurotransmission by improving mitochondrial function. During excitotoxic stimulation, however, Bcl-xL undergoes post-translational cleavage to ∆N-Bcl-xL, and accumulation of ∆N-Bcl-xL causes mitochondrial dysfunction and neuronal death. In this study, we hypothesized that the generation of reactive oxygen species (ROS) during excitotoxicity leads to formation of ∆N-Bcl-xL. We further proposed that the application of an antioxidant with neuroprotective properties such as α-tocotrienol (TCT) will prevent ∆N-Bcl-xL-induced mitochondrial dysfunction via its antioxidant properties. Primary hippocampal neurons were treated with α-TCT, glutamate, or a combination of both. Glutamate challenge significantly increased cytosolic and mitochondrial ROS and ∆N-Bcl-xL levels. ∆N-Bcl-xL accumulation was accompanied by intracellular ATP depletion, loss of mitochondrial membrane potential, and cell death. α-TCT prevented loss of mitochondrial membrane potential in hippocampal neurons overexpressing ∆N-Bcl-xL, suggesting that ∆N-Bcl-xL caused the loss of mitochondrial function under excitotoxic conditions. Our data suggest that production of ROS is an important cause of ∆N-Bcl-xL formation and that preventing ROS production may be an effective strategy to prevent ∆N-Bcl-xL-mediated mitochondrial dysfunction and thus promote neuronal survival.

Sm-p80-based schistosomiasis vaccine: double-blind preclinical trial in baboons demonstrates comprehensive prophylactic and parasite transmission-blocking efficacy.

Schistosomiasis is of public health importance to an estimated one billion people in 79 countries. A vaccine is urgently needed. Here, we report the results of four independent, double-blind studies of an Sm-p80-based vaccine in baboons. The vaccine exhibited potent prophylactic efficacy against transmission of Schistosoma mansoni infection and was associated with significantly less egg-induced pathology, compared with unvaccinated control animals. Specifically, the vaccine resulted in a 93.45% reduction of pathology-producing female worms and significantly resolved the major clinical manifestations of hepatic/intestinal schistosomiasis by reducing the tissue egg-load by 89.95%. A 35-fold decrease in fecal egg excretion in vaccinated animals, combined with an 81.51% reduction in hatching of eggs into the snail-infective stage (miracidia), demonstrates the parasite transmission-blocking potential of the vaccine. Substantially higher Sm-p80 expression in female worms and Sm-p80-specific antibodies in vaccinated baboons appear to play an important role in vaccine-mediated protection. Preliminary analyses of RNA sequencing revealed distinct molecular signatures of vaccine-induced effects in baboon immune effector cells. This study provides comprehensive evidence for the effectiveness of an Sm-p80-based vaccine for schistosomiasis.

Cross-species prophylactic efficacy of Sm-p80-based vaccine and intracellular localization of Sm-p80/Sm-p80 ortholog proteins during development in Schistosoma mansoni, Schistosoma japonicum, and Schistosoma haematobium.

Schistosomiasis remains a major global health problem. Despite large-scale schistosomiasis control efforts, clear limitations such as possible emergence of drug resistance and reinfection rates highlight the need for an effective schistosomiasis vaccine. Schistosoma mansoni large subunit of calpain (Sm-p80)-based vaccine formulations have shown remarkable efficacy in protecting against S. mansoni challenge infections in mice and baboons. In this study, we evaluated the cross-species protective efficacy of Sm-p80 vaccine against S. japonicum and S. haematobium challenge infections in rodent models. We also elucidated the expression of Sm-p80 and Sm-p80 ortholog proteins in different developmental stages of S. mansoni, S. haematobium, and S. japonicum. Immunization with Sm-p80 vaccine reduced worm burden by 46.75% against S. japonicum challenge infection in mice. DNA prime/protein boost (1 + 1 dose administered on a single day) resulted in 26.95% reduction in worm burden in S. haematobium-hamster infection/challenge model. A balanced Th1 (IFN-γ, TNF-α, IL-2, and IL-12) and Th2 (IL-4, IgG1) type of responses were observed following vaccination in both S. japonicum and S. haematobium challenge trials and these are associated with the prophylactic efficacy of Sm-p80 vaccine. Immunohistochemistry demonstrated that Sm-p80/Sm-p80 ortholog proteins are expressed in different life cycle stages of the three major human species of schistosomes studied. The data presented in this study reinforce the potential of Sm-p80-based vaccine for both hepatic/intestinal and urogenital schistosomiasis occurring in different geographical areas of the world. Differential expression of Sm-p80/Sm-p80 protein orthologs in different life cycle makes this vaccine potentially useful in targeting different levels of infection, disease, and transmission.

Insertions within the actin core of actin-related protein 3 (Arp3) modulate branching nucleation by Arp2/3 complex.

The Arp2/3 (actin-related protein 2/3) complex nucleates branched actin filaments involved in multiple cellular functions, including endocytosis and cellular motility. Two subunits (Arp2 and Arp3) in this seven-subunit assembly are closely related to actin and upon activation of the complex form a "cryptic dimer" that stably mimics an actin dimer to nucleate a new filament. Both Arps contain a shared actin core structure, and each Arp contains multiple insertions of unknown function at conserved positions within the core. Here we characterize three key insertions within the actin core of Arp3 and show that each one plays a distinct role in modulating Arp2/3 function. The ß4/ß5 insert mediates interactions of Arp2/3 complex with actin filaments and "dampers" the nucleation activity of the complex. The Arp3 hydrophobic plug plays an important role in maintaining the integrity of the complex but is not absolutely required for formation of the daughter filament nucleus. Deletion of the αK/ß15 insert did not constitutively activate the complex, as previously hypothesized. Instead, it abolished in vitro nucleation activity and caused defects in endocytic actin patch assembly in fission yeast, indicating a role for the αK/ß15 insert in the activated state of the complex. Biochemical characterization of each mutant revealed steps in the nucleation pathway influenced by each Arp3-specific insert to provide new insights into the structural basis of activation of the complex.

Mechanism of a concentration-dependent switch between activation and inhibition of Arp2/3 complex by coronin.

Arp2/3 complex is a key actin filament nucleator that assembles branched actin networks in response to cellular signals. The activity of Arp2/3 complex is regulated by both activating and inhibitory proteins. Coronins make up a large class of actin-binding proteins previously shown to inhibit Arp2/3 complex. Although coronins are known to play a role in controlling actin dynamics in diverse processes, including endocytosis and cell motility, the precise mechanism by which they regulate Arp2/3 complex is unclear. We conducted a detailed biochemical analysis of budding yeast coronin, Crn1, and found that it not only inhibits Arp2/3 complex but also activates it. We mapped regions required for activation and found that Crn1 contains a sequence called CA, which is conserved in WASp/Scar proteins, the prototypical activators of Arp2/3 complex. Point mutations in CA abolished activation of Arp2/3 complex by Crn1 in vitro. Confocal microscopy and quantitative actin patch tracking showed that these mutants had defective endocytic actin patch dynamics in Saccharomyces cerevisiae, indicating that activation of Arp2/3 complex by coronin is required for normal actin dynamics in vivo. The switch between the dual modes of regulation by Crn1 is controlled by concentration, and low concentrations of Crn1 enhance filament binding by Arp2/3 complex, whereas high concentrations block binding. Our data support a direct tethering recruitment model for activation of Arp2/3 complex by Crn1 and suggest that Crn1 indirectly inhibits Arp2/3 complex by blocking it from binding actin filaments.