Adult fruit flies are strongly attracted to odors emitted by the microbial fermentation of sugars in fruits. Such microbes, particularly yeasts, also serve as an essential nutritional source for fruit flies and influence their reproduction and fecundity. Despite being known as natural symbionts, it was previously observed that sucrose-fermenting Saccharomyces cerevisiae can be lethal to Drosophila melanogaster. This study characterizes the conditions that affect the lethality of S. cerevisiae in fruit flies. We showed that the insecticidal activity of yeast was equal in five tested S. cerevisiae strains. Another yeast, Pichia anomala, killed fruit flies, although to a lesser degree than S. cerevisiae. Higher concentrations of yeast resulted in faster killing of fruit flies, although the killing rate was capped at two days of continuous exposure. Both sexes were similarly sensitive to yeast at 30, whereas males were less sensitive to yeast at 25 degrees Celsius. Higher sucrose amounts in food slightly decreased the sensitivity of male but not female flies to yeast. This study characterizes a relatively uncharted side of the intricate interaction between fruit flies and yeast and highlights the possibility for manipulation aimed at mitigating the agricultural impact of these pests.

Fruit flies are naturally attracted to fermenting yeast because yeasts are necessary for Drosophila courtship and egg production and serve as a food source for larval development. Therefore, fermentation-based traps have been used to lure fruit fly pests. Such traps contain sucrose solution as the sole source of carbohydrates for yeast fermentation. However, the volatile organic compounds (VOCs) produced during sucrose fermentation by yeast have yet to be determined. We used SPME-GC/MS analysis and identified VOCs produced by Saccharomyces cerevisiae grown on 50 mM sucrose for 1 h, 1 day, and 4 days. A total of 101 VOCs were identified, representing acids, alcohols, aldehydes, and ketones. The abundance of VOCs varied, with five major VOCs (2-methylbutanol, 3-methylbutanol, ethanol, 2-phenethyl alcohol, and ethyl octanoate) representing 80% of all volatile abundance. The abundance of some VOCs increases over time, whereas others decrease or do not change over time. Future studies will determine specific VOCs that contribute to attracting fruit flies to sucrose-fermenting yeast.

Mycotoxin contamination of food may lead to cancer and is caused by fungal pathogens. In this study, we discovered that two natural products, phenylacetate and acetophenone, inhibit the growth of mycotoxin-producing fungi of agricultural importance. Using disc diffusion assays, we observed that both chemicals demonstrated antifungal activity against mycotoxin-producing Aspergillus flavus. The efficacies of acetophenone and phenylacetate synergized against A. flavus and four additional mycotoxin-producing fungi such as A. parasiticus, Penicillium expansum, Fusarium oxysporum, and F. verticillioides. Using growth kinetic assay, we observed that phenylacetate and acetophenone inhibited growth rates of human fungal pathogens, Candida albicans and C. auris. High-performance liquid chromatography analysis of aflatoxins extracted from A. flavus demonstrated that acetophenone treatment inhibited the production of aflatoxins, while phenylacetate did not have such an effect. This study identifies the phenylacetate-acetophenone combination as a potential antifungal for the mycotoxin-producing fungal treatment of food.

Insects that ingest microbial pathogens are also exposed to their toxins. The sensitivity of insects to ingested toxins of human pathogens and the potential mechanism of toxin resistance has not been thoroughly studied. We tested the survival of Drosophila melanogaster orally fed with exotoxins and endotoxins of ten human bacterial pathogens. We discovered that only a few toxins adversely affect fly survival, and that most toxins either do not affect or paradoxically extend insect survival (hormetic effect) at the dosages tested. We found that in Drosophila, Jra, a homolog of stress response transcription factor Jun, mediates a broad-spectrum toxin response, since the survival of Jra mutants was shortened in the presence of most of the tested toxins. This study begins to uncover the mechanism of the response of insects to toxins. It describes how a toxin-induced Jun stress response system helps insects reduce their sensitivity to toxins of human pathogens. Keywords: toxin; sensitivity; resistance; survival; immunity; Drosophila melanogaster Abbreviations: Jun related antigen (Jra); immune deficiency (Imd); antimicrobial peptides (AMP); mitogen-activated protein kinase (MAPK); Bloomington Drosophila stock center (BDSC); wild type (WT); lipopolysaccharide (LPS); adenosine diphosphate (ADP); adenosine triphosphate (ATP); adenosine monophosphate (cAMP); Jun N-terminal Kinase (JNK); Nuclear Factor-κB (NF-κB); absorption, distribution, metabolism, and excretion (ADME); absorption, distribution, metabolism, excretion, and toxicity (ADMET); confidence interval (CI); soluble N-ethylmale-imide-sensitive factorattachment protein receptors (SNARE); basic leucine zipper (bZIP); deoxyribonucleic acid (DNA); eukaryotic elongation factor (eEF); programmed death-ligand (PD-L); cluster of differentiation (CD).

Flies naturally contain microbes in their intestines after eating microbe-rich food like decaying fruits. When ingesting microbes, insects are also exposed to their toxins. The sensitivity of insects to ingested microbial toxins and their mechanism of response to toxins has not been thoroughly studied. Transcriptional regulator c-Myc has been shown to regulate the response to some but not all microbial toxins in mammals. We tested the sensitivity of wild-type and Myc mutant Drosophila melanogaster strains to two exotoxins, Clostridium perfringens α-toxin and Vibrio cholerae toxin, and two endotoxins, lipopolysaccharides (LPS) of Salmonella minnesota and S. typhimurium. We observed that both sexes of wild-type flies were insensitive to tested toxins. Similarly, Myc mutant males were insensitive to the four toxins. In contrast, female Myc mutants were significantly more sensitive to all tested toxins than wild-type females. The median survival of female Myc mutants was shortened by at least 54 hours in the presence of bacterial toxins. The component of LPS, lipid A, shortened the median survival of Myc females by 104 hours, indicating that the toxicity of LPS is caused by lipid A. This study demonstrates a sex-specific mechanism of the response of insects to toxins and describes that Myc protects female fruit flies from the tested microbial toxins. Keywords: bacterial toxins; sensitivity; resistance; survival; immunity; Drosophila melanogaster Abbreviations: Immune deficiency (Imd); antimicrobial peptides (AMP); mitogen-activated protein kinase (MAPK); Bloomington Drosophila stock center (BDSC); wild type (WT); lipopolysaccharide (LPS); adenosine diphosphate (ADP); Jun N-terminal Kinase (JNK); Nuclear Factor-κB (NF-κB); absorption, distribution, metabolism, and excretion (ADME); absorption, distribution, metabolism, excretion, and toxicity (ADMET); confidence interval (CI); deoxyribonucleic acid (DNA); intestinal stem cells (ISCs); Janus kinases (JAK); signal transducer and activator of transcription (STAT); epidermal growth factor EGF; and Wingless (Wg).

The genders of Drosophila melanogaster vary in their sensitivities to microbial pathogens. While many of the immunity-related genes are located on the X chromosome, the polymorphisms within the Y chromosome were also shown to affect the immunity of flies. In this study, we investigated the necessity of individual genes on the Y chromosome (Y-genes) for male sensitivity to microbes. We identified several Y-genes whose genetic inactivation either increases or decreases the sensitivity of males to gastrointestinal infections with fungal Saccharomyces cerevisiae and bacterial Serratia liquefaciens. Specifically, the loss of function mutations in fly kl-5 and Ppr-Y Y-genes lead to increased and decreased sensitivity of males to fungal challenge, respectively, compared to female sensitivity. In contrast, mutations in Drosophila Pp1-Y1, kl-5, kl-3, Ppr-Y, CCY, and FDY Y-genes lead to increased sensitivity of males to bacterial infection, compared to females. Moreover, while these Y-genes are necessary, the Y chromosome is not sufficient for the sensitivity of males to microbes, since the sensitivity of XXY females to fungal and bacterial challenges was not different from the sensitivity of wild-type female flies, compared to males. This study assigns a new immunity-related function to numerous Y-genes in D.melanogaster.

Anthrax is caused by Bacillus anthracis and can result in nearly 100% mortality due in part to anthrax toxin. Antimalarial amodiaquine (AQ) acts as a host-oriented inhibitor of anthrax toxin endocytosis. Here, we determined the pharmacokinetics and safety of AQin mice, rabbits, and humans as well as the efficacy in the fly, mouse, and rabbit models of anthrax infection. In the therapeutic-intervention studies, AQ nearly doubled the survival of mice infected subcutaneously with a B. anthracis dose lethal to 60% of the animals (LD60). In rabbits challenged with 200 LD50 of aerosolized B. anthracis, AQ as a monotherapy delayed death, doubled the survival rate of infected animals that received a suboptimal amount of antibacterial levofloxacin, and reduced bacteremia and toxemia in tissues. Surprisingly, the anthrax efficacy of AQ relies on an additional host macrophage-directed antibacterial mechanism, which was validated in the toxin-independent Drosophila model of Bacillus infection. Lastly, a systematic literature review of the safety and pharmacokinetics of AQin humans from over 2 000 published articles revealed that AQ is likely safe when taken as prescribed, and its pharmacokinetics predicts anthrax efficacy in humans. Our results support the future examination of AQ as adjunctive therapy for the prophylactic anthrax treatment.

Of the numerous infectious diseases afflicting humans, anthrax disease, caused by Bacillus anthracis, poses a major threat in its virulence and lack of effective treatment. The currently lacking standards of care, as well as the lengthy drug approval process, demonstrate the pressing demand for treatment for B. anthracis infections. The present study screened 1586 clinically approved drugs in an attempt to identify repurposable compounds against B. cereus, a relative strain that shares many physical and genetic characteristics with B. anthracis. Our study yielded five drugs that successfully inhibited B. cereus growth: dichlorophen, oxiconazole, suloctidil, bithionol, and hexestrol. These drugs exhibited varying levels of efficacy in broad-spectrum experiments against several Gram-positive and Gram-negative bacterial strains, with hexestrol showing the greatest inhibition across all tested strains. Through tests for the efficacy of each drug on B. cereus, bithionol was the single most potent compound on both solid and liquid media and exhibited even greater eradication of B. cereus in combination with suloctidil on solid agar. This multifaceted in vitro study of approved drugs demonstrates the potential to repurpose these drugs as treatments for anthrax disease in a time-efficient manner to address a global health need.

Trypanosoma brucei are unicellular parasites endemic to Sub-Saharan Africa that cause fatal disease in humans and animals. Infection with these parasites is caused by the bite of the tsetse fly vector, and parasites living extracellularly in the blood of infected animals evade the host immune system through antigenic variation. Existing drugs for Human and Animal African Trypanosomiasis are difficult to administer and can have serious side effects. Resistance to some drugs is also increasing, creating an urgent need for alternative trypanosomiasis therapeutics. We screened a library of 1,585 U.S. or foreign-approved drugs and identified 154 compounds that inhibit trypanosome growth. As all of these compounds have already undergone testing for human toxicity, they represent good candidates for repurposing as trypanosome therapeutics. In addition to identifying drugs that inhibit trypanosome growth, we wished to identify small molecules that can induce bloodstream form parasites to differentiate into forms adapted for the insect vector. These insect stage parasites lack the immune evasion mechanisms prevalent in bloodstream forms, making them vulnerable to the host immune system. To identify drugs that increase transcript levels of an invariant, insect-stage specific surface protein called procyclin, we engineered bloodstream reporter parasites that express Green Fluorescent Protein (GFP) following induction or stabilization of the procyclin transcript. Using these bloodstream reporter strains in combination with automated flow cytometry, we identified eflornithine, spironolactone, and phenothiazine as small molecules that increase abundance of procyclin transcript. Both eflornithine and spironolactone also affect transcript levels for a subset of differentiation associated genes. While we failed to identify compounds that increase levels of procyclin protein on the cell surface, this study is proof of principle that these fluorescent reporter parasites represent a useful tool for future small molecule or genetic screens aimed at identifying molecules or processes that initiate remodeling of the parasite surface during life cycle stage transitions.

Anthrax is a major zoonotic disease of wildlife, and in places like West Africa, it can be caused by Bacillus anthracis in arid nonsylvatic savannahs, and by B. cereus biovar anthracis (Bcbva) in sylvatic rainforests. Bcbva-caused anthrax has been implicated in as much as 38% of mortality in rainforest ecosystems, where insects can enhance the transmission of anthrax-causing bacteria. While anthrax is well-characterized in mammals, its transmission by insects points to an unidentified anthrax-resistance mechanism in its vectors. In mammals, a secreted anthrax toxin component, 83 kDa Protective Antigen (PA83), binds to cell-surface receptors and is cleaved by furin into an evolutionary-conserved PA20 and a pore-forming PA63 subunits. We show that PA20 increases the resistance of Drosophila flies and Culex mosquitoes to bacterial challenges, without directly affecting the bacterial growth. We further show that the PA83 loop known to be cleaved by furin to release PA20 from PA63 is, in part, responsible for the PA20-mediated protection. We found that PA20 binds directly to the Toll activating peptidoglycan-recognition protein-SA (PGRP-SA) and that the Toll/NF-κB pathway is necessary for the PA20-mediated protection of infected flies. This effect of PA20 on innate immunity may also exist in mammals: we show that PA20 binds to human PGRP-SA ortholog. Moreover, the constitutive activity of Imd/NF-κB pathway in MAPKK Dsor1 mutant flies is sufficient to confer the protection from bacterial infections in a manner that is independent of PA20 treatment. Lastly, Clostridium septicum alpha toxin protects flies from anthrax-causing bacteria, showing that other pathogens may help insects resist anthrax. The mechanism of anthrax resistance in insects has direct implications on insect-mediated anthrax transmission for wildlife management, and with potential for applications, such as reducing the sensitivity of pollinating insects to bacterial pathogens.

Background Zika virus (ZIKV) is a major human pathogen and member of the Flavivirus genus. Previous studies have identified neutralizing antibodies from Zika patients that bind to quaternary epitopes across neighboring envelope (E) proteins, called E dimer epitopes (EDE). An asparagine-linked glycan on the “glycan loop” (GL) of the ZIKV envelope protein protects the functionally important “fusion loop” on the opposite E subunit in the dimer, and EDE antibodies have been shown to bind to both of these loops. Human EDE antibodies have been divided into two subclasses based on how they bind to the glycan loop region: EDE1 antibodies do not require glycosylation for binding, while EDE2 antibodies strongly rely on the glycan for binding. Methods ZIKV GL was expressed on tobacco mosaic virus nanoparticles. Mice were immunized with GL or full-length monomeric E and the immune response was analyzed by testing the ability of sera and monoclonal antibodies to bind to GL and to neutralize ZIKV in in vitro cellular assay. Results We report here the existence of ZIKV moderately neutralizing antibodies that bind to E monomers through epitopes that include the glycan loop. We show that sera from human Zika patients contain antibodies capable of binding to the unglycosylated glycan loop in the absence of the rest of the envelope protein. Furthermore, mice were inoculated with recombinant E monomers and produced neutralizing antibodies that either recognize unglycosylated glycan loop or require glycan for their binding to monomeric E. We demonstrate that both types of antibodies neutralize ZIKV to some extent in a cellular virus neutralization assay. Conclusions Analogous to the existing EDE antibody nomenclature, we propose a new classification for antibodies that bind to E monomer epitopes (EME): EME1 and EME2 for those that do not require and those that do require glycan for binding to E, respectively.

The rising prevalence of multidrug-resistant hospital-acquired infections has increased the need for new antibacterial agents. In this study, a library of 1586 FDA-approved drugs was screened against A. calcoaceticus, a representative of the Acinetobacter calcoaceticus-baumannii complex. Three compounds were found to have previously undiscovered antibacterial properties against A. calcoaceticus: antifungal Miconazole, anthelminthic Dichlorophen, and Bithionol. These three drugs were tested against a wide range of Gram-positive and Gram-negative bacteria and confirmed to have broad-spectrum antibacterial properties. Combinations of these three drugs were also tested against the same bacteria, and two novel combination therapies with synergistic effects were discovered. In the future, antibacterial properties of these three drugs and two combination therapies will be evaluated against pathogenic bacteria using an animal model.

Control of fungal pathogens is increasingly problematic due to the limited number of effective drugs available for antifungal therapy. Conventional antifungal drugs could also trigger human cytotoxicity associated with the kidneys and liver, including the generation of reactive oxygen species. Moreover, increased incidences of fungal resistance to the classes of azoles, such as fluconazole, itraconazole, voriconazole, or posaconazole, or echinocandins, including caspofungin, anidulafungin, or micafungin, have been documented. Of note, certain azole fungicides such as propiconazole or tebuconazole that are applied to agricultural fields have the same mechanism of antifungal action as clinical azole drugs. Such long-term application of azole fungicides to crop fields provides environmental selection pressure for the emergence of pan-azole-resistant fungal strains such as Aspergillus fumigatus having TR34/L98H mutations, specifically, a 34 bp insertion into the cytochrome P450 51A (CYP51A) gene promoter region and a leucine-to-histidine substitution at codon 98 of CYP51A. Altogether, the emerging resistance of pathogens to currently available antifungal drugs and insufficiency in the discovery of new therapeutics engender the urgent need for the development of new antifungals and/or alternative therapies for effective control of fungal pathogens. We discuss the current needs for the discovery of new clinical antifungal drugs and the recent drug repurposing endeavors as alternative methods for fungal pathogen control.

BACKGROUND:Vertebrate glucocorticoid receptor (GR) is an evolutionary-conserved cortisol-regulated nuclear receptor that controls key metabolic and developmental pathways. Upon binding to cortisol, GR acts as an immunosuppressive transcription factor. Drosophila melanogaster, a model organism to study innate immunity, can also be immunosuppressed by glucocorticoids. However, while the genome of fruit fly harbors 18 nuclear receptor genes, the functional homolog of vertebrate GR has not been identified.RESULTS:In this study, we demonstrated that while D. melanogaster is susceptible to Saccharomyces cerevisiae oral infection, the oral exposure to cortisol analogs, cortisone acetate or estrogen, increases fly sensitivity to yeast challenge. To understand the mechanism of this steroid-induced immunosuppression, we identified the closest genetic GR homolog as D. melanogaster Estrogen Related Receptor (ERR) gene. We discovered that Drosophila ERR is necessary for cortisone acetate- and estrogen-mediated increase in sensitivity to fungal infection: while ERR mutant flies are as sensitive to the fungal challenge as the wildtype flies, the yeast-sensitivity of ERR mutants is not increased by these steroids. Interestingly, the fungal cortisone analog, ergosterol, did not increase the susceptibility of Drosophila to yeast infection. The immunosuppressive effect of steroids on the sensitivity of flies to fungi is evolutionary conserved in insects, as we show that estrogen significantly increases the yeast-sensitivity of Culex quinquefasciatus mosquitoes, whose genome contains a close ortholog of the fly ERR gene.CONCLUSIONS:This study identifies a D. melanogaster gene that structurally resembles vertebrate GR and is functionally necessary for the steroid-mediated immunosuppression to fungal infections.

Trypanosoma brucei are unicellular parasites endemic to Sub-Saharan Africa that cause fatal disease in humans and animals. Infection with these parasites is caused by the bite of the tsetse fly vector, and parasites living extracellularly in the blood of infected animals evade the host immune system through antigenic variation. Existing drugs for Human and Animal African Trypanosomiasis are difficult to administer and can have serious side effects. Resistance to some drugs is also increasing, creating an urgent need for alternative trypanosomiasis therapeutics. In addition to identifying drugs that inhibit trypanosome growth, we wish to identify small molecules that can induce bloodstream form parasites to differentiate into forms adapted for the insect vector. These insect stage parasites do not vary proteins on their cell surface, making them vulnerable to the host immune system. To identify drugs that trigger differentiation of the parasite from bloodstream to insect stages, we engineered bloodstream reporter parasites that express Green Fluorescent Protein (GFP) following induction of the invariant insect-stage specific procyclin transcript. Using these bloodstream reporter strains in combination with high-throughput flow cytometry, we screened a library of 1,585 U.S. or foreign-approved drugs and identified eflornithine, spironolactone, and phenothiazine as small molecules that induce transcription of procylin. Both eflornithine and spironolactone also affect transcript levels for a subset of differentiation associated genes. We further identified 154 compounds that inhibit trypanosome growth. As all of these compounds have already undergone testing for human toxicity, they represent good candidates for repurposing as trypanosome therapeutics. Finally, this study is proof of principle that fluorescent reporters are a useful tool for small molecule or genetic screens aimed at identifying molecules or processes that initiate remodeling of the parasite surface during life cycle stage transitions. Author Summary African trypanosomes are unicellular parasites that infect humans and animals, causing a fatal disease known as sleeping sickness in humans and nagana in cattle. These diseases impose a severe economic burden for people living in Sub-Saharan Africa, where parasites are transmitted to humans and animals through the bite of the tsetse fly. Parasites living outside cells in humans and animals are attacked by the antibodies of the host immune system, but they can evade this attack by varying the proteins on their cell surface. In contrast, because flies do not have an antibody-mediated immune response, parasites living in flies do not vary the proteins on their cell surface. In this study, we performed a small molecule screen to identify compounds that might force bloodstream parasites to move forward in their life cycle to become more similar to parasites living in flies, causing them to express a protein on their cell surface that does not vary. This invariant protein on the surface of bloodstream parasites would make bloodstream parasites vulnerable to the host antibodies. We found 3 compounds that increased RNA levels for an invariant insect-stage surface protein and 154 compounds that inhibit parasite growth. We hope these compounds might have potential as novel trypanosomiasis therapeutics.

Exploiting the host endocytic trafficking pathway is a common mechanism by which bacterial exotoxins gain entry to exert virulent effects upon the host cells. A previous study identified a small-molecule, 1-(2,6-dimethyl-1-piperidinyl)-3-[(2-isopropyl-5-methylcyclohexyl)oxy]-2-propanol, that blocks the process of anthrax lethal toxin (LT) cytotoxicity. Here, we report the characterization of the bioactivity of this compound, which we named RC1. We found that RC1 protected host cells independently of LT concentration and also blocked intoxication by other bacterial exotoxins, suggesting that the target of the compound is a host factor. Using the anthrax LT intoxication pathway as a reference, we show that while anthrax toxin is able to bind to cells and establish an endosomal pore in the presence of the drug, the toxin is unable to translocate into the cytosol. We demonstrate that RC1 does not inhibit the toxin directly but rather reduces the enzymatic activity of host cathepsin B that mediates the escape of toxins into the cytoplasm from late endosomes. We demonstrate that the pathogenicity of Human cytomegalovirus and Herpes simplex virus 1, which relies on cathepsin B protease activity, is reduced by RC1. This study reveals the potential of RC1 as a broad-spectrum host-oriented therapy against several aggressive and deadly pathogens.

Inflammasomes activate caspase-1 in response to molecular signals from pathogens and other dangerous stimuli as a part of the innate immune response. A previous study discovered a small-molecule, 4-fluoro- N'-[1-(2-pyridinyl)ethylidene]benzohydrazide, which we named DN1, that reduces the cytotoxicity of anthrax lethal toxin (LT). We determined that DN1 protected cells irrespectively of LT concentration and reduced the pathogenicity of an additional bacterial exotoxin and several viruses. Using the LT cytotoxicity pathway, we show that DN1 does not prevent LT internalization and catalytic activity or caspase-1 activation. Moreover, DN1 does not affect the proteolytic activity of host cathepsin B, which facilitates the cytoplasmic entry of toxins. PubChem Bioactivities lists two G protein-coupled receptors (GPCR), type-1 angiotensin II receptor and apelin receptor, as targets of DN1. The inhibition of phosphatidylinositol 3-kinase, phospholipase C, and protein kinase B, which are downstream of GPCR signaling, synergized with DN1 in protecting cells from LT. We hypothesize that DN1-mediated antagonism of GPCRs modulates signal transduction pathways to induce a cellular state that reduces LT-induced pyroptosis downstream of caspase-1 activation. DN1 also reduced the susceptibility of Drosophila melanogaster to toxin-associated bacterial infections. Future experiments will aim to further characterize how DN1 modulates signal transduction pathways to inhibit pyroptotic cell death in LT-sensitive macrophages. DN1 represents a novel chemical probe to investigate host cellular mechanisms that mediate cell death in response to pathogenic agents.

Diverse pathogenic agents often utilize overlapping host networks, and hub proteins within these networks represent attractive targets for broad-spectrum drugs. Using bacterial toxins, we describe a new approach for discovering broad-spectrum therapies capable of inhibiting host proteins that mediate multiple pathogenic pathways. This approach can be widely used, as it combines genetic-based target identification with cell survival-based and protein function-based multiplex drug screens, and concurrently discovers therapeutic compounds and their protein targets. Using B-lymphoblastoid cells derived from the HapMap Project cohort of persons of African, European, and Asian ancestry we identified host caspases as hub proteins that mediate the lethality of multiple pathogenic agents. We discovered that an approved drug, Bithionol, inhibits host caspases and also reduces the detrimental effects of anthrax lethal toxin, diphtheria toxin, cholera toxin, Pseudomonas aeruginosa exotoxin A, Botulinum neurotoxin, ricin, and Zika virus. Our study reveals the practicality of identifying host proteins that mediate multiple disease pathways and discovering broad-spectrum therapies that target these hub proteins.

The major limitations of pathogen-directed therapies are the emergence of drug-resistance and their narrow spectrum of coverage. A recently applied approach directs therapies against host proteins exploited by pathogens in order to circumvent these limitations. However, host-oriented drugs leave the pathogens unaffected and may result in continued pathogen dissemination. In this study we aimed to discover drugs that could simultaneously cross-inhibit pathogenic agents, as well as the host proteins that mediate their lethality. We observed that many pathogenic and host-assisting proteins belong to the same functional class. In doing so we targeted a protease component of anthrax toxin as well as host proteases exploited by this toxin. We identified two approved drugs, ascorbic acid 6-palmitate and salmon sperm protamine, that effectively inhibited anthrax cytotoxic protease and demonstrated that they also block proteolytic activities of host furin, cathepsin B, and caspases that mediate toxin's lethality in cells. We demonstrated that these drugs are broad-spectrum and reduce cellular sensitivity to other bacterial toxins that require the same host proteases. This approach should be generally applicable to the discovery of simultaneous pathogen and host-targeting inhibitors of many additional pathogenic agents.