Botulinum neurotoxins (BoNTs) are some of the most poisonous natural toxins. Botulinum neurotoxins associate with neurotoxin-associated proteins (NAPs) forming large complexes that are protected from the harsh environment of the gastrointestinal tract. However, it is still unclear how BoNT complexes as large as 900 kDa traverse the epithelial barrier and what role NAPs play in toxin translocation. In this study, we examined the transit of BoNT serotype A (BoNT/A) holotoxin, complex and recombinantly purified NAP complex through cultured and polarized Caco-2 cells and, for the first time, in the small mouse intestine. Botulinum neurotoxin serotype A and NAPs in the toxin complex were detectable inside intestinal cells beginning at 2 h post intoxication. Appearance of the BoNT/A holotoxin signal was slower, with detection starting at 4-6 h. This indicated that the holotoxin alone was sufficient for entry but the presence of NAPs enhanced the rate of entry. Botulinum neurotoxin serotype A detection peaked at approximately 6 and 8 h for complex and holotoxin, respectively, and thereafter began to disperse with some toxin remaining in the epithelia after 24 h. Purified HA complexes alone were also internalized and followed a similar time course to that of BoNT/A complex internalization. However, recombinant HA complexes did not enhance BoNT/A holotoxin entry in the absence of a physical link with BoNT/A. We propose a model for BoNT/A toxin complex translocation whereby toxin complex entry is facilitated by NAPs in a receptor-mediated mechanism. Understanding the intestinal uptake of BoNT complexes will aid the development of new measures to prevent or treat oral intoxications.

Botulinum neurotoxins (BoNTs) cause the disease botulism manifested by flaccid paralysis that could be fatal to humans and animals. Oral ingestion of the toxin with contaminated food is one of the most common routes for botulism. BoNT assembles with several auxiliary proteins to survive in the gastrointestinal tract and is subsequently transported through the intestinal epithelium into the general circulation. Several hemagglutinin proteins form a multi-protein complex (HA complex) that recognizes host glycans on the intestinal epithelial cell surface to facilitate BoNT absorption. Blocking carbohydrate binding to the HA complex could significantly inhibit the oral toxicity of BoNT. Here, we identify lactulose, a galactose-containing non-digestible sugar commonly used to treat constipation, as a prototype inhibitor against oral BoNT/A intoxication. As revealed by a crystal structure, lactulose binds to the HA complex at the same site where the host galactose-containing carbohydrate receptors bind. In vitro assays using intestinal Caco-2 cells demonstrated that lactulose inhibits HA from compromising the integrity of the epithelial cell monolayers and blocks the internalization of HA. Furthermore, co-administration of lactulose significantly protected mice against BoNT/A oral intoxication in vivo. Taken together, these data encourage the development of carbohydrate receptor mimics as a therapeutic intervention to prevent BoNT oral intoxication.

Botulinum neurotoxins (BoNTs) comprise seven agreed-on serotypes, A through G. In 2014, a novel chimeric neurotoxin produced by clostridial strain IBCA10-7060 was reported as BoNT/H, with subsequent names of BoNT/FA or BoNT/HA based on sequence homology of the N-terminus to BoNT/F, the C-terminus to BoNT/A and neutralization studies. The purpose of this study was to define the immunologic identity of the novel BoNT. monoclonal antibodies (mAbs) to the novel BoNT/H N-terminus were generated by antibody repertoire cloning and yeast display after immunization with BoNT/H LC-HN or BoNT/F LC-HN. 21 unique BoNT/H LC-HN mAbs were obtained; 15 from the BoNT/H LC-HN immunized library (KD 0.78 nM to 182 nM) and six from the BoNT/F-immunized libraries (KD 20.5 nM to 1490 nM). A total of 15 of 21 mAbs also bound catalytically inactive BoNT/H holotoxin. The mAbs bound nine non-overlapping epitopes on the BoNT/H LC-HN. None of the mAbs showed binding to BoNT serotypes A-G, nor any of the seven subtypes of BoNT/F, except for one mAb that weakly bound BoNT/F5. The results, combined with the chimeric structure and neutralization by anti-A, but not anti-F antitoxin indicate that immunologically the novel BoNT is BoNT/HA. This determination has significant implications for existing countermeasures and potential vulnerabilities.

Current antifungal interventions have often limited efficiency in treating fungal pathogens, particularly those resistant to commercial drugs or fungicides. Antifungal drug repurposing is an alternative intervention strategy, whereby new utility of various marketed, non-antifungal drugs could be repositioned as novel antifungal agents. In this study, we investigated "chemosensitization" as a method to improve the efficiency of antifungal drug repurposing, wherein combined application of a second compound (viz., chemosensitizer) with a conventional, non-antifungal drug could greatly enhance the antifungal activity of the co-applied drug. Redox-active natural compounds or structural derivatives, such as thymol (2-isopropyl-5-methylphenol), 4-isopropyl-3-methylphenol, or 3,5-dimethoxybenzaldehyde, could serve as potent chemosensitizers to enhance antifungal activity of the repurposed drug bithionol. Of note, inclusion of fungal mutants, such as antioxidant mutants, could also facilitate drug repurposing efficiency, which is reflected in the enhancement of antifungal efficacy of bithionol. Bithionol overcame antifungal (viz., fludioxonil) tolerance of the antioxidant mutants of the human/animal pathogen Aspergillus fumigatus. Altogether, our strategy can lead to the development of a high efficiency drug repurposing design, which enhances the susceptibility of pathogens to drugs, reduces time and costs for new antifungal development, and abates drug or fungicide resistance.

Parasitic infections like amebiasis, trichomoniasis, and giardiasis are major health threats in tropical and subtropical regions of the world. Metronidazole (MTZ) is the current drug of choice for amebiasis, giardiasis, and trichomoniasis but it has several adverse effects and potential resistance is a concern. In order to develop alternative antimicrobials, a library of 1H-1,2,3-triazole-tethered metronidazole-isatin conjugates was synthesized using Huisgens azide-alkyne cycloaddition reaction and evaluated for their amebicidal, anti-trichomonal, and anti-giardial potential. Most of the synthesized conjugates exhibited activities against Trichomonas vaginalis, Tritrichomonas foetus, Entamoeba histolytica, and Giardia lamblia. While activities against T. vaginalis and T. foetus were comparable to that of the standard drug MTZ, better activities were observed against E. histolytica and G. lamblia. Conjugates 9d and 10a were found to be 2-3-folds more potent than MTZ against E. histolytica and 8-16-folds more potent than MTZ against G. lamblia. Further analysis of these compounds on fungi and bacteria did not show inhibitory activity, demonstrating their specific anti-protozoal properties.

A series of N-alkyl tethered C-5 functionalized bis-isatins were synthesized and evaluated for antimicrobial activity against pathogenic microorganisms. The preliminary evaluation studies revealed the compound 4t, with an optimal combination of bromo-substituent at the C-5 position of isatin ring along with propyl chain linker being most active among the synthesized series exhibiting an IC50 value of 3.72 μM against Trichomonas vaginalis while 4j exhibited an IC50 value of 14.8 μM against Naegleria fowleri, more effective than the standard drug Miltefosine. The compound 3f with an octyl spacer length was the most potent among the series against Giardia lamblia with an IC50 of 18.4 μM while 3d exhibited an IC50 of 23 μM against Entamoeba histolytica. This library was also screened against the fungal pathogen Aspergillus parasiticus. A number of the compounds demonstrated potency against this fungus, illustrating a possible broad-spectrum activity. Furthermore, an evaluation of these synthesized compounds against a panel of normal flora bacteria revealed them to be non-cytotoxic, demonstrating the selectivity of these compounds. This observation, in combination with previous studies that isatin is non-toxic to humans, presents a new possible scaffold for drug discovery against these important protozoal pathogens of humans and animals.

Globally, mushroom poisonings cause about 100 human deaths each year, with thousands of people requiring medical assistance. Dogs are also susceptible to mushroom poisonings and require medical assistance. Cyclopeptides, and more specifically amanitins (or amatoxins, here), are the mushroom poison that causes the majority of these deaths. Current methods (predominantly chromatographic, as well as antibody-based) of detecting amatoxins are time-consuming and require expensive equipment. In this work, we demonstrate the utility of the lateral flow immunoassay (LFIA) for the rapid detection of amatoxins in urine samples. The LFIA detects as little as 10 ng/mL of α-amanitin (α-AMA) or γ-AMA, and 100 ng/mL of β-AMA in urine matrices. To demonstrate application of this LFIA for urine analysis, this study examined fortified human urine samples and urine collected from exposed dogs. Urine is sampled directly without the need for any pretreatment, detection from urine is completed in 10 min, and the results are read by eye, without the need for specialized equipment. Analysis of both fortified human urine samples and urine samples collected from intoxicated dogs using the LFIA correlated well with liquid chromatography-mass spectrometry (LC-MS) methods.

Equine-derived antitoxin (BAT^®) is the only treatment for botulism from botulinum neurotoxin serotype G (BoNT/G). BAT^® is a foreign protein with potentially severe adverse effects and is not renewable. To develop a safe, more potent, and renewable antitoxin, humanized monoclonal antibodies (mAbs) were generated. Yeast displayed single chain Fv (scFv) libraries were prepared from mice immunized with BoNT/G and BoNT/G domains and screened with BoNT/G using fluorescence-activated cell sorting (FACS). Fourteen scFv-binding BoNT/G were isolated with K_D values ranging from 3.86 nM to 103 nM (median K_D 20.9 nM). Five mAb-binding non-overlapping epitopes were humanized and affinity matured to create antibodies hu6G6.2, hu6G7.2, hu6G9.1, hu6G10, and hu6G11.2, with IgG K_D values ranging from 51 pM to 8 pM. Three IgG combinations completely protected mice challenged with 10,000 LD₅₀s of BoNT/G at a total mAb dose of 6.25 μg per mouse. The mAb combinations have the potential for use in the diagnosis and treatment of botulism due to serotype G and, along with antibody combinations to BoNT/A, B, C, D, E, and F, provide the basis for a fully recombinant heptavalent botulinum antitoxin to replace the legacy equine product.

The standard of treatment for botulism, equine antitoxin, is a foreign protein with associated safety issues and a short serum half-life which excludes its use as a prophylactic antitoxin and makes it a less-than-optimal therapeutic. Due to these limitations, a recombinant monoclonal antibody (mAb) product is preferable. It has been shown that combining three mAbs that bind non-overlapping epitopes leads to highly potent botulinum neurotoxin (BoNT) neutralization. Recently, a triple human antibody combination for BoNT/A has demonstrated potent toxin neutralization in mouse models with no serious adverse events when tested in a Phase I clinical trial. However, a triple antibody therapeutic poses unique development and manufacturing challenges. Thus, potentially to streamline development of BoNT antitoxins, we sought to achieve the potency of multiple mAb combinations in a single IgG-based molecule that has a long serum half-life. The design, production, and testing of a single tri-epitopic IgG1-based mAb (TeAb) containing the binding sites of each of the three parental BoNT/A mAbs yielded an antibody of nearly equal potency to the combination. The approach taken here could be applied to the design and creation of other multivalent antibodies that could be used for a variety of applications, including toxin elimination.