Toxins

Skeletal muscle regeneration is often impaired after acute muscle damage induced by viperid snake venoms, such as that of Bothrops asper, a medically-relevant species in Latin America. It has been shown that traces of venom that remain in the damaged muscle affect myogenic cells in culture, raising the possibility of inhibition of these toxins during the regenerative process as a way to improve regeneration. Using a mouse model of myonecrosis and regeneration, we evaluated the effects of Varespladib (a phospholipase A2 inhibitor) or Marimastat (a metalloproteinase inhibitor) on muscle regeneration when administered intravenously 24 h after the onset of myonecrosis, i.e., after muscle damage has occurred. The regenerative process was evaluated 14 and 28 days after venom injection. Results show that Marimastat, or a combination of both inhibitors, improved the extent of skeletal muscle regeneration and reduced the extent of tissue fibrosis when compared to tissue from mice receiving venom and no inhibitors, as judged by qualitative and quantitative histological assessment. Results underscore the deleterious role of traces of venom components in the damaged muscle during muscle regeneration and suggest that the administration of metalloproteinase inhibitors, or a combination of metalloproteinase and phospholipase A2 inhibitors, even when muscle damage has developed, may be a therapeutic alternative for improving the extent of muscle regeneration.

Bacillus cereus is a Gram-positive bacterium widely distributed in food, soil, and plants. It is a spore-forming, facultative anaerobic bacterium known as a food-borne opportunistic human pathogen responsible for causing gastrointestinal and non-gastrointestinal infections, including wound-associated infections. In the present study, we report that among single-component, bi-component, and tripartite alpha pore-forming toxin (-PFT) producing bacteria, the tripartite NheABC toxin produced by B. cereus induced the maximum cell death in infected epithelial cells. Similar to its effects in 2D monolayers, NheABC exhibited potent cell toxicity in 3D spheroids and intestinal organoids, targeting their cell membrane and mitochondria. Moreover, using erythrocytes as a model system, we found that the cytolytic activity of NheABC is pH-dependent, and was markedly reduced at acidic pH (5.5). The pH-dependent biological activity of NheABC was further confirmed by a liposome leakage assay. Importantly, NheABC enhanced the colonization of B. cereus in a non-gastrointestinal murine wound infection model. Overall, our study highlights the role of pH in regulating NheABC-mediated cytotoxicity in mammalian cells, which may lead to the development of novel therapeutic strategies for managing B. cereus gastrointestinal and non-gastrointestinal infections.

Historically, venom potencies have been assessed using measures of lethality, such as the median lethal dose (LD50). However, venoms may be selected primarily for their ability to rapidly incapacitate rather than cause mortality, meaning LD50 may not capture the efficacy of venoms in an ecological and evolutionary context. To capture this context, recent studies have adapted measures that assess venoms ability to rapidly incapacitate, such as the median effective dose (ED50). However, while ED50 values are expected to provide a more proximate assessment of ecological variation in venom potency, it is unknown whether historically available LD50 values are still useful proxies of ecologically relevant potency or whether they capture independent axes of venom variation. Here, we test the relationship between LD50 and ED50 in spider venoms by experimentally estimating LD50 and ED50 for 12 species and collating additional potency data for 40 species retrieved from the literature. We observed an isometric relationship between LD50 and ED50 in both analyses, showing these potency measures are both strongly coupled, with an increase in paralysis efficiency associated with a similar increase in lethality. Our results suggest that the functional aspects of venom potency, paralysis and lethality, are intrinsically linked, and due to this strong mechanistic coupling, historically available LD50 values may be used to compare general venom potencies in spiders, provided that they are based on the same prey model.

The ion channel KCa3.1 plays a role in immune regulation, red blood cell function, and is linked to numerous types of cancer. Various animal toxins, such as maurotoxin, bind to the extracellular side of KCa3.1, providing a potential starting point for inhibitor development. We report in this work the discovery of a novel, small-molecule inhibitor, with a micromolar IC50, which was specifically designed to target plasma-membrane KCa3.1 channels from the extracellular side. This compound can serve as a starting point for the development of more selective inhibitors and probes. For the identification of new extracellular inhibitors, molecular dynamics simulations were performed using the experimental structures of KCa3.1 and maurotoxin. The simulations produced a validated binding mode, highlighting key residues involved in the interaction between the toxin and the channel. These findings laid the foundation for the structure-based identification of novel extracellular small-molecule inhibitors of KCa3.1. The Molport database, containing approximately 50 million compounds, was screened using protein-ligand docking, yielding a hit molecule that was experimentally confirmed using patch clamp assays.

The evolution of Mullerian or Batesian mimicry depends on the relative unpalatability of participating taxa, yet the mechanistic basis of such unpalatability remains poorly explored in many systems. Unpalatability in the Corydoradinae catfishes, known for their striking mimetic diversity, is underpinned by sharp, lockable spines, armoured bodies and the production of venoms/toxins. Here, we assess the contribution of the axillary gland, a structure at the base of the pectoral spine, to toxicity in two Corydoradinae genera, Corydoras and Hoplisoma. Using a brine shrimp cytotoxicity assay, we demonstrate that axillary gland extracts are significantly more toxic than muscle extracts in both genera, but toxicity does not differ significantly between genera. Transcriptomic analyses identified 539 candidate toxin genes upregulated in axillary gland tissue, relative to scute tissue, containing signal peptides and had predicted toxin functions based on amino acid sequences. Notably, candidate genes include domains typical of piscine venoms, such as lectins and peptidase S1. Although significant differences in gene expression were detected between genera in candidate toxins, log-fold changes were small, and predicted toxin potency was not significantly different between genera. Together, our findings indicate that axillary gland toxins are likely to contribute to unpalatability in Corydoradinae catfishes and provide support for Mullerian mimicry in this system.

Many people are affected by post-acute sequelae of COVID-19 (PASC/long COVID, LC). LC has severely affected public health. Features of LC including blood pressure dysregulation, coagulopathies, hyperinflammation, and neuropsychiatric complaints. Mechanisms responsible for LC pathogenesis are not clear. The receptor for SARS-CoV-2 is human angiotensin converting enzyme 2 (ACE2), which binds SARS-CoV-2 spike protein receptor-binding domain (RBD) to initiate infection. We hypothesized that some people produce anti-RBD antibodies that sufficiently resemble ACE2 structure to have ACE2-like catalytic activity. Those antibodies, ACE2-like abzymes, may contribute to LC pathogenesis. We previously showed that ACE2-like activity was associated with immunoglobulin in some people with acute and convalescent COVID-19. ACE2-like catalytic activity correlated with blood pressure changes following moderate exercise challenge in convalescents. We screened human monoclonal antibodies (mAbs) against SARS-CoV-2 spike protein from 4 sources. We identified 4 human mAbs with ACE2-like catalytic activity. The activity was not inhibited by MLN-4760, a compound that inhibits native human ACE2, nor by EDTA, unlike native ACE2, a Zinc metalloprotease, but was inhibited by an overlapping pool of Spike peptides. Enzyme kinetic studies showed that the mAbs had lower Vmax and Km values than ACE2. The data suggested that the antibodies cleave angiotensin II via a different mechanism than ACE2. Identification of mAbs with ACE2-like catalytic activity supports the hypothesis that antibodies induced by SARS-CoV-2 infection could help mediate the pathogenesis of COVID-19 and LC, and more generally, the hypothesis that catalytic antibodies induced by infectious agents can contribute to disease pathogenesis.

Candida albicans is an opportunistic human fungal pathogen found in the oral cavity. Human saliva contains a 24-amino acid peptide called histatin 5 (Hst5) that has activity against C. albicans, but degradation of Hst5 by secreted aspartyl proteases (Saps) produced by C. albicans and by salivary proteases can reduce its antifungal efficacy. Building on our previous work that identified K13 and K17 as important residues for stability and activity of Hst5, we systematically investigated amino acid modification at these sites. Modifications explored the influence of hydrophobicity, charge, polarity, size, and aromaticity on Hst5s interaction with Saps and saliva. The K13R variant retained proteolytic stability and antifungal activity after incubation with Sap1, Sap2, Sap3, and Sap9, while other K13 variants generally had reduced stability and activity, emphasizing the importance of a positive charge at this position. At K17, substitutions generally enhanced proteolytic stability and improved antifungal activity after incubation with Saps. We introduced the normalized intact peptide (NIP) parameter as a tool for identifying Hst5 variants with improved stability in the presence of multiple Saps, and NIP revealed K17W as the most proteolytically stable variant overall. Additionally, we observed modest differences in peptide stability in saliva, and the K17W variant was the only variant that retained more activity than Hst5 following incubation with saliva. We further assessed the K17W variants ability to prevent biofilm formation and found it to be more effective than the parent peptide Hst5. Our findings highlight the interactions between the Hst5 K13 and K17 residues with Saps and saliva and provide a strong foundation for future Hst5 engineering efforts to improve proteolytic stability and antifungal efficacy in diverse proteolytic environments.

The cryptococcal Amt family of ammonium transporters have been identified from our previous studies as one of the most highly upregulated proteins during transmigration in an in vitro blood-brain barrier (BBB) model, however, the role of this gene family has never been reported. Therefore, this study aimed to investigate the role of the Amt2 gene in the transmigration of C. neoformans across the BBB, examine its association with other common virulence factors, and assess its relevance to morphological changes in C. neoformans. The results showed that the C. neoformans mutant strain lacking the Amt2 gene (amt2{Delta}) exhibited a significantly reduced ability to transmigrate across the BBB in an in vitro model. Our findings suggest that C. neoformans primarily utilizes a transcellular mechanism for invasion, as indicated by the FITC-dextran permeability assays. Additionally, the size of polysaccharide capsule was significantly smaller in the mutant strain compared to the wild-type. In conclusion, our study proposed that the Amt2 gene plays a crucial role in both the transmigration process and capsule production in C. neoformans, without affecting morphological changes. Our study provides a foundation for future research into the underlying mechanisms of the Amt2 gene in C. neoformans pathogenesis. Author summaryCryptococcus neoformans transmigrates the blood-brain barrier through various mechanisms, with transcellular migration being the major route leading to cryptococcal meningitis. In this study, we identified the Amt2 gene, a member of the Amt family of ammonium transporters, as playing a crucial role in the funguss transmigration process. Our findings indicate that the Amt2 gene promotes capsule production and facilitates the transmigration of C. neoformans, all while not causing damage to human endothelial cells.

Glutathione has important roles in diverse infections, yet its involvement in the interaction between the deadly fungal pathogen Batrachochytrium dendrobatidis (Bd) and its amphibian hosts is still unclear. Using in vitro assays and a cell infection model, we examined how glutathione influences Bd virulence traits and cellular host disease resistance. For Bd, inhibition of glutathione reductase rapidly killed zoospores, indicating that glutathione is essential for this pathogen. In addition, exposure to exogenous glutathione promoted the potential for virulence through accelerated and increased zoospore release. In host amphibian cells, Bd infection decreased intracellular glutathione content and increased reactive oxygen species, suggesting that chytridiomycosis pathogenesis may involve oxidative stress. Depletion of host glutathione before exposure to Bd increased infection severity and Bd growth, whereas amphibian cells with slightly elevated glutathione levels were partially protected against Bd. However, manipulation of host glutathione levels after the establishment of Bd infection did not impact its intracellular growth, implying that the host glutathione-mediated resistance only occurs during the initial Bd invasion process. Importantly, this effect of glutathione on host resistance is not a general response to pathogens, as it was not observed in cells exposed to viral pathogen FV3. As glutathione increased both infectious zoospore production and host resistance to zoospore infection, our study suggests that this antioxidant may play an important role in the host/pathogen interaction during chytridiomycosis. Thus, environmental conditions and therapeutic approaches that affect glutathione systems in the host and/or pathogen have the potential to alter chytridiomycosis dynamics and should be further explored.

Non-front-fanged snakes are abundant, diverse and represent approximately 70% of extant snakes. However, there is limited knowledge about most species and their venoms, in part due to the technical and welfare challenges associated with venom extraction, low venom yields, and the lack of cellular models available. Organoids represent an excellent opportunity to overcome these challenges. Here, we establish, for the first time, venom gland organoids from snakes of the Colubridae family and demonstrate the in vitro production of toxins.

Nicotinic acetylcholine receptors (nAChRs) belong to the pentameric ligand-gated ion channel superfamily (pLGICs). Among them, the neuronal homomeric 7 nAChR is highly permeable to calcium and plays critical roles in synaptic transmission, cell signaling, and inflammation modulation. The biogenesis of 7 nAChRs is enhanced by the chaperone proteins RIC-3 and NACHO. Previously, we reported a motif in the 5-HT3A receptor, another pLGIC, involved in RIC-3 modulation. Residues in this motif are conserved and also found within the L1-MX segment of the 7 nACh subunit. We therefore explored the regulatory roles of these conserved residues in the biogenesis of 7 nAChRs using multiple approaches, including heterologous expression in Xenopus laevis oocytes, mutagenesis, pull-down assays, cell-surface labeling, and two-electrode voltage-clamp (TEVC) recordings. We find that synthetic 7 L1-MX peptide interacts with both RIC-3 and NACHO. In particular, conserved residues W330, R332, and L336 in the L1-MX positively regulates the assembly of 7 oligomers and the biogenesis of 7nAChR. In presence of residues W330, R332, and L336, NACHO promotes an assembly of an 7 pentamer which is resistant to strong denaturing conditions. NACHO-promoted 7 pentamer is also resistant to Endo H enzyme. Sensitivity of the pentamer to moderate temperatures (37 {degrees}C, 45 {degrees}C, and 50 {degrees}C) suggests that NACHO stabilizes the pentamer via non-covalent interactions. In contrast, Ala replacements at these residues disrupt the biogenesis and abolish 7 current. NACHO and RIC-3 co-expression yields partial rescue of functional expression for some Ala replacement constructs. SUMMARYThis work identifies regulatory roles of conserved residues W330, R332, and L336 in the biogenesis of 7 nAChR. This discovery positions MX subdomain as a promising target for future drug development that can minimize adverse effects.

Mycoplasma synoviae is an avian pathogen that causes respiratory disease and synovitis, and its hemagglutinin plays a critical role in host cell adhesion. However, the key residues and structural mechanisms underlying hemagglutination remain unclear. In this study, domain analysis of the hemagglutinin family of Mycoplasma synoviae revealed that it contains long-chain and short-chain types, among which LAM HA (VY93_RS01465) was selected as the bait protein due to its complete C-terminal conserved domain. Through yeast two-hybrid screening, 18 host proteins interacting with LAM HA were identified. Furthermore, five key amino acid residues S83, R85, Y88, N124, and K192 were found to mediate hemagglutination activity. Deletion of these residues reduced the hemagglutination titer of LAM HA under acidic conditions. Secondary structure analysis showed that the deletion mutation decreased the -helix content while increasing the proportions of {beta}-sheet and random coil. Molecular dynamics simulations revealed that the mutant exhibited generally higher root mean square deviation and root mean square fluctuation values than the wild-type under different pH conditions, with a marked decrease in structural stability particularly at pH 5.0 and 6.0. These findings indicate that LAM HA, as a critical adhesin, exerts its hemagglutination function dependent on specific key residues and pH-sensitive conformational stability. IMPORTANCEMycoplasma synoviae (M. synoviae) causes significant economic losses to the poultry industry worldwide. Lipid-related membrane protein hemagglutinin (LAM HA) is a surface adhesin essential for host cell attachment, but its precise amino acid residues and structural features have not been defined. In this study, five key residues (S83, R85, Y88, N124, and K192) were identified as critical for LAM HA-mediated hemagglutination activity. Deletion of these residues altered the secondary structure composition, reduced conformational stability under acidic pH conditions, and decreased hemagglutination activity. These findings reveal a previously unknown structure-function relationship of M. synoviae LAM HA, demonstrating that its hemagglutination activity depends on specific residues and pH-sensitive structural integrity. This provides new insights into the molecular mechanisms of M. synoviae adhesion and offers potential targets for the development of novel intervention strategies against avian mycoplasmosis.

Nociception is an essential response for organisms to avoid potential harm and promote survival. Its molecular determinants are largely conserved across Eumetazoa. TRPV receptors are polymodal ion channels exhibiting selective peripheral expression and functional coupling that underpins nociception and pain modulation in complex organisms. However, the execution of protective behaviours triggered by TRPVs is also found in species with a simpler nervous organisation, thus encouraging their investigation in invertebrate model organisms to increase understanding of animal nociception. Cephalopods represent an interesting invertebrate phylum with respect to the evolution of the nervous system, whose complexity suggests it might support pain-like states that exist in vertebrates. This possibility is reflected by the inclusion of cephalopods in the UK and EU animal welfare legislations. Despite this, there is poor characterisation of cephalopod molecular nociceptors. For this reason, we used in silico analysis to identify two TRPV channels in Octopus vulgaris genome (Ovtrpv1 and Ovtrpv2). We validated the putative transcript sequences and highlighted prevalent expression in sensory tissues. We investigated the functional competence of these TRPVs by heterologously expressing Ovtrpv1 and Ovtrpv2 cDNA into Caenorhabditis elegans null mutants of the orthologous genes, ocr-2 and osm-9 respectively. Ovtrpvs successfully rescued the aversive response to chemical and mechanical noxious stimuli in the C. elegans mutants, suggesting these receptors are polymodal nociceptors. Additionally, complementary investigation using Xenopus laevis oocytes showed Ovtrpv1 and Ovtrpv2 form an active heteromeric channel gated by nicotinamide. This study highlights Ovtrpvs as an important route to better understand nociceptive detection in cephalopods.

The supernatant of Bacillus toyonensis biovar thuringiensis Bto_UNVM-42 exhibits nematicidal activity, although its molecular basis remains unclear. While pesticidal proteins in Bacillus thuringiensis and related species are classically considered to be intracellular and associated with parasporal crystals, their potential presence in the extracellular fraction has been largely unexplored. Here, LC-MS/MS analysis of the secretome from LB-grown cultures revealed the extracellular presence of pesticidal protein homologs related to Cry32-, Cyt1-, and Mpp3-like protein families, together with degradative enzymes including collagenase, chitinase, proteases, and cytolysins. Signal peptide prediction supported classical secretion for several proteins, while others were consistent with non-classical secretion pathways. The consistent detection of these proteins in cell-free supernatants provides strong proteomic evidence for their extracellular localization. These findings challenge the prevailing crystal-centric paradigm of Bt-like pesticidal proteins and support an expanded model in which soluble extracellular components contribute to pathogenicity. This work highlights the value of secretome analysis for the characterization of Bt-like strains and provides new insights into the molecular basis of nematicidal activity in B. toyonensis. O_LIFirst report of Cry32-, Cyt-, and Mpp-like homologs in the Bto_UNVM-42 secretome. C_LIO_LIExtracellular detection challenges classical intracellular Bt-like toxin paradigm. C_LIO_LILC-MS/MS reveals toxin homologs in culture supernatant. C_LIO_LIEvidence supports secretion beyond crystal-associated proteins. C_LIO_LISecretome suggests expanded functional repertoire in Bt-related strains. C_LI

The greater wax moth, Galleria mellonella, is an increasingly important invertebrate model for infection biology, yet silk extrusion during handling complicates larval injections and hampers survival assessment. Here, we develop and characterise a simple cold-shock method that reliably inhibits silk production without compromising larval viability. Larvae exposed to -20 {degrees}C for 10 minutes completely suppress silk extrusion with 100% survival, representing a substantial improvement over previous chilling approaches. Cold-shocked larvae successfully remained capable of completing development, although pupation and adult emergence were delayed, body weight and fecundity were reduced, and wing deformities were more common. While cold-shock did not alter silk gland morphology, spinneret structure, or fibroin gene expression, confocal imaging revealed pronounced disorganisation of F-actin and -tubulin networks within silk gland cells, indicating cytoskeletal disruption as a likely mechanism underlying silk inhibition. When challenged with Escherichia coli, cold-shocked larvae responded comparably to controls, with survival influenced primarily by feeding status. Together, these findings demonstrate that short-term cold-shock provides an efficient, reproducible, and easy implemented method for preventing silk extrusion in Galleria larvae, markedly improving handling and experimental safety while preserving their suitability as a model host for pathogen research. HighlightsO_LICold-shock at -20 {degrees}C for 10 minutes inhibits silk extrusion. C_LIO_LILarvae survive treatment with no loss of suitability for infection studies. C_LIO_LIDevelopment slows and adult weight, fecundity, and wing quality decline. C_LIO_LISilk glands stay intact; gene expression remains unchanged after cold-shock. Cytoskeletal disruption likely drives the failure of silk secretion. C_LI

New approaches to mitigate the reduction of crop yields by plant parasitic nematodes are needed in the face of increasing concerns of the impact of nematicides on precious ecosystems. One approach is to target receptors in the parasitic nematode that are vital for their survival that less widely expressed in non-target organisms. Nematodes express a phylogenetically restricted 5-HT-gated chloride channel, MOD-1, activation of which causes paralysis in Caenorhabditis elegans. We show that MOD-1 is expressed in the motor nervous system of the plant parasitic nematode Globodera pallida and its functional characterisation is validated by 5-HT activation when reconstituted in Xenopus laevis oocytes. To evaluate MOD-1 as a nematicide target we utilised a previously described platform called PhaGeM4 for PharmacoGenetic targeting of M4 neurone in which MOD-1 is expressed in transgenic C. elegans and nematode development in the face of MOD-1 chemical modulation is tracked. We screened Pathogen Box, a chemical library of 400 diverse drug-like molecules, using PhaGeM4. This identified 10 putative hits for C. elegans MOD-1. These hits were pursued through a sequential, iterative pipeline encompassing mod-1 dependent C. elegans motility and G.pallida motility assays in combination with pharmacological interrogation of G. pallida mod-1 in PhaGeM4. This approach highlights 3 compounds with a mod-1 dependent action (quipazine, our benchmark compound; MMV687251, a vancomycin-like compound; MMV688774, an antifungal with common name posaconazole) and one compound that acts through an undetermined target (MMV002816, also known as the antifilarial drug, diethylcarbamazine). Each of these compounds had a significant inhibitory effect on G. pallida J2 root invasion. Overall, this lends confidence that the PhaGeM4 screening platform can delivery new chemical leads for crop protection and highlights four new chemistries of interest. More generally, this approach could be applied to other ligand-gated ion channels of interest as targets.

The tobacco hornworm moth (Manduca sexta) is evaluated as a model of bacterial virulence and host-pathogen dynamics. Infections of Pseudomonas aeruginosa were established by injection of 5th-instar larvae, and multiple assays of virulence were evaluated. Infected larvae exhibited dose-dependent mortality, reduced growth, melanization, behavioral changes, and altered frass constitution. Even low-dose infections that were not fatal exhibited impaired growth, but individual growth trajectories revealed considerable heterogeneity among worms given the same dose. Twice-daily antibiotic treatment with gentamicin or cefepime improved survival four- to five-fold but did not rescue 100%. Heat-killed cells and filtered culture supernatant alone induced significant morbidity and mortality, suggesting secreted bacterial products are important to pathogenesis. Bacterial burden analysis revealed a shifting bacterial distribution over time, with decreasing hemolymph titers and increasing localization in fat body, gut, and carcass. Hornworms thus offer a more sensitive analysis of bacterial infection dynamics and consequences than do larvae of the more commonly used wax moth.

Marine Aspergillus terreus has been explored as an important chitinase-producing fungal strain for-N-Acetyl-D-Glucosamine (GlcNAc) production from chitin substrates. Here, a purified extracellular 45 kDa chitinase of marine Aspergillus terreus (accession number JQ248076) was characterized in terms of substrate specificity. Conventionally, endochitinase cleaves the chitin substrate randomly to produce GlcNAc and its different multimers. So, it requires at least tetramer to characterize the endochitinases; whereas, exochitinases cleaves the chitin substrate from its reducing end and produce either GlcNAc or chitobiose (GlcNAc dimer). In present chitinase characterization, the HPLC followed by HRMS analyses revealed differential product formation from the chitin substrates of varied chain length. With swollen chitin polymer, the enzyme produced GlcNAc as a sole product; whereas with chitohexaose substrate, a mixture of GlcNAc and its oligomers were obtained. Although, mass spectrometry-based proteomics analysis identified the isolated chitinase as an endochitinase 1 precursor (Accession XP_001217186). However, the enzyme kinetic study exhibited higher catalytic efficiency for exochitinase specific dimeric chromogenic substrate in comparison to endochitinase specific tetrameric fluorogenic substrate, which indicated predominantly exochitinase behavior of the enzyme. Further, the in-silico study predicted the differential cleavage pattern of the enzyme, which could be due to different mode of substrate binding and processive mechanism through the tunnel shaped binding cleft of the enzyme. The dual mode of catalytic activity of the present chitinase was further confirmed by a molecular docking study with different lengths of substrates. With the unique dual mode of action, the chitinase of marine Aspergillus terreus offers a great promise towards its utility in the production of GlcNAc.

Xylazine is a 2-adrenergic agonist typically used in as a sedative and analgesic in veterinary medicine. For some years, xylazine has been reported as an additive to fentanyl on the illicit drug market and has been associated with severe side-effects including severe ulcerations and potential amputations at the sites of injection along with an increased risk of respiratory depression and death. We recently reported that xylazine has modest {kappa} opioid agonist activity in vitro and in vivo and asked if other 2-adrenergic agonists had similar off-target activities. To test this hypothesis, we profiled US FDA-approved 2-adrenergic agonists at 320 G protein coupled receptors (GPCRs) to identify potentially deleterious and/or beneficial off-targets. Although all other tested 2-adrenergic agonists were devoid of {kappa} opioid agonist activity, each had a distinct pattern of activity at various GPCRs and differential patterns of signaling bias at 2-receptor subtypes. These findings suggest potential molecular targets for both side-effects and therapeutic activities among known 2-adrenergic agonists.

Ehrlichia canis is primarily a Rhipicephalus sanguineus tick-borne rickettsial pathogen initially identified as causing canine monocytic ehrlichiosis, and infections in people have also been reported in Venezuela, Mexico, and parts of Europe. It is of high importance to have a vaccine suitable in protecting the canine host, which will aid in lessening E. canis infections also in people. Gene inactivation mutations in the phage head-to-tail connector protein genes (phtcp) from E. chaffeensis and A. marginale caused attenuated growth, and prior infection with the mutated bacteria induced protective immunity against wild-type bacterial infections in natural hosts, independent of blood-borne infection or tick-transmission infection. In the current study, we describe the development of targeted mutagenesis for the first time in E. canis genome and with a novel modification to avoid introducing antibiotic resistance cassettes to delete the phtcp ortholog from E. canis. The mutated E. canis was then assessed for its in vivo growth and the induction of host immunity exerted following the mutant infection aiding to protect against wild-type infection challenge in the canine host. We assessed systemic pathogen loads, hematological parameters, IgG immune responses, and plasma cytokines following the mutant infection relative to uninfected dogs. Similarly, the assessments were carried out following wild-type pathogen infections in dogs with or without prior mutant infection challenges. The study demonstrates that prior infection of dogs with the mutant induces immunity to prevent infection establishment by wild-type E. canis. Similarly, the mutant infection resulted in clear biological differences compared to the wild-type infection. This study establishes that the molecular genetic methods are broadly applicable to pathogens belonging to the family Anaplasmataceae and that the modified live vaccines with phtcp gene orthologs are valuable in reducing the diseases caused by the tick-borne rickettsial pathogens belong to Anaplasmataceae, including E. canis.