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.

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 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.

GPI-anchored proteins are crucial cell surface proteins with diverse, organism-specific functions, in eukaryotes. They are produced when the GPI transamidase (GPIT), a five-subunit membrane-bound enzyme complex, attaches a pre-formed GPI anchor to the C-terminal end of nascent proteins on the lumenal face of the endoplasmic reticulum. This process requires the removal of a C-terminal signal sequence (SS) on the substrate protein by the action of an endopeptidase subunit of the GPIT, Gpi8/ PIG-K. Using an AMC-tagged peptide in a cell free (post-mitochondrial fraction) assay, this manuscript studies the steady state kinetics of enzymatic cleavage of the substrate by GPIT of the human pathogenic fungus, C. albicans. We show that Mn+2 enhances activity by improving substrate binding but plays no direct role in substrate cleavage per se. Molecular dynamics simulations suggest that the divalent cation binds at a site away from the active site but provides compactness and stability to Gpi8. It also enables a conformation in which a flexible loop (219-244 residues) in the vicinity of the catalytic pocket is able to interact with and position the scissile bond for cleavage by Cys202. Steady state kinetics also indicate that peptides of lengths 7-mer to 9-mer are better bound than 4-mer or 15-mer peptide substrates. A bulky residue at the site of cleavage reduces the catalytic activity of the GPIT. This is the first detailed steady state kinetics study on the endopeptidase activity of a GPIT from any organism.

Human malaria infections begin with the injection of Plasmodium sporozoites via mosquito saliva. Whole sporozoite immunizations have been used as a model to study immune responses to malaria parasites, having culminated in circumsporozoite protein (CSP)-targeting vaccines and monoclonal antibodies (mAbs). However, antibody responses targeting non-CSP antigens on the sporozoite surface remain poorly characterized. Here, we isolated single B cells from a human volunteer immunized by Plasmodium falciparum-infected mosquito bites, who had acquired non-CSP-specific antibodies that recognize sporozoites. We identified two mAbs that recognize the surface of P. falciparum sporozoites, but do not bind to CSP. Using immunoprecipitation followed by mass-spectrometry, we found that the target of these mAbs is not a P. falciparum protein but the mosquito salivary protein SG1L3. We observed that recombinant SG1L3 binds to P. falciparum sporozoites. However, the SG1L3-specific mAbs and SG1L3-specific polyclonal antibodies from this volunteer, as well as polyclonal antibodies raised against recombinant SG1L3 in rabbits, fail to block liver stage infection in vitro, making this an unlikely target for functional antibodies. We observed that inhabitants from an area with intense Anopheles exposure in Burkina Faso can have antibodies against SG1L3, and that antibody titers increase with age. In conclusion, we identified the first human mAbs against a mosquito saliva protein that binds to the surface of sporozoites. Future work should assess whether naturally acquired antibodies against this protein may be used as a serological marker of mosquito exposure.

Campylobacter jejuni is a leading global cause of acute foodborne gastroenteritis however, C. jejuni lacks some of the classic virulence determinants associated with other common enteric bacterial pathogens. In recent years an increasing number of C. jejuni isolates have been identified to encode Type Six Secretion System (T6SS), an apparatus utilised by Gram-negative bacteria to secrete toxic bacterial effectors into neighbouring cells. Despite the prevalence of the T6SS and previous investigations, the roles of the C. jejuni T6SS are still not well characterised especially when compared to our knowledge of other clinically relevant T6SS-positive bacterial species. Additionally, as of yet, no C. jejuni T6SS cargo effectors have been characterised. In this study, we show the C. jejuni 488 strain T6SS displays contact-dependent antagonistic behaviour towards T6SS-negative C. jejuni, Campylobacter coli, Escherichia coli and Enterococcus faecium strains suggesting the presence of the T6SS contributes to the competitive capacity of this C. jejuni T6SS-positive strain. Moreover, this antagonistic activity is linked to the functionality of CJ488_0980 and CJ488_0982, two novel putative Tox-REase-7 domain-containing effectors, which were identified through bioinformatical analysis of the C. jejuni 488 strain genome. Additionally, our investigations propose the C. jejuni 488 T6SS contributes to interaction, invasion and intracellular survival in human intestinal epithelial cells (IEC). Collectively, these initial findings are the first examples of in vitro investigation of putative cargo effectors in Campylobacter spp. and provide valuable insights into the roles of C. jejuni T6SS effectors in bacterial competition and pathogenesis. This study highlights the importance of T6SS as an emerging virulence determinant in Campylobacter spp. warranting further investigation.

BackgroundIdiopathic epilepsy (IE) is the most common chronic nervous system disorder of dogs, and its cause is poorly understood. Emerging evidence suggests that microbiome alterations can occur with IE via the microbiota-gut-brain axis. Therefore, we analyzed the fecal microbiomes of 98 dogs (49 IE, 49 control) in a pairwise case-control observational study using 16S rRNA gene sequencing. ResultsAlthough the microbial community was mostly similar between groups, IE was associated with a modest but significant shift in Weighted-Unifrac distance (P = 0.042). We used six differential abundance (DA) methods to identify differentially abundant amplicon sequencing variants (ASVs) between IE and control groups. Notably, one Collinsella ASV was found to be significantly more abundant in IE dogs by all six methods. The gut microbial compositions varied drastically across households (accounting for about 69% of the total variation), but did not have significant differences between sex, age, or breed. Phenobarbital administration in IE dogs had a significant effect on seizure control, and was not associated with changes in the microbiome. ConclusionOur findings suggest a relationship between gut microbiomes and IE. However, the specific mechanism needs to be further investigated.

BackgroundShigellosis morbidity and mortality, combined with the increase in multidrug-resistant infections make Shigella vaccine development a global imperative. Glycoconjugate vaccines that couple immunogenic O-antigen to protein derived from Shigella may provide broader protection across Shigella species and serogroups. Such an approach also circumvents immunotolerance arising from repeated use of the same carrier. Here we use bioconjugation, exploiting an oligosaccharyltransferase (OST) enzyme to couple O-antigen and carrier protein in vivo, to generate a "double-hit" Shigella glycoconjugate vaccine. MethodGlycoconjugates were synthesised in E. coli SDB1 cells expressing S. sonnei O-antigen, the OST PglS, and one of two Shigella carrier proteins. Recombinant glycoconjugate was purified using anion exchange chromatography and then used to immunise mice. Antibody responses were measured and compared by ELISA. ResultsWhen co-produced in E. coli, PglS was able to transfer the cloned S. sonnei O-antigen onto three carrier proteins, modified to accept glycans from the PglS transferase enzymes- the standard bioconjugate carrier ExoA and two immunogenic Shigella-specific outer membrane proteins, EmrK and MdtA. Production of MdtA or ExoA glycoconjugates for immunisation studies utilised successive rounds of anion exchange chromatography, to remove unglycosylated material and obtain highly purified glycoconjugate proteins for us in vaccination. Analysis of murine sera following immunisation revealed an IgG response was raised against both carrier protein and the S. sonnei O-antigen for each glycoconjugate. ConclusionA novel, conserved Shigella protein can be utilised as an effective carrier for the generation of a "double-hit", immunogenic Shigella glycoconjugate vaccine that elicits IgG responses to both carrier protein and S. sonnei O-antigen.

Synaptic vesicle glycoproteins 2 (SV2) are integral membrane proteins essential for neurotransmitter release and are implicated in neurological disorders including epilepsy and Parkinsons disease. In the attempt to develop a ligand selective for SV2C, and in collaboration with UCB, UCB-F was identified as a potential candidate. However, the affinity of UCB-F to SV2C was found to be temperature dependent, decreasing by about 10-fold from +4 to 37 degrees. UCB1A was subsequently identified as SV2C ligand displaying in vitro a 100-fold selectivity for SV2C compared with SV2A. In this study we investigated whether the binding of UCB-1A to SV2A and SV2C was affected by the temperature. A combination of experimental binding assay data and molecular dynamics (MD) simulations were used. The binding studies revealed that UCB1A affinity for SV2A decreased significantly at 37 {degrees}C compared with 4 {degrees}C, whereas binding to SV2C remained largely unchanged. MD simulations reproduced these observations, namely that ligand RMSD values at 310 K showed that UCB1A binding fluctuated markedly in the SV2A complex, with many trajectories exceeding the 3.0 [A] stability cutoff, whereas UCB1A remained relatively well-anchored in SV2C under the same conditions. Structural analysis showed that, while UCB1A adopts a conserved binding pose across all isoforms stabilized by {pi}- {pi} stacking and a hydrogen bond with Asp, SV2C possesses a unique stabilizing feature. In SV2C, Tyr298 is less exposed to the solvent and engages in a persistent hydrogen bond with Asparagine, a structural feature that reinforces pocket stability and limits temperature-induced destabilization. This interaction is absent in SV2A, consistent with its greater temperature sensitivity. Together, these findings provide a mechanistic explanation for the experimentally observed temperature independence of UCB1A binding to SV2C. More broadly, the results highlight the importance of incorporating physiologically relevant temperatures into SV2 ligand evaluation and demonstrate how combining experiments with simulations can uncover isoform-specific mechanisms of ligand recognition and stability.

Venom is an important functional trait that helps predatory animals capture prey. Centipede predatory venoms are complex cocktails of multiple proteins, such as neurotoxins (scoloptoxins), cytotoxins, {beta}-pore-forming toxins, and enzymes. We examined venom phenotypes in two closely related and co-occurring centipede species, Scolopendra morsitans (n=28) and S. hardwickei (n=11), in peninsular India to determine whether their venoms are similar or dissimilar. An integrated proteo-transcriptomic approach was used to characterise the venom phenotypes of the two species across multiple individuals in peninsular India. We used species occurrence records and species distribution models to assess the distributional overlap among these species within the peninsular Indian region. The species showed significant overlap in their current and projected geographical ranges, corresponding with their co-occurrence. We characterised the venom profiles of both species and found that the venoms were cocktails of enzymes, {beta}-pore-forming toxins, and neurotoxins comprising 110 and 84 proteins in S. morsitans and S. hardwickei, respectively. However, the venom composition of both species differed significantly in toxin abundance and species-specific protein repertoires. This indicates trait divergence in venom phenotypes, suggesting that distinct venom compositions may facilitate coexistence among ecologically similar predatory centipedes. The observed variation in venom phenotypes among co-distributed species opens up important avenues for future research into their ecological roles and functional significance. In this study, we provided a detailed account of venom composition across multiple individuals from the species geographic range and highlighted the importance of investigating the role of venom as a trait that could influence species interactions and shape communities in these diverse tropical forests.

Cetacean pathology is a cornerstone for population and marine ecosystem health monitoring, allowing clear differentiation among natural and anthropogenic threats. Previous studies in the Canary Islands reported natural causes of death in 59.4% (1999-2005) and 81% (2006-2012) of stranded cetaceans, versus anthropogenic causes in 33.3% and 19%, respectively. This study aimed to determine the causes of death (CD), pathologic findings, and epidemiological patterns of 316 cetaceans stranded in the Canary Islands between 2013 and 2018. The CDs were classified in pathologic entities (PEs) emphasizing natural versus anthropic origins. Of 316 animals, 224 (70.9%) from 18 species were suitable for pathological investigations. Among natural PEE, natural pathology associated with good nutritional status (NP-GNS) and natural pathology associated with significant loss of nutritional status (NP-LNS) represented 43/224 (19.2%) and 36/224 (16%) cases, respectively. Natural pathology with undetermined nutritional status (NP-UNS) occurred in 19/224 (8.5%) animals. Intra- and interspecific traumatic interactions (ITI) represented 30/224 (13.4%) cases, followed by neonatal/perinatal pathology (NPN) 19/224 (8.5%) and live-stranding stress and/or capture myopathy (LS-CM) 18/224 (8%). Infectious and parasitic diseases predominated in natural PEs. Anthropogenic PEs included interaction with fishing activities (IFA) in 17/224 (7.6%) cases, vessel collisions (VC) in 9/22 (4%) cases, and foreign body-associated pathology (FBAP) in 3/224 (1.3%) animals. Overall, anthropogenic causes accounted for 12.9% of deaths, natural causes for 73.6%, and the CD could not be established in 30/194 (13.4%) cases. This study reaffirms the trends concerning recognized PEs (NP-GNS, NP-LNS, NP-UNS, ITI, NPN, LS-CM, IFA, VC, and FBAP), expands the body of knowledge on cetacean pathology in the Canary Islands, and reports novel findings including mixed infections, clostridiosis in uncommon species, uremic syndrome secondary to urethral nematodiasis, gas embolism in unusual species, epibiont stomatitis, congenital musculo-skeletal malformations, or neoplastic processes. These findings advance understanding of cetacean mortality patterns and support conservation and management strategies.

N-glycosylation is an essential post-translational modification required for proper protein folding, stability, trafficking, and secretion in eukaryotes. In such organisms, an efficient endoplasmic reticulum (ER) quality control, such as the ER-associated degradation (ERAD) pathway, is critical for maintaining cellular homeostasis. During ERAD, terminally misfolded glycoproteins undergo N-deglycosylation prior to proteasomal degradation, a process typically mediated by peptide N-glycanase (PNGase). However, in the filamentous fungi, the PNGase seems to be catalytically inactive, indicating evolutionary divergence from the canonical PNGase pathway. Filamentous fungi also encode endo-{beta}-N-acetylglucosaminidases (ENGases), particularly members of glycoside hydrolase family 18 (GH18), which may compensate for the loss of canonical PNGase activity. Here, we investigated the roles of the cytosolic GH18 ENGase and a putative acidic PNGase in N. crassa using transcriptomic and functional approaches. Our results demonstrate that the cytosolic GH18 ENGase is an active deglycosylating enzyme likely associated with the ERAD pathway, whereas no deglycosylation activity was detected for the acidic PNGase. Deletion of the ENGase severely compromises tolerance to diverse stress conditions and induces substantial transcriptomic reprogramming, including upregulation of a GH20 exo-{beta}-N-acetylhexosaminidase under ER stress. These findings identify cytosolic ENGase as a key component of fungal proteostasis and suggest that N. crassa activates alternative compensatory mechanisms to maintain protein quality control when canonical deglycosylation pathways are impaired.

1Canine atopic dermatitis (CAD) is a chronic inflammatory skin condition sometimes associated with microbial dysbiosis, including alterations in colonization by the lipophilic yeast Malassezia pachydermatis. This study investigated the population diversity of M. pachydermatis in the ear canals of healthy and CAD-affected dogs using Fourier-transform infrared (FTIR) spectroscopy and whole genome sequencing (WGS). Among 60 dogs, M. pachydermatis prevalence was significantly higher in CAD cases than in healthy controls. FTIR spectroscopy revealed greater strain heterogeneity in CAD-affected dogs, often with distinct genotypes in each ear, while healthy dogs exhibited more homogeneous populations. Using a previously developed FTIR-based artificial neural network classifier, we assigned strains to three phylogroups. Strains from phylogroups I and III were significantly enriched in CAD-affected dogs, while phylogroup II was most prevalent overall and the dominant phylogroup in healthy controls. This suggests that CAD-associated inflammation may favor specific M. pachydermatis phylogroups and sub-clusters within phylogroups, shaping colonization dynamics. FTIR-based typing showed full concordance with WGS across 35 sequenced isolates, recapitulating relationships among phylogenetically related isolates and their similar phenotypic profiles. Overall, our findings reveal strain-level shifts in M. pachydermatis populations associated with CAD and establish FTIR spectroscopy as a rapid, cost-effective tool for large-scale epidemiological studies.

BackgroundVirtual colony count is a kinetic, 96-well turbidimetric assay that has been used since 2003 to determine the antimicrobial activity of antimicrobial peptides including the defensin HNP1. Virtual colony count results differed from traditional colony counting results in studies of the antimicrobial activity of the human cathelicidin LL-37 and related peptides. The difference could possibly have been caused by an inoculum effect. MethodsThe virtual colony count assay was conducted using inocula that varied from 1250 to 1x108 virtual colony forming units (CFUv) per milliliter. ResultsThe virtual colony count assay demonstrated a pronounced inoculum effect of HNP1 against Staphylococcus aureus ATCC 29213, accompanied by biofilm formation observed in the wells of the 96 well plates at all inocula. The S. aureus inoculum effect was not as drastic as previously reported for Escherichia coli. ConclusionsThe inoculum effect is further evidence that biofilm formation is a resistance mechanism used by a variety of bacteria against antimicrobial peptides such as HNP1.

The emergence of antimicrobial resistance (AMR) has driven the search for alternative therapeutic strategies, including antivirulence approaches targeting bacterial quorum sensing (QS). Azelaic acid (AzA), a naturally occurring dicarboxylic acid with known antimicrobial properties, has not previously been characterized as a QS inhibitor in Gram-negative pathogens. This study evaluated the dual antimicrobial and antivirulence activity of AzA against reference strains and clinical isolates of Pseudomonas aeruginosa, Enterobacteriaceae, and Staphylococcus aureus through in vitro assays and molecular docking analyses. Minimum inhibitory concentration (MIC) values ranged from 250 to 1000 {micro}g/mL, with lower MICs observed in clinical isolates of E. coli and S. aureus. Subinhibitory concentrations (250, 500 and 750 {micro}g/mL) were used to assess QS-regulated virulence factors in P. aeruginosa, including pyocyanin, elastase, alginate, and protease production. AzA exhibited a significant, dose-dependent inhibition of all evaluated virulence factors across both reference and multidrug-resistant (MDR) and pan-drug-resistant (PDR) clinical strains (p < 0.001), achieving inhibition levels exceeding 90% in several cases, particularly for protease activity. Molecular docking analyses revealed that AzA interacts with key QS-related proteins (LasI, LasR, PqsD, and PqsR), showing moderate binding affinities (-5.3 to -6.5 kcal/mol) and stable interactions within conserved ligand-binding domains. These findings suggest a multitarget modulatory mechanism affecting interconnected QS pathways. Overall, this study demonstrates, for the first time, that AzA acts as a quorum sensing inhibitor in P. aeruginosa, attenuating virulence without directly affecting bacterial growth, highlighting its potential as a promising antivirulence therapeutic strategy.

Plant parasitic nematodes (PPNs) are microscopic soil dwelling pests that infect crops, using a lance-like organ, the stylet, to hatch, invade plant roots, and establish feeding sites. Stylet function is underpinned by pharyngeal muscle contraction and relaxation cycles, making it an attractive route to disrupt the PPN lifecycle. However, knowledge of pharyngeal regulation in PPNs is relatively limited. In the free-living nematode Caenorhabditis elegans, the nicotinic receptor EAT-2 stimulates pharyngeal contraction to facilitate feeding. Here we hypothesize that EAT-2 orthologues may regulate a similar function in PPNs. A phylogenetic analysis reveals that EAT-2 and its orthologues in other nematode species cluster as a distinct group suggesting that EAT-2 is exclusive of other animal species. We identified eat-2 in the genome of the potato cyst nematode Globodera rostochiensis and used in situ hybridization to establish an anterior expression pattern consistent with a pharyngeal function. In vitro pharmacological assays directly compared the response of C. elegans pharynx and G. rostochiensis stylet to cholinergic compounds. Both pharyngeal and stylet activity were stimulated by acetylcholine and nicotine, and these responses were blocked by the nicotinic receptor antagonists, mecamylamine and tubocurarine. These data are consistent with a conserved cholinergic pathway mediated by EAT-2 regulating pharyngeal muscle function. It highlights EAT-2 as a potential determinant of stylet thrusting and a promising pharmacological target to selectively mitigate PPN infections.