Understanding bacterial pathogen diversity: A proteogenomic analysis and use of an array of genome assemblies to identify novel virulence factors of the honey bee bacterial pathogen Paenibacillus larvae.

Mass spectrometry proteomics data are typically evaluated against publicly available annotated sequences, but the proteogenomics approach is a useful alternative. A single genome is commonly utilized in custom proteomic and proteogenomic data analysis. We pose the question of whether utilizing numerous different genome assemblies in a search database would be beneficial. We reanalyzed raw data from the exoprotein fraction of four reference Enterobacterial Repetitive Intergenic Consensus (ERIC) I-IV genotypes of the honey bee bacterial pathogen Paenibacillus larvae and evaluated them against three reference databases (from NCBI-protein, RefSeq, and UniProt) together with an array of protein sequences generated by six-frame direct translation of 15 genome assemblies from GenBank. The wide search yielded 453 protein hits/groups, which UpSet analysis categorized into 50 groups based on the success of protein identification by the 18 database components. Nine hits that were not identified by a unique peptide were not considered for marker selection, which discarded the only protein that was not identified by the reference databases. We propose that the variability in successful identifications between genome assemblies is useful for marker mining. The results suggest that various strains of P. larvae can exhibit specific traits that set them apart from the established genotypes ERIC I-V.

Proteomic insight into the interaction of Paenibacillus larvae with honey bee larvae before capping collected from an American foulbrood outbreak: Pathogen proteins within the host, lysis signatures and interaction markers.

American foulbrood (AFB) is a devastating disease of honey bees. There remains a gap in the understanding of the interactions between the causative agent and host, so we used shotgun proteomics to gain new insights. Nano-LC-MS/MS analysis preceded visual description and Paenibacillus larvae identification in the same individual sample. A further critical part of our methodology was that larvae before capping were used as the model stage. The identification of the virulence factors SplA, PlCBP49, enolase, and DnaK in all P. larvae-positive samples was consistent with previous studies. Furthermore, the results were consistent with the array of virulence factors identified in an in vitro study of P. larvae exoprotein fractions. Although an S-layer protein and a putative bacteriocin were highlighted as important, the microbial collagenase ColA and InhA were not found in our samples. The most important virulence factor identified was isoform of neutral metalloproteinase (UniProt: V9WB82), a major protein marker responsible for the shift in the PCA biplot. This protein is associated with larval decay and together with other virulence factors (bacteriocin) can play a key role in protection against secondary invaders. Overall, this study provides new knowledge on host-pathogen interactions and a new methodical approach to study the disease.

Probiotic candidates for controlling Paenibacillus larvae, a causative agent of American foulbrood disease in honey bee.

BACKGROUND: American foulbrood (AFB) disease caused by Paenibacillus larvae is dangerous, and threatens beekeeping. The eco-friendly treatment method using probiotics is expected to be the prospective method for controlling this pathogen in honey bees. Therefore, this study investigated the bacterial species that have antimicrobial activity against P. larvae. RESULTS: Overall, 67 strains of the gut microbiome were isolated and identified in three phyla; the isolates had the following prevalence rates: Firmicutes 41/67 (61.19%), Actinobacteria 24/67 (35.82%), and Proteobacteria 2/67 (2.99%). Antimicrobial properties against P. larvae on agar plates were seen in 20 isolates of the genus Lactobacillus, Firmicutes phylum. Six representative strains from each species (L. apis HSY8_B25, L. panisapium PKH2_L3, L. melliventris HSY3_B5, L. kimbladii AHS3_B36, L. kullabergensis OMG2_B25, and L. mellis OMG2_B33) with the largest inhibition zones on agar plates were selected for in vitro larvae rearing challenges. The results showed that three isolates (L. apis HSY8_B25, L. panisapium PKH2_L3, and L. melliventris HSY3_B5) had the potential to be probiotic candidates with the properties of safety to larvae, inhibition against P. larvae in infected larvae, and high adhesion ability. CONCLUSIONS: Overall, 20 strains of the genus Lactobacillus with antimicrobial properties against P. larvae were identified in this study. Three representative strains from different species (L. apis HSY8_B25, L. panisapium PKH2_L3, and L. melliventris HSY3_B5) were evaluated to be potential probiotic candidates and were selected for probiotic development for the prevention of AFB. Importantly, the species L. panisapium isolated from larvae was identified with antimicrobial activity for the first time in this study.

Use of Dicranum polysetum extract against Paenibacillus larvae causing American Foulbrood under in vivo and in vitro conditions.

Recent research shows that Dicranum species can be used to ameliorate the negative effects of honeybee bacterial diseases and that novel compounds isolated from these species may have the potential to treat bacterial diseases. This study aimed to investigate the efficacy of Dicranum polysetum Sw. against American Foulbrood using toxicity and larval model. The effectiveness of D. polysetum Sw. ethanol extract in combating AFB was investigated in vitro and in vivo. This study is important in finding an alternative treatment or prophylactic method to prevent American Foulbrood disease in honey bee colonies. Spore and vegetative forms of Paenibacillus larvae PB31B with ethanol extract of D. polysetum were tested on 2040 honey bee larvae under controlled conditions. Total phenolic and flavonoid contents of D. polysetum ethanol extracts were determined as 80.72 mg/GAE(Gallic acid equivalent) and 303.20 µg/mL, respectively. DPPH(2,2-diphenyl-1-picrylhydrazyl) radical scavenging percent inhibition value was calculated as 4.32%. In Spodoptera frugiperda (Sf9) and Lymantria dispar (LD652) cell lines, the cytotoxic activities of D. polysetum extract were below 20% at 50 µg/mL. The extract was shown to considerably decrease infection in the larvae, and the infection was clinically halted when the extract was administered during the first 24 h after spore contamination. The fact that the extract contains potent antimicrobial/antioxidant activity does not reduce larval viability and live weight, and does not interact with royal jelly is a promising development, particularly regarding its use to treat early-stage AFB infection.

Beneficial bacteria as biocontrol agents for American foulbrood disease in honey bees (Apis mellifera).

American foulbrood (AFB) is a cosmopolitan bacterial disease that affects honey bee (Apis mellifera) larvae and causes great economic losses in apiculture. Currently, no satisfactory methods are available for AFB treatment mainly due to the difficulties to eradicate the tenacious spores produced by the etiological agent of AFB, Paenibacillus larvae (Bacillales, Paenibacillaceae). This present review focused on the beneficial bacteria that displayed antagonistic activities against P. larvae and demonstrated potential in AFB control. Emphases were placed on commensal bacteria (genus Bacillus and lactic acid bacteria in particular) in the alimentary tract of honey bees. The probiotic roles lactic acid bacteria play in combating the pathogenic P. larvae and the limitations referring to the application of these beneficial bacteria were addressed.

A Comparison of the Antibacterial Efficacy of Carbohydrate Lipid-like (Thio)Ether, Sulfone, and Ester Derivatives against Paenibacillus larvae.

Paenibacillus larvae is the causative agent of American foulbrood (AFB), the most serious bacterial disease affecting developing honeybee larvae and pupas. In this study, a library of 24 (thio)glycosides, glycosyl sulfones, 6-O-esters, and ethers derived from d-mannose, d-glucose, and d-galactose having C10 or C12 alkyl chain were evaluated for their antibacterial efficacy against two P. larvae strains. The efficacy of the tested compounds determined as minimal inhibitory concentrations (MICs) varied greatly. Generally, dodecyl derivatives were found to be more potent than their decylated analogs. Thioglycosides were more efficient than glycosides and sulfones. The activity of the 6-O-ether derivatives was higher than that of their ester counterparts. Seven derivatives with dodecyl chain linked (thio)glycosidically or etherically at C-6 showed high efficacy against both P. larvae strains (MICs ranged from 12.5 µM to 50 µM). Their efficacies were similar or much higher than those of selected reference compounds known to be active against P. larvae-lauric acid, monolaurin, and honeybee larval food components, 10-hydroxy-2-decenoic acid, and sebacic acid (MICs ranged from 25 µM to 6400 µM). The high efficacies of these seven derivatives suggest that they could increase the anti-P. larvae activity of larval food and improve the resistance of larvae to AFB disease through their application to honeybee colonies.

Total Synthesis and Biological Evaluation of Paenilamicins from the Honey Bee Pathogen Paenibacillus larvae.

Paenilamicins are a group of complex polycationic peptide secondary metabolites with antibacterial and antifungal activities produced by the devastating honey bee brood pathogen Paenibacillus larvae causing the lethal brood disease American Foulbrood (AFB). Here, we report the convergent total synthesis and structural revision of paenilamicin B2. Specific stereoisomers of paenilamicin B2 were synthesized for unambiguous confirmation of the natural product structure and for evaluation of biological activities. These studies revealed the N-terminal fragment of paenilamicin as an important pharmacophore. Infection assays using bee larvae and the insect pathogen Bacillus thuringiensis demonstrated that paenilamicins outcompete bacterial competitors in the ecological niche of P. larvae. Finally, we show first data that classifies paenilamicins as potential ribosome inhibitors. Hence, our synthesis route is a further step for understanding the pathogenicity of P. larvae and for thorough structure-activity-relationship as well as mode-of-action studies in the near future.

Development and evaluation of a core genome multilocus sequence typing scheme for Paenibacillus larvae, the deadly American foulbrood pathogen of honeybees.

Paenibacillus larvae is the causative agent of the fatal American foulbrood disease in honeybees (Apis mellifera). Strain identification is vital for preventing the spread of the disease. To date, the most accessible and robust scheme to identify strains is the multilocus sequence typing (MLST) method. However, this approach has limited resolution, especially for epidemiological studies. As the cost of whole-genome sequencing has decreased and as it becomes increasingly available to most laboratories, an extended MLST based on the core genome (cgMLST) presents a valuable tool for high-resolution investigations. In this study, we present a standardized, robust cgMLST scheme for P. larvae typing using whole-genome sequencing. A total of 333 genomes were used to identify, validate and evaluate 2419 core genes. The cgMLST allowed fine-scale differentiation between samples that had the same profile using traditional MLST and allowed for the characterization of strains impossible by MLST. The scheme was successfully used to trace a localized Swedish outbreak, where a cluster of 38 isolates was linked to a country-wide beekeeping operation. cgMLST greatly enhances the power of a traditional typing scheme, while preserving the same stability and standardization for sharing results and methods across different laboratories.

Discovery of Paenibacillus larvae ERIC V: Phenotypic and genomic comparison to genotypes ERIC I-IV reveal different inventories of virulence factors which correlate with epidemiological prevalences of American Foulbrood.

Paenibacillus larvae is the etiological agent of American Foulbrood (AFB), a highly contagious brood disease of honey bees (Apis mellifera). AFB requires mandatory reporting to the veterinary authority in many countries and until now four genotypes, P. larvae ERIC I-IV, have been identified. We isolated a new genotype, ERIC V, from a Spanish honey sample. After a detailed phenotypic comparison with the reference strains of the ERIC I-IV genotypes, including spore morphology, non-ribosomal peptide (NRP) profiling, and in vivo infections of A. mellifera larvae, we established a genomic DNA Macrorestriction Fragment Pattern Analysis (MRFPA) scheme for future epidemiologic discrimination. Whole genome comparison of the reference strains and the new ERIC V genotype (DSM 106052) revealed that the respective virulence gene inventories of the five genotypes corresponded with the time needed to kill 100 % of the infected bee larvae (LT100) in in vivo infection assays. The rarely isolated P. larvae genotypes ERIC II I-V with a fast-killing phenotype (LT100 3 days) harbor genes with high homology to virulence factors of other insect pathogens. These virulence genes are absent in the epidemiologically prevalent genotypes ERIC I (LT100 12 days) and ERIC II (LT100 7 days), which exhibit slower killing phenotypes. Since killing-retardation is known to reduce the success of hygienic cleaning by nurse bees, the identified absence of virulence factors might explain the epidemiological prevalences of ERIC genotypes. The discovery of the P. larvae ERIC V isolate suggests that more unknown ERIC genotypes exist in bee colonies. Since inactivation or loss of a few genes can transform a fast-killing phenotype into a more dangerous slow-killing phenotype, these rarely isolated genotypes may represent a hidden reservoir for future AFB outbreaks.

Feeding Honeybee Colonies with Honeybee-Specific Lactic Acid Bacteria (Hbs-LAB) Does Not Affect Colony-Level Hbs-LAB Composition or Paenibacillus larvae Spore Levels, Although American Foulbrood Affected Colonies Harbor a More Diverse Hbs-LAB Community.

The main current methods for controlling American Foulbrood (AFB) in honeybees, caused by the bacterial pathogen Paenibacillus larvae, are enforced incineration or prophylactic antibiotic treatment, neither of which is fully satisfactory. This has led to an increased interest in the natural relationships between the pathogenic and mutualistic microorganisms of the honeybee microbiome, in particular, the antagonistic effects of Honeybee-Specific Lactic Acid Bacteria (hbs-LAB) against P. larvae. We investigated whether supplemental administration of these bacteria affected P. larvae infection at colony level over an entire flowering season. Over the season, the supplements affected neither colony-level hbs-LAB composition nor naturally subclinical or clinical P. larvae spore levels. The composition of hbs-LAB in colonies was, however, more diverse in apiaries with a history of clinical AFB, although this was also unrelated to P. larvae spore levels. During the experiments, we also showed that qPCR could detect a wider range of hbs-LAB, with higher specificity and sensitivity than mass spectrometry. Honeybee colonies are complex super-organisms where social immune defenses, natural homeostatic mechanisms, and microbiome diversity and function play a major role in disease resistance. This means that observations made at the individual bee level cannot be simply extrapolated to infer similar effects at colony level. Although individual laboratory larval assays have clearly demonstrated the antagonistic effects of hbs-LAB on P. larvae infection, the results from the experiments presented here indicate that direct conversion of such practice to colony-level administration of live hbs-LAB is not effective.

Meliponamycins: Antimicrobials from Stingless Bee-Associated Streptomyces sp.

Social insects establish complex interactions with microorganisms, some of which play defensive roles in colony protection. The important role of pollinators such as the stingless bee Melipona scutellaris in nature encouraged us to pursue efforts to study its associated microbiota. Here we describe the discovery of two novel cyclic hexadepsipeptides, meliponamycin A (1) and meliponamycin B (2), from Streptomyces sp. ICBG1318 isolated from M. scutellaris nurse bees. Their structures were established by interpretation of NMR and MS data, and the absolute configuration of the constituent amino acids was determined by the advanced Marfey's method. Compounds 1 and 2 showed strong activity against the entomopathogen Paenibacillus larvae and human pathogens Staphylococcus aureus and Leishmania infantum.

In Vitro Antagonistic Effect of Gut Bacteriota Isolated from Indigenous Honey Bees and Essential Oils against Paenibacillus Larvae.

The aim of study was to isolate and identify the gut bacteria of Apis mellifera and to evaluate antagonistic effect of the bacteriota against Paenibacillus larvae, which causes American foulbrood (AFB) in honeybees. The dilution plating method was used for the quantification of selected microbial groups from digestive tract of bees, with an emphasis on the bacteriota of the bees' intestines. Bacteria were identified using mass spectrometry (MALDI-TOF-MS Biotyper). Overall, five classes, 27 genera and 66 species of bacteria were identified. Genera Lactobacillus (10 species) and Bacillus (8 species) were the most abundant. Gram-negative bacteria were represented with 16 genera, whereas Gram-positive with 10 genera. Delftia acidovorans and Escherichia coli were the most abundant in the digestive tract of honey bee. Resistance to a selection of antimicrobials was assessed for the bacterial isolates from bee gut and confirmed against all antimicrobials included in the study, with the exception of cefepime. Lactobacillus spp., especially L. kunkeei, L. crispatus and L. acidophilus. showed the strongest antimicrobial activity against P. larvae, the causal pathogen of AFB. Antimicrobial activity of essential oils against isolated bacteria and two isolates of P. larvae were assessed. Application of a broad selection of plant essential oils indicated that Thymus vulgaris had the highest antimicrobial activity against P. larvae.

Determination of Chloramphenicol in Honey Using Salting-Out Assisted Liquid-Liquid Extraction Coupled with Liquid Chromatography-Tandem Mass Spectrometry and Validation According to 2002/657 European Commission Decision.

Honey is a natural food widely consumed due to its high content in nutrients and bioactive substances. In order to prevent hive infections, xenobiotics such as pesticides and antibiotics are commonly used. Chloramphenicol (CAP) is a broad-spectrum antibiotic used to treat honeybee larvae diseases. However, CAP has toxic and nondose-dependent effects in sensitive subjects; for this reason, its use has been prohibited in food-producing animals, such as the honeybee. In this study, we proposed a rapid, simple, and cheap analytical method, based on salting-out assisted liquid-liquid extraction coupled with UHPLC MS/MS detection for the accurate determination of CAP in honey to be used in routine analyses. The parameters that influence the extraction efficiency have been optimized using an experimental design in order to maximize the recovery of the analyte by reducing the matrix effects. Therefore, the developed method was internally validated according to the 2002/657/EC Decision guidelines and applied to the analysis of 96 honey samples.

The biological role of the enigmatic C3larvinAB toxin of the honey bee pathogenic bacterium Paenibacillus larvae.

Paenibacillus larvae is the causative agent of the notifiable epizootic American foulbrood, a fatal bacterial disease of honey bee larvae. The species P. larvae has been classified into four differentially virulent and prevalent genotypes (ERIC I-IV), which also differ in their virulence factor equipment. Recently, a novel P. larvae toxin, the C3-like C3larvin, has been described. Genome analysis now revealed that the C3larvin gene is actually a part of a toxin locus encompassing two genes encoding a binary AB toxin with the A subunit being C3larvin (C3larvinA) and a putative B subunit (C3larvinB) encoded by the second gene. Sequence and structural analyses demonstrated that C3larvinB is a homologue of the Bacillus anthracis protective antigen (PA), the B subunit of anthrax toxin. The C3larvinAB toxin locus was interrupted by point mutations in all analysed P. larvae ERIC I and ERIC II strains. Only one P. larvae ERIC III/IV strain harboured an uninterrupted toxin locus comprising full-length genes for C3larvinA and B. Exposure bioassays did not substantiate a role as virulence factor for C3larvinAB in P. larvae ERIC I/II. However, the PA homologue C3larvinB had an influence on the virulence of the unique P. larvae strain expressing the functional C3larvinAB locus.

Honeybee-Specific Lactic Acid Bacterium Supplements Have No Effect on American Foulbrood-Infected Honeybee Colonies.

Paenibacillus larvae, the causative agent of American foulbrood (AFB), is the primary bacterial pathogen affecting honeybees and beekeeping. The main methods for controlling AFB are incineration of diseased colonies or prophylactic antibiotic treatment (e.g., with tylosin), neither of which is fully satisfactory. The search for superior means for controlling AFB has led to an increased interest in the natural relationships between the honeybee-pathogenic and mutualistic microorganisms and, in particular, the antagonistic effects of honeybee-specific lactic acid bacteria (hbs-LAB) against P. larvae These effects have been demonstrated only on individual larvae in controlled laboratory bioassays. Here we investigated whether supplemental administration of hbs-LAB had a similar beneficial effect on P. larvae infection at colony level. We compared experimentally AFB-infected colonies treated with hbs-LAB supplements to untreated and tylosin-treated colonies and recorded AFB symptoms, bacterial spore levels, and two measures of colony health. To account for the complexity of a bee colony, we focused on (Bayesian) probabilities and magnitudes of effect sizes. Tylosin reduced AFB disease symptoms but also had a negative effect on colony strength. The tylosin treatment did not, however, affect P. larvae spore levels and might therefore "mask" the potential for disease. hbs-LAB tended to reduce brood size in the short term but was unlikely to affect AFB symptoms or spores. These results do not contradict demonstrated antagonistic effects of hbs-LAB against P. larvae at the individual bee level but rather suggest that supplementary administration of hbs-LAB may not be the most effective way to harness these beneficial effects at the colony level.IMPORTANCE The previously demonstrated antagonistic effects of honeybee-derived bacterial microbiota on the infectivity and pathogenicity of P. larvae in laboratory bioassays have identified a possible new approach to AFB control. However, honeybee colonies are complex superorganisms where social immune defenses play a major role in resistance against disease at the colony level. Few studies have investigated the effect of beneficial microorganisms on bee diseases at the colony level. Effects observed at the individual bee level do not necessarily translate into similar effects at the colony level. This study partially fills this gap by showing that, unlike at the individual level, hbs-LAB supplements did not affect AFB symptoms at the colony level. The inference is that the mechanisms regulating the honeybee microbial dynamics within a colony are too strong to manipulate positively through supplemental feeding of live hbs-LAB and that new potential remedies identified through laboratory research have to be tested thoroughly in situ, in colonies.

Large-scale cultivation of the bumblebee gut microbiota reveals an underestimated bacterial species diversity capable of pathogen inhibition.

A total of 1940 isolates from gut samples of 60 bumblebees representing Bombus pascuorum, Bombus terrestris, Bombus lucorum and Bombus lapidarius was collected and identified through state-of the-art taxonomic methods. The bacterial species diversity in these Bombus species exceeded that suggested by phylotype analysis through 16S rRNA amplicon sequencing, and revealed that B. pascuorum and B. terrestris had a unique microbiota composition, each. Representatives of most phylotypes reported earlier and detected in the present study were effectively isolated, and included several novel bacterial taxa and species reported for the first time in the bumblebee gut. Isolates were screened in pectin degradation assays and growth inhibition assays against the honeybee pathogens Paenibacillus larvae, Melissococcus plutonius and Ascosphaera apis and the bumblebee parasite Crithidia bombi. While inhibitory activity against each of these pathogens was observed, only one single culture was able to degrade pectin and polygalacturonic acid in vitro. The availability of accurately identified microbial isolates will facilitate future evaluation of the functional potential of the bumblebee gut microbiota.

A PCR-Based Method for Distinguishing between Two Common Beehive Bacteria, Paenibacillus larvae and Brevibacillus laterosporus.

Paenibacillus larvae and Brevibacillus laterosporus are two bacteria that are members of the Paenibacillaceae family. Both are commonly found in beehives and have historically been difficult to distinguish from each other due to related genetic and phenotypic characteristics and a shared ecological niche. Here, we discuss the likely mischaracterization of three 16S rRNA sequences previously published as P. larvae and provide the phylogenetic evidence that supported the GenBank reassignment of the sequences as B. laterosporus We explore the issues that arise by using only 16S rRNA or other single-gene analyses to distinguish between these bacteria. We also present three sets of molecular markers, two sets that distinguish P. larvae from B. laterosporus and other closely related species within the Paenibacillus genus and a third set that distinguishes B. laterosporus from P. larvae and other closely related species within the Brevibacillus genus. These molecular markers provide a tool for proper identification of these oft-mistaken species.IMPORTANCE 16S rRNA gene sequencing in bacteria has long been held as the gold standard for typing bacteria and, for the most part, is an excellent method of taxonomically identifying different bacterial species. However, the high level of 16S rRNA sequence similarity of some published strains of P. larvae and B. laterosporus, as well as possible horizontal gene transfer events within their shared ecological niche, complicates the use of 16S rRNA sequence as an effective molecular marker for differentiating these two species. Additionally, shared characteristics of these bacteria limit the effectiveness of using traditional phenotypic identification assays, such as the catalase test. The results from this study provide PCR methods to quickly differentiate between these two genera and will be useful when studying Brevibacillus, Paenibacillus, and other disease-relevant bacteria commonly found in beehives.

Proteinase pattern of honeybee prepupae from healthy and American Foulbrood infected bees investigated by zymography.

American foulbrood disease (AFB) is the main devastating disease that affects honeybees' brood, caused by Paenibacillus larvae. The trend of the research on AFB has addressed the mechanisms by which P. larvae bacteria kill honeybee larvae. Since prepupae could react to the infection of AFB by increasing protease synthesis, the aim of this work was to compare protease activity in worker prepupae belonging to healthy colonies and to colonies affected by AFB. This investigation was performed by zymography. In gel, proteolytic activity was observed in prepupae extracts belonging only to the healthy colonies. In the prepupae extracts, 2D zimography followed by protein identification by MS allowed to detect Trypsin-1 and Chymotrypsin-1, which were not observed in diseased specimens. Further investigations are needed to clarify the involvement of these proteinases in the immune response of honeybee larvae and the mechanisms by which P. larvae inhibits protease production in its host.

Functional characterization of the uracil transporter from honeybee pathogen Paenibacillus larvae.

The genome of the Honeybee bacterial pathogen, Paenibacillus larvae, encodes for protein a with substantial amino acid sequence similarity to the canonical Escherichia coli uracil transporter UraA. P. larvae expresses the uracil permease (PlUP) locus, and is sensitive to the presence of the toxic uracil analog 5-fluorouracil under vegetative growth conditions. The solute transport and binding profile of PlUP was determined by radiolabeled uptake experiments via heterologous expression in nucleobase transporter-deficient Saccharomyces cerevisiae strains. PlUP is specific for the transport of uracil and competitively binds xanthine and uric acid. Further biochemical characterization reveals that PlUP has a strong affinity for uracil with a Km 19.5 ±â€¯1.6 µM. Uracil transport is diminished in the presence of the proton disruptor carbonyl cyanide m-chlorophenylhydrazone, but not by the sodium gradient disruptor Ouabain.

Silencing of Apis mellifera dorsal genes reveals their role in expression of the antimicrobial peptide defensin-1.

Like all other insects, two key signalling pathways [Toll and immune deficiency (Imd)] regulate the induction of honey bee immune effectors that target microbial pathogens. Amongst these effectors are antimicrobial peptides (AMPs) that are presumed to be produced by the nuclear factors kappa B (NF-κB) Dorsal and Relish from the Toll and Imd pathways, respectively. Using in silico analysis, we previously proposed that the honey bee AMP defensin-1 was regulated by the Toll pathway, whereas hymenoptaecin was regulated by Imd and abaecin by both the Toll and Imd pathways. Here we use an RNA interference (RNAi) assay to determine the role of Dorsal in regulating abaecin and defensin-1. Honey bees have two dorsal genes (dorsal-1 and dorsal-2) and two splicing isoforms of dorsal-1 (dorsal-1A and dorsal-1B). Accordingly, we used both single and multiple (double or triple) isoform knockdown strategies to clarify the roles of dorsal proteins and their isoforms. Down-regulation of defensin-1 was observed for dorsal-1A and dorsal-2 knockdowns, but abaecin expression was not affected by dorsal RNAi. We conclude that defensin-1 is regulated by Dorsal (Toll pathway).