Core proteome mediated subtractive approach for the identification of potential therapeutic drug target against the honeybee pathogen Paenibacillus larvae

Introduction

The majority of plant species crucial for our food supply rely on insect pollinators. Honey bees (scientific name: Apis mellifera) play a crucial role in global fruit cultivation, with over 90% of crops depending on their pollination services (Zheng et al., 2018). This vital service has a direct impact on human food consumption and sustains biodiversity by supporting the pollination of flowering plants and crops. Approximately 35% of the food consumed by people depends on bee-mediated pollination (Klein et al., 2007). Products such as beeswax and royal jelly, in addition to honey, are produced by bees and have been utilized for ages in food, medicine, and cosmetics (Dumitru et al., 2022).

Honeybees confront various disease-causing agents, including bacteria, viruses, protozoa, fungi, and parasitic mites (Neov et al., 2019). Notable among these pathogens is Paenibacillus larvae, the causative agent of American foulbrood disease (AFB), a highly destructive and contagious disease affecting Apis mellifera larvae (Ye et al., 2023). This Gram-positive, facultative anaerobic, spore-forming bacterium is capable of infecting honeybee brood within the first three days of their lives. It can also impact the pupal stage (Daisley et al., 2023). This pathogen has a global presence, with viable spores persisting in hives for extended periods, even decades, posing a continuous threat to honeybee colonies and their health. Given its virulence and significant impact on honey bee colonies, AFB is internationally classified as a notifiable disease, requiring mandatory reporting worldwide (Dickel et al., 2022). Considering the severity of the infection, a frequent occurrence, rapid and easy spread, epizooty and enzooty are common (Ebeling et al., 2023). The classification of AFB as a highly dangerous infectious animal disease by the World Organization for Animal Health underscores the severity and rapid spread of this disease within apiaries (Matovic et al., 2023).

When clinical signs of the disease become apparent in a hive, a common practice to control its spread is by resorting to extreme measures such as burning the hive, equipment, and the entire colony. Additionally, antibiotics like oxytetracycline hydrochloride (marketed as Terramycin) or tylosin (a macrolide) are sometimes employed to inhibit the proliferation of vegetative cells of P. larvae in the midgut of honeybee larvae. However, in some regions, the prophylactic use of these antibiotics has led to the emergence of antibiotic-resistant strains of P. larvae (Alippi et al., 2007; Genersch et al., 2010). Consequently, due to the risk of developing resistant strains and potential contamination of honeybee products, several European Union nations have prohibited the use of antibiotics in beekeeping practices.

It is essential to clarify that antibiotics are primarily utilized for antimicrobial metaphylaxis of AFB, aiming to prevent outbreaks rather than treating established clinical infections in hives affected by AFB. Furthermore, it’s worth noting that there are no Maximum Residue Limits (MRLs) established for antibiotics in honey, according to European Community laws (Mutinelli, 2003), which prohibits the sale of honey containing antibiotic residues. This highlights the need for alternative, sustainable approaches to manage P. larvae. Integrated pest management (IPM) principles advocate for a judicious combination of methods, promoting the health of honeybee colonies while minimizing the environmental impact. By integrating IPM into beekeeping practices, we can strive for more effective and environmentally conscious strategies in the management and prevention of AFB outbreaks. IPM offers a comprehensive and sustainable approach that considers various factors such as biological, cultural, and mechanical controls alongside chemical interventions (Bava et al., 2023). It emphasizes ecological balance and minimizes the impact on non-target organisms. Additionally, it involves the utilization of substances known for their anti-P. larvae properties, encompassing both synthetic compounds such as (Thio) ether, sulfone, ester derivatives (Šamšulová et al., 2023) and naturally derived agents like fatty acids (such as algal metabolite lauric acid) (Lopes et al., 2016), essential oils (Ansari et al., 2016), plant extracts (Isidorov et al., 2018), propolis (along with its constituents) (Wilson et al., 2015), as well as probiotic bacteria (Truong et al., 2023).

It also calls for investigation of newer approaches and techniques to find effective treatments based on novel therapeutic targets for managing AFB by inhibiting P. larvae. In this context, computer-aided drug design (CADD), a swift method for efficiently identifying viable therapeutic candidates, can help to reduce the usage of animal models in pharmacological research as well as help with the rational design of new and safe drug candidates (Niazi & Mariam, 2023). It can facilitate repositioning of already-marketed medications. This support to medicinal chemists and pharmacologists is instrumental throughout the drug development process (Brogi et al., 2020). Furthermore, the exploration of plant-derived components as potential agents against AFB aligns with the rich historical utilization of traditional herbal medicine. Plant-derived compounds may offer effective and safe alternatives for managing AFB (Abdallah et al., 2023), contributing to a broader understanding of sustainable and eco-friendly strategies in apiculture. Recently, the antibacterial activity of inflorescences and roots extracts of Cannabis (Cannabis sativa L) against P. larvae has been reported (Giselle et al., 2023). Researchers have also reported tannin extract to be potent antimicrobials against P. larvae (Giménez et al., 2021). The extract of Dicranum polysetum has also shown a significant reduction in infection among honey bee larvae, with clinical cessation of infection upon extract administration within the initial 24 h after spore ingestion. The antimicrobial activity of these compounds does not compromise larval viability, live weight, or interact adversely with royal jelly and shows promise in addressing early-stage AFB infection without negative impacts on essential aspects of larval health. Further research is warranted to investigate the potential reduction of risk of developing resistance to these novel compounds (Karaoğlu et al., 2023).

This is the first subtractive proteomic study that aims to identify potential novel drug targets in P. larvae. Utilizing a target that has not been previously employed in antimicrobial therapy against a selected species, offers reduced risk of resistance development, as the bacteria may not have encountered selective pressures against these novel targets. The exploration of new targets also provides an opportunity for identifying compounds with unique mechanisms of action (Aljeldah, 2022). The chosen target was used to identify potent inhibitors using natural product libraries by virtual screening, molecular docking and MD simulation. We hope that our research will contribute to the development of innovative drugsfor combating AFB and safeguarding honeybee populations.

Materials & Methods

Results

ACP synthase III3D structure determination and active site prediction

The 3D structure of ACP synthase III was predicted employing the Alpha-Fold software, and its depiction is presented in Fig. 1A. The obtained structure achieved a commendable MolProbityScore of 1.78. Additionally, the Ramachandran plot (depicted in Fig. 1B) revealed an impressive 95.20% of residues situated within the highly favored region, with a mere 0.31% falling into the outlier category.

Table 4:

List of P. larvae druggable proteins.

List of P. larvae proteins with similarities to Drug-Bank proteins.

Protein name	DEG identifier	Accession	Similarity to bee proteome	Similarity with the Human Proteome	Drugs available in Drug Bank
Iron-containing alcohol dehydrogenase	DEG10430416	WP_192807335.1	No significant similarity	No significant similarity	Carbaphosphonate
Ketoacyl-ACP synthase III	DEG10570076	WP_174567582.1	No significant similarity	No significant similarity	Cerulenin
Isochorismatase	DEG10470205	WP_024093894.1	No significant similarity	No significant similarity	Malonyl-CoA Formic acid Isochorismic Acid

DOI: 10.7717/peerj.17292/table-4

The active site (binding pockets) of the modeled protein was predicted using the CASTp online prediction tool. Approximately 23 binding pockets were predicted for the protein. The binding cavity with the highest volume (1,392.832) was considered in the next stage of our study. Selecting the binding cavity with the highest volume is based on the assumption that a larger cavity can accommodate ligands of various sizes and shapes (Das et al., 2020; Gazgalis et al., 2020). The amino acids residing in this cavity can play a major role in binding ligand molecules during docking analysis (Melnikova et al., 2023).

Virtual screening for the discovery of ACP synthase III inhibitors

Molecular docking, is a computational technique employed to ascertain the optimal conformation of a ligand within its receptor, based on factors such as position (x, y, z), orientation (qx, qy, qz, qw), and torsion angles (T1, T2…Tn) within the ligand’s binding site. Subsequently, the ligands were categorized according to their binding affinities with the target. Among a pool of over 36,000 compounds, 4,267 exhibited interactions with the target. The three ligands demonstrating the lowest binding energies (Fig. S1) are shown with ACP synthase III interactions in Fig. 2.

One of these ligands, ZINC95910054, is derived from celery (Apiumgraveolens), a wetland plant belonging to the Apiaceae family, with a longstanding history of cultivation as a culinary vegetable. Comprising a total of 54 atoms, including 26 heavy atoms, its chemical formula is C18H28O8. Notably, this ligand displayed a binding energy of −10.84 Kcal/mol.

Another ligand, ZINC59586481, demonstrated a docking binding energy of −9.71 Kcal/mol. In contrast, ZINC14444861, originating from the plant Angelicasinensis an herbaceous member of the Apiaceae family with a rich medicinal tradition in East Asia exhibited a binding energy of −9.51 Kcal/mol. Comprising a total of 41 atoms, half of which are heavy atoms, this ligand featured 17 carbon atoms in its structure.

MD simulation

The stability of top ranked docked complexes was inferred by MD simulation studies. The MD simulation study showed a stable system for ZINC95910054 and ACP synthase III, as confirmed by RMSD and RMSF values during 100 ns trajectories (Fig. 3). On the average the RMSD values were less than 3 Å for all runs and RMSF reached upto 7.5 Å  around residue 203 (comprising loop-region). Loop regions are more flexible than rest of the protein and depict higher RMSF.

Discussion

The worldwide contamination of bee colonies by AFB disease underscores its severity, with the causative agent being the spore-forming bacterium P. larvae (Papić, Diricks & Kušar, 2021). This contagious disease causes great economic losses in apiculture (Locke, Low & Forsgren, 2019). The economic consequences of AFB include reduced honey production and compromised pollination services (Popovska Stojanov et al., 2021; Wakgari & Yigezu, 2021). Beekeepers may experience financial losses due to the destruction of infected colonies, decreased hive productivity, and the costs associated with disease management measures (Stanimirović et al., 2019).

Application and supplementary feeding of antibiotics to restrain clinical AFB symptoms and infection is a common practice in beekeeping (Masood et al., 2022; Zabrodski, 2022), although it raises concerns about the development of antibiotic-resistant strains of P. larvae. The widespread use of antibiotics in beekeeping to control AFB facilitates the dissemination of antibiotic resistance genes and contributes to compromised immunity in honey bees (Li et al., 2019). Daisley et al. (2020) have reported that administration of oxytetracycline led to an increase in efflux pump resistance (tetB gene abundance) and depletion of crucial symbionts like Frischella perrera and Lactobacillus strains, known for their roles in regulating immune function and nutrient metabolism. The observed microbial changes manifested in decreased capped brood counts, indicative of compromised hive nutritional status and productivity. Additionally, the antibiotic-induced alterations were associated with a reduction in the antimicrobial capacity of adult hemolymph, highlighting a decline in immune competence among the honey bees. These findings underscore the intricate relationship between antibiotic exposure, changes in gut microbiota, and the consequential impact on honey bee health and immune functionality (Ortiz-Alvarado, 2019). In addition to this, the presence of antibiotic residues in beehive products destined for human consumption are undesirable (Ye et al., 2023).

AFB control measures, such as burning infected colonies to prevent the spread of the disease, can result in the loss of equipment and resources invested in the affected hives (Forsgren et al., 2013). The economic impact extends beyond individual beekeepers to the broader agricultural sector, as honey bees play a crucial role in pollinating crops, contributing to the overall food production chain. AFB disease control measures also involve relocating adult honey bees to new foundation, eliminating bees and equipment with contamination (Zabrodski, 2022), treating colonies with substances like sulphathiazole (Cervera-Chiner et al., 2020) or oxytetracycline (Puvača, 2022), and sterilizing equipment through methods such as gamma radiation, hot paraffin wax dipping, scorching, or ethylene oxide fumigation (Kisil & Fotina, 2020; Matheson & Reid, 1992).

Essential oils of several plants have been beneficial in curbing P. larvae (Ansari et al., 2016; Puvača, 2022). Bacteriophages and their lytic enzymes have also emerged as a promising alternative for the treatment and prevention of AFB (Jończyk-Matysiak et al., 2020). Probiotic strains have also demonstrated antimicrobial properties against P. larvae (Truong et al., 2023). Here, we aimed to identify a potent therapeutic target and inhibitor molecules from TCM against the ACP synthase III of P. larvae. This was accomplished through the utilization of an integrative computational subtractive proteomics approach, associated with a pan-proteomic assessment of various strains of P. larvae.

It resulted in the identification of 1,438 core proteins. Subtractive proteome analysis was also used to identify a collection of 24 proteins non-homologous to humans and honeybees but crucial for the pathogen survival. Ketoacyl-ACP synthase III was chosen as a possible therapeutic target out of the three druggable proteins. The 3D structure of the Ketoacyl-ACP synthase III was modeled and validated and further subjected to molecular docking against TCM compounds. Based on the lowest binding energy, ZINC95910054, ZINC59586481, and ZINC14444861 were prioritized as inhibitors. ZINC95910054 displayed least binding energy of −10.84 Kcal/mol, making it the highest-scoring inhibitor and possibly, the most promising candidate for inhibition of fatty acid synthesis and associated cellular processes in P. larvae. Further experimental screening is recommended to identify the most potential ligand among the three.

Different natural strategies based on the application of essential oils, plant extracts, propolis, royal jelly, nonconventional natural molecules, bacteria, and bacteriocines, have been studied in vitro and in vivo for the prevention and the control of P. larvae (Alonso-Salces et al., 2017). For instance, Floris, Carta & Moretti (1996) tested the antimicrobial activity of 21 types of essential oils against six strains of P. larvae using the agar diffusion method. The in vitro results showed that the most effective treatment was the cinnamon oil. Furthermore, Giménez et al. (2021) found that among thirteen tested natural molecules, menadione, lauric acid, monoglyceride of lauric acid and naringenin showed antimicrobial activity against ten P. larvae isolates. This study demonstrated the in vitro bactericidal activity of these molecules, specifically menadione and lauric acid, at concentrations practical for field application.

On the other hand, other researchers studied the inhibition of ACP synthase III enzyme in numerous bacterial species. The ACP synthase III (encoded by gene FabH) is one of functional enzymes in fatty acid biosynthesis (FAB), which initiates the FAB cycle by catalyzing the first condensation step between acetyl-CoA and malonyl-ACP (Zhang, Li & Zhu, 2012). Thiolactomycin (TLM), a natural compound produced by Actinomycetes has depicted an IC50 value of >100 against ACP synthase of S. aureus (He & Reynolds, 2002), 110 M against ACP synthase of the Escherichia coli (Price et al., 2001). Hence, the obtained compound if synthesized and tested in vivo, seems promising for the inhibition of P. larvae and the control of the AFB.

We acknowledge several limitations of our study. The exclusion of strains with draft or incomplete genomes means that a portion of relevant data was omitted from our analysis. It is also essential to recognize that the strains studied may not fully represent the global P. larvae population, given that sequences from various regions worldwide have not been universally sequenced and submitted to NCBI. This potential lack of global diversity in the dataset could impact the overall comprehensiveness of our pan-genome analysis.Future studies incorporating a more diverse range of strains could provide a more comprehensive understanding of the pan-proteome dynamics within this bacterial species. Furthermore, while computational predictions serve as valuable tools, their biological relevance should be experimentally validated to ensure accuracy and applicability. The lack of experimental confirmation may impose limitations on the translational impact of our findings. It is also important to note that computational models may not fully capture unforeseen biological complexities, such as post-translational modifications or protein-protein interactions. Antibiotics used in metaphylaxis are intended to prevent outbreaks of AFB, meaning they are applied in honeybee colonies that do not exhibit any clinical symptoms of the disease. However, the pharmacokinetic profiles of these prioritized compounds are not well-established for honeybees, posing a challenge in predicting their behavior in vivo. Furthermore, the absence of comparative data with established antibiotic metaphylaxis makes it difficult to assess the advantages or disadvantages of the prioritized compounds. Despite these constraints, our study serves as a foundational step, paving the way for future research to systematically address these limitations. We recommend further exploration into identifying phytochemicals with similar properties, aiming for a more nuanced inhibition of both P. larvae and AFB.

Conclusion

In conclusion, this bioinformatics exploration of essential proteins within P. larvae has yielded the identification of three promising candidates as potential therapeutic targets, with potential applications in pharmacology. Notably, ACP synthase III emerged as the primary focus of our research, chosen based on rigorous criteria. Through virtual screening by molecular docking with Autodock software, we scrutinized inhibitor ligands from the traditional Chinese library, ultimately singling out ZINC95910054 as a promising inhibitor for ACP synthase III. Encouragingly, MD simulation affirmed the stability of this complex, bolstering its candidacy as a prospective target for future AFB control. However, the exclusion of certain genotypic information and the reliance on strains with complete genome sequences may influence the generalizability of our findings to the broader P. larvae population. Hence, we propose further studies incorporating more diverse strains, compounds and experimental techniques as well.

Supplemental Information