Team:Tec-Chihuahua/Description

Erwinions






<






Production of antimicrobial peptides in Escherichia coli for Paenibacillus larvae and Melissococcus plutonius inhibition

Abstract

American and European Foulbrood are diseases that affect bee (Apis mellifera) larvae worldwide. In the last two years, 53 countries suffered from these diseases, 6 of them are among the top 10 honey producers. The causal agents of these ailments are Gram-positive bacteria: Paenibacillus larvae and Melissococcus plutonius respectively. Nowadays, two techniques for the treatment of Foulbrood are used: antibiotics and incineration of hives. The former promotes the development of antibiotic resistance in bacteria while the latter results unprofitable for beekeepers. Therefore, we propose the production of bee antimicrobial peptides (AMPs) in Escherichia coli to treat P. larvae and M. plutonius infections. Defensin 1, abaecin, defensin 2, and apidaecin are each expressed in a different BL21 (DE3) culture. PelB leader peptide and a 6X His-tag foster adequate expression and further purification. Through mathematical modeling, the diffusivity of PLGA-nanoencapsulated apidaecin is evaluated for future in vivo delivery in the bee system.



Introduction

The most significant agricultural management practice is, without a doubt, pollination. Crops that directly rely on this activity are estimated to have a global price tag of between US$235 and US$577 billion a year. In fact, 87% of the food we consume depends on pollination.1 Apis mellifera ranks as the most frequent species of pollinator for crops worldwide2 making possible and is responsible for the production of strawberry, alfalfa, avocado, coffee, apples, lemons, among many others.3 Bees improve the food production of 2 billion small farmers around the world helping guarantee the food security of the world population.4


Pollination is the greatest strength of bees, but that's not all they do; the world honey market reported historical records during 2015, with volumes of

operation exceeding 2.3 billion dollars. In 2016, Mexico contributed 55,358 tons to the world market, with a value of 2,279 million Mexican pesos.5 Beekeeping in Mexico has great socio-economic importance since it is considered as one of the main livestock activities generating foreign income6, emphasizing that the third part of Mexico’s agricultural production depends on bees.7



Not properly pollinated cucumber, promoting a poor development


Yet in the midst of the highly demanded bee population, beekeepers of multiple continents have suffered severe colony losses in recent years and this issue is ascribed to Colony Collapse Disorder, that corresponds to the 30% annual lost in the number of hives worldwide. The cause of this collapse is unclear, and it is attributed to an infectious synergy of multiple factors including pesticides, nutritional complications due to changes in climate patterns and diseases.9 While viruses and fungal pathogens have been identified as good indicators of this condition, these pathogens, on their own, are not able to explain all losses, suggesting that honey bee colonies are suffering from compromised immune systems which pathogens

are able to take advantage of.8 Two highly contagious diseases that affect bee (Apis mellifera) larvae worldwide demonstrates the magnitude of this problem: American and European Foulbrood. In the last two years, 53 countries suffered from these diseases10 and 6 of them are among the top 10 honey producers.11

Foulbrood presence around the world from 2016 to 2018.10


American Foulbrood

American foulbrood (AFB) is the most virulent bacterial disease of honey bee brood worldwide.12 An infected hive will suffer a significant loss of larvae and without the new breed, to replace the older workers, the entire colony is in danger, in addition, the spores of the causal agent, Paenibacillus larvae, can remain active for more than 30 years.13


The spread starts when larvae ingest food contaminated by the spores of the pathogen. The spores reach the lumen in the midgut where they germinate, transform to its vegetative form and multiply before beginning the attack the epithelium; the secondary metabolites (black and white hexagons in the image) produced by P. larvae help to overcome the microbial competitors and to conquer the midgut, then P. larvae secrete an enzyme (red stars in the image) that degrades chitin in order to digest the peritrophic matrix (pink structure that has

Pathogenesis of P. larvae.14

the objective of protecting the midgut epithelium (gray cells with blue nuclei) against pathogens. This step marks the transition from the non-invasive phase to the invasive phase of the infection. Finally, the bacteria attack the epithelial cells and manage to reach the hemolymph, here the destructive phase starts, and larvae die by septicemia. During this last phase, the nutrients are scarce, and the bacteria begin to sporulate to form a mass of spores that are latter scattered by the adult bees inside and outside. the colonies.14



European Foulbrood

As it is very difficult to eradicate, European foulbrood (EFB) maintains the colonies weak and vulnerable for many years instead of showing a sudden high mortality rate. Melissococcus plutonius, the bacteria responsible for this ailment, can remain in the body of those larvae that survived the disease during its pupal stage and reached the adult stage or can infect the bees responsible for cleaning the cells when larvae die. The adult bees expel the bacteria through the feces or from the food they provide to the larvae; M. plutonius enters the larva until it reaches the midgut where it reproduces exponentially. Later, the pathogen is situated at the interface of the peritrophic membrane and competes for nutrients causing larval death by starvation.15

Nowadays, there are two deficient treatment methods used worldwide against these two Gram-positive bacteria. First, antibiotics like oxytetracycline and chloramphenicol lead bacteria to mutate, making them resistant and harder to fight. In addition, innocuousness is a determining factor for the acceptance of honey in importing countries;16 it is necessary to comply with the zero-tolerance policy for antibiotics that limits waste to 1 part per billion exercised by the European Union, the United States and now, by markets that previously were more tolerant in terms of waste.17 More drastically, beekeepers can incinerate everything in the hive. Despite it being the most effective way to end with the

Pathogenesis of M. plutonius

infection, it has many negative implications: it abruptly disposes of years-worth of beekeeper investment, requires extensive authorization processes, and brings a stressful halt to the bees’ lives.



Detailed Project Description

Honey bee immune responses are composed of a complex suite of individual immune mechanisms that divide in three levels of resistance: physical barriers as the first line of defense, cell-mediated immunity, and cell-free humoral immunity. Antimicrobial peptides (AMPs) are recognized as key components of humoral immunity and their two basic mechanisms of action are: generation of leaks into prokaryotic membranes and inhibition of bacterial protein translation or folding.18

Only young larvae are susceptible to AFB and regarding EFB, any larval stage results in vulnerability, but the older the larva is the less it is affected by the infection.15 In AFB, the hemocoel of challenged larvae is flooded with an extremely high dose of P. larvae that might demand too much from the humoral and cellular immune system at least from very young larvae. The susceptibility of larvae is attributed to the age-dependent development and composition of the peritrophic matrix that represents a barrier for P. larvae to reach the

gut epithelium and the fat body being the major tissue for the synthesis of antimicrobial compounds results too small in first instar larvae to produce enough amounts of AMPs to defend against P. larvae and M. plutonius.19


Our project objective is to produce AMPs in E. coli (BL21) and then provide larvae the sufficient amount of these key humoral immunity components for it to defend against P. larvae and M. plutonius. AMPs display antimicrobial activity at lower concentrations than conventional antibiotics, bacterial resistance is less likely to develop and they have a broad antimicrobial activity.23 Four AMPs, previously reported to be effective at inhibiting these bacteria, are considered: apidaecin, abaecin, defensin 1 and defensin 2.



Methodology

Here we present the 8 steps our project involves, all the way from the beginning until the end. For additional information of each step, click on the images!

<b>Step 1: Genetic Design Assembly</b> <br>DNA sequences encoding for each AMP are cohesive enzyme restriction digested and later ligated with other DNA sequences to obtain a composite; it containing the required parts in order to foster an adequate transcription, translation, peptide viability in a foreign microenvironment and peptide purification. Four functional genetic designs, each dedicated to one of the AMPs, result assembled.
<b>Step 2: Bacterial Transformation</b> <br>Calcium competent <i>E. coli</i> BL21 (DE3) cells, specialized in protein expression, are transformed with the four assembled genetic designs; separate cultures for each one.
<b>Step 3: IPTG Induction</b> <br><i>E. coli.</i> BL21 (DE3) is designed to generate the high activity transcription T7 RNA polymerase after isopropyl ß-D-1-thiogalactopyranoside (IPTG) induction; T7 RNA polymerase is now present in order to start transcription in a T7 promoter. <i>E. coli</i> BL21 (DE3) expression fundament is considered pursuing a plausible control for peptide expression.
<b>Step 4: Bacteria Sonication</b> <br> Bacterial cell membrane rupture is performed through fast and strong pulses to liberate the produced peptides in the milieu.
<b>Step 5: Protein Purification</b> <br> Affinity chromatography purification, based on the interactions between a transition metal ion (Co²+, Ni²+, Cu²+, Zn²+) immobilized on a matrix and specific amino acid side chains,<sup>20</sup> is performed thanks to the 6X His-tag fused downstream each peptide. Isolated peptides are obtained.
<b>Step 6: Antimicrobial Assays</b> <br> Synergies between AMPs have been previously reported. The possible permutations, as well as isolated AMPs, are evaluated in a liquid antimicrobial assay against <i>P. larvae</i> and <i>M. plutonius</i> separately. Peptides participating in the assays showing the best results are selected.
<b>Step 7: PLGA Microencapsulation</b> <br>Poly Lactic-co-Glycolic Acid (PLGA) is a biocompatible and biodegradable FDA approved polymer that has been extensively studied as a delivery vehicle for drugs, proteins and various other macromolecules.<sup>21</sup> PLGA nanoparticles are reported to control drug release, to protect the compounds from inactivation before reaching their site of action<sup>22</sup> and they also exhibit a wide range of pH-dependent erosion times.<sup>21</sup> Selected AMPs are nanoencapsulated for posterior application in the beehive milieu.
<b>Step 8: In-Liquid-Diet Incorporation</b> <br>Nanocapsules are added to the liquid sugar-water diet beekeepers commonly provide to the bees. Nurse bees are in charge of feeding larvae; first, they consume the nanocapsules and subsequently they deliver them to the larvae.

×

Once the AMP’s are inside the larvae system they complete the overall objective of the project, inhibiting both pathogenic bacteria, and they use different mechanisms of action to do it.



Apidaecin

Apidaecin is mostly lethal to Gram-negative bacteria, nevertheless, in the previous investigation by Khilnani, J. in 201523 apidaecin by itself showed antimicrobial activity against P. larvae and worked in synergy with defensin 2 effectively inhibiting this pathogen. Apidaecin appears to have no effect toward eukaryotic cells, and there is little to no bacterial resistance. The mechanism by which apidaecin kills bacteria starts with a non-specific binding of the peptide to an outer membrane component, which is a substantial lipopolysaccharide (LPS) component. Apidaecin later invades the periplasmic space thanks to a specific receptor/docking molecule, which is a component of the transport system on the inner membrane. The peptide is then translocated into the interior of the cell.24 It is transported through into the cytosol by SbmA.25 Once the peptide is inside of the cell it has two possible targets: DnaK and the 70s ribosome. Apidaecin leads the protein synthesis inhibition by targeting the ribosome. However, it appears that the ultimate target is the DnaK, this is the major bacterial Hsp70 (70 kDa heat shock proteins). DnaK has several functions that end up inhibited by the peptide; the ATPase activity, which is involved in the initiation of DNA synthesis, and the refolding of misfolded proteins.24 DnaK is also indispensable for the viability of the cell in stress conditions like heat shock at 42ºC.26


Defensin 1 & 2

Defensins have selective activity against Gram-positive bacteria. They disrupt, by forming channels, the permeability barrier of the cytoplasmic membrane, resulting in a loss of cytoplasmic K+, a partial depolarization of the inner membrane, a decrease in cytoplasmic ATP, and inhibition of respiration processes.27


Abaecin

Abaecin shows activity against both Gram-negative and positive bacteria28. It inhibits protein biosynthesis by targeting 70s ribosomes and also inhibits DnaK29. Abaecin requires a compromised cell envelope or the presence of a pore-forming peptide such as hymenoptaecin or defensin before it can penetrate the membrane and gain access to its intracellular targets.30



  1. FAO. (2018). The importance of bees and other pollinators for food and agriculture. Retrieved from http://www.fao.org/3/I9527EN/i9527en.PDF
  2. Hung K-LJ, Kingston JM, Albrecht M, Holway DA, Kohn JR. 2018. The worldwide importance of honey bees as pollinators in natural habitats. Proc. R. Soc. B 285: 20172140. http://dx.doi.org/10.1098/rspb.2017.2140
  3. Pariona, A. (2017). Which Crops and Plants Are Pollinated by Honey Bees? Retrieved from https://www.worldatlas.com/articles/which-crops-plants-are-pollinated-by-honey-bees.html
  4. FAO. (2018). El poder de los polinizadores: por qué más abejas significan mejores alimentos. Retrieved from http://www.fao.org/zhc/detail-events/es/c/430002/
  5. PROCCyT. (2018). La apicultura; actividad fundamental para alcanzar la seguridad alimentaria en México. Retrieved from http://proccyt.org.mx/noticias/303-la-apicultura-actividad-fundamental-para-alcanzar-la-seguridad-alimentaria-en-mexico-2
  6. Soto, L., Elizarraras, R. & Soto, I. (2017). Situación apícola en México y perspectiva de la producción de miel en el Estado de Veracruz. Retrieved from http://www.ecorfan.org/spain/researchjournals/Estrategias_del_Desarrollo_Empresarial/vol3num7/Revista_de_Estrategias_del_Desarrollo_Empresarial_V3_N7_5.pdf
  7. FORDECyT. (2018). Demanda 2018-01. Retrieved from https://www.conacyt.gob.mx/index.php/el-conacyt/convocatorias-y-resultados-conacyt/convocatorias-fordecyt/convocatorias-abiertas-fordecyt/fordecyt-2018-01/16743-anexo-5-3-demanda-2018-01/file
  8. vanEngelsdorp, D., Hayes, J., Underwood, R. M., & Pettis, J. (2008). A Survey of Honey Bee Colony Losses in the U.S., Fall 2007 to Spring 2008. PLoS ONE, 3(12), e4071. doi:10.1371/journal.pone.0004071
  9. Cornman, R. S., Tarpy, D. R., Chen, Y., Jeffreys, L., Lopez, D., Pettis, J. S., … Evans, J. D. (2012). Pathogen Webs in Collapsing Honey Bee Colonies. PLoS ONE, 7(8), e43562. doi:10.1371/journal.pone.0043562
  10. OIE Database. (2018) Detailed country (ies) disease incidence. Retrieved, September 20, 2018 from http://www.oie.int/wahis_2/public/wahid.php/Diseaseinformation/statusdetail
  11. OEC. (2016). Miel. Retrieved, September 20, 2018 from https://atlas.media.mit.edu/es/profile/hs92/0409/
  12. Khezri, M., Moharrami, M., Modirrousta, H., Torkaman, M., Rokhzad, B. & Khanbabaie, H. (2018). Prevalence of American foulbrood in asymptomatic apiaries of Kurdistan, Iran. Retrieved from https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5891840/
  13. Morse, R. & Steinkraus, H. (1992). American foulbrood incidence in some US and Canadian honeys. Retrieved https://www.apidologie.org/articles/apido/pdf/1992/06/Apidologie_0044-8435_1992_23_6_ART0001.pdf
  14. Poppinga, L., & Genersch, E. (2015). Molecular pathogenesis of American Foulbrood: how Paenibacillus larvae kills honey bee larvae. Current Opinion In Insect Science, 10, 29-36. doi: 10.1016/j.cois.2015.04.01
  15. Forsgren, E. (2010). European foulbrood in honey bees. Journal of Invertebrate Pathology, 103, S5–S9. doi:10.1016/j.jip.2009.06.016
  16. Sánchez, M., Martínez, E., Fabela, M., Pacheco, N. y González-Flores, T. (2017). Inocuidad de la miel: Producción y comercialización de miel y sus derivados en México: Desafío y oportunidades para la exportación. Retrieved from https://ciatej.repositorioinstitucional.mx/jspui/bitstream/1023/409/1/Inocuidad%20de%20la%20miel.pdf
  17. Inforural. (2014). Persiste uso de antibióticos prohibidos en la producción de miel peninsular. Retrieved from https://www.inforural.com.mx/persiste-uso-de-antibioticos-prohibidos-en-la-produccion-de-miel-peninsular/
  18. Danihlík, J., Aronstein, K., & Petřivalský, M. (2015). Antimicrobial peptides: a key component of honey bee innate immunity. Journal of Apicultural Research, 54(2), 123–136. doi:10.1080/00218839.2015.1109919
  19. Gätschenberger, H., Azzami, K., Tautz, J., & Beier, H. (2013). Antibacterial Immune Competence of Honey Bees (Apis mellifera) Is Adapted to Different Life Stages and Environmental Risks. PLoS ONE, 8(6), e66415. https://doi.org/10.1371/journal.pone.0066415
  20. Bornhorst, J. A., & Falke, J. J. (2000). [16] Purification of proteins using polyhistidine affinity tags. Applications of Chimeric Genes and Hybrid Proteins Part A: Gene Expression and Protein Purification, 245–254. doi:10.1016/s0076-6879(00)26058-8
  21. Makadia, H. & Siegel, S. (2011). Poly Lactic-co-Glycolic Acid (PLGA) as Biodegradable Controlled Drug Delivery Carrier. Retrieved from https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3347861/
  22. Teixeira, M., Alonso, M., Pinto, M. & Barbosa, C. (2004). Development and characterization of PLGA nanospheres and nanocapsules containing xanthone and 3-methoxyxanthone. Retrieved from https://pdfs.semanticscholar.org/ea0a/bca0c6a6d983260b5bef2a7b7c92409ecb38.pdf
  23. Khilnani, Jasmin Camille. (2015). The Effects of Honeybee (Apis mellifera) Antimicrobial Peptides on Paenibacillus larvae. UNLV Theses, Dissertations, Professional Papers, and Capstones. 2486. https://digitalscholarship.unlv.edu/thesesdissertations/2486
  24. Li, W. F., Ma, G. X., & Zhou, X. X. (2006). Apidaecin-type peptides: biodiversity, structure–function relationships and mode of action. Peptides, 27(9), 2350-2359.
  25. Krizsan, A., Volke, D., Weinert, S., Sträter, N., Knappe, D., & Hoffmann, R. (2014). Insect-Derived Proline-Rich Antimicrobial Peptides Kill Bacteria by Inhibiting Bacterial Protein Translation at the 70 S Ribosome. Angewandte Chemie International Edition, 53(45), 12236–12239.
  26. Calloni, G., Chen, T., Schermann, S. M., Chang, H., Genevaux, P., Agostini, F., et al. (2012). DnaK Functions as a Central Hub in the E. coli Chaperone Network. Cell Reports, 1(3), 251–264. https://doi.org/10.1016/j.celrep.2011.12.007
  27. Cociancich, S., Ghazi, A., Hetru, C., Hoffman, J. A., & Letellier, L. (1993). Insect Defensin, an Inducible Antibacterial Peptide, Forms Voltage-dependent Channels in Micrococcus luteus. The Journal of Biological Chemistry, 268(26), 19239-19245
  28. Shen, X., Ye, G., Cheng, X., Yu, C., Hu, C., Allosaar, I. (2010). Characterization of an abaecin-like antimicrobial peptide identified from a Pteromalus puparum cDNA clone. Retrieved from https://www.sciencedirect.com/science/article/pii/S0022201110001114
  29. Mishra, A., Choi, J., Moon, E. & Baek, K.-H. (2018). Tryptophan-Rich and Proline-Rich Antimicrobial Peptides. Molecules, 23 (4), 815. doi:10.3390/molecules23040815
  30. Rahnamaeian, M., Cytrynska, M., Zdybicka-Barabas, A., Dobslaff, K., Wiesner, J., Twyman, R., et al. (2015). Insect antimicrobial peptides show potentiating functional interactions against Gram-negative bacterial. Retrieved from http://rspb.royalsocietypublishing.org/content/282/1806/20150293


Follow us on
social media:


Instagram Facebook Twitter Snapchat Gmail

We thank our
sponsors:


Thermo Neb IDT Snap QTEc ITESM