Team:Tec-Chihuahua/description

Erwinions

Abstract

American and European Foulbrood are diseases affecting honey bee (Apis mellifera) larvae all around the world. The causal agents of these are two gram-positive bacteria: Paenibacillus larvae and Melissococcus plutonius. Nowadays, two techniques for the treatment of AFB and EFB are used: antibiotics and incineration of affected hives. The former promotes the development of antibiotic resistance in bacteria while the latter results unprofitable for beekeepers. The production of native bee antimicrobial peptides (AMPs) in Escherichia coli is proposed to treat P. larvae and M. plutonius infections. Defensin 1, abaecin, defensin 2, and apidaecin will each be expressed in a different culture. A 2⁴ factorial design will be used to identify the optimal AMP combination. The final product, AMPs with a specific packaging, will be available for beekeepers to apply in their beehives and inhibit the proliferation of pathogenic bacteria.

Detailed description

American and European Foulbrood are diseases that affect honey bee (Apis mellifera) larvae all around the world. The causal agents of these are two Gram-positive bacteria: Paenibacillus larvae and Melissococcus plutonius , respectively. P. larvae is an endospore-forming bacterium which infects larvae through nurse bees and consumes them until only a biomass of spores is left in the cell. M. plutonius, named after the Greek god of death, outcompetes larval honeybees for food and leaves them starving before “melting” them.

These two diseases cause enormous losses in the apiculture industry and contribute to the factors that threaten all bees. Nowadays, two techniques for the treatment of AFB and EFB are used: antibiotics and incineration of affected hives. There are harsh regulations on the use of antibiotics and doubly so when talking about exported goods. Antibiotics not only make the product harder to sell, but also promote the development of resistance in bacteria. Incineration is unprofitable for beekeepers and often requires extensive authorization processes.

However, insects possess several antimicrobial peptides (AMPs) as part of their innate immune response. Native bee AMPs produced in Escherichia coli could be an effective alternative for the treatment of P. larvae and M. plutonius infections.

Defensin 1 and 2, abaecin, and apidaecin are suggested for this purpose. These AMPs possess different mechanisms of action. The defensins depolarize the membrane, open channels in it, and allow the efflux of potassium ions, which would either be compensated with contaminating cations or efflux of anions, destabilizing biochemical processes. There also is evidence that defensins stop the respiratory activity and reduce the level of ATPs present. Abaecin and apidaecin, on the other hand, stop protein synthesis through the interference of the 70S ribosome and inhibits DnaK activity. Apidaecin binds to LPS and disrupts the ABC transport system, as well.

The project’s first objective is to express these AMPs in a different bacterial culture each, with an inducible promoter. T7 RNA polymerase expression is regulated by the Lac operon, which is induced through the presence of IPTG. Therefore, the usage of T7 promoters allows us to induce AMP production. Proteins will be extracted through sonication of bacterial cultures and then isolated through a His-tag purification process.

Once all four AMPs are purified, all possible combinations will be tested against P. larvae and M. plutonius . A 2⁴ factorial design will be used to evaluate individual effects, quantify interactions, and identify the optimal AMP combination.

PLGA-microencapsulated AMPs would be added to bees’ diet so nurses can bring the AMPs to the infected larvae. This microencapsulated AMPs would be available for beekeepers to apply in their hives and thus inhibit the proliferation of the pathogenic bacteria.

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!