1. Mentor: Kirsten Crossgrove, PhD
Mutation of predicted AKT phosphorylation sites in Brugia malayi DAF-16
Amanda Danno
Mentor: Kirsten Crossgrove, PhD
University of Wisconsin – Whitewater, Department of Biological Sciences
Introduction
Brugia malayi is a nematode that causes lymphatic filariasis, also known as elephantiasis, in humans (Epidemiology & Risk Factors, 2013). Caenorhabditis elegans is a free-living nematode that is considered a well-studied genetic model system (Wood, 1988). Both nematodes have a similar stage of life; in Caenorhabditis elegans it is the dauer stage, and in Brugia malayi it is the iL3 infective stage (Burglin et al., 1998). The dauer stage in Caenorhabditis elegans is an alternative life-cycle stage. This stage allows the nematode to endure harsh environments (Hu, 2007). When the environment permits favorable living conditions the nematode moves into the next life cycle stage. In Brugia malayi the infective stage allows for life inside the mosquito while it waits for its next human host, before moving into the L4 life stage (Denham &McGreevy, 1977).
Results
Through the technique of site directed mutagenesis we will change the Bma-DAF-16a and Bma-DAF-16b isoforms so that the protein can not be phosphorylated at specific predicted phosphorylation sites. There are 3 predicted phosphorylation sites for AKT in each isoform and we have individually mutated each and are currently making combination mutants.
Positive clones were sequenced to make sure the selected sites got mutated properly. We then transfected mammalian cells with the plasmid constructs to look at Bma-DAF-16 cellular localization, and the ability of Bma-DAF-16 protein to activate expression of a reporter gene (luciferase), in the presence and absence of serum. The serum contains insulin and therefore reflects the amount of signaling; high serum is high signaling (DAF-16 inactive) and low serum is low signaling (DAF-16 is active). The localization of the proteins will become visible under a confocal microscope because of the use of a fluorescent ligand, which binds to a Halotag domain that is fused to the DAF-16 protein.
Figure 5: The predicted Akt-1 phosphorylation sites in the two Bma-DAF-16 isoforms. Threonine (T) or Serine (S) were mutated to Alanine (A) at each indicated position, preventing phosphorylation.
Figure 6: The site directed mutagenesis protocol. The initial plasmid containing a Bma-DAF-16 isoform was PCR amplified with forward and reverse primers. The product was then digested by DpnI and transformed in JM109 competent cells. Image from: ttps://web.stanford.edu/~loening/protocols/Site_Directed_Mutagenesis.pdf
Figure 2: C. elegans, like B. malayi, endures a series of molts throughout development. C. elegans can enter dauer (arrow), an alternative life stage, which is thought to be similar to B. malayi iL3.
Image adapted from Jorgensen and Mango (2002).
Figure 1: B. malayi undergoes a series of cuticular molts and passes through mosquito and human hosts. Image source:
Figure 9. Transfections and luciferase assays were performed as described in figure 8. Single or double phosphorylation site mutations were used as indicated. Activity is shown relative to empty vector and transfection efficiency was normalized by co-transfection of a constitutive Renilla luciferase vector. The average of three experiments is shown. Error bars represent standard error. Contrary to predictions, the phosphorylation site mutants behaved similarly to the wild type proteins. Both showed lower reporter gene activity in the presence of serum. Serum responsiveness may not be controlled by AKT phosphorylation. However, triple mutants have not been tested yet. A B
Figure 3: Examples of lymphatic filariasis and elephantiasis caused by B. malayi, B. timori, and Wuchereria bancrofti.
Thickening of skin.
(B) Elephantiasis of the leg.
Figure 7: Gel electrophoresis showing that the mutagenesis PCR amplified the expected product (arrow).
Conclusions
Bma-DAF-16a and Bma-DAF-16b can activate transcription of a reporter gene.
We have generated single and double phosphorylation site mutants for both isoforms.
Mutation of predicted AKT phosphorylation sites does not reduce serum responsiveness of Bma-DAF-16 transactivation activity, though in some cases overall activity is lower.
The insulin/insulin like growth factor signaling (IIS) pathway limits dauer formation (Hu, 2007). The target of this pathway is the DAF-16 Forkhead Box O (FOXO) transcription factor (Murphy & Hu, 2013). Insulin signaling that turns off DAF-16 prevents dauer formation and is required for dauer recovery (Murphy and Hu, 2013). DAF-16 activity in Caenorhabditis elegans is known to be regulated by phosphorylation (Murphy, and Hu, 2013). When phosphorylation takes place, DAF-16 moves from the nucleus, where it regulates target genes, to the cytoplasm, where it does not. Most of the genes that make up the Caenorhabditis elegans signaling pathway have been identified in Brugia malayi (Garland, 2010).
Future Directions and Significance
Experiments with all single, double and triple mutants need to be completed to determine if phosphorylation at this site is necessary for Bm-daf-16 regulation and to see if the proteins are localized to the nucleus or cytoplasm under different conditions.
Understanding the role of Bm-DAF-16a in the B. malayi life cycle may lead to better treatment options for filarial infections.
Figure 4:
The insulin signaling pathway in C. elegans regulates DAF-16. When insulin signaling is not present DAF-16 stays in the nucleus. When insulin signaling is present, AKT phosphorylates DAF-16, causing DAF-16 to move out of the nucleus of the cell and into the cytoplasm.
Acknowledgments
Brenda Garland, Katherine Stanford, Alexius Folk, Lee Smith, and Jenna Maiorelle for identification and cloning of the Bma-daf-16a and Bma-daf-16b isoforms.
Bio364 students (fall 2016), Science Academy students (2017) and Alanna Gould for assistance in generating phosphorylation site mutants.
Danielle Zgoba, Austin Chriske, Kristin Hausen, Johnna Dykstra, and Bernard Mulu for general help in lab
UW-Whitewater Undergraduate Research Program for funding
UW-Whitewater Department of Biological Sciences
Dr. John Hawdon, George Washington University, for DAF-16 DBE reporter plasmid
References
Denham, D., & McGreevy, P. (1977). Brugian Filariasis: Epidemiological and Experimental Studies. Advances in Parasitology, 17,
Epidemiology & Risk Factors. (2013). Retrieved February 27, 2015, from lymphaticfilariasis/epi.html
Garland, B. (2010). Identification of orthologs of Caenorhabditis dauer formation genes in the parasitic nematode Brugia malayi. Honors thesis. Department of Biological Sciences. UW-
Hu, P.J., Dauer (2007). WormBook, ed. The C. elegans Research Community, WormBook, doi/ /wormbook ,
Murphy C.T., Hu P.J. (2013). Insulin/insulin-like growth factor signaling in C. elegans, WormBook, ed. The C. elegans Research Community, WormBook, doi/ /wormbook ,
Wood, W. (1988). Introduction to C. elegans Biology, 1. In The Nematode Caenorhabditis elegans. Cold Spring Harbor, New York: Cold Spring Harbor Laboratory Press
No Insulin Signaling
Insulin Signaling
Hypothesis
We predict that insulin signaling regulates the transition from iL3 to L4, and that Bma-DAF-16 is regulated by phosphorylation.
Research Question
Are phosphorylation sites in Bma-DAF-16 required for cytoplasmic localization in response to insulin signaling?
Figure 8: The protocol used during the transfection experiments.