1. Destabilization of the Bacterial Accumulation Associated Protein (AAP)
Through Mutation of Phenylalanine-20 to Modify Antigen Recognition
Against Biofilm Production
Sarah Albrecht, Matthew Berhe, Serena LaBounty, Elijah Lazo
Mentors: Ruying Chen, Jenny Tran; PI: Dr. Richard To
Stipends for Training Aspiring Researchers (STAR), Teach Lab, University of Washington Seattle, WA
Abstract
During the development of biofilm, bacteria produce AAP to survive
antibiotic treatment. Biofilm related infections are difficult to treat with
conventional methods due to cellular accumulation. Antibodies targeting
the AAP have been reported to reduce biofilm production. Within AAP,
each G5 repeat is coiled around a hydrophobic core and is considered an
antigenic fragment that the immune system targets. G5 presentation by
the antigen presenting cells may be improved with structural
modification. Changing the amino acid sequence may cause the
breakdown of the hydrophobic core that can destabilize the protein to
improve G5 presentation as an antigen. The hydrophobic Phenylalanine-
20 in G5 is changed into alanine by site–directed mutagenesis and the
mutated peptide sequence is inserted into the pET21a+ vector for the
peptide expression. The mutated AAP-G5 peptide will be tested with a
mice model in the future to examine immune response.
Background
• Biofilm is the leading cause of infections related to biomaterial.
S. epidermidis is the most common bacteria involved in
nosocomial infections and affects millions of people each year
[1,2].
kb
• The formation of biofilm occurs in two phases, bacteria
adhesion to a surface followed by bacterial accumulation due
to AAP secretion. [3].
• Within this AAP, there are repeated G5 domains which have
been used as vaccine to generate antibodies against AAP.
• We have mutated one G5 domain in order to increase immune
response against the formation of biofilm.
• The G5 domain has the capacity of being hydrophobic or
hydrophilic due to an excess glycine, the smallest amino acid
that has a single hydrogen side chain.
• Phenylalanine-20 is a hydrophobic amino acid.
When grouped with surrounding hydrophobic
amino acids, it creates a dense stack.
(See figure A)
• We attempted to mutate Phenylalanine-20
into alanine that is less hydrophobic.
• Due to the decrease in hydrophobicity, the mutated G5 is
expected to break down easier by antigen presenting cells,
potentially leading to a more effective transfer by MHCII to the
T cells.
pVAX
pVAX
pVAX
pET21a+
p-VAX-MutAAP
Miniprep
- Extract pVAX-AAP plasmid from cellwtAAPG5
Restriction Analysis
Mutagenesis
-Change Phenylalanie-20 to
Alanine
MutG5
XL-10 Blue
Miniprep
-Extract mutated plasmidMutAAP
DNA Sequencing
-Used the Sangers method
Polymerase Chain
Reaction (PCR)
-Amplify mutated AAP
domain
Ligation
-Splice mutated AAP domain
into pET21a+ plasmid
T7 Promoter
MutAAP
pET-MutAAP
Transform to BL21DE3 cells
for Protein Expression
Top10F’ Cells
Transformation
-Insert mutated plasmid into
XL-10 Blue cells
Methods
Acknowledgements:
This research was made possible by the NIH National Heart Lung & Blood Institute Grant #5R25HL103180-05 "UW STAR Program”. Thank you to Dr. Richard To, our lab mentor and
professor, for showing us how to conduct research and for teaching us how to work in a scientific manor. Thank you to Karlotta Rosebaugh and Teri Ward, for giving us this opportunity
to broaden our horizons. Thank you to our lab assistants Ruying Chen and Jenny Tran for educational guidance and support. Finally, thank you to all the lab participants for making this
internship an experience to remember.
References:
1. J. C. Linnes, K. Mikhova, and J. D. Bryers, “Adhesion of Staphylococcus epidermidis to biomaterials is inhibited by fibronectin and albumin.,” J. Biomed. Mater. Res. A, vol. 100, no. 8,
pp. 1990–7, Aug. 2012.
2. T. Bjarnsholt, “The role of bacterial biofilms in chronic infections.,” APMIS. Suppl., no. 136, pp. 1–51, May 2013.
3. D. Sun, M. A. Accavitti, J. D. Bryers, D. Sun, M. A. Accavitti, and J. D. Bryers, “Inhibition of Biofilm Formation by Monoclonal Antibodies against Staphylococcus epidermidis RP62A
Accumulation-Associated Protein Inhibition of Biofilm Formation by Monoclonal Antibodies against Staphylococcus epidermidis RP62A Accumulation- Associated Protein,” 2005.
Genomic DNA pVAX-AAP
ResultsResults
• Extracted and isolated the pET 21
• Restriction analysis proves pET 21 is present (Figure 1)
• pET 21 this vector was amplified and purified
~1.8 µg/mL (Figure 2)
• pVAX Mutagenesis was unsuccessful
~kanamycin resistant bacteria colony was not present in
the subsequent transformation
• Used alternative pVAX mutagenesis as a control (Figure 3)
B Region
Colony Polymerase Chain Reaction (PCR)
- Amplify the mutated pET21a+
with the inserted pVAX.
Conclusion
• The pET 21 was uncontaminated and it was confirmed the
presence of DNA with a UV spectrometry and gel purification.
• The transformation of pVAXAAP was unsuccessful and the
problem potentially occurred some point after mutagenesis.
~Mutagenesis and Transformation: Primers didn’t match
up and couldn’t make copies or could not mutate the
plasmid.
• The ligation of pET21 was successful. Future experiments are
needed to study the mutagenesis and transformation of the
pVAX. (Figure 2)
The future plan is to perform in vivo in mice with the mutated G-5
and observe the immune response. The immunization with
mutated G5 may increase the antibody production against AAP
and should hinder biofilm formation. The hope is to be able to
destroy biofilms much more efficiently to decrease nosocomial
infections.
Figure 1 Figure 3Figure 2 Figure 3