Sid Rode's FliC aptamer project (2014)

Selection of Aptamer against Surface Protein FliC for research against

Melioidosis causing bacteria B. pseudomallei

Sid Rode – September 16^th Fall 2014

N40B RNA Pool - FliC (BPSL 3319)

Abstract

Melioidosis, caused by the gram-negative saprophyte Burkholderia pseudomallei, is a disease of public health importance in Southeast Asia and northern Australia that is associated with high case-fatality rates in animals and humans.[i] Definitive diagnosis requires positive bacterial culture and confirmation of the organism, which usually takes several days. Melioidosis can be dormant for many years before manifesting, making diagnosis even further complicated.[ii] Furthermore, B. pseudomallei is resistant to many standard antibiotics used in empirical therapy for sepsis.[iii]

Nucleic acid aptamers are oligonucleotides (RNA or ssDNA) which bind to their targets (protein) with high selectivity and sensitivity. An aptamer against B. pseudomallei would be utilized to diagnose Melioidosis before the bacteria has any time to metastasize throughout the infected host. The aptamer selected against FliC, a protein present in the flagellum of the organism, could be used as a biomarker in diagnostic purposes and potentially to inhibit the pathogenesis of the infection.

FliC as a flagellar filament structural protein was identified in a variety of organisms. The Flagellin interacts with the structure of the entire organelle, forming filaments from thin organized strands.[iv] With a known molecular weight of 39 kDa the protein monomer functions as a multimer in full length, giving the flagellum a whip-like characteristic that propels the bacterium forward (Winsor, 2008). Such is so, that an aptamer may also be used to block the binding site of the protein to the rest of the bacterium, essentially immobolizing the organism preventing its proliferation into full-blown Melioidosis. However, such a technique is yet to be tested and little is known about the protein’s affinity for nucleic acid molecules. The SELEX process of in vitro selection of such an aptamer against the protein has already commenced with one initial round. FliC is being provided by Dr. Katy Brown of the University of Texas’ Institute for Cellular and Molecular Biology and her laboratory.

Specific Aim 1: To find an aptamers against FliC selected through in-vitro aptamer selection. This is done by exposing a library random nucleic acid sequences to the target protein i.e. FliC. Those sequences that bind the target are retained, amplified, and utilized in further rounds of selection. Following multiple rounds of stringent filter-based selection, individual aptamers are cloned and sequenced in order to identify the highest affinity binding aptamers from the pool. The aptamer may be further modified to make it more specific to the target or to add fluorescence to track levels of FliC in the body. The aptamer obtained through the above procedure can then be used for diagnostics and finding early traces of Melioidosis in screening tests.

Specific Aim 2: To modify found aptamer against FliC to prevent the binding of the protein to B.pseudomallei’s, thereby potentially inhibiting the spread of infection in the body.

Budget Costs

The protein itself is provided at no cost by Dr. Katy Brown from the Institute for Cellular and Molecular Biology of the University of Texas. Her lab works with B. pseudomallei strains.

Link to full proposal

References

[i] Melioidosis: Epidemiology, Pathophysiology, and Management

Allen C. Cheng, Bart J. Currie

Clin Microbiol Rev. 2005 April; 18(2): 383–416. doi: 10.1128/CMR.18.2.383-416.2005

Correction in: Clin Microbiol Rev. 2007 July; 20(3): 533.

[ii] Ngauy V, Lemeshev Y, Sadkowski L, Crawford G (2005). "Cutaneous Melioidosis in a Man Who Was Taken as a Prisoner of War by the Japanese during World War II". J Clin Microbiol 43

[iii] Melioidosis.

White NJ Lancet. 2003 May 17; 361(9370):1715-22.

[iv] Matzke, N. J. (2003, November 10). Evolution in (brownian) space: a model for the origin of the bacterial flagellum. Retrieved from http://www.talkdesign.org/faqs/flagellum.html