Dustin Taylor - Aptamer Selection Against Burkholderia pseudomallei Fimbria Protein for Efficient Diagnosis of Melioidosis

Aptamer Selection Against Burkholderia pseudomallei Fimbria Protein for Efficient Diagnosis of Melioidosis

Dustin Taylor – Fall 2011

September 16^th, 2011

N58 RNA – B. pseudomallei fimbriae

Burkholderia pseudomallei is a gram negative bacterium that causes the disease melioidosis, marked by the presence of joint pain, cough, skin infections, lung nodules, and pneumonia. It has a niche in soil and surface water, and as a consequence is endemic in Southeast Asian populations where contact with these environments is essentially inevitable.⁴ It is most commonly acquired through a break in the skin, or by inhaling the aerosolized form of the bacterium.

Most current diagnostics include a complete screening involving numerous cultures and a throat swab; this is very time consuming, and only adds to the 50% mortality rate. Additionally, rapid discovery methods, such as antibody and antigen detection, have relatively low sensitivities averaging around 70%, and as a consequence are not highly trusted.³ The goal of this experiment, then, was to develop an aptamer to more efficiently reveal the presence of a B. pseudomallei infection.

Fimbriae from this particular bacterium have been isolated in order to more easily develop aptamer binders. The specific aim was to select for RNA fragments that demonstrate a high binding affinity for the fimb

riae; as this could lead to the eventual development of aptamers for faster diagnostics of the presence of this bacterium in a particular sample.

Figure 1: Illustration of aptamer binding. The specific aim for this selection is to find an aptamer that will bind a bacterial fimbria and provide a quicker diagnostic test for the presence of

B. pseudomallei.

The fimbriae proteins have been procured by Kate McCaul of Dr. Kate Brown’s lab. The only remaining step with cost is to functionalize the proteins with biotin. This cost is unknown as the amount of biotin used is target-specific, and has not been recorded.

Introduction and Background

Aptamers are RNA fragments with high binding affinity that are selected to “stick” to certain substances. However, only ~1 in 10¹⁰ RNA fragments folds to accommodate the specific binding properties of a given ligand. (Ellington) In order to obtain these very rare molecules, RNA pools are purified through a rigorous SELEX process that removes those that will not bind the selected target.⁹

Figure 2: SELEX, or systematic evolution of ligands by exponential enrichment, is a process starts with a very large library of R0 pool RNA. The target and the RNA library are allowed to bind, and a series of washes is performed to remove the unbound fragments. The bound oligonucleotides are eluted and amplified by RT-PCR. The resulting amplified DNA is transcribed, and this RNA is used to perform another round of selection, adapted from Stoltenburg (2007).

The binding segment’s functionality can be attributed to the streptavidin-biotin complex, one of the strongest naturally occurring binding affinities. The target of interest is treated with biotin, and when this is combined with a solution of streptavidin beads, a very strong attraction forms. This then allows for highly specialized selection against the RNA library, only leaving those with robust affinities. After 8-15 rounds of the SELEX process, there is a possibility of finding an aptamer specific to the target, which in this case consisted of fimbriae proteins from the bacterium B. pseudomallei.⁹

As previously mentioned, Burkholderia pseudomallei is a gram-negative bacterium endemic to Southeast Asia. It is contracted by direct contact with the organism in the environment, and is the causative agent of melioidosis. Acute infections manifest themselves as septicemia or pulmonary illness, while chronic infections will mostly involve organ abscesses.¹ The issue with acute cases is that the symptoms and signs are often very conducive with influenza, and therefore are treated inaccurately. Even more frightening is the possibility of a 62-year dormancy period in some chronic occurrences.¹ Due to the aforementioned mischaracterizations of this disease, many cases are fatal, even with treatment – this is why a better diagnostic is imperative for the native populations of these affected areas.

This specific bacterium is a facultative intracellular organism, and so will carry out its life cycle within a host cell if allowed. Possible sources of its pathogenicity include its flagella, exoproteins, fimbriae and pili.² In this instance, the bacterial fimbriae were selected against, with the hopes of developing a more efficient diagnostic procedure for this terrible illness. In bacteria, fimbriae are short strands of protein filament on the bacterial surface. As this particular bacterium is gram negative and possesses an outer membrane, the fimbriae are located there rather than the inner plasma membrane. They are somewhat similar to flagella, with the exception being that they are shorter and stiffer. These fimbriae are used for surface attachment – this is of utmost importance in Burkholderia pseudomallei as it allows the pathogens to bind to bodily tissues and begin the infection process.¹⁰ There is no current aptamer against this target; labs at The University of Texas and the University of Georgia are the main researchers of new diagnostic assays for B. pseudomallei, but these mostly involve antibodies at this point.¹

Figure 3: Illustration of bacterial fimbriae.The fimbria proteins are indicated as thin, “hair-like” strands emanating from the bacterium E. coli, adapted from Connell (1996).

An aptamer against fimbria proteins in B. pseudomallei would have enormous diagnostic applications. As stated previously, the current diagnostic methods are simply inadequate. The working standard is positive culture growth, and it is just too slow with respect to detection, as some melioidosis infections have an incubation period of merely 48 hours.¹ A marketable aptamer would be more sensitive and cheaper than antibody treatments, and would work much more rapidly than culturing. Concurrent projects involved selecting aptamers against the flagella and pili as well – due to the specificities involved with binding the very unique protein synthesized by B. pseudomallei, this work excluded false positives identifying the presence of other bacteria. A potential downstream application is the combination of these binding RNA fragments for each protein (multimeric and monomeric flagellin, pilin, and fimbriae) into a veritable “super-aptamer” that would operate even more quickly. In addition, a step could be added to attach green fluorescent protein so as to better indicate the presence of the bacterium in infected samples. Upon success with this venture, a supplementary function would be drug delivery to infected sites, possibly to inhibit the bacterium’s innate intracellular motility.

Experimental Design

These proteins are 176 amino acids in length, weigh approximately 17.46 kD, and normally function as monomers.¹¹ Fimbriae isolated from B. pseudomallei have not been shown to be stable in any particular buffer; because the isoelectric point is about 4.67, and due to the potential downstream applications of this aptamer in the body, PBS buffer was used. At a pH of 7.4 in the PBS buffer, the protein carries a charge of approximately -9.1.⁸ Because of this highly negative charge, RNA does not readily bind, and the protein was functionalized with biotin in order to utilize the biotin-streptavidin attraction during the bead immobilization step. Salt concentrations in the buffer were increased as well to “bridge the negative charge.” (Adapted from Aptamer Protocol)

As an aptamer for the detection of Burkholderia pseudomallei would be most useful in serum samples, incubations for this target were held at 37° C. Initially, reactions were incubated for 45 minutes to give the RNA fragments the greatest chance of binding; in future rounds, incubation times might be reduced to only allow the faster, more effective binders. (Adapted from Aptamer Protocol) Kate McCaul’s advice was sought with respect to the protein’s stability at neutral pH and in a freezer, but she did not immediately answer this request. The N58 RNA pool was used, as it has not shown any contamination in previous rounds; additionally, N58 primers were much more readily available. Nevertheless, a no template negative selection was run in order to identify any amplifying artifacts contaminating the solution.

Budget

As the fimbria proteins have been isolated in Dr. Katy Brown’s lab, there were no purchasing expenses. However, as stated above, the fimbrial filaments need to be functionalized with biotin. As the amount of biotin used will be target-specific, the cost for this step is unknown; there are no documented cases of biotinylating fimbriae proteins. Dr. Brown’s email address is Kate01@mail.utexas.edu.

References

· 1. Brown, Kate. “Rapid Diagnostic Assays for B. pseudomallei and B. mallei.”

· 2. “Burkholderia pseudomallei.” The Institute for Genomic Research (2006). http://pathema.jcvi.org/pathema/b_pseudomallei.shtml

· 3. Cheng, AC. (2010) “Melioidosis – advances in diagnosis and treatment.” Curr Opin Infect Dis. 6: 554-559.

· 4. Cheng, AC, Currie, Bart J. (2005) “Melioidosis: Epidemiology, Pathophysiology, and Management.” Clinical Microbiology Reviews. 18: 383-416.

· 5. Connell, H, Agace, W, et. al. (1996) “Type 1 fimbrial expression enhances Escherichia coli virulence for the urinary tract.” Proceedings of the National Academy of Sciences. 93: 9827-9832.

· 6. Ellington, Andrew, Szostak, Jack W. (1990) “In vitro selection of RNA molecules that bind specific ligands.” Nature. 346: 818.

· 7. NCBI. “Fimbrial protein – [Burkholderia pseudomallei 406e].” http://www.ncbi.nlm.nih.gov/protein/EDO86848.1

· 8. Scripps. Protein calculator v3.3. http://www.scripps.edu/cgi-bin/cdputnam/protcalc3

· 9. Stoltenburg, Regina, Reinemann, Christine, Strehlitz, Beate. (2007). “SELEX-A revolutionary method to generate high-affinity nucleic acid ligands.” Biomolecular Engineering. 24: 2504

· 10. Todar, Kenneth. (2008). “Bacterial Structure in Relationship to Pathogenicity.” Todar’s Online Textbook of Bacteriology. http://www.textbookofbacteriology.net/BSRP.html

· 11. Uniprot. (2007). “Fimbrial protein - Burkholderia pseudomallei 406e.” http://www.uniprot.org/uniprot/A8EDF5