Investigating the role of the Neisseria meningitidis Fic family protein (2014)
Research For Life
School of Biological Sciences
The objective of this project is to elucidate the mechanism of action of the Fic family protein, which was identified as a Neisseria meningitidis gene associated with more virulent isolates. Bacterial proteins containing a Fic domain are a recently-described novel class of bacterial toxin. Like many other bacterial toxins, they disable the function of key host cell signalling molecules, including those belonging to the Rho GTPase family. A gene encoding a protein with a Fic domain was identified in a genomic locus present in disease- but not carriage-associated isolates of N. meningitidis. We are interested in determining the role of this protein in meningococcal pathogenesis, by further analysing its function in host cells, particularly human bronchial epithelial cells, which represent the normal host for N. meningitidis in the upper respiratory tract. Fic family proteins exert their toxic effects by transferring an AMP moiety (i.e., AMPylation) onto target proteins; the presence of the AMP moiety inhibits interaction with downstream signalling partners, resulting in loss of function.
In order to achieve this goal, we are aiming to purify the Fic family protein and perform reactions with labelled ATP, identifying targeted host cell proteins by immunoprecipitation with an antibody that recognises the label on the transferred AMP.
The gene encoding the Fic family protein has been cloned into an expression plasmid, pET28a(+). The gene was amplified with primers that incorporated restriction sites to enable directional cloning into the expression vector. One of the enzymes used is Nde I; incorporation of the ATG start of the gene into the ATG of the Nde I sequence results in the insertion of the coding region in the appropriate reading frame, with a 6-histidine tag added to the C-terminus of the protein. The correct plasmid construct has been confirmed by restriction digest and sequencing. The correct plasmid was transferred to BL-21, an E. coli strain that is optimal for protein expression. Trial runs were conducted to ensure that the E. coli containing the correct plasmid made the correct protein when induced by the addition of IPTG.
We chose four BL-21 colonies with the plasmid to test. The strains were cultured overnight in liquid and diluted back the following morning. The fresh cultures were grown to mid-log phase, at which point the protein was induced by the addition of IPTG. Samples were taken from the bacteria prior to induction as a control. After four hours of induction, cells were pelleted and lysed in SDS-PAGE loading buffer and loaded on a 12.5% gradient SDS-PAGE gel. The gel was stained with Coomassie blue to confirm the cells were producing a protein of the correct size (~52 kDa) after induction.
Once the construct had been made and transferred to BL-21 cells, we scaled up protein production. A large batch of the E. coli strain containing the expression plasmid was grown overnight, diluted back the following morning into fresh medium, and then induced with IPTG once a mid-range OD was reached. After 4 hours of induction, the cells were pelleted and lysed, using either Bugbuster (Millipore) or B-Per reagent (Pierce). The cell lysates were run over a prepared Nickel (Ni-NTA) column, according to standard protocols. The purified protein contains a 6-histidine tag to mediate binding of the protein to the nickel resin. The columns were washed with a series of wash buffers, containing higher concentrations of imidazole; finally the columns were washed with elution buffer, containing a high concentration of imidazole (to compete with the histidine tag). The purified protein was eluted, and the elution buffer was removed by running the protein sample on a Zeba desalting column (Pierce). Finally, the protein was concentrated on a Microcon centrifugal filter with a 10-kDa cutoff (Millipore). The total amount of protein was quantified using a Coomassie (Bradford) assay (Pierce) and stored at -80°C.
To identify host cell proteins altered by the Fic family protein, whole cell extracts (WCE) were prepared. Bronchial epithelial cells (16HBE cells) were cultured until they reached confluence, then were lysed with RIPA buffer in ice for 30 minutes, scraped and centrifuged. The protein concentration of the supernatant was determined.
A reaction was set up, containing WCE, purified Fic family protein, reaction buffer, and FITC-labelled ATP, as the Fic family protein substrate. Transfer of an AMP moiety (derived from the labelled ATP molecule) to a target protein will result in the fluorescein moiety also being transferred to any altered host proteins. The reaction was incubated at 37°C for 40 minutes.
Following the reaction, proteins containing the FITC label were immunoprecipitated. A crosslinked anti-fluorescein antibody, conjugated to protein G sepharose beads, was added to the reaction mixture. Following incubation, the beads were washed extensively and heated in SDS-PAGE loading buffer, then run on a 12.5% gradient SDS-PAGE gel. To date, we have attempted to visualise the modified proteins, using either Commassie blue stain, which is not adequately sensitive, or by scanning the gel with a Fuji Scanner fluorescence scanner, with a FITC setting for the filter. To date we have been able to visualise protein bands following the reaction, but are repeating the experiment to obtain clearer bands on the SDS-PAGE gels. Any identified modified proteins will be excised and analysed by mass spectrometry, to identify the modified proteins. Targeted proteins will be confirmed, and the nature of their modification will also be determined using proteomics techniques.