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Investigating the role of the Neisseria meningitidis Fic family protein


Reports of research work funded by grants prior to 2016

Victoria University of Wellington

Investigating the role of the Neisseria meningitidis Fic family protein

JK MacKichan
School of Biological Sciences

The overall objective of this work was to elucidate the mechanism of action of the Fic domain protein (NMBNZ0533_1733) from Neisseria meningitidis.  This protein has homologues in other bacterial pathogens, where it has been demonstrated to constitute a novel class of bacterial virulence factor.  The Fic domain proteins from various bacterial pathogens have been shown to alter the function of the Rho family of cell signalling molecules.  These alterations can affect various host defense mechanisms, including phagocytosis, phagosome trafficking, apoptosis, and cytoskeletal dynamics; by targeting Rho GTPases, bacteria can inhibit or redirect many of these function to their benefit.

The Nm Fic family protein is present and expressed in the New Zealand N. meningitidis epidemic strain type, but is absent from multiple carriage-associated isolates.  In addition, this gene is part of a large (~40-kb) genomic island that includes several putative two-partner secretion systems (TPSS).  The Fic domain protein includes a signal sequence that is characteristic of proteins secreted by TPSS.  The Fic domain itself is a catalytic domain that carries out adenylylation of target proteins; the resulting covalent attachment of an AMP moiety often results in inhibition of the protein’s normal function and interaction with binding partners.

In order to elucidate the mechanism of action of the N. meningitidis serogroup B Fic domain protein, a Fic protein deletion mutant was constructed in the wild type serogroup B N. meningitidis strain, NZ98/254.  The mutant was compared with the wild type for its ability to adhere to, invade, and survive intracellularly in tissue culture epithelial cells.  In addition, the Fic domain protein was expressed heterologously in an E. coli expression strain (BL-21), purified, and used for in vitro interactions with whole cell extracts from a human bronchial epithelial cell line.

Our experiments confirmed that the Nm Fic domain protein is expressed in the presence of human epithelial cells, and that it is key to the interaction between the bacteria and host cells.  Although the N. meningitidis Type IV pili have been described as the major adhesin for attachment to host cells, we found that deletion of the Fic domain protein resulted in reduced levels of adherence to a bronchial epithelial cell line (16HBE), but not an epithelial-like lung cell line (A549).  Reduced adherence also resulted in lower levels of invasion, as determined by a gentamicin protection assay.  Interesting preliminary data was obtained when looking at long-term intracellular survival and escape of the mutant and the wild type from epithelial cells.  The Fic domain mutant survived better, and was recovered in higher numbers, relative to the wild type. One explanation for this is that the Fic domain protein is cytotoxic to cells harbouring intracellular bacteria, an observation that is consistent with the reported role of Fic domain proteins as bacterial cytotoxins.  However, further investigations are needed to confirm this observation.

The Fic domain protein from N. meningitidis, containing a His6 tag, was expressed under an inducible promoter in the E. coli expression strain, BL-21.  The protein was successfully purified, following optimisation steps.  The purified protein was combined with whole cell extracts of the bronchial epithelial cell line, 16HBE, together with a commercially-available fluorescein-labelled ATP analogue (F1-ATP).  The goal of these experiments was to identify the host cell protein or proteins that acquired a fluorescently-labelled AMP moiety, following the enzymatic activity of the Fic domain protein.  Control reactions (lacking the Fic domain protein) were also included, as human cells are known to have endogenous adenylylation activity.  Our results were inconclusive, however.  A band of about 12-kDa was seen in the reactions containing the Fic domain protein, but was only seen before the immunoprecipitation.  However, we are following up on these results, as this size is consistent with members of the Rho GTPase family of proteins.  Alternative experiments currently underway include morphology studies of mammalian cells expressing the bacterial protein, combined with cytotoxicity and apoptosis assays. We are also currently making a panel of site directed mutants of the Fic domain protein, to determine the role of the putative secretion signal and the Fic catalytic domain.  These mutants, expressed in E. coli, are being tested for their ability to adhere to host cells, and for any cytotoxicity effects on tissue culture host cells.

Investigating the role of the Neisseria meningitidis Fic family protein

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