Streptococcus pneumoniae (pneumococcus) is a highly infectious pathogen and the main cause of bacterial meningitis in children and older adults. In addition, pneumococcus is increasingly resistant to antibiotics, highlighting the need for more effective therapeutics. Elucidating the pathways pneumococcus exploits in initiating host infection would lead to improvement in available treatments, diminishing pneumococcal meningitis-related deaths. Importantly, pneumococcus produces glycan-modifying enzymes, which it uses to alter host glycosylation and promote infection. Of these, three are neuraminidases, which cleave sialic acid from cell surface glycans. Modification of sialic acid expression plays a critical role in regulating inter- and intracellular signaling. As such, pneumococcal neuraminidase function has the potential to facilitate progression of pathogenesis. I hypothesize that pneumococcal neuraminidases unmask glycoproteins during infection, initiating a signaling cascade to facilitate invasion of the host. In order to identify proteins de-sialylated by pneumococcal neuraminidases in meningitis infection, I have applied a recently developed glycoproteomics technique to the blood-brain barrier model cell line, hCMEC/D3. In brief, galactose residues exposed upon neuraminidase treatment of hCMEC/D3 cells are oxidized to introduce aldehyde groups, which are then labeled with aminooxy biotin using an aniline-catalyzed ligation. Tagged proteins are subsequently streptavidin purified and subjected to mass spectrometry for identification. Using this technique, I have detected enrichment of ~ 60 proteins in neuraminidase-treated hCMEC/D3 cells compared to untreated cells. Of particular interest are numerous genes involved in the host immune response as well as several proteins associated with �-catenin signaling, a pathway implicated in pneumococcal infection of the lung. To determine the functional significance of substrates on infection, I am assessing adherence, invasion, and transcytosis of pneumococcus in hCMEC/D3 cells in which expression of individual neuraminidase substrates has been silenced. Overall, this work will provide unique insights into signaling pathways utilized in pneumococcal infections.

Role of pneumococcal neuraminidases in facilitating meningitis infection

247th National Meeting (2014)

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