Characterization of Polynucleotide Phosphorylase (PNPase) and the Virulence of Streptococcus pyogenes

Polynucleotide phosphorylase (PNPase) is a 3’-5’ exoribonuclease that phosphorylyticly degrades mRNA molecules in bacteria and in eukaryotic organelles.Streptococcus pyogenes (a group A streptococcus [GAS]) is a Gram-positive human pathogen responsible for a plethora of syndromes ranging from the less-invasive pharyngitis (a.k.a strep throat) to severely invasive cases such as necrotizing fascitis and streptococcal toxic shock syndrome. Recently, PNPase was shown to be required for the decay of 2 S. pyogenes transcripts during late exponential growth: sagA and sda. The aforementioned transcripts were found to be stabilized (more abundant) in a pnpA mutant strain. Furthermore, the pnpA transcript was found to be up-regulated 3.5-fold in S. mutans after exposure to a low pH acid-shock. In this work, two cell culture infection assays were employed to further characterize the S. pyogene pnpA mutant strain. Interestingly, in a cell proliferation assay using RAW cells (murine macrophage-like cells), the pnpA mutant demonstrated a 3.5-fold reduced proliferation over the course of a 7-hour infection relative to its isogenic wild type strain. More importantly, complementation of the pnpA gene expressed on a plasmid restored wild-type-like levels of proliferation. In a separate cell cytotoxicty assay that evaluates bacteria mediated host- cell death and rounding, HeLa cells, Shed (dental pulp) cells, and Periodontal stem cells are being used in S. pyogenes infections. Preliminary data is inconclusive, and additional experiments are underway in efforts of determining whether the pnpA mutant is diminished in its ability to induce host-cell rounding and death