Dynamic Protein Phosphorylation in Streptococcus pyogenes during Growth, Stationary Phase, and Starvation.

Phosphorylation of proteins at serine, threonine, and tyrosine residues plays an important role in physiological processes of bacteria, such as cell cycle, metabolism, virulence, dormancy, and stationary phase functions. Little is known about the targets and dynamics of protein phosphorylation in Streptococcus pyogenes, which possesses a single known transmembrane serine/threonine kinase belonging to the class of PASTA kinases. A proteomics and phosphoproteomics workflow was performed with S. pyogenes serotype M49 under different growth conditions, stationary phase, and starvation. The quantitative analysis of dynamic phosphorylation, which included a subset of 463 out of 815 identified phosphorylation sites, revealed two main types of phosphorylation events. A small group of phosphorylation events occurred almost exclusively at threonine residues of proteins related to the cell cycle and was enhanced in growing cells. The majority of phosphorylation events occurred during stationary phase or starvation, preferentially at serine residues. PASTA kinase-dependent cell cycle regulation processes found in related bacteria are conserved in S. pyogenes. Increased protein phosphorylation during the stationary phase has also been described for some other bacteria, and could therefore be a general feature in the physiology of bacteria, whose functions and the kinases involved need to be elucidated in further analyses.

Synthetic mRNA delivered to human cells leads to expression of Cpl-1 bacteriophage-endolysin with activity against Streptococcus pneumoniae.

Endolysins are bacteriophage-encoded hydrolases that show high antibacterial activity and a narrow substrate spectrum. We hypothesize that an mRNA-based approach to endolysin therapy can overcome some challenges of conventional endolysin therapy, namely organ targeting and bioavailability. We show that synthetic mRNA applied to three human cell lines (HEK293T, A549, HepG2 cells) leads to expression and cytosolic accumulation of the Cpl-1 endolysin with activity against Streptococcus pneumoniae. Addition of a human lysozyme signal peptide sequence translocates the Cpl-1 to the endoplasmic reticulum leading to secretion (hlySP-sCpl-1). The pneumococcal killing effect of hlySP-sCpl-1 was enhanced by introduction of a point mutation to avoid N-linked-glycosylation. hlySP-sCpl-1N215D, collected from the culture supernatant of A549 cells 6 h post-transfection showed a significant killing effect and was active against nine pneumococcal strains. mRNA-based cytosolic Cpl-1 and secretory hlySP-sCpl-1N215D show potential for innovative treatment strategies against pneumococcal disease and, to our best knowledge, represent the first approach to mRNA-based endolysin therapy. We assume that many other bacterial pathogens could be targeted with this novel approach.