Comparing genome-scale metabolic models of the non-resistant Enterococcus faecalis ATCC 19433 and the multi-resistant Enterococcus faecalis V583.

Enterococcus faecalis is a versatile lactic acid bacterium with a large variety of implications for humans. While some strains of this species are pathobionts being resistant against most of the common antibiotics, other strains are regarded as biological protectants or even probiotics. Accordingly, E. faecalis strains largely differ in the size and content of their accessory genome. In this study, we describe the genome-scale metabolic network reconstruction of E. faecalis ATCC 19433, a non-resistant human-associated strain. A comparison of the genome-scale metabolic model (GSM) of E. faecalis ATCC 19433 with a previously published GSM of the multi-resistant pathobiontic E. faecalis V583 reveals high similarities in the central metabolic abilities of these two human associated strains. This is reflected, e.g., in the identical amino acid auxotrophies. The ATCC 19433 strain, however, has a 14.1 % smaller genome than V583 and lacks the multiple antibiotic resistance genes and genes involved in capsule formation. Based on the measured metabolic fluxes at different growth rates, the energy demand at zero growth was calculated to be about 40 % lower for the ATCC 19433 strain compared to V583. Furthermore, the ATCC 19433 strain seems less prone to the depletion of amino acids utilizable for energy metabolism. This might hint at a lower overall energy demand of the ATCC 19433 strain as compared to V583.

PEGylation increases antitumoral activity of arginine deiminase of Streptococcus pyogenes.

Arginine auxotrophy is a metabolic defect that renders tumor cells vulnerable towards arginine-depleting substances, such as arginine deiminase (ADI) from Streptococcus pyogenes (SpyADI). Previously, we confirmed SpyADI susceptibility on patient-derived glioblastoma multiforme (GBM) models in vitro and in vivo. For application in patients, serum half-life of the enzyme has to be increased and immunogenicity needs to be reduced. For this purpose, we conjugated the S. pyogenes-derived SpyADI with 20 kDa polyethylene glycol (PEG20) moieties, achieving a PEGylation of seven to eight of the 26 accessible primary amines of the SpyADI. The PEGylation reduced the overall activity of the enzyme by about 50% without affecting the Michaelis constant for arginine. PEGylation did not increase serum stability of SpyADI in vitro, but led to a longer-lasting reduction of plasma arginine levels in mice. Furthermore, SpyADI-PEG20 showed a higher antitumoral capacity towards GBM cells in vitro than the native enzyme. KEY POINTS: • PEGylation has no effect on the affinity of SpyADI for arginine • PEGylation increases the antitumoral effects of SpyADI on GBM in vitro • PEGylation prolongs plasma arginine depletion by SpyADI in mice.