Date Published: January 31, 2019
Publisher: Public Library of Science
Author(s): Marcel-Lino Dangel, Johann-Christoph Dettmann, Steffi Haßelbarth, Martin Krogull, Miriam Schakat, Bernd Kreikemeyer, Tomas Fiedler, Jens Kreth.
5’-nucleotidases are widespread among all domains of life. The enzymes hydrolyze phosphate residues from nucleotides and nucleotide derivatives. In some pathobiontic bacteria, 5’-nucleotidases contribute to immune evasion by dephosphorylating adenosine mono-, di-, or tri-phosphates, thereby either decreasing the concentration of pro-inflammatory ATP or increasing the concentration of anti-inflammatory adenosine, both acting on purinergic receptors of phagocytic cells. The strict human pathogen Streptococcus pyogenes expresses a surface-associated 5’-nucleotidase (S5nA) under infection conditions that has previously been discussed as a potential virulence factor. Here we show that deletion of the S5nA gene does not significantly affect growth in human blood, evasion of phagocytosis by neutrophils, formation of biofilms and virulence in an infection model with larvae of the greater wax moth Galleria mellonella in S. pyogenes serotypes M6, M18 and M49. Hence, the surface-associated 5’-nucleotidase S5nA seems dispensable for evasion of phagocytosis and biofilm formation in S. pyogenes.
Streptococcus pyogenes (Group A Streptococcus) is a Gram-positive human pathogen primarily causing purulent infections of the skin (impetigo) and throat (pharyngitis, tonsillitis) and frequently also severe invasive or systemic diseases such as necrotizing fasciitis, sepsis or streptococcal toxic shock-like syndrome . The list of newly identified factors contributing to virulence mechanisms of S. pyogenes is constantly growing. To date, more than 50 virulence factors have been described in S. pyogenes . Recently, the 5’-nucleotidase S5nA has been added to that list of potential virulence factors .
5’NT have been described to contribute to virulence in several bacteria [3, 7–10]. S5nA, a recombinant 5’NT of S. pyogenes has been shown to increase survival of L. lactis in the presence of phagocytes in vitro . Therefore, we aimed to elucidate whether deletion of the S5nA gene actually affects the virulence of S. pyogenes. For this purpose the S5nA genes were replaced by a kanamycin resistance gene in three different S. pyogenes strains, i.e. the serotype M49 strain 591 (reference sequence ORF spy49_0686c), the M18 serotype strain MGAS8232 (reference sequence ORF spyM18_0933) and the M6 serotype strain K006 (reference sequence ORF M6_Spy0695). Furthermore, complementation strains expressing the S5nA gene from a pAT19-based plasmid under the control of the native promoter were constructed.
5’NT have been associated with virulence of certain bacteria, as it has been shown that they hydrolyze phosphate residues of adenosine phosphates and hence can interfere with the immunologically relevant ATP/adenosine ratio in human infections. This may happen via production of the anti-inflammatory adenosine and/or degradation of the pro-inflammatory ATP, both acting on phagocytic cells via purinergic receptors [9, 11, 12, 29–33].
Our study shows that deletion of the 5’NT encoding gene S5nA does not affect S. pyogenes with respect to phagocytosis by neutrophils, growth of the bacteria in human blood, survival rates of the G. mellonella larvae in the infection model or biofilm formation capacity in three different strains covering the serotypes M6, M18 and M49. Hence, although an immunomodulatory capacity of the S. pyogenes S5nA was demonstrated by Zheng and colleagues , for the evasion of phagocytosis and biofilm formation this enzyme seems to be dispensable for S. pyogenes.