Orthologues of Streptococcus pyogenes nuclease A (SpnA) and Streptococcal 5′-nucleotidase A (S5nA) found in Streptococcus iniae

Abstract

Streptococcus pyogenes nuclease A (SpnA) and streptococcal 5' nucleosidase A (S5nA) are two recently described virulence factors from the human pathogen S. pyogenes. In vitro studies have shown that SpnA is a nuclease that cleaves ssDNA and dsDNA, including the DNA backbone of neutrophil extracellular traps. S5nA was shown to hydrolyse AMP and ADP, but not ATP, to generate the immunomodulatory molecule adenosine. S5nA also generates the macrophage-toxic deoxyadenosine from dAMP. However, detailed in vivo studies of the two enzymes have been hampered by difficulties with using current animal models for this exclusive human pathogen. Here we report the identification of two novel enzymes from the fish pathogen Streptococcus iniae that show similarities to SpnA and S5nA in amino acid sequence, protein domain structure and biochemical properties. We propose that SpnAi and S5nAi are orthologues of the S. pyogenes enzymes, providing a rationale to analyse the in vivo function of the two enzymes using a S. iniae-zebrafish infection model.

Cell wall‐anchored 5′‐nucleotidases in Gram‐positive cocci

“…Several streptococcal 5′‐NTs have been functionally characterized , including ecto‐5′‐nucleotidase (Nt5e) from S. sanguinis (Fan et al, ), Ecto‐5‐nucleoside diphosphate phosphohydrolase (NudP) from S. agalactiae (Firon et al, ), streptococcal 5′‐nucleotidase A (S5nA) from S. pyogenes (Zheng et al, ), S5nAi from S. iniae (Soh, Loh, & Proft, ), S. suis adenosine synthase (Ssads) (Liu et al, ) and S. equi subsp. zooepidemicus 5′‐nucleotidase (5Nuc; Ma, Guo, & Fan, ).…”

“…There are also differences between the 5′‐NTs regarding substrate specificity for other nucleosides and metal‐ion requirements. Hydrolysis of other nucleoside monophosphates (GMP, CMP, TMP) was shown for NudP, S5nA and S5nAi (Firon et al, ; Soh et al, ; Zheng et al, ), in contrast to the S. aureus AdsA which only recognizes AMP and GMP, but not CMP and TMP. Furthermore, enzymatic activities for individual substrates also differ.…”

“…We have shown that incubation of rS5nA and rSpnA with DNA as the sole substrate results in the production of dAdo (Zheng et al, ). The same experiment was also used to demonstrate the synergy between the S. iniae enzymes S5nAi and SpnAi (Soh et al, ). The destruction of NETs and generation of dAdo has also been reported for S. equi subsp.…”

“…Notably, the authors found that ENuc and 5Nuc each possess both nuclease and 5′‐nucleotidase activity suggesting a redundant rather than synergistic effect. 5Nuc carries the same protein domains (cl13995 and Pfam02872) as S5nA and S5nAi, which both lack nuclease activity (Soh et al, ; Zheng et al, ) and nuclease activity is also absent in S. aureus AdsA (Thammavongsa et al, ). The molecular mechanism for the dual enzymatic reaction of 5Nuc is not clear, but future protein structural analysis might shed further light on this phenomenon.…”

Functional Analysis of Two Novel Streptococcus iniae Virulence Factors Using a Zebrafish Infection Model

“…Analyzing the complete genome of S. iniae 9117, we recently identified two S. iniae proteins that share some amino acid sequence homology with two important S. pyogenes immune evasion factors, Streptococcus pyogenes nuclease A (SpnA) [16] and streptococcal 5 -nucleotidase A (S5nA) [17]. These were termed SpnAi and S5nAi, respectively [18]. These S. iniae proteins are expressed as precursor proteins with an N-terminal signal peptide and a C-terminal cell wall anchor domain, including the canonical sortase A recognition motif LPXTG [16][17][18].…”