5′‐nucleotidases (5′‐NTs) are enzymes that catalyze the hydrolysis of nucleoside monophosphates to produce nucleosides and phosphate. Since the identification of adenosine synthase A (AdsA) in Staphylococcus aureus in 2009, several other 5′‐NTs have been discovered in Gram‐positive cocci, mainly in streptococci. Despite some differences in substrate specificity, pH range and metal ion requirements, all characterized 5′‐NTs use AMP and ADP, and in some cases ATP, to produce the immunosuppressive adenosine, which dampens pro‐inflammatory immune responses. Several 5′‐NTs are also able to use dAMP as substrate to generate deoxy‐adenosine which is cytotoxic for macrophages. A synergy between 5′‐NTs and exonucleases which are commonly expressed in Gram‐positive cocci has been described, where the nucleases provide dAMP as a cleavage product from DNA. Some of these nucleases produce dAMP by degrading the DNA backbone of neutrophil extracellular traps (NETs) resulting in a “double hit” strategy of immune evasion. This Micro Review provides an overview of the biochemical properties of Gram‐positive cell wall‐anchored 5′‐NTs and their role as virulence factors. A potential use of 5′‐NTs for vaccine development is also briefly discussed.
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.
Streptococcus iniae is a major fish pathogen that contributes to large annual losses in the aquaculture industry, exceeding US$100 million. It is also reported to cause opportunistic infections in humans. We have recently identified two novel S. iniae virulence factors, an extracellular nuclease (SpnAi) and a secreted nucleotidase (S5nAi), and verified their predicted enzymatic activities using recombinant proteins. Here, we report the generation of green fluorescent S. iniae spnAi and s5nAi deletion mutants and their evaluation in a transgenic zebrafish infection model. Our results show nuclease and nucleotidase activities in S. iniae could be attributed to SpnAi and S5nAi, respectively. Consistent with this, larvae infected with the deletion mutants demonstrated enhanced survival and bacterial clearance, compared to those infected with wild-type (WT) S. iniae. Deletion of spnAi and s5nAi resulted in sustained recruitment of neutrophils and macrophages, respectively, to the site of infection. We also show that recombinant SpnAi is able to degrade neutrophil extracellular traps (NETs) isolated from zebrafish kidney tissue. Our results suggest that both enzymes play an important role in S. iniae immune evasion and might present potential targets for the development of therapeutic agents or vaccines.
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