| Summary: | <i>Streptococcus iniae</i> 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 <i>S. iniae</i> 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 <i>S. iniae spnAi</i> and <i>s5nAi</i> deletion mutants and their evaluation in a transgenic zebrafish infection model. Our results show nuclease and nucleotidase activities in <i>S. iniae</i> 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) <i>S. iniae</i>. Deletion of <i>spnAi</i> and <i>s5nAi</i> 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 <i>S. iniae</i> immune evasion and might present potential targets for the development of therapeutic agents or vaccines.
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