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

ABSTRACT: 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.

SUBMITTER: Soh KY

PROVIDER: S-EPMC7564053 | biostudies-literature | 2020 Sep

REPOSITORIES: biostudies-literature

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

Soh Kar Yan KY Loh Jacelyn Mei San JMS Hall Christopher C Proft Thomas T

Microorganisms 20200905 9

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 sp ...[more]

PMID: 32899555

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Project description:BackgroundStreptococcus iniae is an important fish pathogen that cause significant economic losses to the global aquaculture industry every year. Although there have some reports on the genotype of S.iniae and its relationship with virulence, no genome-scale comparative analysis has been performed so far. In our previous work, we characterized 17 isolates of S.iniae from Trachinotus ovatus and divided them into two genotypes using RAPD and rep-PCR methods. Among them, BH15-2 was classified as designated genotype A (in RAPD) and genotype 1 (in rep-PCR), while BH16-24 was classified as genotype B and genotype 2. Herein, we compared the differences in growth, drug resistance, virulence, and genome between BH15-2 and BH16-24.ResultsThe results showed that the growth ability of BH16-24 was significantly faster than that of BH15-2 at the exponential stage. Antimicrobial tests revealed that BH15-2 was susceptible to most of the tested antibiotics except neomycin and gentamycin. In contrast, BH16-24 was resistant to 7 antibiotics including penicillin, sulfasomizole, compound sulfamethoxazole tablets, polymyxin B, spectinomycin, rifampin and ceftazidime. Intraperitoneal challenge of T.ovatus, showed that the LD50 value of BH15-2 was 4.0 × 102 CFU/g, while that of BH16-24 was 1.2 × 105 CFU/g. The genome of S.iniae BH15-2 was 2,175,659 bp with a GC content of 36.80%. Meanwhile, the genome of BH16-24 was 2,153,918 bp with a GC content of 36.83%. Comparative genome analysis indicated that compared with BH15-2, BH16-24 genome had a large-scale genomic inversion fragment, at the location from 502,513 bp to 1,788,813 bp, resulting in many of virulence and resistance genes differentially expression. In addition, there was a 46 kb length, intact phage sequence in BH15-2 genome, which was absent in BH16-24.ConclusionComparative genomic studies of BH15-2 and BH16-24 showed that the main difference is a 1.28 Mbp inversion fragment. The inversion fragment may lead to abnormal expression of drug resistant and virulence genes, which is believed to be the main reason for the multiple resistance and weakened virulence of BH16-24. Our study revealed the potential mechanisms in underlying the differences of multidrug resistance and virulence among different genotypes of S.iniae.

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

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Functional Analysis of Two Novel <i>Streptococcus iniae</i> Virulence Factors Using a Zebrafish Infection Model.

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