Yina S et al. (2016), The first description of complete invertebrate ...

ECB-ART-44604

Sci Rep 2016 Apr 01;6:23783. doi: 10.1038/srep23783.

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The first description of complete invertebrate arginine metabolism pathways implies dose-dependent pathogen regulation in Apostichopus japonicus.

Yina S , Chenghua L , Weiwei Z , Zhenhui W , Zhimeng L .

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In this study, three typical members representative of different arginine metabolic pathways were firstly identified from Apostichopus japonicus, including nitric oxide synthase (NOS), arginase, and agmatinase. Spatial expression analysis revealed that the AjNOS transcript presented negative expression patterns relative to those of Ajarginase or Ajagmatinase in most detected tissues. Furthermore, Vibrio splendidus-challenged coelomocytes and intestine, and LPS-exposed primary coelomocytes could significantly induce AjNOS expression, followed by obviously inhibited Arginase and AjAgmatinase transcripts at the most detected time points. Silencing the three members with two specific siRNAs in vivo and in vitro collectively indicated that AjNOS not only compete with Ajarginase but also with Ajagmatinase in arginine metabolism. Interestingly, Ajarginase and Ajagmatinase displayed cooperative expression profiles in arginine utilization. More importantly, live pathogens of V. splendidus and Vibrio parahaemolyticus co-incubated with primary cells also induced NO production and suppressed arginase activity in a time-dependent at an appropriate multiplicity of infection (MOI) of 10, without non-pathogen Escherichia coli. When increasing the pathogen dose (MOI = 100), arginase activity was significantly elevated, and NO production was depressed, with a larger magnitude in V. splendidus co-incubation. The present study expands our understanding of the connection between arginine''s metabolic and immune responses in non-model invertebrates.

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Genes referenced: LOC100887844 LOC100888042

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	Figure 1. The tissue distribution of AjNOS, Ajarginase and Ajagmatinase in normal sea cucumber detected by quantitative PCR.The transcript levels in coelomocytes, intestine, tentacle, and respiratory tress were normalized to that in muscle. Five biological replicates were performed in the experiment and the obtained data were expressed as the mean ± SD (n = 5). Asterisks indicated significant differences: P < 0.05, *P < 0.01.


	Figure 4. Related data from Apostichopus japonicus primary cultured coelomocytes after each gene silencing.(A,D,G) Silencing efficiency of AjNOS, Ajarginase or Ajagmatinase in primary coelomocytes after specific siRNAs transfection and relative expression of mRNAs after interfering for 24 h, respectively. (B,E,H) NO production in the primary cultured coelomocytes after AjNOS, Ajarginase or Ajagmatinase knock-down, respectively. (C,F,I) arginase activity in the primary cultured coelomocytes after AjNOS, Ajarginase or Ajagmatinase knock-down, respectively. Five biological replicates were performed in the experiment and the obtained data were expressed as the mean ± SD (n = 5).
	Figure 5. Related data from Apostichopus japonicus individual levels after each gene silencing.(A,D,G) Silencing efficiency of AjNOS, Ajarginase or Ajagmatinase in individuals coelomocytes after specific siRNAs transfection and relative expression of mRNAs after interfering for 24 h, respectively. (B,E,H) NO production in the individuals coelomocytes after AjNOS, Ajarginase or Ajagmatinase knock-down, respectively. (C,F,I) arginase activity in the individuals coelomocytes after AjNOS, Ajarginase or Ajagmatinase knock-down, respectively. Five biological replicates were performed in the experiment and the obtained data were expressed as the mean ± SD (n = 5).
	Figure 6. Effects of in vitro stimulation of pathogen or non-pathogen on Apostichopus japonicus primary cultured coelomocytes for 12 and 24 h with an appropriate multiplicity of infection (MOI) of 10 and 100.(A) NO production in the primary cultured coelomocytes after Vibrio splendidus, Vibrio Parahaemolyticus and Escherichia Coli infection. (B) arginase activity in the primary cultured coelomocytes after Vibrio splendidus, Vibrio Parahaemolyticus and Escherichia Coli infection. Five biological replicates were performed in the experiment and the obtained data were expressed as the mean ± SD (n = 5).
	Figure 7. Schematic representation of the arginine metabolic pathways of host-pathogen interaction.CAT was short from cationic amino acid transporter and mediated arginine transport. The extracellular urea released by pathogens (Vsagmatinase or Vparginase). Ajarginase and Ajagmatinase transcribed and translated and generated into urea. AjNOS transcribed and translated and generated into NO.

References [+] :

Abu-Soud, Nitric oxide synthases reveal a role for calmodulin in controlling electron transfer. 1993, Pubmed