Comprehensive identification and profiling of host miRNAs in response to Singapore grouper iridovirus (SGIV) infection in grouper (Epinephelus coioides)

microRNAs (miRNAs) are an evolutionarily conserved class of non-coding RNA molecules that participate in various biological processes. Employment of high-throughput screening strategies greatly prompts the investigation and profiling of miRNAs in diverse species. In recent years, grouper (Epinephelus spp.) aquaculture was severely affected by iridoviral diseases. However, knowledge regarding the host immune responses to viral infection, especially the miRNA-mediated immune regulatory roles, is rather limited. In this study, by employing Solexa deep sequencing approach, we identified 116 grouper miRNAs from grouper spleen-derived cells (GS). As expected, these miRNAs shared high sequence similarity with miRNAs identified in zebrafish (Danio rerio), pufferfish (Fugu rubripes), and other higher vertebrates. In the process of Singapore grouper iridovirus (SGIV) infection, 45 and 43 miRNAs with altered expression (>1.5-fold) were identified by miRNA microarray assays in grouper spleen tissues and GS cells, respectively. Furthermore, target prediction revealed 189 putative targets of these grouper miRNAs. (C) 2015 Elsevier Ltd. All rights reserved

Antitoxin HigA inhibits virulence gene mvfR expression in Pseudomonas aeruginosa

Toxin/antitoxin (TA) systems are ubiquitous in bacteria and archaea and participate in biofilm formation and stress responses. The higBA locus of the opportunistic pathogen Pseudomonas aeruginosa encodes a type II TA system. Previous work found that the higBA operon is cotranscribed and that HigB toxin regulates biofilm formation and virulence expression. In this study, we demonstrate that HigA antitoxin is produced at a higher level than HigB and that higA mRNA is expressed separately from a promoter inside higB during the late stationary phase. Critically, HigA represses the expression of mvfR, which is an important virulence-related regulator, by binding to a conserved HigA palindrome (5 '-TTAAC GTTAA-3 ') in the mvfR promoter, and the binding of HigB to HigA derepresses this process. During the late stationary phase, excess HigA represses the expression of mvfR and higBA. However, in the presence of aminoglycoside antibiotics where Lon protease is activated, the degradation of HigA by Lon increases P. aeruginosa virulence by simultaneously derepressing mvfR and higB transcription. Therefore, this study reveals that the antitoxin of the P. aeruginosa TA system is integrated into the key virulence regulatory network of the host and functions as a transcriptional repressor to control the production of virulence factors

Ubiquitin–Proteasome System Is Required for Efficient Replication of Singapore Grouper Iridovirus

The ubiquitin-proteasome system (UPS) serves as the major intracellular pathway for protein degradation and plays crucial roles in several cellular processes. However, little is known about the potential actions of the UPS during fish virus infection. In this study, we elucidated the possible roles of UPS in the life cycle of Singapore grouper iridovirus (SGIV); a large DNA virus that usually causes serious systemic diseases with high mortality in groupers. Data from transcriptomic analysis of differentially expressed genes illustrated that expression of 65 genes within the UPS pathway, including ubiquitin encoding, ubiquitination, deubiquitination, and proteasome, were up- or down-regulated during SGIV infection. Using different proteasome inhibitors, inhibition of the proteasome decreased SGIV replication in vitro, accompanied by inhibition of virus assembly site formation, and viral gene transcription and protein transportation. Over-expression of ubiquitin partly rescued the inhibitory effect of ubiquitin inhibitor on SGIV replication, suggesting that UPS was required for fish iridovirus infection in vitro. Viral or host proteins regulated by proteasome inhibition during SGIV infection were investigated with two-dimensional gel electrophoresis and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Sixty-two differentially expressed proteins, including 15 viral and 47 host proteins, were identified after SGIV infection. The host proteins were involved in ubiquitin-mediated protein degradation, metabolism, cytoskeleton, macromolecular biosynthesis, and signal transduction. Among them, 11 proteins were negatively regulated upon MG132 treatment during SGIV infection. This is believed to be the first study to provide evidence that UPS was essential for fish virus infection and replication