Search

Tilapia tilapinevirus, or Tilapia Lake Virus (TiLV), is a RNA virus associated with mass morbidity and mortality in tilapia, leading to severe economic losses for global tilapia aquaculture. In this study, we investigated the persistence of TiLV in water by spiking sterile distilled water (SDW), freshwater collected from rearing fish tanks (FW) and natural pond water (PW) at 27°C as a representative of environmental water conditions with 0.6 ml of stock virus (3.18 × 10 7 viral copies/ml of water). The water samples were filtered through an electronegative charge membrane and quantified using reverse transcriptase quantitative PCR at 0, 3, 5, 7, 10 and 14 days post-inoculation. The results revealed that TiLV RNA in SDW was reduced by 1.34 log 10 in 14 days. A similar approximately 4 log 10 removal of the virus in FW and PW was observed at 3 and 7 days, respectively. Moreover, the infectivity of TiLV was further studied; the virus lost its infectivity in E-11 cells after 1 day in SDW, FW and PW water samples, even though the virus was spiked 10 more times than in the viral persistence study. Taken together, the results could be applied to improving biosecurity practices in tilapia farms by disinfecting or resting reservoir water for at least three to five days prior to stocking tilapia, to limit the spread of TiLV., (© 2022 John Wiley & Sons Ltd.)

Candida albicans is an opportunistic yeast that can cause life-threatening systemic infection in immunocompromised individuals. During infections, C. albicans has to cope with genotoxic stresses generated by the host immune system. DNA-protein crosslink (DPC), the covalent linkage of proteins with DNA, is one type of DNA damages that can be caused by the host immune response. DPCs are bulky lesions that interfere with the progression of replication and transcription machineries, and hence threaten genomic integrity. Accordingly, either a DPC tolerance mechanism or a DPC repair pathway is essential for C. albicans to maintain genomic stability and survive in the host. Here, we identified Wss1 (weak suppressor of Smt3) in C. albicans (CaWss1) using bioinformatics, genetic complementation, and biochemical studies. We showed that CaWss1 promotes cell survival under genotoxic stress conditions that generate DPCs and that the catalytic metalloprotease domain of CaWss1 is essential for its cellular function. Interactions of CaWss1 with Cdc48 and small ubiquitin-like modifier, although not strictly required, contribute to the function of CaWss1 in the suppression of the growth defects under DPC-inducing conditions. This report is the first investigation of the role of CaWss1 in DPC tolerance in C. albicans., (© 2020 John Wiley & Sons Ltd.)

Tilapia lake virus (TiLV) is an emerging virus associated with high fish mortality and economic losses. This study investigates the virucidal effects of the following disinfectants (active ingredients) on TiLV: 2.5 ppm iodine, 10 ppm sodium hypochlorite (NaOCl), 300 ppm hydrogen peroxide (H 2 O 2 ), 80 ppm formalin and 5,000 ppm (0.5%) Virkon ® . Factors that affect the disinfectants' efficacy, including temperature, contact time and soiling (organic matter) interference, were examined under conditions mimicking natural aquaculture practices. TiLV inactivation of higher than 5 log 10 TCID 50  ml -1 was achieved after 10 min and at 28°C for all disinfectants except formalin; similar inactivation levels were reached by NaOCl and Virkon ® at 10 min and 4°C. Extended exposure to formalin from 10 to 60 min at 28°C rendered more than 5 log 10 inactivation. Increasing synthetic organic matter in the water to mimic soiling interference reduced the efficacy of NaOCl, iodine and H 2 O 2 when tested at 10 min and 28°C; however, Virkon ® still achieved more than 5 log 10 inactivation. This study demonstrates that most common disinfectants effectively reduced viral loads to minimum levels. To limit the spread of TiLV in aquaculture farms and related facilities, the appropriate use of such disinfectants should therefore be promoted and implemented., (© 2019 John Wiley & Sons Ltd.)

Streptococcosis is an important bacterial disease in Nile tilapia causing severe economic losses to tilapia aquaculture worldwide. The effects of water quality (low- [LS] and high-level [HS] soiling, to mimic clean or dirty surface conditions and temperatures) and disinfectant application (diluted concentrations and exposure time) were characterized on the inactivation of Streptococcus agalactiae isolated from diseased tilapia. Five isolates were tested against three commercial disinfectant products with the main ingredients being povidone iodine (Anidine 100™; AD), benzalkonium chloride (Better BKC 80%™; BKC 80), and a mixture of quaternary ammonium compounds and glutaraldehyde (Chloraldehyde™; CR). CR demonstrated highest efficacy to S. agalactiae inactivation, followed by BKC 80 and AD, respectively. Higher-level soiling, low temperature, diluted concentrations and short exposure time all decreased the disinfectant efficacy. CR and BKC 80 provided more than 5-log inactivation at 1-min exposure at 20°C under HS conditions, and also with ten-fold-diluted concentrations at 60-min exposure time at 30°C. However, AD required 10-min exposure to effectively remove bacteria under LS conditions at 30°C. The results could facilitate aquaculture management planning that leads to operating cost reductions and improvements in biosecurity., (© 2018 John Wiley & Sons Ltd.)

We report the physiological role of OhrR as an organic peroxide sensor and transcription repressor in Xanthomonas campestris pv. phaseoli. In vivo exposure of X. campestris pv. phaseoli to either tert-butyl or cumene hydroperoxides efficiently neutralized OhrR repression of expression from the OhrR-regulated P1 promoter. H2O2 was a weak and non-physiological inducer of the system while other oxidants and metabolites of organic peroxide metabolism did not induce the expression from the P1. Northern blotting results indicated a correlation between concentrations of tert-butyl hydroperoxide used in the treatment and the induction of ohr (an OhrR-regulated gene) expression. In addition, the levels of ohr mRNA in cultures induced by various concentrations of tert-butyl hydroperoxide were reduced in cells with high levels of an organic peroxide metabolising enzyme (AhpC-AhpF) but not in cells with high catalase levels suggesting that organic peroxide interacts with OhrR. DNA band shift experiments using purified OhrR and the P1 promoter fragment showed that organic peroxide treatment prevented binding of the protein to the P1 promoter by oxidation of OhrR, as the inhibition of binding to the P1 promoter was reversed by addition of a reducing agent, DTT. The highly conserved cysteine residue C22 of OhrR is required for organic peroxide inducible gene expression. A mutant protein, OhrRC22S can repress the P1 promoter activity but is insensitive to organic peroxide treatment. Thus, OhrR is the first transcription repressor characterized that appeared to evolve to physiologically sense organic peroxides.

Bacteria adapt to the presence of reactive oxygen species (ROS) by increasing the expression of detoxification enzymes and protein and DNA repair functions. These responses are co-ordinated by transcription factors that regulate target genes in response to ROS. We compare three classes of peroxide-sensing regulators: OxyR, PerR and OhrR. In all three cases, peroxides effect changes in the redox status of cysteine residues, but the molecular details are distinct. OxyR is converted into a transcriptional activator by the formation of a disulphide bond between two reactive cysteine residues. PerR is a metalloprotein that functions as a peroxide- sensitive repressor. Oxidation is modulated by metal ion composition and may also involve disulphide bond formation. OhrR represses an organic peroxide resistance protein and mediates derepression in response to organic peroxides. Peroxide sensing in this system requires a single conserved cysteine, which is oxidized to form a cysteine-sulphenic acid derivative.

ohrR encodes a novel organic peroxide-inducible transcription repressor, and we have demonstrated that ohrR is regulated at the transcriptional and the post-transcriptional levels. Primer extension results show that ohrR transcription initiates at the A residue of the ATG translation initiation codon for the ohrR coding sequence. Thus, the gene has a leaderless mRNA. The ohrR promoter (P1) has high homology to the consensus sequence for Xanthomonas promoters, which is reflected in the high in vivo promoter activity of P1. Deletion of a 139 bp fragment containing the P1 promoter showed that the sequences upstream of -35 regions were required for neither the promoter activity nor OhrR autoregulation. In vitro, purified OhrR specifically binds to the P1 promoter. DNase I footprinting of OhrR binding to the P1 revealed a 44 bp region of protection on both DNA strands. The protected regions include the -35 and -10 regions of P1. We suggest that OhrR represses gene expression by blocking RNA polymerase binding to the promoter. There are two steps in the post-transcriptional regulation of ohrR, namely differential stability and inefficient translation of the mRNA. The bicistronic ohrR-ohr mRNA was highly labile and underwent rapid processing in vivo to give only stable monocistronic ohr mRNA and undetectable ohrR mRNA. Furthermore, the ohrR mRNA was inefficiently translated. We propose that, in uninduced cells, the concentration of OhrR is maintained at low levels by the autoregulation mechanism at the transcriptional levels and by the ohrR mRNA instability coupled with inefficient translation at the post-transcriptional level. Upon exposure to an organic peroxide, the compound probably interacts with OhrR and prevents it from repressing the P1 promoter, thus allowing high-level expression of the ohrR-ohr operon. The rapid processing of bicistronic mRNA gives highly stable ohr mRNA and corresponding high levels of Ohr, which remove an organic per-oxide. Once the peroxide has been removed, the autoregulation mechanism feeds back to inhibit the expression of the operon.

In Xanthomonas campestris pv. phaseoli, a gene for the alkyl hydroperoxide reductase subunit C (ahpC) had unique patterns of regulation by various forms of OxyR. Reduced OxyR repressed expression of the gene, whereas oxidized OxyR activated its expression. This dual regulation of ahpC is unique and unlike all other OxyR-regulated genes. The ahpC transcription start site was determined. Analysis of the region upstream of the site revealed promoter sequences that had high homology to the Xanthomonas consensus promoter sequence. Data from gel shift experiments indicated that both reduced and oxidized OxyR could bind to the ahpC regulatory region. Moreover, the reduced and the oxidized forms of OxyR gave different DNase I footprint patterns, indicating that they bound to different sites. The oxidized OxyR binding site overlapped the -35 region of the ahpC promoter by a few bases. This position is consistent with the role of the protein in activating transcription of the gene. Binding of reduced OxyR to the ahpC promoter showed an extended DNase I footprint and DNase I hypersensitive sites, suggesting that binding of the protein caused a shift in the binding site and bending of the target DNA. In addition, binding of reduced OxyR completely blocked the -35 region of the ahpC promoter and prevented binding of RNA polymerase, leading to repression of the gene. Monitoring of the ahpC promoter activity in vivo confirmed the location of the oxidized OxyR binding site required for activation of the promoter. A mutant that separated OxyR regulation from basal ahpC promoter activity was constructed. The mutant was unable to respond to oxidants by increasing ahpC expression. Physiologically, it had a slower aerobic growth rate and was more sensitive to organic peroxide killing. This indicated that oxidant induction of ahpC has important physiological roles in normal growth and during oxidative stress.