Postbiotics Applications as Infectious Disease Control Agent in Aquaculture.

Aquaculture is developing so fast that infectious disease outbreak happens regularly. Antibiotic treatment results in development of antibiotic resistance pathogens, thus cause urgent action in searching of other alternative treatment method. Postbiotic was one of the explored strategies among various proposed alternatives. Due to its benefits in agriculture industry, it may be useful in aquaculture industry. Although many reviews were reported on other alternative strategies, the review on postbiotic in aquaculture is limited. This mini review provides an overview of different postbiotics as aquaculture disease control agents. Peptides and exopolysaccharides have antimicrobial properties against bacterial pathogens. Then, short chain fatty acids have both antimicrobial activities against bacterial pathogens and immunostimulating effects to aquatic organism. Vitamins, peptidoglycan and lipopolysaccharide are reported as immunostimulants. Finally, cell surface proteins and teichoic acid can act as vaccine.

Characterization and safety evaluation of partially purified bacteriocin produced by Escherichia coli E isolated from fermented pineapple Ananas comosus (L.) Merr.

Antibacterial activity of cell-free supernatant from Escherichia coli E against selected pathogenic bacteria in food and aquaculture was the highest against Edwardsiella tarda 3, a significant aquaculture pathogen. Biochemical properties of the bacteriocins were studied and bacteriocin was found to be sensitive to proteinase K, demonstrating its proteinaceous nature. In addition, pH and temperature affected bacteriocin activity and stability. The bacteriocins were partially purified by ammonium sulfate precipitation. The antibacterial activity was only detected in 20% ammonium sulfate fraction and direct detection of its activity was performed by overlaying on the indicator strains. The inhibition zone associated with the antibacterial activity was detected in the sample overlaid by E. tarda 3 and Staphylococcus aureus DMST8840 with the relative molecular mass of about 27 kDa and 10 kDa, respectively. Bacteriocin showed no cytotoxic effect on NIH-3T3 cell line; however, two virulence genes, aer and sfa, were detected in the genome of E. coli E by PCR. The characteristics of bacteriocins produced by E. coli E exhibited the antibacterial activity against both Gram-positive and Gram-negative pathogenic bacteria and the safe use determined by cytotoxicity test which may have interesting biotechnological applications.

Fibroblasts and extracellular matrix differently modulate MMP activation by primary and metastatic head and neck cancer cells.

A genetically related pair of human head and neck cancer (HNSCC) cell lines derived from the same patient at different stages of disease was used to investigate the role of extracellular matrix, integrin, and CXCL12-CXCR4 receptor interactions and their signal pathways in MMP-2 and MMP-9 activation and cell invasion. We found that collagen I enhanced MMP-2 and MMP-9 secretion in both primary and metastatic HNSCC cells. Collagen I acted through α(2)ß(1) integrin to activate tyrosine kinases, protein kinase C, ERK1/2, and p38, which in turn activated MMP-2 and MMP-9 production. The signaling function was also involved in the enhancement of cell invasion. Experiments using cocultures between live and fixed cells demonstrated that direct contact between tumor and fibroblast cells was required to activate MMP-2 and MMP-9 secretion in both tumor cells and fibroblasts. The augmentation appears specific for MMP-2. Fibroblasts seem to be responsible for the increased MMP-2 in the coculture. In addition, fibroblast or tumor cell-conditioned media upregulated the secretion of MMP-2 and MMP-9 in HNSCC cells. These findings indicate that autocrine and paracrine factors are involved in the augmented secretion of MMPs in coculture. We also found that CXCL12-enhanced HNSCC cell invasion through paracrine-activated CXCR4, which triggered MMP-dependent cell invasion. Together, our results suggest that cell-matrix and cell-cell interactions including autocrine and paracrine factors play important roles in the invasive behavior of HNSCC via upregulation of MMP-2 and MMP-9.

Regulation of quinone detoxification by the thiol stress sensing DUF24/MarR-like repressor, YodB in Bacillus subtilis.

Recently, we showed that the MarR-type repressor YkvE (MhqR) regulates multiple dioxygenases/glyoxalases, oxidoreductases and the azoreductase encoding yvaB (azoR2) gene in response to thiol-specific stress conditions, such as diamide, catechol and 2-methylhydroquinone (MHQ). Here we report on the regulation of the yocJ (azoR1) gene encoding another azoreductase by the novel DUF24/MarR-type repressor, YodB after exposure to thiol-reactive compounds. DNA binding activity of YodB is directly inhibited by thiol-reactive compounds in vitro. Mass spectrometry identified YodB-Cys-S-adducts that are formed upon exposure of YodB to MHQ and catechol in vitro. This confirms that catechol and MHQ are auto-oxidized to toxic ortho- and para-benzoquinones which act like diamide as thiol-reactive electrophiles. Mutational analyses further showed that the conserved Cys6 residue of YodB is required for optimal repression in vivo and in vitro while substitution of all three Cys residues of YodB affects induction of azoR1 transcription. Finally, phenotype analyses revealed that both azoreductases, AzoR1 and AzoR2 confer resistance to catechol, MHQ, 1,4-benzoquinone and diamide. Thus, both azoreductases that are controlled by different regulatory mechanisms have common functions in quinone and azo-compound reduction to protect cells against the thiol reactivity of electrophiles.

The MarR-type repressor MhqR (YkvE) regulates multiple dioxygenases/glyoxalases and an azoreductase which confer resistance to 2-methylhydroquinone and catechol in Bacillus subtilis.

Catechol and 2-methylhydroquinone (2-MHQ) cause the induction of the thiol-specific stress response and four dioxygenases/glyoxalases in Bacillus subtilis. Using transcription factor arrays, the MarR-type regulator YkvE was identified as a repressor of the dioxygenase/glyoxalase-encoding mhqE gene. Transcriptional and proteome analyses of the DeltaykvE mutant revealed the upregulation of ykcA (mhqA), ydfNOP (mhqNOP), yodED (mhqED) and yvaB (azoR2) encoding multiple dioxygenases/glyoxalases, oxidoreductases and an azoreductase. Primer extension experiments identified sigma(A)-type promoter sequences upstream of mhqA, mhqNOP, mhqED and azoR2 from which transcription is elevated after thiol stress. DNase I footprinting analysis showed that YkvE protects a primary imperfect inverted repeat with the consensus sequence of tATCTcgaAtTCgAGATaaaa in the azoR2, mhqE and mhqN promoter regions. Analysis of mhqE-promoter-bgaB fusions confirmed the significance of YkvE binding to this operator in vivo. Adjacent secondary repeats were protected by YkvE in the azoR2 and mhqN promoter regions consistent with multiple DNA-protein binding complexes. DNA-binding activity of YkvE was not directly affected by thiol-reactive compounds in vitro. Mutational analyses showed that MhqA, MhqO and AzoR2 confer resistance to 2-MHQ. Moreover, the DeltaykvE mutant displayed a 2-MHQ and catechol resistant phenotype. YkvE was renamed as MhqR controlling a 2-MHQ and catechol-resistance regulon of B. subtilis.

Dual negative control of spx transcription initiation from the P3 promoter by repressors PerR and YodB in Bacillus subtilis.

The spx gene encodes an RNA polymerase-binding protein that exerts negative and positive transcriptional control in response to oxidative stress in Bacillus subtilis. It resides in the yjbC-spx operon and is transcribed from at least five promoters located in the yjbC regulatory region or in the yjbC-spx intergenic region. Induction of spx transcription in response to treatment with the thiol-specific oxidant diamide is the result of transcription initiation at the P(3) promoter located upstream of the spx coding sequence. Previous studies conducted elsewhere and analyses of transcription factor mutants using transformation array technology have uncovered two transcriptional repressors, PerR and YodB, that target the cis-acting negative control elements of the P(3) promoter. Expression of an spx-bgaB fusion carrying the P(3) promoter is elevated in a yodB or perR mutant, and an additive increase in expression was observed in a yodB perR double mutant. Primer extension analysis of spx RNA shows the same additive increase in P(3) transcript levels in yodB perR mutant cells. Purified YodB and PerR repress spx transcription in vitro when wild-type spx P(3) promoter DNA was used as a template. Point mutations at positions within the P(3) promoter relieved YodB-dependent repression, while a point mutation at position +24 reduced PerR repression. DNase I footprinting analysis showed that YodB protects a region that includes the P(3) -10 and -35 regions, while PerR binds to a region downstream of the P(3) transcriptional start site. The binding of both repressors is impaired by the treatment of footprinting reactions with diamide or hydrogen peroxide. The study has uncovered a mechanism of dual negative control that relates to the oxidative stress response of gram-positive bacteria.

Transcription from the P3 promoter of the Bacillus subtilis spx gene is induced in response to disulfide stress.

The spx gene of Bacillus subtilis encodes a global regulator that controls transcription initiation in response to oxidative stress by interaction with RNA polymerase (RNAP). It is located in a dicistronic operon with the yjbC gene. The spx gene DNA complements an spx null mutation with respect to disulfide stress resistance, suggesting that spx is transcribed from a promoter located in the intergenic region of yjbC and spx. Transcription of the yjbC-spx operon has been reported to be driven by four promoters, three (P(1), P(2), and P(B)) residing upstream of yjbC and one (P(M)) located in the intergenic region between yjbC and spx. Primer extension analysis uncovered a second intergenic promoter, P(3), from which transcription is elevated in cells treated with the thiol-specific oxidant diamide. P(3) is utilized by the sigma(A) form of RNA polymerase in vitro without the involvement of a transcriptional activator. Transcriptional induction from P(3) did not require an Spx-RNAP interaction and was observed in a deletion mutant lacking DNA upstream of position -40 of the P(3) promoter start site. Deletion mutants with endpoints 3' to the P(3) transcriptional start site (positions +5, +15, and +30) showed near-constitutive transcription at the induced level, indicating the presence of a negative control element downstream of the P(3) promoter sequence. Point mutations characterized by bgaB fusion expression and primer extension analyses uncovered evidence for a second cis-acting site in the P(3) promoter sequence itself. The data indicate that spx transcription is under negative transcriptional control that is reversed when disulfide stress is encountered.

The global regulator Spx functions in the control of organosulfur metabolism in Bacillus subtilis.

Spx is a global transcriptional regulator of the oxidative stress response in Bacillus subtilis. Its target is RNA polymerase, where it contacts the alpha subunit C-terminal domain. Recently, evidence was presented that Spx participates in sulfate-dependent control of organosulfur utilization operons, including the ytmI, yxeI, ssu, and yrrT operons. The yrrT operon includes the genes that function in cysteine synthesis from S-adenosylmethionine through intermediates S-adenosylhomocysteine, ribosylhomocysteine, homocysteine, and cystathionine. These operons are also negatively controlled by CymR, the repressor of cysteine biosynthesis operons. All of the operons are repressed in media containing cysteine or sulfate but are derepressed in medium containing the alternative sulfur source, methionine. Spx was found to negatively control the expression of these operons in sulfate medium, in part, by stimulating the expression of the cymR gene. In addition, microarray analysis, monitoring of yrrT-lacZ fusion expression, and in vitro transcription studies indicate that Spx directly activates yrrT operon expression during growth in medium containing methionine as sole sulfur source. These experiments have uncovered additional roles for Spx in the control of gene expression during unperturbed, steady-state growth.

A regulatory protein that interferes with activator-stimulated transcription in bacteria.

Transcriptional activator proteins in bacteria often operate by interaction with the C-terminal domain of the alpha-subunit of RNA polymerase (RNAP). Here we report the discovery of an "anti-alpha" factor Spx in Bacillus subtilis that blocks transcriptional activation by binding to the alpha-C-terminal domain, thereby interfering with the capacity of RNAP to respond to certain activator proteins. Spx disrupts complex formation between the activator proteins ResD and ComA and promoter-bound RNAP, and it does so by direct interaction with the alpha-subunit. ResD- and ComA-stimulated transcription requires the proteolytic elimination of Spx by the ATP-dependent protease ClpXP. Spx represents a class of transcriptional regulators that inhibit activator-stimulated transcription by interaction with alpha.