PepA binds to and negatively regulates esrB to control virulence in the fish pathogen Edwardsiella piscicida.

As an important marine fish pathogen, Edwardsiella piscicida infects a broad range of fish species and causes substantial economic losses. The EsrA-EsrB two-component system is essential for the expression of type III and type VI secretion systems (T3/T6SSs), the key virulence determinants in the bacterium. In this study, a pull-down assay with the esrB promoter as bait was performed to identify the upstream regulators of esrB. As a result, PepA, a leucyl aminopeptidase, was identified as a repressor of EsrB and T3/T6SS expression. PepA bound to the esrB promoter region and negatively regulated the production of T3/T6SS proteins in early stages. Moreover, PepA was found to affect the in vivo colonization of E. piscicida in turbot livers through the regulation of EsrB expression. Collectively, our results enhance the understanding of the virulence regulatory network and in vivo colonization mechanism of E. piscicida. One sentence summary: PepA regulates EsrB expression in Edwardsiella piscicida.

DNA methylation and NF-Y regulate Piwil1 expression during chicken spermatogenesis.

The P-element induced wimpy testis (Piwi) protein family, a subfamily of the Argonaute protein family, is involved in gene silencing and shows specific expression in spermatogenic cells. To reveal the transcriptional regulatory mechanisms of Piwil1 in chickens, we cloned sequences of the chicken Piwil1 promoter region and performed luciferase reporter and electrophoretic mobility shift assays to analyze the transcriptional activity and identify important transcriptional regulatory elements. The results showed that the region from -90 to -43 in the 5'-flanking region of Piwil1 contains a transcriptional regulatory CCAAT box that was necessary for the transcriptional activity of the Piwil1 promoter. Moreover, the transcription factor nuclear factor Y (NF-Y) was bound to the Piwil1 promoter CCAAT box specifically in germ cells. In addition, bisulfite sequencing to determine the methylation profile of the Piwil1 promoter CpG island in different spermatogenic and non-germ cell populations was performed. Compared with germ cells, non-germ cells showed increased methylation of the promoter region containing the CCAAT box, loss of NF-Y binding, and silencing of the Piwil1 locus. It is demonstrated that the specific expression of Piwil1 in chicken germ cells is regulated by the transcription factor NF-Y and differential CpG island methylation.

Promoter analysis and transcriptional regulation of a Gig2 gene in grass carp (Ctenopharyngodon idella).

Grass carp reovirus (GCRV)-induced gene 2 (Gig2) is recognized as a new antiviral factor involved in response to viral infection. However, little is known about the mechanisms behind the transcriptional regulation of Gig2 when infected by virus. In this study, the upstream promoter region of grass carp (Ctenopharyngodon idella) Gig2 gene (CiGig2) was identified by homology cloning strategy. CiGig2 promoter sequence was found to be 859 bp in length and contained three scattered IFN-stimulated response elements (ISRE). In addition, some grass carp IRFs (CiIRF1, CiIRF2 and CiIRF3) ORF sequences were subcloned into the expression plasmids pET-32a and expressed in Escherichia coli BL21, then the expressed proteins were purified by affinity chromatography with the Ni-NTA His-Bind Resin. Gel mobility shift assay was employed to screen the transcriptional regulatory factor for CiGig2. The results revealed that the recombinant polypeptides of CiIRF1, CiIRF2 and CiIRF3 bound to CiGig2 promoter with high affinity; indicating that IRF1, IRF2 and IRF3 could be the potential transcriptional regulatory factors for Gig2. Subsequently, CiGig2 promoter sequence was cloned into pGL3-Basic vector and the ORFs of CiIRF1, CiIRF2 and CiIRF3 were cloned into the expression plasmids pcDNA3.1 (+). Then, pGL3-CiGig2 promoter sequence and pcDNA3.1-CiIRFs were co-transfected into C. idella kidney (CIK) cells. The in vivo effects of CiIRFs on CiGig2 promoter were measured by dual-luciferase assays in the transfected CIK cells. Our results showed that the roles of CiIRFs were diversified in regulating CiGig2 transcription, e.g., CiIRF3 played a positive role in during this process; on the contrary CiIRF1 worked as a suppressor; however the effect of CiIRF2 on CiGig2 transcription was not obvious. For further study the roles of the three ISREs in CiGig2 transcription, we cloned three mutant CiGig2 promoters called ISRE1mut-luc (deleted ISRE1), ISRE2mut-luc (deleted ISRE2) and ISRE3mut-luc (deleted ISRE3), respectively. In vitro, gel mobility shift assays showed that all three mutant promoters also were combined with CiIRFs. CIK cells were co-transfected with CiGig2 promoter mutants (ISRE1mut-luc, ISRE2mut-luc or ISRE3mut-luc, respectively) and pcDNA3.1-IRFs. The results suggested that different ISRE played the diverse roles. ISRE2 is more important than ISRE1 and ISRE3 to the transcription of CiGig2 induced by CiIRF1. ISRE1 and ISRE3 are important to the transcription of CiGig2 induced by CiIRF2 and CiIRF3.

Activation of NF-κB contributes to production of pig-major acute protein and serum amyloid A in pigs experimentally infected with porcine circovirus type 2.

Acute phase proteins (APPs) have protective and regulatory roles in the inflammatory response. Previous studies indicate that APPs in serum change after pigs are infected with porcine circovirus type 2 (PCV2), but the mechanisms underlying APP production have remained unclear. In this present study, 35-day-old pigs were challenged with PCV2 and responses compared to an uninfected control group. To investigate the concentrations of APPs in serum and the activity of NF-κB in the liver, five pigs in the PCV2-infected group were euthanized at 14, 21 and 35days post inoculation (dpi) while four pigs were sacrificed in the control group at 0, 14, 21 and 35 days, respectively. The concentrations of pig-major acute protein (Pig-MAP), C-reactive protein (CRP) and serum amyloid A (SAA) in infected animals were increased at 14 and 21 dpi, while the concentration of alpha-1 acid glycoprotein (AGP) was lower at 35 dpi, indicating that PCV2 induced the production of APPs. Moreover, the DNA binding activity of NF-κB and expression levels of NF-κB p65 subunit (NF-κB p65) from the cytoplasm to nucleus were increased at 14 and 21 dpi in the liver of infected pigs, while the phosphorylation of IκBα (p-IκBα) in the liver was also increased at 21dpi. This demonstrated that PCV2 infection induced the activation of NF-κB. Both SAA and Pig-MAP concentrations correlated significantly with expression levels of NF-κB p65, indicating that activation of NF-κB contributes to the production of SAA and Pig-MAP.

Classical swine fever virus suppresses maturation and modulates functions of monocyte-derived dendritic cells without activating nuclear factor kappa B.

Classical swine fever virus (CSFV) compromises the host immune system, causing the severe disease of pigs. Dendritic cells (DCs) are the most potent inducers of immune responses. In the present study, we investigated the functional properties of porcine monocyte-derived DCs (Mo-DCs) affected by CSFV. Results showed that the expression of surface markers of DCs such as major histocompatibility complex class II (MHC-II), CD80, CD83 and CD86 were unimpaired, but an obviously increased expression of CD172a in DCs was noticed 48 h after CSFV infection. The expression profiles of cytokines were detected in cultured Mo-DCs after various treatments for 48 h by Q-RT-PCR. The findings suggested that CSFV infection significantly increased the mRNA expression of IL-10 and TNF-α, and inhibited IL-12 expression, with little effect on IFN-α and IFN-γ expression. We further demonstrated that CSFV was incapable of activating the nuclear factor kappa B (NF-κB) in infected DCs, which was characterized by an unvaried DNA binding activity of NF-κB, the lack of translocation of p65/RelA from the cytoplasm to the nucleus and the stabilization of p65/RelA expression. Furthermore, Western blot analysis indicated that the inactivation of NF-κB was due to the failure of IκBα degradation. The data demonstrated that CSFV could be replicated in DCs and CSFV infection could modulate the secretion of crucial co-stimulatory molecules and cytokines which down-regulated maturation of DCs, without activating NF-κB in DCs. Thus, the results suggested a possible mechanism for CSFV evasion of innate host defenses, providing the basis for understanding molecular pathways in CSFV pathogenesis.

Predictive markers in calpastatin for tenderness in commercial pig populations.

The identification of predictive DNA markers for pork quality would allow US pork producers and breeders to select genetically superior animals more quickly and efficiently for the production of consistent, high-quality meat. Genome scans have identified QTL for tenderness on SSC 2, which have been fine-mapped to the calpastatin locus. The objectives of this study were to identify the sequence variation in calpastatin that likely affects tenderness in commercial-level pig populations and to develop definitive DNA markers that are predictive of pork tenderness for use in marker-assisted selection programs. We resequenced the calpastatin regulatory and transcribed regions in pigs with divergently extreme shear force values to identify possible mutations that could affect tenderness. A total of 194 SNP were identified in this sequence, and 31 SNP were found in predicted transcription factor binding sites. We tested 131 polymorphisms in our research population and a subset (40) of these in samples of industry pigs for their association with objective measures of tenderness. We identified 4 SNP that were consistently associated with pork tenderness in all the populations studied, representing 2,826 pigs from 4 distinct populations. Gel shift assays were designed for these SNP and 12 other polymorphic sites. Six sites demonstrated a gel shift when probes were incubated with nuclear extract from muscle, heart, or testis. Four of these sites, a specificity protein 1 (Sp1) site around nucleotides 12978 and 12979, a potential thyrotroph embryonic factor (Tef) site at nucleotide 25587, an unknown site at nucleotide 48699, and myocyte enhancer factor-2 (Mef-2)/TATA sites with SNP at positions 49223 and 49228 were allele specific in binding nuclear proteins. The allele frequencies for the tender alleles were similar (0.11 to 0.36) in the 4 different commercial populations. These 4 SNP were not in complete linkage disequilibrium with each other and may independently affect calpastatin expression, tenderness, or both. These markers should be predictive of pork tenderness in industry populations.

Hemolysin EthA in Edwardsiella tarda is essential for fish invasion in vivo and in vitro and regulated by two-component system EsrA-EsrB and nucleoid protein HhaEt.

Edwardsiella tarda is a Gram-negative pathogen for hemorrhagic septicemia in fish. Recently, two-component system (TCS) EsrA-EsrB in E. tarda has been found to play key roles in regulating type III secretion system (TTSS) and type VI secretion system (T6SS). In this study, a markedly attenuated ΔesrB mutant was investigated to exhibit enhanced cell-invasion capability, as well as the increased cytotoxicity of its extracellular products (ECPs). Compared with the parental strain, the ΔesrB mutant unexpectedly displayed the significantly increased hemolytic activity, and the restoration of hemolysin production was observed in the complemented strain esrB(+). A hemolysis-associated 147 kDa protein, EthA, was found to be up-regulated in the ECPs of ΔesrB. The deletion of ethA gene in E. tarda wild type and ΔesrB strains drastically decreased their capacities in internalization of epithelial papilloma of carp (EPC) cells. These results indicated that the increased production of EthA was responsible for the enhanced cell-invasion related capabilities in ΔesrB. Furthermore, the expression of EthA in ΔesrB exhibited a temperature-induced manner, and a nucleoid protein Hha(Et) was identified to mediate ethA expression by directly binding to its promoter. These results demonstrated that the virulence determinant EthA was fully required for invasion abilities of E. tarda and was subjected to the control of a complicated and precisely regulated network primed for its invasion, colonization and infection process in fish.

Effects of fenbendazole on the murine humoral immune system.

Pinworms are highly contagious parasites that have been effectively treated in laboratory rodents with fenbendazole (FBZ). Whether FBZ has any detrimental side effects that may compromise experimental results is unknown. Here we asked whether the immune systems from young and aged mice are altered under FBZ treatment. We compared control and FBZ-treated groups of young (age, 2 to 4 mo) and old (age, 22 to 24 mo) BALB/cN mice. The treated mice received a total of 4 wk (alternating-week treatment regimen) of FBZ-medicated feed. Spleen and bone marrow were collected for immunologic assays, and heart, stomach, intestines, kidneys, and liver were evaluated by histopathology. Our results indicate that FBZ treatment has significant effects on the immune systems of mice; these effects are greater in aged mice. FBZ treatment adversely affected mRNA and protein expression of E2A (a transcription factor crucial for B lymphocytes) in activated precursor B lymphocytes obtained from the bone marrow of young and old mice. These effects were reversed by 6 wk on regular feed after the end of treatment. Activated B lymphocytes from the spleens of young and old mice showed decreased function (cell proliferation, E2A mRNA and protein expression) through the last time point of FBZ treatment but recovered by 2 to 4 wk after treatment. Our findings suggest that FBZ treatment may alter sensitive immune and molecular measures as presented here, and postponing the experimental use of mice until at least 6 wk after treatment should be considered.

Cloning and characterization of 5'-untranslated region of porcine beta casein gene (CSN2).

beta-Casein (CSN2) is a major milk protein in most mammals. The CSN2 gene is generally induced by lactogenic hormones bound to its promoter. The expression of this gene can be enhanced by signal transducers and activators of transcription (STAT) and glucocorticoid receptor (GR). Here, we analyzed the promoter and intron 1 regions of the porcine CSN2 gene. The porcine CSN2 promoter and intron 1 regions (-3098bp to +2446bp) were cloned into the pGL3-Basic vector containing the luciferase reporter gene (pCSN2-PEI). Lactogenic signals induced the transcription of porcine CSN2. By using AG490, a Janus kinase (JAK) inhibitor, we demonstrated that STAT5 positively regulates the transcription of porcine CSN2. Further, seven STAT mutants were generated by site-directed mutagenesis. By performing electrophoretic mobility shift assays (EMSAs), we located a critical element for pCSN2-PEI transcription bound to STAT5 in the -102bp to -84bp region. The construct containing only the promoter region (pCSN2-P), however, did not exert any promotive effects on transcription in two cell types-a mouse mammary epithelial cell line (HC11) and porcine mammary gland epithelial cells (PMECs). Thus, the construct containing intron 1 of porcine CSN2 exerts an elevating effect on transcription. We suggest that the transcription of porcine CSN2 is regulated by lactogenic signals via the STAT5 site (-102bp to -84bp) and intron 1.

Effects of nuclear factor-kappaB on regulation of cytokine expression and apoptosis in bovine monocytes exposed to Mycobacterium avium subsp paratuberculosis.

OBJECTIVE: To evaluate the role of the nuclear factor-kappaB (NF-kappaB) in the response of bovine monocytes to exposure to Mycobacterium avium subsp paratuberculosis (MAP). SAMPLE POPULATION: Monocytes from healthy adult Holstein cows that were known to be negative for MAP infection. PROCEDURES: Monocytes were incubated with MAP organisms with or without a specific inhibitor of the NF-kappaB pathway (pyrrolidine dithiocarbamate), and activation of the NF-kappaB pathway was detected by use of an electrophorectic mobility shift assay. The capacities of monocytes to express tumor necrosis factor (TNF)-alpha, interleukin (IL)-10, and IL-12; to acidify phagosomes; to phagocytize and kill MAP organisms; and to undergo apoptosis were evaluated. RESULTS: Addition of MAP organisms to monocytes activated the NF-kappaB pathway as indicated by increased NF-kappaB-DNA binding. Addition of pyrrolidine dithiocarbamate prevented nuclear translocation of NF-kappaB, decreased expression of TNF-alpha and IL-10, and increased IL-12 expression. Treatment of MAP-exposed monocytes with pyrrolidine dithiocarbamate increased the rate of apoptosis but failed to alter phagosome acidification, organism uptake, or organism killing by those cells. CONCLUSIONS AND CLINICAL RELEVANCE: Results indicated that NF-kappaB rapidly translocated to the nucleus after exposure of bovine monocytes to MAP organisms. These data suggest that NF-kappaB is involved in initiation of inflammatory cytokine transcription and inhibition of apoptosis but that it is not directly involved in phagosome acidification or organism killing.

Tumour necrosis factor-alpha (TNF-alpha) increases nuclear factor kappaB (NFkappaB) activity in and interleukin-8 (IL-8) release from bovine mammary epithelial cells.

Epithelia play important immunological roles at a variety of mucosal sites. We examined NFkappaB activity in control and TNF-alpha treated bovine mammary epithelial monolayers (BME-UV cells). A region of the bovine IL-8 (bIL-8) promoter was sequenced and a putative kappaB consensus sequence was identified bioinformatically. We used this sequence to analyse nuclear extracts for IL-8 specific NFkappaB activity. As a surrogate marker of NFkappaB activation, we investigated IL-8 release in two models. Firstly in BME-UV monolayers, IL-8 release in the presence of pro- and anti-inflammatory agents was determined by enzyme-linked immunosorbent assay (ELISA). Secondly, we measured IL-8 secretion from a novel model of intact mucosal sheets of bovine teat sinus. IL-8 release into bathing solutions was assessed following treatment with pro- and anti-inflammatory agents. TNF-alpha enhanced NFkappaB activity in bovine mammary epithelial monolayers. p65 NFkappaB homodimer was identified in both control and TNF-alpha treated cells. Novel sequencing of the bovine IL-8 promoter identified a putative kappaB consensus sequence, which specifically bound TNF-alpha inducible p50/p65 heterodimer. TNF-alpha induced primarily serosal IL-8 release in the cell culture model. Pre-treatment with anti-TNF or dexamethasone inhibited TNF-alpha induced IL-8 release. High dose interleukin-1beta (IL-1beta) induced IL-8 release, however significantly less potently than TNF-alpha. Bovine mammary mucosal tissue released high basal levels of IL-8 which were unaffected by TNF-alpha or IL-1beta but inhibited by both dexamethasone and anti-TNF. These data support a role for TNF-alpha in activation of NFkappaB and release of IL-8 from bovine mammary epithelial cells.

DNA binding activity of transcription factors in bronchial cells of horses with recurrent airway obstruction.

Horses with recurrent airway obstruction (RAO) present many similarities with human asthmatics including airway inflammation, hyperresponsiveness, reversible obstruction, and increased NF-kappaB expression. Studies in experimental asthma models have shown that transcriptions factors such as activator protein-1 (AP-1), GATA-3, cyclic AMP response element binding protein (CREB) and CAAT/enhancer binding protein (C/EBP) may also play an important role in airway inflammation. The purpose of this study was to measure DNA binding activity of these transcription factors in the airways of horses with RAO and to compare it to pulmonary function and bronchoalveolar lavage fluid (BALF) cytology. Seven horses with RAO and six control animals were studied during a moldy hay challenge and after 2 months at pasture. Pulmonary function, BALF cytology and transcription factors' activities in bronchial brushings were measured during hay and pasture exposures. During moldy hay challenge, RAO-affected horses developed severe airway obstruction and inflammation and a significantly higher airway AP-1 binding activity than in controls. After 2 months on pasture, pulmonary function and airway AP-1 binding activity were not different between RAO and control horses. The DNA binding activity of CREB in airways of RAO-affected horses increased significantly after 2 months at pasture and became higher than in controls. A significant positive correlation was detected between AP-1 binding activity and indicators of airway obstruction and inflammation. Airway GATA-3, CEBP and CREB binding activities were negatively correlated with indices of airway obstruction. However, contrarily to CREB binding activity, GATA-3 and CEBP binding activities were not different between RAO and control horses and were unaffected by changes in environment. These data support the view that AP-1 and CREB play a role in modulating airway inflammation in horses with RAO.

Functional characterisation of the Japanese flounder, Paralichthys olivaceus, Mx promoter.

The Japanese flounder, Paralichthys olivaceus, genome appears to encode a single Mx gene based on Southern blotting and previous cDNA studies. The 5' flanking region of the Japanese flounder Mx gene was cloned and analysed for its regulatory regions. A TATA box (-24 to -30), two interferon-stimulated response elements (ISREs) (-69 to -80 and -508 to -521) and two Sp1 sites (-563 to -572 and -994 to -1003) were identified relative to the transcription start site. The effects of various stimuli, as well as the effects of various promoter mutations, were investigated in a transient expression system using Japanese flounder (hirame) natural embryo (HINAE) cells and luciferase reporter gene constructs. Although not sensitive to LPS, ConA or PMA, reporter gene expression increased more than 10-fold after stimulation by polyinosinic:polycytidilic acid (poly I:C), an established inducer of interferon. Deletion mutational analyses revealed the ISRE closest to the transcription start site to be crucial for promoter activity. The distal ISRE, despite its relatively distant location, contributed to induce maximal promoter activity, but when alone was not sufficient by itself to elicit any significant promoter activity. An electrophoretic mobility shift assay confirmed the binding of transcription factors to both ISREs. Induction of luciferase by poly I:C was inhibited by 2-Aminopurine, a protein kinase (PKR) inhibitor, in a dose-dependent (1-10 mM) manner, suggesting that PKR may be required as a signal transducer for type I IFN signaling in fish. This Mx reporter assay may be useful for quantifying the responses and elucidating the regulation pathways of IFN type I.

Characterization of native and recombinant bovine pregnancy-associated glycoproteins.

Pregnancy-associated glycoproteins (PAGs) are products of the ruminant placenta that belong to the aspartic proteinase family. Extensive glycosylation may account for the size and heterogeneity of their molecules. To assess this we investigated the effect of glycosidase and tunicamycin treatments on native (n) and mammalian-cell generated recombinant (r) bovine PAGs. Native PAG came from explant culture conditioned medium (150 days pregnancy) while rPAG was obtained by transfection of HEK 293 cells with the bPAG-1 gene employing the PRcRSV expression vector. The undigested nPAG gave a homogenous band at 67 kDa after one-dimensional SDS-PAGE, silver staining and Western blotting, but rPAG gave dual bands at 54 and 52 kDa. PNGase F digestion of nPAG gave five bands ranging from 60 to 37 kDa and digestion of rPAG gave three bands ranging from 54 to 37 kDa. On two-dimensional electrophoresis, the undigested pI ranges of n- and rPAGs were 4.7-5.6 and 7.3-8.8, respectively. The digested isoforms of n- and rPAGs had pI ranges from 5.1 to 8.5 and 7.9-8.5, respectively. Tunicamycin treatment had no effect on the mobility of nPAG but it had a pronounced time-dependant effect on the mobility of rPAG. Our findings indicate that both n- and rPAGs have principally N-linked oligosacharides.

Differential activation of interferon regulatory factors-3 and -7 by non-cytopathogenic and cytopathogenic bovine viral diarrhoea virus.

Non-cytopathogenic bovine viral diarrhoea virus (ncpBVDV) has previously been shown to inhibit the function of interferon regulatory factor-3 in cultured cells [J. Virol. 76 (2002) 8979]. In this study, we show that, like ncpBVDV, when cells were previously exposed to cytopathogenic BVDV (cpBVDV) the appearance of an IRF-3-DNA complex from nuclear extracts that can be induced by heterologous virus infection was not observed. Infection of cells with ncpBVDV or cpBVDV resulted in neither the translocation of IRF-7 from the cytoplasm to the nucleus of infected cells, nor an inhibition of its nuclear translocation in cells super-infected by Semliki Forest Virus. We conclude that cpBVDV and ncpBVDV both share the ability to inhibit the full function of IRF-3 but neither stimulate or block the nuclear uptake of IRF-7.

Transcriptional regulation of the bovine oxytocin receptor gene.

The oxytocin receptor (OTR) is expressed in the cow uterus at high levels at estrus and at term of pregnancy. This expression appears to be controlled mostly at the transcriptional level and correlates with increasing estrogen concentration and progesterone withdrawal. Approximately 3200 base pairs of the upstream region of the bovine OTR gene were cloned and analyzed using a combination of bioinformatic, electrophoretic mobility shift (EMSA), and transfection analyses. Using nuclear proteins from high- and low-expressing tissues, EMSA indicated no significant quantitative or qualitative changes in specific DNA-protein binding, suggesting that transcription is probably controlled by signalling systems targeting constitutive factors. Using various cell types, including primary and immortalized ruminant endometrial epithelial cells, as hosts for transfection of promoter-reporter constructs showed that endogenous activity resided only in the longest, i.e., 3.2-kb, construct but not in those shorter than 1.0 kb. While estrogen appears to be important in vivo, no effect of estradiol was found on any construct directly; only when the longest 3.2-kb construct was used in combination with some cotransfected steroid receptor cofactors, e.g., SRC1e, was an estradiol-dependent effect observed. A putative interferon-responsive element (IRE) was found at approximately -2,400 from the transcription start site. This element was shown to bind mouse IRF1 and IRF2 as well as similar proteins from bovine endometrial and myometrial nuclear extracts. This element also responded to these factors when cotransfected into various cell types. The bovine equivalents to IRF1 and IRF2 were molecularly cloned from endometrial tissue and shown to be expressed in a temporal fashion, supporting the role of interferon-tau in maternal recognition of pregnancy. Of many factors tested or analyzed, these components of the IFN system are the only ones found to significantly influence the transcription of the bovine OTR gene.