A Sensitive Immunodetection Assay Using Antibodies Specific to Staphylococcal Enterotoxin B Produced by Baculovirus Expression.

Staphylococcal enterotoxin B (SEB) is a potent bacterial toxin that causes inflammatory stimulation and toxic shock, thus it is necessary to detect SEB in food and environmental samples. Here, we developed a sensitive immunodetection system using monoclonal antibodies (mAbs). Our study is the first to employ a baculovirus expression vector system (BEVS) to produce recombinant wild-type SEB. BEVS facilitated high-quantity and pure SEB production from suspension-cultured insect cells, and the SEB produced was characterized by mass spectrometry analysis. The SEB was stable at 4 °C for at least 2 years, maintaining its purity, and was further utilized for mouse immunization to generate mAbs. An optimal pair of mAbs non-competitive to SEB was selected for sandwich enzyme-linked immunosorbent assay-based immunodetection. The limit of detection of the immunodetection method was 0.38 ng/mL. Moreover, it displayed higher sensitivity in detecting SEB than commercially available immunodetection kits and retained detectability in various matrices and S. aureus culture supernatants. Thus, the results indicate that BEVS is useful for producing pure recombinant SEB with its natural immunogenic property in high yield, and that the developed immunodetection assay is reliable and sensitive for routine identification of SEB in various samples, including foods.

Transcriptomic analysis of genes modulated by cyclo(L-phenylalanine-L-proline) in Vibrio vulnificus.

Diketopiperazine is produced by various organisms, including bacteria, fungi, and animals, and has been suggested as a novel signal molecule involved in the modulation of genes with various biological functions. Vibrio vulnificus, which causes septicemia in humans, produces cyclo(L-phenylalanine-L-proline) (cFP). To understand the biological roles of cFP, the effect of the compound on the expression of the total mRNA in V. vulnificus was assessed by nextgeneration sequencing. Based on the transcriptomic analysis, we classified the cFP-regulated genes into functional categories and clustered them according to the expression patterns resulted from treatment with cFP. From a total of 4,673 genes, excepting the genes encoding tRNA in V. vulnificus, 356 genes were up-regulated and 602 genes were down-regulated with an RPKM (reads per kilobase per million) value above 3. The genes most highly induced by cFP comprised those associated with the transport and metabolism of inorganic molecules, particularly iron. The genes negatively regulated by cFP included those associated with energy production and conversion, as well as carbohydrate metabolism. Noticeably, numerous genes related with biofilm formation were modulated by cFP. We demonstrated that cFP interferes significantly with the biofilm formation of V. vulnificus.

The fur-iron complex modulates expression of the quorum-sensing master regulator, SmcR, to control expression of virulence factors in Vibrio vulnificus.

The gene vvpE, encoding the virulence factor elastase, is a member of the quorum-sensing regulon in Vibrio vulnificus and displays enhanced expression at high cell density. We observed that this gene was repressed under iron-rich conditions and that the repression was due to a Fur (ferric uptake regulator)-dependent repression of smcR, a gene encoding a quorum-sensing master regulator with similarity to luxR in Vibrio harveyi. A gel mobility shift assay and a footprinting experiment demonstrated that the Fur-iron complex binds directly to two regions upstream of smcR (-82 to -36 and -2 to +27, with respect to the transcription start site) with differing affinities. However, binding of the Fur-iron complex is reversible enough to allow expression of smcR to be induced by quorum sensing at high cell density under iron-rich conditions. Under iron-limiting conditions, Fur fails to bind either region and the expression of smcR is regulated solely by quorum sensing. These results suggest that two biologically important environmental signals, iron and quorum sensing, converge to direct the expression of smcR, which then coordinates the expression of virulence factors.

Iron and quorum sensing coordinately regulate the expression of vulnibactin biosynthesis in Vibrio vulnificus.

Vibrio vulnificus is a halophilic marine pathogen associated with human diseases such as septicemia and serious wound infections. Genes vvsA and vvsB, which are co-transcribed and encode a member of the nonribosomal peptide synthase family, are required for vulnibactin biosynthesis in V. vulnificus. In this study, we found that quorum sensing represses the transcription of a vvsAB-lux reporter fusion. Gel shift assay and DNaseI footprinting experiments show that the main regulator of quorum sensing, SmcR, binds to a 22-bp region located between -40 and -19 with respect to the vvsA transcription start site. Mutation of the SmcR binding site abolishes the repression of vvsA::luxAB by SmcR. Fur represses vvsAB transcription in the presence of iron by binding to a 47-bp region located between -45 and +2 with respect to the vvsA transcription start site. A competition gel shift assay and footprinting experiment using Fur and SmcR showed that Fur binds to the vvsA promoter region with higher affinity than SmcR. Studies with the vvsAB::luxAB transcriptional fusion demonstrate that in the presence of iron, Fur is the key repressor of vvsAB transcription, whereas in iron-limited conditions, SmcR is the key regulator repressing vvsAB transcription. This study demonstrates that the Fe-Fur complex and quorum sensing cooperate to repress the transcription of vvsAB in response to iron conditions, suggesting that fine tuning of the intracellular iron level is important for the survival and pathogenicity of V. vulnificus.

Identification of a peptide that interacts with Nestin protein expressed in brain cancer stem cells.

Glioma stem cells (GSCs) are presumably major culprits for brain tumor initiation, progression, and recurrence after conventional therapies. Thus, selective targeting and eradication of GSCs may provide a promising and effective therapeutic approach. Here, we isolated a GSC-targeting (GSCT) peptide that demonstrated selective binding affinity for many undifferentiated GSCs using in vitro phage display technology. This GSCT peptide binds to isotypes of Nestin proteins specifically expressed in GSCs, enabling it to target Nestin-positive cells in human glioblastoma tissues. In human glioblastoma tissue specimens, the fluorescence-conjugated GSCT peptide could visualize putative GSC populations, showing its possible use as a diagnostic agent. GSCT peptide is also internalized into undifferentiated GSCs specifically in vitro, and moreover, intravenously injected GSCT peptide effectively penetrated into tissues, specifically accumulated in gliomas that arise from subcutaneous and orthotopic implantation, and predominantly targeted Nestin-positive cells in these tumors. Thus, our GSCT peptide may be useful for the development of more promising therapeutic and diagnostic modalities that target GSCs in brain tumors.

Comparison of the antibacterial properties of phage endolysins SAL-1 and LysK.

In spite of the high degree of amino acid sequence similarity between the newly discovered phage endolysin SAL-1 and the phage endolysin LysK, SAL-1 has an approximately 2-fold-lower MIC against several Staphylococcus aureus strains and higher bacterial cell-wall-hydrolyzing activity than LysK. The amino acid residue change contributing the most to this enhanced enzymatic activity is a change from glutamic acid to glutamine at the 114th residue.

Antibacterial and biofilm removal activity of a podoviridae Staphylococcus aureus bacteriophage SAP-2 and a derived recombinant cell-wall-degrading enzyme.

Antibacterial and biofilm removal activity of a new podoviridae Staphylococcus aureus bacteriophage (SAP-2), which belongs to the phi29-like phage genus of the Podoviridae family, and a cell-wall-degrading enzyme (SAL-2), which is derived from bacteriophage SAP-2, have been characterized. The cell-wall-degrading enzyme SAL-2 was expressed in Escherichia coli in a soluble form using a low-temperature culture. The cell-wall-degrading enzyme SAL-2 had specific lytic activity against S. aureus, including methicillin-resistant strains, and showed a minimum inhibitory concentration of about 1 microg/ml. In addition, this enzyme showed a broader spectrum of activity within the Staphylococcus genus compared with bacteriophage SAP-2 in its ability to remove the S. aureus biofilms. Thus, the cell-wall-degrading enzyme SAL-2 can be used to prevent and treat biofilm-associated S. aureus infections either on its own or in combination with other cell-wall-degrading enzymes with anti-S. aureus activity.

Opposite roles of MRF4 and MyoD in cell proliferation and myogenic differentiation.

The basic helix-loop-helix myogenic regulatory factors play critical roles in skeletal myogenesis. Among the myogenic regulatory factors (MRFs), MRF4 shows a biphasic expression pattern during the formation of myotomes, although its function remains unclear. In this study, we used BEF (spontaneously immortalized bovine embryonic fibroblast that shows myogenic differentiation by overexpression of MyoD) and C2C12 cells to investigate the function of MRF4. Ectopic expressions of MRF4 did not stimulate myogenic differentiation in the BEF and C2C12 cells, but did show a marked increase of cell proliferation, upregulation of cyclin E, and downregulation of p21WAF1. Furthermore, MRF4 was found to induce degradation of the MyoD protein, which acts as a transcriptional activator for p21WAF1, and thus indicates that MRF4 accelerates cell proliferation by suppressing MyoD-dependent p21WAF1 expression. However, forced expression of MyoD in the MRF4-overexpressing cells inhibited cell proliferation and partially induced myogenic differentiation, which suggests that MyoD is a potential negative intercessor of MRF4 in the regulation of the cell cycle. Taken together, these results indicate that MRF4 and MyoD play competitive roles in myogenesis by stimulating cell proliferation and differentiation, respectively.