The Vibrio cholerae var regulon encodes a metallo-β-lactamase and an antibiotic efflux pump, which are regulated by VarR, a LysR-type transcription factor

The genome sequence of V. cholerae O1 Biovar Eltor strain N16961 has revealed a putative antibiotic resistance (var) regulon that is predicted to encode a transcriptional activator (VarR), which is divergently transcribed relative to the putative resistance genes for both a metallo-β-lactamase (VarG) and an antibiotic efflux-pump (VarABCDEF). We sought to test whether these genes could confer antibiotic resistance and are organised as a regulon under the control of VarR. VarG was overexpressed and purified and shown to have β-lactamase activity against penicillins, cephalosporins and carbapenems, having the highest activity against meropenem. The expression of VarABCDEF in the Escherichia coli (ΔacrAB) strain KAM3 conferred resistance to a range of drugs, but most significant resistance was to the macrolide spiramycin. A gel-shift analysis was used to determine if VarR bound to the promoter regions of the resistance genes. Consistent with the regulation of these resistance genes, VarR binds to three distinct intergenic regions, varRG, varGA and varBC located upstream and adjacent to varG, varA and varC, respectively. VarR can act as a repressor at the varRG promoter region; whilst this repression was relieved upon addition of β-lactams, these did not dissociate the VarR/varRG-DNA complex, indicating that the de-repression of varR by β-lactams is indirect. Considering that the genomic arrangement of VarR-VarG is strikingly similar to that of AmpR-AmpC system, it is possible that V. cholerae has evolved a system for resistance to the newer β-lactams that would prove more beneficial to the bacterium in light of current selective pressures

Ectodomain shedding of human Nogo-66 receptor homologue-1 by zinc metalloproteinases.

The Nogo-66 receptor (NgR) plays a pivotal role in the inhibition of neuroregeneration as the receptor for multiple neurite outgrowth inhibitors such as Nogo-A. We have previously shown that NgR undergoes zinc metalloproteinase-mediated ectodomain shedding in neuroblastoma cells. Here, we demonstrate that the NgR-related protein NgR homologue-1 is released from neuroblastoma cells as a full-length ectodomain (NgRH1-ecto) and an N-terminal fragment (NTF-NgRH1) containing the leucine-rich repeat region of the protein. Inhibitors of the major protease classes failed to block the release of NgRH1-ecto, suggesting that this occurs via a protease-independent mechanism, presumably by a phospholipase-like enzyme. The release of NTF-NgRH1 was blocked by a hydroxamate-based zinc metalloproteinase inhibitor and tissue inhibitor of metalloproteinases-2 and -3, but not -1, implicating the involvement of membrane-type matrix metalloproteinases in this process. Our findings thus highlight the parallels between the ectodomain shedding of NgRH1 and that previously described for NgR

VarR binds to the varBC intergenic region

<b>EMSA of 50ng VarR/ 0.08ng 25bp varBC IR DNA (labelled complexed with titrations of unlabelled 30bp varBC DNA. </b><div>Lanes 1 and 2, 0ng and 50ng VarR with 0.08ng 30bp <i>varRG </i>IR DNA (control). Lanes 3 to 12, competitive assay of 50ng VarR/0.08ng 25bp <i>varBC</i> complexed with titrations of unlabelled 0.08ng 25bp <i>varBC</i> IR DNA (0, 0.125, 0.25, 0.5, 1, 2, 5, 10, 20, 40ng, respectively).</div><div><br></div><div><b><br></b></div

Erythromycin does not dissociate the VarR/varBC DNA complex

<b>EMSA of VarR/25bp varBC IR DNA complex with increasing concentrations of erythromycin. </b>Lane 1 0.08ng 25bp varBC IR DNA only. Lanes 2 to 16 50ng VarR/0.08ng 25 bp varBC IR DNA complex with increasing titrations erythromycin (0, 0.5, 1, 2, 4, 8, 16, 32, 64, 128, 256,512, 1024ng, 10, 100ug, respectively).<div><br></div

Penicillin G does not dissociate the VarR/varRG DNA complex

<div><b>EMSA of VarR/30bp <i>varRG</i> IR DNA complex with increasing titration of Penicillin G. </b></div><div>Lane 1 0.08ng 30bp <i>varRG </i>IR DNA only. Lanes 2 to 16 50ng VarR/0.08ng 30bp varRG DNA complex with increasing titrations of Penicillin G (0, 0.5, 1, 2, 4, 8, 16, 32, 64, 128, 256, 512, 1024ng, 10, 100 ug, respectively).</div

Characterization of white matter damage in animal models of multiple sclerosis by magnetization transfer ratio and quantitative mapping of the apparent bound proton fraction f.

Quantitative magnetization transfer magnetic resonance imaging (qMT-MRI) can be used to improve detection of white matter tissue damage in multiple sclerosis (MS) and animal models thereof. To study the correlation between MT parameters and tissue damage, the magnetization transfer ratio (MTR), the parameter f* (closely related to the bound proton fraction) and the bound proton transverse relaxation time T(2B) of lesions in a model of focal experimental autoimmune encephalomyelitis (EAE) were measured on a 7T animal scanner and data were compared with histological markers indicative for demyelination, axonal density, and tissue damage. A clear spatial correspondence was observed between reduced values of MTR and demyelination in this animal model. We observed two different levels of MTR and f* reduction for these lesions. One was characterized by a pronounced demyelination and the other corresponded to a more severe loss of the cellular matrix. Changes in f* were generally more pronounced than those of MTR in areas of demyelination. Moreover, a reduction of f* was already observed for tissue where MTR was virtually normal. No changes in T(2B) were observed for the lesions. We conclude that MTR and qMT mapping are efficient and reliable readouts for studying demyelination in animal models of MS, and that the analysis of regional f* might be even superior to the analysis of MTR values. Therefore, quantitative mapping of f* from human brains might also improve the detection of white matter damage in MS

Zinc metalloproteinase-mediated cleavage of the human Nogo-66 receptor.

The central nervous system myelin components oligodendrocyte-myelin glycoprotein, myelin-associated glycoprotein and the Nogo-66 domain of Nogo-A inhibit neurite outgrowth by binding the neuronal glycosyl-phosphatidylinositol-anchored Nogo-66 receptor (NgR) that transduces the inhibitory signal to the cell interior via a transmembrane co-receptor, p75NTR. Here, we demonstrate that human NgR expressed in human neuroblastoma cells is constitutively cleaved in a post-ER compartment to generate a lipid-raft associated C-terminal fragment that is present on the cell surface and a soluble N-terminal fragment that is released into the medium. Mass spectrometric analysis demonstrated that the N-terminal fragment terminated just after the C-terminus of the ligand-binding domain of NgR. In common with other shedding mechanisms, the release of this fragment was blocked by a hydroxamate-based inhibitor of zinc metalloproteinases, but not by inhibitors of other protease classes and up-regulated by treatment with the cellular cholesterol depleting agent methyl-beta-cyclodextrin. The N-terminal fragment bound Nogo-66 and blocked Nogo-66 binding to cell surface NgR but failed to associate with p75NTR, indicative of a role as a Nogo-66 antagonist. Furthermore, the N- and C-terminal fragments of NgR were detectable in human brain cortex and the N-terminal fragment was also present in human cerebrospinal fluid, demonstrating that NgR proteolysis occurs within the human nervous system. Our findings thus identify a potential cellular mechanism for the regulation of NgR function at the level of the receptor

A diagrammatic representation of the <i>var</i> operon.

<p>The locality of the β-lactamase, <i>varG</i>, the MDR <i>varABCDEF</i> transporter complex and the divergently transcribed regulatory <i>varR</i> genes are shown. Arrows indicate orientation of transcription. Three intergenic regions <i>varRG</i>, <i>varGA</i> and <i>varBC</i> to which VarR is hypothesised to regulate transcription are also illustrated.</p

VarR regulation of <i>Cm</i>^<i>R</i> expression.

<p>VarR regulation of <i>Cm</i>^<i>R</i> expression.</p