Conjugal transfer of the nopaline-type Agrobacterium Ti plasmid pTiC58 is regulated by a trasriptional activator, TraR, and a diffusible signal molecule, conjugation factor (CF). CF is a member of a family of substituted homoserine lactones (HSLs) that act as coinducers for regulating gene expression in diverse Gram-negative bacteria by a mechanism called antoinduction. In Vibrio fischeur HSL production is conferred by the luxI gene. Homologues of this gene are responsible for HSL production by other Gram-negative bacteria. A gene that we call tral, conferring production of material with CF activity, was localized to a 1-kb region at the upstream end of tra of pTiC58. Spectroscopy showed that the activity was authentic CF. Sequence analysis showed that Otn could encode a protein of 211 amino acids, TraI, that is related to the proteins responsible for HSL production by other bacteria. A second, partial open reading frame immiatel downstream of tral could encode a protein related to TrbB of plasmid RP4, which is required for conjugal transer. Transcription of tnal and of the downstream tra3 genes requires TraR and CF and initiates from the tral promoter. The results show that tnd is responsible for CF production, that it is the first gene of the n3 operon, and that expression of this operon is regulated by autoinduction.

Plasmid conjugation systems are composed of two components, the DNA transfer and replication system, or Dtr, and the mating pair formation system, or Mpf. During conjugal transfer an essential factor, called the coupling protein, is thought to interface the Dtr, in the form of the relaxosome, with the Mpf, in the form of the mating bridge. These proteins, such as TraG from the IncP1 plasmid RP4 (TraG RP4 ) and TraG and VirD4 from the conjugal transfer and T-DNA transfer systems of Ti plasmids, are believed to dictate specificity of the interactions that can occur between different Dtr and Mpf components. The Ti plasmids of Agrobacterium tumefaciens do not mobilize vectors containing the oriT of RP4, but these IncP1 plasmid derivatives lack the trans-acting Dtr functions and TraG RP4 . A. tumefaciens donors transferred a chimeric plasmid that contains the oriT and Dtr genes of RP4 and the Mpf genes of pTiC58, indicating that the Ti plasmid mating bridge can interact with the RP4 relaxosome. However, the Ti plasmid did not mobilize transfer from an IncQ relaxosome. The Ti plasmid did mobilize such plasmids if TraG RP4 was expressed in the donors. Mutations in traG RP4 with defined effects on the RP4 transfer system exhibited similar phenotypes for Ti plasmid-mediated mobilization of the IncQ vector. When provided with VirD4, the tra system of pTiC58 mobilized plasmids from the IncQ relaxosome. However, neither TraG RP4 nor VirD4 restored transfer to a traG mutant of the Ti plasmid. VirD4 also failed to complement a traG RP4 mutant for transfer from the RP4 relaxosome or for RP4-mediated mobilization from the IncQ relaxosome. TraG RP4 -mediated mobilization of the IncQ plasmid by pTiC58 did not inhibit Ti plasmid transfer, suggesting that the relaxosomes of the two plasmids do not compete for the same mating bridge. We conclude that TraG RP4 and VirD4 couples the IncQ but not the Ti plasmid relaxosome to the Ti plasmid mating bridge. However, VirD4 cannot couple the IncP1 or the IncQ relaxosome to the RP4 mating bridge. These results support a model in which the coupling proteins specify the interactions between Dtr and Mpf components of mating systems.Plasmid conjugation conceptually can be divided into two functions. In the first, the DNA is processed by a complex of proteins, one of which introduces a single-strand nick at the nic site within the oriT recognition sequence. Called the relaxosome, the proteins of this complex are coded for by genes of the Dtr (DNA transfer and replication) component of the transfer system. In the second, the nucleoprotein transfer intermediate comprised of the nicked strand covalently linked at the 5Ј end to the relaxase is secreted from the donor directly into the recipient via a bridge that forms between the mating pair. This translocation apparatus is a complex membraneassociated structure coded for by the Mpf (mating pair formation) genes.The relaxosome of one conjugal plasmid may or may not be transferrable by the Mpf system of another. Specificity is conferred, in part, by a...

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Objective To characterize long-term repopulation of peripheral immune cells following alemtuzumab-induced lymphopenia in relapsing-remitting MS (RRMS), with a focus on regulatory cell types, and to explore associations with clinical outcome measures. Methods The project was designed as a multicenter add-on longitudinal mechanistic study for RRMS patients enrolled in CARE-MS II, CARE-MS II extension at the University of Southern California and Stanford University, and an investigator-initiated study conducted at the Universities of British Columbia and Chicago. Methods involved collection of blood at baseline, prior to alemtuzumab administration, and at months 5, 11, 17, 23, 36, and 48 post-treatment. T cell, B cell, and natural killer (NK) cell subsets, chemokine receptor expression in T cells, in vitro cytokine secretion patterns, and regulatory T cell (Treg) function were assessed. Clinical outcomes, including expanded disability status score (EDSS), relapses, conventional magnetic resonance imaging (MRI) measures, and incidents of secondary autoimmunity were tracked. Results Variable shifts in lymphocyte populations occurred over time in favor of CD4+ T cells, B cells, and NK cells with surface phenotypes characteristic of regulatory subsets, accompanied by reduced ratios of effector to regulatory cell types. Evidence of increased Treg competence was observed after each treatment course. CD4+ and CD8+ T cells that express CXCR3 and CCR5 and CD8+ T cells that express CDR3 and CCR4 were also enriched after treatment, indicating heightened trafficking potential in activated T cells. Patterns of repopulation were not associated with measures of clinical efficacy or secondary autoimmunity, but exploratory analyses using a random generalized estimating equation (GEE) Poisson model provide preliminary evidence of associations between pro-inflammatory cell types and increased risk for gadolinium (Gd+) enhancing lesions, while regulatory subsets were associated with reduced risk. In addition, the risk for T2 lesions correlated with increases in CD3+CD8+CXCR3+ cells. Conclusions Lymphocyte repopulation after alemtuzumab treatment favors regulatory subsets in the T cell, B cell, and NK cell compartments. Clinical efficacy may reflect the sum of interactions among them, leading to control of potentially pathogenic effector cell types. Several immune measures were identified as possible biomarkers of lesion activity. Future studies are necessary to more precisely define regulatory and effector subsets and their contributions to clinical efficacy and risk for secondary autoimmunity in alemtuzumab-treated patients, and to reveal new insights into mechanisms of immunopathogenesis in MS. Trial registration Parent trials for this study are registered with ClinicalTrials.gov: CARE-MS II: NCT00548405, CARE-MS II extension: NCT00930553 and ISS: NCT01307332.

Biofilms contribute to persistent bacterial infections as well as formidable resistances to conventional antibiotics. However, it is still a major challenge to establish an advanced antibacterial nanoplatform that can efficiently eradicate biofilms while overcoming bacterial resistances. Taking advantage of the stimuli-responsive technique and the magnetic guidance strategy, here we present a highly efficient nanoplatform for planktonic inactivation and biofilm disruption. The multilayer films consisting of antibiotic gentamicin (Gen), tannic acid, and silver nanoparticles (AgNPs) were fabricated and coated on magnetic nanoparticles via electrostatic interactions. To achieve controlled drug release and improved biocompatibility, biodegradable hyaluronic acid was capped on the outer surface as a responsive shell. In vitro release profiles suggested that the nanocomposites showed both enzyme and pH-responsive release properties. The nanoplatform could be employed as a powerful nanocarrier for small molecular Gen and AgNPs delivery and on-demand release in response to bacterial infection microenvironment. The nanocomposites also showed satisfying antibacterial capacities against planktonic Gram-positive Staphylococcus aureus and Gram-negative Escherichia coli. Intriguingly, with magnetic field navigation (NdFeB, 2000 gauss), the nanocomposites could be guided to handily penetrate into S. aureus biofilm and performed dual-responsive release, showing significantly enhanced biofilm disruption. Moreover, excess reactive oxygen species production resulting from the nanocomposites contributed to the decomposition of biofilm matrix and ultimate biofilm eradication. As a consequence, the ingenious antibacterial nanoplatform could be promising for combating biofilm infections while overcoming bacterial resistances with extra environmental factors such as magnetic field.

Carbon quantum dots derived from gentamicin sulfate show low drug resistance, eradication of mature Staphylococcus aureus biofilm and low toxicity to mammalian cells.

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