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Tra Proteins Characteristic of F-Like Type IV Secretion Systems Constitute an Interaction Group by Yeast Two-Hybrid Analysis. Robin L. Harris, 2004.Using yeast two-hybrid screens, we have defined an interaction group of six Tra proteins encoded by the F plasmid and required by F+ cells to elaborate F pili . The six proteins are TraH, TraF, TraW, TraU, TrbI, and TrbB . Except for TrbI, these proteins were all identified as hallmarks of F-like type IV secretion systems (TFSSs), with no homologues among TFSS genes of P-type or I-type systems (T . Lawley, W . Klimke, M . Gubbins, and L . Frost, FEMS Microbiol . Lett . 224:1-15, 2003) . Also with the exception of TrbI, which is an inner membrane protein, the remaining proteins are or are predicted to be periplasmic . TrbI consists of one membrane-spanning segment near its N terminus and an 88-residue, hydrophilic domain that extends into the periplasm . Hence, the proteins of this group probably form a periplasmic cluster in Escherichia coli . The interaction network identifies TraH as the most highly connected node, with two-hybrid links to TrbI, TraU, and TraF . As measured by transcriptional activation of lacZ, the TrbI-TraH interaction in Saccharomyces cerevisiae requires the TraH amino acid segment from residues 193 to 225 . The TraU and TraF interactions are localized to C-terminal segments of TraH (amino acids 315 to 458 for TraF and amino acids 341 to 458 for TraU) . The TrbI-TraH interaction with full-length (less the signal peptide) TraH is weak but increases 40-fold with N-terminal TraH deletions; the first 50 amino acids appear to be critical for inhibiting TrbI binding in yeast . Previous studies by others have shown that, with the exception of trbB mutations, which do not affect the elaboration or function of F pili under laboratory conditions, a mutation in any of the other genes in this interaction group alters the number or length distribution of F pili . We propose a model whereby one function of the TraH interaction group is to control F-pilus extension and retraction . Mouse Model of Cervicovaginal Toxicity and Inflammation for Preclinical Evaluation of Topical Vaginal Microbicides. Bradley J. Catalone, 2004.Clinical trials evaluating the efficacy of nonoxynol-9 (N-9) as a topical microbicide concluded that N-9 offers no in vivo protection against human immunodeficiency virus type 1 (HIV-1) infection, despite demonstrated in vitro inactivation of HIV-1 by N-9 . These trials emphasize the need for better model systems to determine candidate microbicide effectiveness and safety in a preclinical setting . To that end, time-dependent in vitro cytotoxicity, as well as in vivo toxicity and inflammation, associated with N-9 exposure were characterized with the goal of validating a mouse model of microbicide toxicity . In vitro studies using submerged cell cultures indicated that human cervical epithelial cells were inherently more sensitive to N-9-mediated damage than human vaginal epithelial cells . These results correlated with in vivo findings obtained by using Swiss Webster mice in which intravaginal inoculation of 1% N-9 or Conceptrol gel (containing 4% N-9) resulted in selective and acute disruption of the cervical columnar epithelial cells 2 h postapplication accompanied by intense inflammatory infiltrates within the lamina propria . Although damage to the cervical epithelium was apparent out to 8 h postapplication, these tissues resembled control tissue by 24 h postapplication . In contrast, minimal damage and infiltration were associated with both short- and long-term exposure of the vaginal mucosa to either N-9 or Conceptrol . These analyses were extended to examine the relative toxicity of polyethylene hexamethylene biguanide (PEHMB), a polybiguanide compound under evaluation as a candidate topical microbicide . In similar studies, in vivo exposure to 1% PEHMB caused minimal damage and inflammation of the genital mucosa, a finding consistent with the demonstration that PEHMB was >350-fold less cytotoxic than N-9 in vitro . Collectively, these studies highlight the murine model of toxicity as a valuable tool for the preclinical assessment of toxicity and inflammation associated with exposure to candidate topical microbicides . Overexpression of the waaZ Gene Leads to Modification of the Structure of the Inner Core Region of Escherichia coli Lipopolysaccharide, Truncation of the Outer Core, and Reduction of the Amount of O Polysaccharide on the Cell Surface. Emilisa Frirdich, 2003.The waa gene cluster is responsible for the biosynthesis of the lipopolysaccharide (LPS) core region in Escherichia coli and Salmonella. Homologs of the waaZ gene product are encoded by the waa gene clusters of Salmonella enterica and E . coli strains with the K-12 and R2 core types . Overexpression of WaaZ in E . coli and S . enterica led to a modified LPS structure showing core truncations and (where relevant) to a reduction in the amount of O-polysaccharide side chains . Mass spectrometry and nuclear magnetic resonance spectroscopy were used to determine the predominant LPS structures in an E . coli isolate with an R1 core (waaZ is lacking from the type R1 waa gene cluster) with a copy of the waaZ gene added on a plasmid . Novel truncated LPS structures, lacking up to 3 hexoses from the outer core, resulted from WaaZ overexpression . The truncated molecules also contained a KdoIII residue not normally found in the R1 core .
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