High throughput method for analysis of repeat number for 28 phase variable loci of C. jejuni strain NCTC11168

Mutations in simple sequence repeat tracts are a major mechanism of phase variation in several bacterial species including Campylobacter jejuni. Changes in repeat number of tracts located within the reading frame can produce a high frequency of reversible switches in gene expression between ON and OFF states. The genome of C. jejuni strain NCTC11168 contains 29 loci with polyG/polyC tracts of seven or more repeats. This protocol outlines a method for rapidly determining ON/OFF states of these 28 phase-variable loci in a large number of individual colonies. The method combines a series of multiplex PCR assays with a GeneScan assay and automated extraction of tract length, repeat number and expression state. This high throughput, multiplex assay has utility for detecting shifts in phase variation states within and between populations over time and for exploring the effects of phase variation on adaptation to differing selective pressures. An important output of this assay is combinatorial expression states that cannot be determined by other methods. This method can be adapted to analysis of phase variation in other C. jejuni strains and in a diverse range of bacterial species

The fibronectin-binding motif within FlpA facilitates Campylobacter jejuni

Campylobacter jejuni is a gram-negative, curved and rod-shaped bacterium that causes human gastroenteritis. Acute disease is associated with C. jejuni invasion of the intestinal epithelium. Epithelial cells infected with C. jejuni strains containing mutations in the FlpA and CadF fibronectin (Fn)-binding proteins exhibit reduced invasion of host cells and a C. jejuni CadF FlpA double mutant is impaired in the activation of epidermal growth factor receptor (EGFR) and Rho GTPase Rac1. Although these observations establish a role for Fn-binding proteins during C. jejuni invasion, their mechanistic contributions to invasion-associated signaling are unclear. We examined FlpA, a C. jejuni Fn-binding protein composed of three FNIII-like repeats D1, D2 and D3, to identify the interactions required for cellular adherence on pathogen-induced host cell signaling. We report that FlpA binds the Fn gelatin-binding domain via a motif within the D2 repeat. Epithelial cells infected with a flpA mutant exhibited decreased Rac1 activation and reduced membrane ruffling that coincided with impaired delivery of the secreted Cia proteins and reduced cell association. Phosphorylation of the Erk1/2 kinase, a downstream effector of EGFR signaling, was specifically associated with FlpA-mediated activation of β(1)-integrin and EGFR signaling. In vivo experiments revealed that FlpA is necessary for C. jejuni disease based on bacterial dissemination to the spleen of IL-10(−/−) germ-free mice. Thus, a novel Fn-binding motif within FlpA potentiates activation of Erk1/2 signaling via β(1)-integrin during C. jejuni infection

Sol-gel based optically active phenolphthalein encapsulated nanomatrices for sensing application

In this work, synthesis and characterization of phenolphthalein-immobilized titania (T-phph) and silica–titania (ST-phph) nanomatrix is reported. The thin films are deposited by sol–gel method at low temperature. The effect of host–guest chemistry in matrices, on the surface structures, optical and sensing activity of the resultant thin films is studied. The phenolphthalein-immobilized fabricated nanoparticles/nanomatrices are analyzed by field emission scanning electron microscope, energy-dispersive X-ray spectroscopy, atomic-force microscopy, X-ray diffraction, Fourier transform infrared spectroscopy, surface analysis, thermogravimetric analysis, and UV–Vis spectroscopy. Thermally stable and high surface area homogeneous nanoparticles, containing nanocrystalline anatase phase with low refractive index (1.58), low roughness (5.5 nm), and high transparency (95 %) are obtained for phenolphthalein-immobilized ST-phph nanomatrix. Moreover, smaller nanoparticles (56–121 nm) with good incorporation of dye and good response of sensing are obtained. The sensor response is optimized at pH 12 with 10.1 pKa value at 555 nm. Graphical Abstract: [Figure not available: see fulltext