CagI Is an Essential Component of the Helicobacter pylori Cag Type IV Secretion System and Forms a Complex with CagL

Helicobacter pylori, the causative agent of type B gastritis, peptic ulcers, gastric adenocarcinoma and MALT lymphoma, uses the Cag type IV secretion system to induce a strong proinflammatory response in the gastric mucosa and to inject its effector protein CagA into gastric cells. CagA translocation results in altered host cell gene expression profiles and cytoskeletal rearrangements, and it is considered as a major bacterial virulence trait. Recently, it has been shown that binding of the type IV secretion apparatus to integrin receptors on target cells is a crucial step in the translocation process. Several bacterial proteins, including the Cag-specific components CagL and CagI, have been involved in this interaction. Here, we have examined the localization and interactions of CagI in the bacterial cell. Since the cagI gene overlaps and is co-transcribed with the cagL gene, the role of CagI for type IV secretion system function has been difficult to assess, and conflicting results have been reported regarding its involvement in the proinflammatory response. Using a marker-free gene deletion approach and genetic complementation, we show now that CagI is an essential component of the Cag type IV secretion apparatus for both CagA translocation and interleukin-8 induction. CagI is distributed over soluble and membrane-associated pools and seems to be partly surface-exposed. Deletion of several genes encoding essential Cag components has an impact on protein levels of CagI and CagL, suggesting that both proteins require partial assembly of the secretion apparatus. Finally, we show by co-immunoprecipitation that CagI and CagL interact with each other. Taken together, our results indicate that CagI and CagL form a functional complex which is formed at a late stage of secretion apparatus assembly

Imaging atomizing sprays with high visibility using two-photon fluorescence laser sheet imaging

Two-photon excitation laser induced fluorescence (2p-LIF) is used here for imaging through an optically dense spray system. The main advantage of the approach is that a low level of unwanted fluorescence signal originating from multiple-light scattering is generated. This leads to high visibility and image contrast even through scattering media, thus providing faithful descriptions of the imaged fluid structures. While 2p-LIF imaging is a well-known point measurement approach in the field of life science microscopy [1], it has, to the best of the authors' knowledge, never been tested for observing atomizing sprays. We take advantage of this process here, at a macroscopic scale, by imaging a light sheet of ~1cm height. To generate enough 2p-LIF signal at such large scale and for single-shot detection, ultra-short laser pulses of high pulse energy are needed. This is obtained by using a laser system providing 25 fs pulses centered at 800 nm wavelength and having 2.5 mJ pulse energy. The technique is demonstrated by imaging a single spray plume from a 6 hole commercial Gasoline Direct Injection (GDI) system running at 200 bar injection pressure. The injected liquid is water mixed with Fluorescein dye. We show the important image contrast improvement of 2p-LIF light sheet imaging in comparison with the traditional shadowgraphy, laser sheet Mie scattering and back-fluorescence imaging. The proposed approach is very promising as a future imaging tool for detailed analysis of the dynamics of atomizing spray in the spray formation region