Hierarchies of Host Factor Dynamics at the Entry Site of Shigella flexneri during Host Cell Invasion

Abstract

<jats:title>ABSTRACT</jats:title><jats:p><jats:named-content xmlns:xlink="http://www.w3.org/1999/xlink" content-type="genus-species" xlink:type="simple">Shigella flexneri</jats:named-content>, the causative agent of bacillary dysentery, induces massive cytoskeletal rearrangement, resulting in its entry into nonphagocytic epithelial cells. The bacterium-engulfing membrane ruffles are formed by polymerizing actin, a process activated through injected bacterial effectors that target host small GTPases and tyrosine kinases. Once inside the host cell,<jats:named-content xmlns:xlink="http://www.w3.org/1999/xlink" content-type="genus-species" xlink:type="simple">S. flexneri</jats:named-content>escapes from the endocytic vacuole within minutes to move intra- and intercellularly. We quantified the fluorescence signals from fluorescently tagged host factors that are recruited to the site of pathogen entry and vacuolar escape. Quantitative time lapse fluorescence imaging revealed simultaneous recruitment of polymerizing actin, small GTPases of the Rho family, and tyrosine kinases. In contrast, we found that actin surrounding the vacuole containing bacteria dispersed first from the disassembling membranes, whereas other host factors remained colocalized with the membrane remnants. Furthermore, we found that the disassembly of the membrane remnants took place rapidly, within minutes after bacterial release into the cytoplasm. Superresolution visualization of galectin 3 through photoactivated localization microscopy characterized these remnants as small, specular, patchy structures between 30 and 300 nm in diameter. Using our experimental setup to track the time course of infection, we identified the<jats:named-content xmlns:xlink="http://www.w3.org/1999/xlink" content-type="genus-species" xlink:type="simple">S. flexneri</jats:named-content>effector IpgB1 as an accelerator of the infection pace, specifically targeting the entry step, but not vacuolar progression or escape. Together, our studies show that bacterial entry into host cells follows precise kinetics and that this time course can be targeted by the pathogen.</jats:p>

Maintenance of vacuole integrity by bacterial pathogens

“…The first assay takes advantage of the observation that cytosolic galectins are specifically recruited to the remnants of ruptured vacuoles [48,49]. Cells expressing GFPGalectin 3 have been successfully used to follow the escape of Shigella into the cytosol in real time [50,51]. The second technique also utilizes fluorescent microscopy to detect vacuole rupture and takes advantage of the ability of Gram negative bacteria to display β-lactamases on their surface.…”

Molecular Mechanisms of Host Cytoskeletal Rearrangements by Shigella Invasins

“…In separate functional studies, the invasive capacity and the pace of invasion were reduced by 50% and 75%, respectively, by Shigella strains with IpgB1 deletions [22,72]. However, deletion of IpgB2 did not cause any phenotypic change in the invasive capacity [22,71].…”

Bacterial Internalization, Localization, and Effectors Shape the Epithelial Immune Response during Shigella flexneri Infection

“…Human epithelial HeLa cells (ATCC) were grown in Dulbecco's modified Eagle's medium (DMEM) containing 10% decomplemented fetal calf serum (FCS) at 37°C, 5% CO 2 . Transfection of HeLa cells with p-mOrangeempty (7,8), p-mOrange-bla, p-mOrange-Actin, or p-YFP-p65 (a kind gift of R. Weil) was performed using Fugene 6 transfection reagent (Promega) according to the manufacturer's instructions 48 h prior to experimentation.…”

“…Shigella uses a virulence plasmid-encoded type 3 secretion system (T3SS) that injects sets of primary and secondary effectors into the host cytosol, which are directly induced by the bacterial regulators/effectors VirB and MxiE, respectively, and which interfere with diverse host cell signaling pathways (5,6). It attaches to the epithelial cell membrane, where it induces actin focus formation and subsequent internalization into a pathogencontaining vacuole (7). Then, within 5 to 15 min, Shigella triggers vacuolar rupture and invades the host cytosolic compartment as a niche for replication and intracellular spread (8).…”