Nexilin is a dynamic component of Listeria monocytogenes and enteropathogenic Escherichia coli actin-rich structures

Simon Fraser University, Department of Biological Sciences, Shrum Science Centre, Room B8276, Burnaby, BC, V5A 1S6, Canada.
Cellular Microbiology (Impact Factor: 4.92). 03/2012; 14(7):1097-108. DOI: 10.1111/j.1462-5822.2012.01781.x
Source: PubMed


The bacterial pathogens Listeria monocytogenes and enteropathogenic Escherichia coli (EPEC) generate motile actin-rich structures (comet tails and pedestals) as part of their infectious processes. Nexilin, an actin-associated protein and a component of focal adhesions, has been suggested to be involved in actin-based motility. To determine whether nexilin is commandeered during L. monocytogenes and EPEC infections, we infected cultured cells and found that nexilin is crucial for L. monocytogenes invasion as levels of internalized bacteria were significantly decreased in nexilin-targeted siRNA-treated cells. In addition, nexilin is a component of the machinery that drives the formation of L. monocytogenes comet tails and EPEC pedestals. Nexilin colocalizes with stationary bacteria and accumulates at the distal portion of comet tails and pedestals of motile bacteria. We also show that nexilin is crucial for efficient comet tail formation as cells pre-treated with nexilin siRNA generate malformed comet tails, whereas nexilin is dispensable during EPEC pedestal generation. These findings demonstrate that nexilin is required for efficient infection with invasive and adherent bacteria and is key to the actin-rich structures these microbes generate.

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    ABSTRACT: Enteropathogenic Escherichia coli (EPEC) co-opt host signaling pathways and recruit numerous host proteins to motile morphological structures, called pedestals, at sites of bacterial attachment. These pedestals are hallmarks of EPEC-based disease and the identification and characterization of the functions of pedestal proteins continue to steadily increase. To identify additional constituents in an unbiased manner, we developed a strategy where EPEC pedestals were elongated artificially, severed, and then concentrated prior to their analysis by mass spectrometry (MS)-based proteomics. We identified >90 unique mammalian proteins over multiple experimental trials from our preparations. 17 predicted molecules were significantly higher in abundance (p < 0.05) when compared to both the negative controls and sample means. Validation of two identified proteins (cyclophilin A [non-actin-associated] and transgelin [actin-associated]) by immunolocalization was used to confirm our analysis and both showed enrichment at EPEC pedestals. The EPEC pedestal concentration technique developed here together with the identification of novel pedestal proteins provides not only a resource for the further characterization of molecular components within these structures, but also demonstrates that EPEC pedestals can be used as a model system for the identification of novel functions of proteins not normally thought to be at actin-based structures.
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