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Shigella flexneri is an intracellular pathogen that disseminates in colonic epithelial cells through actin-based motility and formation of membrane protrusions at cell-cell contacts, that project into adjacent cells and resolve into vacuoles, from which the pathogen escapes, thereby achieving cell-to-cell spread. Actin nucleation at the bacterial pole relies on the recruitment of the nucleation-promoting factor N-WASP, which activates the actin nucleator ARP2/3. In cells, the vast majority of N-WASP exists as a complex with WIP. The involvement of WIP in N-WASP-dependent actin-based motility of various pathogens, including vaccinia virus and S. flexneri, has been highly controversial. Here, we show that WIPF2 was the only WIP family member expressed in the human colonic epithelial cell line HT-29, and its depletion impaired S. flexneri dissemination. WIPF2 depletion increased the number of cytosolic bacteria lacking actin tails (non-motile) and decreased the velocity of motile bacteria. This correlated with a decrease in the recruitment of N-WASP to the bacterial pole, and among N-WASP-positive bacteria, a decrease in actin tail-positive bacteria, suggesting that WIPF2 is required for N-WASP recruitment and activation at the bacterial pole. In addition, when motile bacteria formed protrusions, WIPF2 depletion decreased the number of membrane protrusions that successfully resolved into vacuoles.

The Actin-Related Protein 2/3 (ARP2/3) complex is an actin nucleator that generates a branched actin network in mammalian cells. In addition to binding nucleation promoting factors, LeClaire et al. demonstrated that its phosphorylation state is essential key for its activity ( LeClaire et al., 2008 ). In cells, the ARP2/3 complex is phosphorylated on threonine and tyrosine residues of the ARP2, ARP3, and ARPC1 subunits ( Vadlamudi et al., 2004 ; LeClaire et al., 2008 ; Narayanan et al., 2011 ; LeClaire et al., 2015 ). In particular, phosphorylation of threonine 237 and 238 of the ARP2 subunit is necessary to allow a change in the ARP2/3 complex structure to its active conformation ( Narayanan et al., 2011 ; LeClaire et al., 2015 ). While important for many functions in eukaryotic cells, ARP2/3 complex activity also benefits several cellular pathogens (Haglund and Welch, 2011; Welch and Way, 2013). Recently, we demonstrated that the bacterial pathogen, Legionella pneumophila, manipulates ARP2/3 complex phosphorylation state using a bacterial protein kinase injected in host cell cytoplasm ( Michard et al., 2015 ). Here, we describe how to test the ability of a bacterial protein kinase or another protein kinase to phosphorylate the ARP2/3 complex in an in vitro context. First, the ARP2/3 complex and the bacterial protein kinase are produced and purified. Then, the purified proteins are incubated in the presence of ATP, and the ARP2/3 phosphorylation level is analyzed by Western blot.

Legionella pneumophila, the etiological agent of legionellosis, replicates within phagocytic cells. Crucial to biogenesis of the replicative vacuole is the Dot/Icm type 4 secretion system, which translocates a large number of effectors into the host cell cytosol. Among them is LegK2, a protein kinase that plays a key role in Legionella infection. Here, we identified the actin nucleator ARP2/3 complex as a target of LegK2. LegK2 phosphorylates the ARPC1B and ARP3 subunits of the ARP2/3 complex. LegK2-dependent ARP2/3 phosphorylation triggers global actin cytoskeleton remodeling in cells, and it impairs actin tail formation by Listeria monocytogenes, a well-known ARP2/3-dependent process. During infection, LegK2 is addressed to the Legionella-containing vacuole surface and inhibits actin polymerization on the phagosome, as revealed by legK2 gene inactivation. Consequently, LegK2 prevents late endosome/lysosome association with the phagosome and finally contributes to remodeling of the bacterium-containing phagosome into a replicative niche. The inhibition of actin polymerization by LegK2 and its effect on endosome trafficking are ARP2/3 dependent since it can be phenocopied by a specific chemical inhibitor of the ARP2/3 complex. Thus, LegK2-ARP2/3 interplay highlights an original mechanism of bacterial virulence with an unexpected role in local actin remodeling that allows bacteria to control vesicle trafficking in order to escape host defenses., IMPORTANCE Deciphering the individual contribution of each Dot/Icm type 4 secretion system substrate to the intracellular life-style of L. pneumophila remains the principal challenge in understanding the molecular basis of Legionella virulence. Our finding that LegK2 is a Dot/Icm effector that inhibits actin polymerization on the Legionella-containing vacuole importantly contributes to the deciphering of the molecular mechanisms evolved by Legionella to counteract the endocytic pathway. Indeed, our results highlight the essential role of LegK2 in preventing late endosomes from fusing with the phagosome. More generally, this work is the first demonstration of local actin remodeling as a mechanism used by bacteria to control organelle trafficking. Further, by characterizing the role of the bacterial protein kinase LegK2, we reinforce the concept that posttranslational modifications are key strategies used by pathogens to evade host cell defenses.

International audience; Legionella pneumophila is a paradigm of highly adapted intravacuolar pathogens that acquired the rare ability to replicate within a phagocytic cell. Here, we review recent progress about the role of Type 4 secretion system effectors involved in the biogenesis of the replicative niche, the Legionella containing vacuole.