The Shigella flexneri type 3 secretion system is required for tyrosine kinase-dependent protrusion resolution, and vacuole escape during bacterial dissemination.

Carole J Kuehl,Ana-Maria Dragoi,Hervé Agaisse,Raymond Schuch

doi:10.1371/journal.pone.0112738

Abstract

Shigella flexneri is a human pathogen that triggers its own entry into intestinal cells and escapes primary vacuoles to gain access to the cytosolic compartment. As cytosolic and motile bacteria encounter the cell cortex, they spread from cell to cell through formation of membrane protrusions that resolve into secondary vacuoles in adjacent cells. Here, we examined the roles of the Type 3 Secretion System (T3SS) in S. flexneri dissemination in HT-29 intestinal cells infected with the serotype 2a strain 2457T. We generated a 2457T strain defective in the expression of MxiG, a central component of the T3SS needle apparatus. As expected, the ΔmxiG strain was severely affected in its ability to invade HT-29 cells, and expression of mxiG under the control of an arabinose inducible expression system (ΔmxiG/pmxiG) restored full infectivity. In this experimental system, removal of the inducer after the invasion steps (ΔmxiG/pmxiG (Ara withdrawal)) led to normal actin-based motility in the cytosol of HT-29 cells. However, the time spent in protrusions until vacuole formation was significantly increased. Moreover, the number of formed protrusions that failed to resolve into vacuoles was also increased. Accordingly, the ΔmxiG/pmxiG (Ara withdrawal) strain failed to trigger tyrosine phosphorylation in membrane protrusions, a signaling event that is required for the resolution of protrusions into vacuoles. Finally, the ΔmxiG/pmxiG (Ara withdrawal) strain failed to escape from the formed secondary vacuoles, as previously reported in non-intestinal cells. Thus, the T3SS system displays multiple roles in S. flexneri dissemination in intestinal cells, including the tyrosine kinase signaling-dependent resolution of membrane protrusions into secondary vacuoles, and the escape from the formed secondary vacuoles.

Highlights

The intracellular pathogen Shigella flexneri is the causative agent of bacillary dysentery in humans [1]
Generation of the DmxiG strain 2457T To study the roles of the Type 3 Secretion System (T3SS) in the dissemination of S. flexneri serotype 2a strain 2457T in HT-29 intestinal cells, we generated a mutant defective in the expression of MxiG, an essential component of the needle apparatus
The DmxiG strain located in between adjacent HT-29 cells, but was not observed free in the cytosolic compartment, suggesting that the DmxiG strain was defective for invasion and/or primary vacuole escape in polarized HT-29 cells (Figure 1A)

Summary

Introduction

The intracellular pathogen Shigella flexneri is the causative agent of bacillary dysentery in humans [1]. The pathogen rapidly escapes the formed vacuoles and gains access to the cytosolic compartment where it multiplies and displays actin-based motility [3,4]. This process of invasion and escape from the primary vacuole relies on the expression of the bacterial Type 3 Secretion System (T3SS), a needle-like apparatus that translocates T3SS effector proteins directly from the bacterial cytoplasm into the cytoplasm of the eukaryotic cell [2,5]. The bacteria once again escape these vacuolar compartments, gaining access to the cytosol of adjacent cells where they continue the dissemination process. Mutations in IcsA, a central component of actin-based motility, or VirB, a master transcriptional regulator of S. flexneri virulence gene expression, result in the inability to complete this cycle of cell-to-cell spread and drastically decrease the virulence of the corresponding mutant strains [7,8]

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Conclusion