A system for identifying post‐invasion functions of invasion genes: requirements for the Mxi–Spa type III secretion pathway of Shigella flexneri in intercellular dissemination

Abstract

SummaryInvasion and intercellular spread are hallmarks of Shigella pathogenicity. Invasion of the eukaryotic cell cytosol requires a type III secretion system (Mxi± Spa) and its cognate set of secreted Ipa invasins. Once intracellular, the IcsA protein directs a form of actin-based motility that helps to drive intracellular bacterial movement, formation of cellular protrusions and cell-to-cell spread. Work in our laboratory has focused on identifying additional factors required for this intercellular form of dissemination. In this study, we sought to identify novel contributions of the type III secretion pathway to post-invasion-speci®c processes, distinct from its previously characterized roles in invasion. Studies of post-invasion Ipa and Mxi±Spa functions are complicated by an absolute requirement for these virulence proteins in invasion. To circumvent this problem, we developed a system called TIER (for test of intracellular expression requirements), whereby speci®c ipa, mxi or spa loci are transiently expressed before infection of tissue culture cell monolayers (thus supporting invasion), but then repressed after invasion in the intracellular environment. Such invasive type III secretion mutants (called TIER mutants) were severely restricted in their ability to spread intercellularly and form plaques in con¯uent tissue culture cell monolayers. Intercellular spread defects were associated with the repression of most type III pathway components examined, including structural (MxiM and Spa33), secreted effector (IpaB, IpaC and IpaD) and regulatory elements (VirF and VirB). A kinetic analysis of bacterial growth in L2 cell monolayers showed that each of the TIER mutants was defective with respect to long-term intracellular proliferation and viability. Examination of TIER mutant-infected monolayers by electron microscopy revealed that the type III pathway was required for a late step in intercellular spread ± bacterial escape from protrusion-derived, double-membranebound vacuoles. The TIER mutants were eventually degraded in a process involving vacuolar acidi®ca-tion. Based on these ®ndings, we propose that Ipa secretion via Mxi±Spa is required in the protrusion vacuole for double-membrane lysis.

Intracellular type III secretion by cytoplasmic Shigella flexneri promotes caspase-1-dependent macrophage cell death

“…In line with these findings, the Mxi-Spa T3SS is functionally required after initial invasion of epithelial cells, and effector proteins are present in the bacterial cytoplasm prior to and after uptake by these cells (Ménard et al, 1994a;Page et al, 1999;Rathman et al, 2000;Schuch et al, 1999). To study a role for IpaB, IpaC and their chaperone IpgC post-invasion, the ipaB, ipaC, or ipgC genes were expressed under the control of the IPTGinducible lac promoter in the corresponding mutant strains (Page et al, 1999).…”

“…7B). A possible explanation for these observations is that the endogenous chaperone IpgC is limiting the amount of IpaB stored in the bacteria (Schuch et al, 1999). However, even as the total amount of endogenously produced IpaB is apparently not increased by an induction of plasmid-encoded IpaB prior to the infection, continuous replenishment of the bacterial IpaB pool upon induction might allow more IpaB to be secreted, thus promoting cytotoxicity of S. flexneri.…”

“…Thus, IpaB, IpaC as well as IpgC are required for intercellular dissemination, in particular for the lysis of the protrusion membranes during cell-to-cell spread. In a related approach, the requirement of type III secretion postinvasion was demonstrated by a system termed TIER (test of intracellular expression requirements) (Schuch et al, 1999). Here, specific loci were transiently expressed under the control of an arabinose-inducible promoter in the corresponding mutant strains, followed by infection of an epithelial cell monolayer in a glucose-rich medium repressing the genes.…”

Actin-based motility is sufficient for bacterial membrane protrusion formation and host cell uptake

“…The second class consists of mutants affected in their ability to polymerize actin or to move by actin-based motility (Makino et al, 1986;Bernardini et al, 1989;Pal et al, 1989;Nakata et al, 1993;Rathman et al, 2000). A third class contains mutants that are unable to escape into the cytoplasm of the adjacent epithelial cell (Allaoui et al, 1992;Suzuki et al, 1994;Page et al, 1999;Schuch et al, 1999). A fourth class is defective for intercellular spread via unknown mechanisms (Rathman et al, 2000); these may include mutants unable to form membrane protrusions and/or mediate protrusion uptake into adjacent cells.…”

The Inside Story of Shigella Invasion of Intestinal Epithelial Cells

“…Cadherin-based junctions are required for Shigella cell-to-cell spread, presumably by providing the machinery necessary for protrusion formation by the donor cell, and its endocytosis by the recipient cell in a process requiring the activity of the T3SA and myosin II (Sansonetti et al 1994;Schuch et al 1999;Rathman et al 2000). Recently, Shigella was observed to disseminate preferentially at multijunctions, corresponding to sites within the epithelium in which several cells intersect (Fukumatsu et al 2012).…”