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

Invasion and intercellular spread are hallmarks of Shigella pathogenicity. Invasion of the eukaryotic cell cytosol requires a type III secretion syste m (Mxi-Spa) and its cognate set of secreted Ipa invasins. Once intracellula r, the IcsA protein directs a form of actin-based motility that helps to dr ive intracellular bacterial movement, formation of cellular protrusions and cell-to-cell spread. Work in our laboratory has focused on identifying add itional factors required for this intercellular form of dissemination. In t his study, we sought to identify novel contributions of the type III secret ion pathway to post-invasion-specific processes, distinct from its previous ly characterized roles in invasion. Studies of post-invasion Ipa and Mxi-Sp a functions are complicated by an absolute requirement for these virulence proteins in invasion. To circumvent this problem, we developed a system cal led TIER (for test of intracellular expression requirements), whereby speci fic ipa, mxi or spa loci are transiently expressed before infection of tiss ue culture cell monolayers (thus supporting invasion), but then repressed a fter invasion in the intracellular environment. Such invasive type III secr etion mutants (called TIER mutants) were severely restricted in their abili ty to spread intercellularly and form plaques in confluent tissue culture c ell monolayers. Intercellular spread defects were associated with the repre ssion of most type III pathway components examined, including structural (M xiM and Spa33), secreted effector (IpaB, IpaC and IpaD) and regulatory elem ents (VirF and VirB). A kinetic analysis of bacterial growth in L2 cell mon olayers showed that each of the TIER mutants was defective with respect to long-term intracellular proliferation and viability. Examination of TIER mu tant-infected monolayers by electron microscopy revealed that the type III pathway was required for a late step in intercellular spread - bacterial es cape from protrusion-derived, double-membrane-bound vacuoles. The TIER muta nts were eventually degraded in a process involving vacuolar acidification. Based on these findings, we propose that Ipa secretion via Mxi-Spa is requ ired in the protrusion vacuole for double-membrane lysis.