The type III secretion system apparatus determines the intracellular niche of bacterial pathogens

Upon entry into host cells, intracellular bacterial pathogens establish a variety of replicative niches. Although some remodel phagosomes, others rapidly escape into the cytosol of infected cells. Little is currently known regarding how professional intracytoplasmic pathogens, including Shigella, mediate phagosomal escape. Shigella, like many other Gram-negative bacterial pathogens, uses a type III secretion system to deliver multiple proteins, referred to as effectors, into host cells. Here, using an innovative reductionist-based approach, we demonstrate that the introduction of a functional Shigella type III secretion system, but none of its effectors, into a laboratory strain of Escherichia coli is sufficient to promote the efficient vacuole lysis and escape of the modified bacteria into the cytosol of epithelial cells. This establishes for the first time, to our knowledge, a direct physiologic role for the Shigella type III secretion apparatus (T3SA) in mediating phagosomal escape. Furthermore, although protein components of the T3SA share a moderate degree of structural and functional conservation across bacterial species, we show that vacuole lysis is not a common feature of T3SA, as an effectorless strain of Yersinia remains confined to phagosomes. Additionally, by exploiting the functional interchangeability of the translocator components of the T3SA of Shigella, Salmonella, and Chromobacterium, we demonstrate that a single protein component of the T3SA translocon—Shigella IpaC, Salmonella SipC, or Chromobacterium CipC—determines the fate of intracellular pathogens within both epithelial cells and macrophages. Thus, these findings have identified a likely paradigm by which the replicative niche of many intracellular bacterial pathogens is established.Intracellular bacterial pathogens use a variety of elaborate means to survive within host cells. Postinvasion, some such as Legionella, Salmonella, and Chlamydia species modify bacteria-containing vacuoles to avoid death via phagosomal acidification or lysosomal fusion. Others, including Shigella, Listeria, Rickettsia, and Burkholderia species, rapidly escape from phagosomes into the cytosol of infected cells. Although escape from phagosomes by the classic intracytoplasmic Gram-positive bacterium Listeria monocytogenes is well understood (1), much less is known regarding how Gram-negative pathogens, including the model professional intracytoplasmic Shigella species, enter the cytosol.During the course of an infection, many Gram-negative pathogens, including Shigella, Salmonella, enteropathogenic Escherichia coli, and Yersinia species, use type III secretion systems (T3SSs) as injection devices to deliver multiple virulence proteins, referred to as effectors, directly into the cytosol of infected cells (2). T3SSs are composed of ∼20 proteins and sense host cell contact via a tip complex at the distal end of a needle filament, which then acts as a scaffold for the formation of a translocon pore in the host cell membrane. Although components of their type III secretion apparatus (T3SA) are relatively well conserved, each pathogen delivers a unique repertoire of effectors into host cells, likely accounting for the establishment of a variety of replicative niches. For example, Salmonella and Shigella secreted effectors promote the uptake of these bacteria into nonphagocytic cells, whereas those from Yersinia inhibit phagocytosis by macrophages.All four pathogenic Shigella species—Shigella flexneri, Shigella sonnei, Shigella boydii, and Shigella dysenteriae—deliver ∼30 effectors into host cells, the majority of which are encoded on a large virulence plasmid (VP) alongside the genes for all of the proteins needed to form a T3SA (3). These secreted proteins play major roles in Shigella pathogenesis, including host cell invasion and modulation of innate immune response. One effector, IpgD, promotes the efficiency of Shigella phagosomal escape, although it is not absolutely required for this process (4). Interestingly, IpaB and IpaC, components of the Shigella translocon, the portion of the T3SA that inserts into the host cell membrane, have been implicated to mediate phagosomal escape based on the behavior of recombinant proteins (5–7). The physiologic relevance of these findings has not yet been directly addressed, as strains that lack either of these two proteins are completely impaired in the delivery of Shigella effectors into host cells (8).Here, using a reductionist approach, we directly tested a role for the Shigella translocon apparatus in phagosomal escape. Using an innovative reengineering approach, we introduced a functional effectorless Shigella T3SA into a nonpathogenic laboratory strain of DH10B E. coli. Remarkably, upon entry into host epithelial cells, these bacteria efficiently escape from phagosomes. This demonstrates for the first time, to our knowledge, in the context of an infection, a direct role for the Shigella T3SA in mediating vacuole lysis. Despite structural conservation across T3SS families, we further observed that, in the absence of any type III effectors, the Ysc T3SA mediates little to no Yersinia phagosomal escape, suggesting that not all injectisomes have equivalent functions. Lastly, by exploring the functional interchangeability of translocon components of the Shigella, Salmonella, and Chromobacterium T3SA, we demonstrate that one translocon protein controls the extent to which these intracellular pathogens escape into the cytosol of infected cells, thus demonstrating a major role for the T3SA in determining the site of the replicative niche of intracellular bacteria.