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Over the course of an infection, many Gram-negative bacterial pathogens use complex nanomachines to directly inject tens to hundreds of proteins (effectors) into the cytosol of infected host cells. These effectors rewire processes to promote bacterial replication and spread. The roles of effectors in pathogenesis have traditionally been investigated by screening for phenotypes associated with their absence, a top-down approach that can be limited, as effectors often act in a functionally redundant or additive manner. Here we describe a synthetic Escherichia coli -based bottom-up platform to conduct gain-of-function screens for roles of individual Shigella effectors in pathogenesis. As proof of concept, we screened for Shigella effectors that limit cell death induced on cytosolic entry of bacteria into epithelial cells. Using this platform, in addition to OspC3, an effector known to inhibit cell death via pyroptosis, we have identified OspD2 and IpaH1.4 as cell death inhibitors. In contrast to almost all type III effectors, OspD2 does not target a host cell process, but rather regulates the activity of the Shigella type III secretion apparatus limiting the cytosolic delivery (translocation) of effectors during an infection. Remarkably, by limiting the translocation of a single effector, VirA, OspD2 controls the timing of epithelial cell death via calpain-mediated necrosis. Together, these studies provide insight into the intricate manner by which Shigella effectors interact to establish a productive intracytoplasmic replication niche before the death of infected epithelial cells., Competing Interests: The authors declare no conflict of interest.

Numerous Gram-negative bacterial pathogens utilize type III secretion systems (T3SSs) to inject tens of effector proteins directly into the cytosol of host cells. Through interactions with cognate chaperones, type III effectors are defined and recruited to the sorting platform, a cytoplasmic component of these membrane-embedded nanomachines. However, notably, a comprehensive review of the literature reveals that the secretion of most type III effectors has not yet been linked to a chaperone, raising questions regarding the existence of unknown chaperones as well as the universality of chaperones in effector secretion. Here, we describe the development of the first high-throughput type III secretion (T3S) assay, a semiautomated solid-plate-based assay, which enables the side-by-side comparison of secretion of over 20 Shigella effectors under a multitude of conditions. Strikingly, we found that the majority of Shigella effectors are secreted at equivalent levels by wild-type and variants of Shigella that no longer encode one or all known Shigella T3S effector chaperones. In addition, we found that Shigella effectors are efficiently secreted from a laboratory strain of Escherichia coli expressing the core Shigella type III secretion apparatus (T3SA) but no other Shigella - specific proteins. Furthermore, we observed that the sequences necessary and sufficient to define chaperone-dependent and -independent effectors are fundamentally different. Together, these findings support the existence of a major, previously unrecognized, noncanonical chaperone-independent secretion pathway that is likely common to many T3SSs. IMPORTANCE Many bacterial pathogens use specialized nanomachines, including type III secretion systems, to directly inject virulence proteins (effectors) into host cells. Here, we present the first extensive analysis of chaperone dependence in the process of type III effector secretion, providing strong evidence for the existence of a previously unrecognized chaperone-independent pathway. This noncanonical pathway is likely common to many bacteria, as an extensive review of the literature reveals that the secretion of multiple type III effectors has not yet been knowingly linked to a chaperone. While additional studies will be required to discern the molecular details of this pathway, its prevalence suggests that it can likely serve as a new target for the development of antimicrobial agents., (Copyright © 2018 Ernst et al.)

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 themodified 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. [ABSTRACT FROM AUTHOR]