The Shigella flexneri Type Three Secretion System Employs Both Chaperone‐Dependent and Noncanonical Chaperone‐Independent Pathways

The virulence of many bacterial pathogens, such as Shigella flexneri , is dependent on the activity of type three secretion systems (T3SS). T3SSs are complex nanomachines that enable the delivery of tens of proteins, or effectors, directly into the host cell cytosol. The traditional consensus has been that most, if not all, effectors are dependent on a cognate chaperone for secretion. However, a comprehensive review of the literature reveals that cognate chaperones for most effectors have not been identified. Using the Shigella T3SS as a model, we observe that that the majority of effectors are still secreted in strains lacking known chaperones. This strongly suggests the existence of a noncanonical chaperone‐independent (CI) secretion pathway. In order to explore this possibility, I compared the sequences that are necessary and sufficient for the secretion of representative CD and presumed CI effectors using both liquid culture‐ and solid plate‐based assays optimized to monitor the in vitro secretion of Shigella effectors. To determine the sequences that are sufficient for effector secretion, I compared the levels of secreted MyoD, a heterologous mammalian protein, when fused to the first N‐terminal 50, 100, or 200 residues of two CI (VirA and OspF) and two CD (OspB and OspD1) effectors. Fusion of just the first 50 residues of the CD effectors, but not even 200 residues of the CI effectors, were sufficient to enable secretion of the MyoD. Next, to determine the sequences that are necessary for effector secretion in the same CD and CI effectors, I compared the secreted levels of variants engineered to have sequential deletions of 50 or 100 residues. None of the modified CI effectors were secreted, while the modified CD effectors were secreted at levels similar to the corresponding full‐length effectors. Therefore, the sequences that are necessary and sufficient to define CD and CI markedly differ, suggesting that they are recognized and delivered by the T3SS via different pathways. Finally, I have expanded the deletion studies to include two additional effectors, OspC3 and IpaH4.5. The secretion of OspC3 is only moderately impaired in the absence of its cognate chaperone, indicating that it is secreted via both CD and CI pathways. IpaH4.5 is a member of a family of Shigella effectors that are composed of two distinct functional domains: The N‐terminal domain interacts with host proteins, and the conserved C‐terminal domain encodes for E3 ubiquitin ligase. Preliminary results of these studies suggest that the sequences necessary for secretion again markedly differ from those of representative CD effectors, supporting the existence of a CI pathway and potential for a currently unidentified secretion signal. In sum, these experiments support the existence of a noncanonical CI secretion pathway. We hypothesize that this pathway is common to all T3SSs and thus can serve as a target for the development of novel antibiotics. Support or Funding Information NIH R01 AI064285, Brit d'Arbeloff Research Scholar This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .