The virulence of many human bacterial pathogens is dependent on transkingdom nanomachines, includingtype III secretion systems (T3SSs), which act to directly deliver tens of virulence proteins, often referred to aseffectors, into the cytosol of mammalian cells. Many critical gaps exist in our understanding of how type IIIsecreted (T3S) proteins, referred to as effectors, are defined and delivered to the T3S apparatus (T3SA). Whileeach pathogen injects its own unique set of effectors into hosts, components of their machines share a highdegree of similarity. For several decades, the dogma has been that the effector secretion is dependent onsmall acidic T3S chaperones that bind to their N-terminal regions. These chaperones control the hierarchy ofsecretion of proteins by mediating their recruitment to the sorting platform, a complex that cycles between thecytosol and membrane-embedded T3SA. Interestingly, cognate chaperones have not yet been identified forthe majority of T3S effectors, including those from intensively studied Salmonella, Yersinia, Shigella andpathogenic Escherichia T3SSs. Here, we present data that support the existence of a noncanonical T3SSchaperone-independent (CI) pathway likely conserved across numerous phylogenetically distinct T3SSfamilies. Here, using the Shigella flexneri T3SS as a model system, we propose to: 1. Determine how T3S chaperones are recruited to the sorting platform. Using the Protein Interaction 2. Dissect the molecular mechanisms by which CI effectors are recognized and delivered to the T3SA. 3. Investigate the existence of a co-translational ATPase-independent type III secretion pathway.Together the proposed studies shown not only advance our understanding regarding how T3S effectors aredefined and delivered to the T3SA, but also result in the identification of targets for the development of novelantimicrobial agents that target the virulence of the large family of Gram-negative bacterial pathogens whosevirulence is dependent on a functional T3SS.