Identification of Novel Type III Secretion System Determinants in Shigella flexneri

Abstract

Many Gram-negative bacteria, such as Shigella, Salmonella, Yersinia and pathogenic Escherichia spp., utilize type III secretion systems (T3SSs) to inject virulence proteins into the cytosol of eukaryotic host cells. T3SSs are complex nanomachines that assemble within and span the bacterial cellular envelope. While the structure of the type III secretion apparatus (T3SA) is relatively conserved, each pathogen delivers its own unique set of proteins. These translocated virulence proteins, referred to as effectors, modulate host cellular processes to promote bacterial replication and spread. Effectors are traditionally defined by proteinaceous signals and are post- translationally delivered to the membrane embedded T3SA via interactions with type III secretion chaperones. In this study, the requirement of T3S chaperones in effector secretion in Shigella flexneri was assessed. The development of a high-throughput plate-based secretion assay enabled the side-by-side analysis of numerous effectors from mutant strains lacking known chaperones. Strikingly, in the absence of all known T3S chaperones the secretion of the majority of effectors was unaffected, suggesting that unidentified secretion factors or chaperone-independent secretion pathways may exist. Since effector gene expression is tightly regulated, global RNA sequencing was conducted to identify novel factors involved in the type III secretion of effectors. This led to the identification of previously unrecognized T3S determinants. Given the structural conservation of T3SAs and the prevalence of these novel factors, it is possible that they may play a similar role in T3SSs of other Gram-negative pathogens.