Abstract
ABSTRACTNumerous 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.
Highlights
Numerous Gram-negative bacterial pathogens utilize type III secretion systems (T3SSs) to inject tens of effector proteins directly into the cytosol of host cells
Upon contact with host cells, the translocon complex, positioned at the tip of the T3SA, is inserted into the host membrane. This interaction triggers a conformational change, which leads to activation of the T3SA resulting in the injection of translocon components followed by effectors into host cells
In this report, we describe the development of a solid-plate-based secretion assay that enables, for the first time, the side-by-side concurrent analysis of secretion of Ͼ20 different Shigella effectors under multiple conditions
Summary
Numerous Gram-negative bacterial pathogens utilize type III secretion systems (T3SSs) to inject tens of effector proteins directly into the cytosol of host cells. A comprehensive review of the literature reveals that over the past 20 years chaperones have been identified for only a third (38/109) of the effectors of the well-studied Shigella Mxi-Spa (11/31), Salmonella SPI1 (6/11), Salmonella SPI2 (4/20), and Yersinia Ysc (4/6) as well as enteropathogenic Escherichia coli/enterohemorrhagic E. coli (EPEC/EHEC) Esc (13/41) T3SSs [12,13,14,15,16,17,18,19] (see Table S1 in the supplemental material) These observations question the commonly held notion that chaperones play essential roles in effector secretion and raise the possibility of the existence of unidentified chaperones and/or a chaperone-independent T3S pathway
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