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Abstract
Many Gram-negative bacteria colonize and exploit host niches using a protein apparatus called a type III secretion system (T3SS) that translocates bacterial effector proteins into host cells where their functions are essential for pathogenesis. A suite of T3SS-associated chaperone proteins bind cargo in the bacterial cytosol, establishing protein interaction networks needed for effector translocation into host cells. In Salmonella enterica serovar Typhimurium, a T3SS encoded in a large genomic island (SPI-2) is required for intracellular infection, but the chaperone complement required for effector translocation by this system is not known. Using a reverse genetics approach, we identified a multi-cargo secretion chaperone that is functionally integrated with the SPI-2-encoded T3SS and required for systemic infection in mice. Crystallographic analysis of SrcA at a resolution of 2.5 Å revealed a dimer similar to the CesT chaperone from enteropathogenic E. coli but lacking a 17-amino acid extension at the carboxyl terminus. Further biochemical and quantitative proteomics data revealed three protein interactions with SrcA, including two effector cargos (SseL and PipB2) and the type III-associated ATPase, SsaN, that increases the efficiency of effector translocation. Using competitive infections in mice we show that SrcA increases bacterial fitness during host infection, highlighting the in vivo importance of effector chaperones for the SPI-2 T3SS.
Highlights
Many Gram-negative bacteria that colonize host animals use a type III secretion system (T3SS) to deliver effector proteins directly into host cells where their interaction with host proteins and membranes contribute to pathogenesis
Using a bacterial mutant lacking the srcA gene, we found that this chaperone was needed for Salmonella to compete against wild type cells during systemic disease because it controls secretion of at least 2 key proteins involved in immune escape and cell-to-cell transmission
Typhimurium) [22] identified a hypothetical gene, STM2138, that was coregulated with genes in Salmonella Pathogenicity Islands (SPI)-2 and repressed,20-fold in an ssrB mutant compared to wild type
Summary
Many Gram-negative bacteria that colonize host animals use a type III secretion system (T3SS) to deliver effector proteins directly into host cells where their interaction with host proteins and membranes contribute to pathogenesis. Secretion chaperones are proteins required for T3SS function with roles in apparatus assembly and effector delivery, but are not themselves subject to secretion [3]. These chaperones often have common physical features such as low molecular weight (,15 kDa), an acidic isoelectric point and a predicted amphipathic helix at the carboxyl terminus. Class I chaperones have a structural fold of five b-strands and three a-helices, forming homodimers that bind to the CBD in a horseshoe-like structure These chaperones have been further sub-classified based on their substrate repertoire and location with respect to the genes encoding the T3SS [3]. Secondary structure predictions suggest class III chaperones adopt an extended alpha helical structure, which was confirmed by the crystal structure of the CesA chaperone in enteropathogenic E. coli that binds the EspA filament protein [5]
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