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Quantification of real-time Salmonella effector type III secretion kinetics reveals differential secretion rates for SopE2 and SptP.

ABSTRACT: Gram-negative pathogenic bacteria such as Salmonella utilize the type III secretion system to inject bacterial effector proteins into a host cell. Upon entry, these effectors bind mammalian cell proteins, hijack cellular signaling pathways, and redirect cellular function, thus enabling bacterial infection. In this study we use the FlAsH/tetracysteine labeling system to fluorescently tag specific effectors in Salmonella to observe real-time secretion of these proteins into a mammalian host cell. The tetracysteine tag is genomically incorporated, thus preserving endogenous control of bacterial effectors. We demonstrate that two effectors, SopE2 and SptP, exhibit different secretion kinetics, as well as different rates of degradation within the host cell. These proteins respectively activate and suppress GTPase Cdc42, suggesting that there is a temporal hierarchy for effector delivery and persistence within the cell that is directly related to effector function.

SUBMITTER: Van Engelenburg SB

PROVIDER: S-EPMC3297674 | biostudies-literature | 2008 Jun

REPOSITORIES: biostudies-literature

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Quantification of real-time Salmonella effector type III secretion kinetics reveals differential secretion rates for SopE2 and SptP.

Van Engelenburg Schuyler B SB Palmer Amy E AE

Chemistry & biology 20080601 6

Gram-negative pathogenic bacteria such as Salmonella utilize the type III secretion system to inject bacterial effector proteins into a host cell. Upon entry, these effectors bind mammalian cell proteins, hijack cellular signaling pathways, and redirect cellular function, thus enabling bacterial infection. In this study we use the FlAsH/tetracysteine labeling system to fluorescently tag specific effectors in Salmonella to observe real-time secretion of these proteins into a mammalian host cell. ...[more]

PMID: 18559272

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Project description:Strains of the various Salmonella enterica serovars cause gastroenteritis or typhoid fever in humans, with virulence depending on the action of two type III secretion systems (Salmonella pathogenicity island 1 [SPI-1] and SPI-2). SptP is a Salmonella SPI-1 effector, involved in mediating recovery of the host cytoskeleton postinfection. SptP requires a chaperone, SicP, for stability and secretion. SptP has 94% identity between S. enterica serovar Typhimurium and S Typhi; direct comparison of the protein sequences revealed that S Typhi SptP has numerous amino acid changes within its chaperone-binding domain. Subsequent comparison of ΔsptP S Typhi and S. Typhimurium strains demonstrated that, unlike SptP in S. Typhimurium, SptP in S Typhi was not involved in invasion or cytoskeletal recovery postinfection. Investigation of whether the observed amino acid changes within SptP of S Typhi affected its function revealed that S Typhi SptP was unable to complement S. Typhimurium ΔsptP due to an absence of secretion. We further demonstrated that while S. Typhimurium SptP is stable intracellularly within S Typhi, S Typhi SptP is unstable, although stability could be recovered following replacement of the chaperone-binding domain with that of S. Typhimurium. Direct assessment of the strength of the interaction between SptP and SicP of both serovars via bacterial two-hybrid analysis demonstrated that S Typhi SptP has a significantly weaker interaction with SicP than the equivalent proteins in S. Typhimurium. Taken together, our results suggest that changes within the chaperone-binding domain of SptP in S Typhi hinder binding to its chaperone, resulting in instability, preventing translocation, and therefore restricting the intracellular activity of this effector. Studies investigating Salmonella pathogenesis typically rely on Salmonella Typhimurium, even though Salmonella Typhi causes the more severe disease in humans. As such, an understanding of S. Typhi pathogenesis is lacking. Differences within the type III secretion system effector SptP between typhoidal and nontyphoidal serovars led us to characterize this effector within S Typhi. Our results suggest that SptP is not translocated from typhoidal serovars, even though the loss of sptP results in virulence defects in S. Typhimurium. Although SptP is just one effector, our results exemplify that the behavior of these serovars is significantly different and genes identified to be important for S. Typhimurium virulence may not translate to S Typhi.

Dataset Information

Quantification of real-time Salmonella effector type III secretion kinetics reveals differential secretion rates for SopE2 and SptP.

Publications

Quantification of real-time Salmonella effector type III secretion kinetics reveals differential secretion rates for SopE2 and SptP.

Similar Datasets

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