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Soluble Oligomers of the Pore-forming Toxin Cytolysin A from Escherichia coli Are Off-pathway Products of Pore Assembly.

ABSTRACT: The ?-pore-forming toxin Cytolysin A (ClyA) is responsible for the hemolytic activity of various Escherichia coli and Salmonella enterica strains. Soluble ClyA monomers spontaneously assemble into annular dodecameric pore complexes upon contact with membranes or detergent. At ClyA monomer concentrations above ?100 nm, the rate-limiting step in detergent- or membrane- induced pore assembly is the unimolecular reaction from the monomer to the assembly-competent protomer, which then oligomerizes rapidly to active pore complexes. In the absence of detergent, ClyA slowly forms soluble oligomers. Here we show that soluble ClyA oligomers cannot form dodecameric pore complexes after the addition of detergent and are hemolytically inactive. In addition, we demonstrate that the natural cysteine pair Cys-87/Cys-285 of ClyA forms a disulfide bond under oxidizing conditions and that both the oxidized and reduced ClyA monomers assemble to active pores via the same pathway in the presence of detergent, in which an unstructured, monomeric intermediate is transiently populated. The results show that the oxidized ClyA monomer assembles to pore complexes about one order of magnitude faster than the reduced monomer because the unstructured intermediate of oxidized ClyA is less stable and dissolves more rapidly than the reduced intermediate. Moreover, we show that oxidized ClyA forms soluble, inactive oligomers in the absence of detergent much faster than the reduced monomer, providing an explanation for several contradictory reports in which oxidized ClyA had been described as inactive.

SUBMITTER: Roderer D

PROVIDER: S-EPMC4786705 | biostudies-literature | 2016 Mar

REPOSITORIES: biostudies-literature

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Soluble Oligomers of the Pore-forming Toxin Cytolysin A from Escherichia coli Are Off-pathway Products of Pore Assembly.

Roderer Daniel D Benke Stephan S Schuler Benjamin B Glockshuber Rudi R

The Journal of biological chemistry 20160112 11

The α-pore-forming toxin Cytolysin A (ClyA) is responsible for the hemolytic activity of various Escherichia coli and Salmonella enterica strains. Soluble ClyA monomers spontaneously assemble into annular dodecameric pore complexes upon contact with membranes or detergent. At ClyA monomer concentrations above ∼100 nm, the rate-limiting step in detergent- or membrane- induced pore assembly is the unimolecular reaction from the monomer to the assembly-competent protomer, which then oligomerizes ra ...[more]

PMID: 26757820

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Project description:UnlabelledStreptococcus pneumoniae is a leading pathogen with an extracellular lifestyle; however, it is detected by cytosolic surveillance systems of macrophages. The innate immune response that follows cytosolic sensing of cell wall components results in recruitment of additional macrophages, which subsequently clear colonizing organisms from host airways. In this study, we monitored cytosolic access by following the transit of the abundant bacterial surface component capsular polysaccharide, which is linked to the cell wall. Confocal and electron microscopy visually characterized the location of cell wall components in murine macrophages outside membrane-bound organelles. Quantification of capsular polysaccharide through cellular fractionation demonstrated that cytosolic access of bacterial cell wall components is dependent on phagocytosis, bacterial sensitivity to the host's degradative enzyme lysozyme, and release of the pore-forming toxin pneumolysin. Activation of p38 mitogen-activated protein kinase (MAPK) signaling is important for limiting access to the cytosol; however, ultimately, these are catastrophic events for both the bacteria and the macrophage, which undergoes cell death. Our results show how expression of a pore-forming toxin ensures the death of phagocytes that take up the organism, although cytosolic sensing results in innate immune detection that eventually allows for successful host defense. These findings provide an example of how cytosolic access applies to an extracellular microbe and contributes to its pathogenesis.ImportanceStreptococcus pneumoniae (the pneumococcus) is a bacterial pathogen that is a leading cause of pneumonia. Pneumococcal disease is preceded by colonization of the nasopharynx, which lasts several weeks before being cleared by the host's immune system. Although S. pneumoniae is an extracellular microbe, intracellular detection of pneumococcal components is critical for bacterial clearance. In this study, we show that following bacterial uptake and degradation by phagocytes, pneumococcal products access the host cell cytosol via its pore-forming toxin. This phenomenon of cytosolic access results in phagocyte death and may serve to combat the host cells responsible for clearing the organism. Our results provide an example of how intracellular access and subsequent immune detection occurs during infection with an extracellular pathogen.

Dataset Information

Soluble Oligomers of the Pore-forming Toxin Cytolysin A from Escherichia coli Are Off-pathway Products of Pore Assembly.

Publications

Soluble Oligomers of the Pore-forming Toxin Cytolysin A from Escherichia coli Are Off-pathway Products of Pore Assembly.

Similar Datasets

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