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1.
Mucosal Immunol ; 9(1): 124-36, 2016 Jan.
Article in English | MEDLINE | ID: mdl-25993444

ABSTRACT

Uropathogenic Escherichia coli (UPEC) is the main etiological agent of urinary tract infections (UTIs). Little is known about interactions between UPEC and the inflammasome, a key innate immune pathway. Here we show that UPEC strains CFT073 and UTI89 trigger inflammasome activation and lytic cell death in human macrophages. Several other UPEC strains, including two multidrug-resistant ST131 isolates, did not kill macrophages. In mouse macrophages, UTI89 triggered cell death only at a high multiplicity of infection, and CFT073-mediated inflammasome responses were completely NLRP3-dependent. Surprisingly, CFT073- and UTI89-mediated responses only partially depended on NLRP3 in human macrophages. In these cells, NLRP3 was required for interleukin-1ß (IL-1ß) maturation, but contributed only marginally to cell death. Similarly, caspase-1 inhibition did not block cell death in human macrophages. In keeping with such differences, the pore-forming toxin α-hemolysin mediated a substantial proportion of CFT073-triggered IL-1ß secretion in mouse but not human macrophages. There was also a more substantial α-hemolysin-independent cell death response in human vs. mouse macrophages. Thus, in mouse macrophages, CFT073-triggered inflammasome responses are completely NLRP3-dependent, and largely α-hemolysin-dependent. In contrast, UPEC activates an NLRP3-independent cell death pathway and an α-hemolysin-independent IL-1ß secretion pathway in human macrophages. This has important implications for understanding UTI in humans.


Subject(s)
Carrier Proteins/immunology , Inflammasomes/drug effects , Interleukin-1beta/immunology , Macrophages/immunology , Uropathogenic Escherichia coli/immunology , Animals , Bacterial Toxins/toxicity , Carrier Proteins/genetics , Cell Death/drug effects , Gene Expression Regulation , Hemolysin Proteins/toxicity , Host-Pathogen Interactions , Humans , Inflammasomes/immunology , Interleukin-1beta/genetics , Macrophages/drug effects , Macrophages/microbiology , Mice , NLR Family, Pyrin Domain-Containing 3 Protein , Primary Cell Culture , Signal Transduction , Species Specificity , Uropathogenic Escherichia coli/pathogenicity
2.
Article in English | MEDLINE | ID: mdl-20445269

ABSTRACT

Pathogens require protein-folding enzymes to produce functional virulence determinants. These foldases include the Dsb family of proteins, which catalyze oxidative folding in bacteria. Bacterial disulfide catalytic processes have been well characterized in Escherichia coli K-12 and these mechanisms have been extrapolated to other organisms. However, recent research indicates that the K-12 complement of Dsb proteins is not common to all bacteria. Importantly, many pathogenic bacteria have an extended arsenal of Dsb catalysts that is linked to their virulence. To help to elucidate the process of oxidative folding in pathogens containing a wide repertoire of Dsb proteins, Salmonella enterica serovar Typhimurium has been focused on. This Gram-negative bacterium contains three DsbA proteins: SeDsbA, SeDsbL and SeSrgA. Here, the expression, purification, crystallization and preliminary diffraction analysis of these three proteins are reported. SeDsbA, SeDsbL and SeSrgA crystals diffracted to resolution limits of 1.55, 1.57 and 2.6 A and belonged to space groups P2(1), P2(1)2(1)2 and C2, respectively.


Subject(s)
Bacterial Proteins/chemistry , Oxidoreductases Acting on Sulfur Group Donors/chemistry , Protein Disulfide-Isomerases/chemistry , Salmonella typhimurium/enzymology , Bacterial Proteins/genetics , Bacterial Proteins/isolation & purification , Crystallization , Crystallography, X-Ray , Gene Expression , Oxidoreductases Acting on Sulfur Group Donors/genetics , Oxidoreductases Acting on Sulfur Group Donors/isolation & purification , Protein Disulfide-Isomerases/genetics , Protein Disulfide-Isomerases/isolation & purification
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