ABSTRACT
Various pathogenic clostridia produce binary protein toxins associated with enteric diseases of humans and animals. Separate binding/translocation (B) components bind to a protein receptor on the cell surface, assemble with enzymatic (A) component(s), and mediate endocytosis of the toxin complex. Ultimately there is translocation of A component(s) from acidified endosomes into the cytosol, leading to destruction of the actin cytoskeleton. Our results revealed that CD44, a multifunctional surface protein of mammalian cells, facilitates intoxication by the iota family of clostridial binary toxins. Specific antibody against CD44 inhibited cytotoxicity of the prototypical Clostridium perfringens iota toxin. Versus CD44(+) melanoma cells, those lacking CD44 bound less toxin and were dose-dependently resistant to C. perfringens iota, as well as Clostridium difficile and Clostridium spiroforme iota-like, toxins. Purified CD44 specifically interacted in vitro with iota and iota-like, but not related Clostridium botulinum C2, toxins. Furthermore, CD44 knockout mice were resistant to iota toxin lethality. Collective data reveal an important role for CD44 during intoxication by a family of clostridial binary toxins.
Subject(s)
ADP Ribose Transferases/toxicity , Bacterial Toxins/toxicity , Endocytosis/physiology , Hyaluronan Receptors/metabolism , Animals , Blotting, Western , Cell Line, Tumor , Chlorocebus aethiops , Dithiothreitol/pharmacology , Dose-Response Relationship, Drug , Hyaluronan Receptors/genetics , Immunoprecipitation , Mice , Mice, Knockout , Vero CellsABSTRACT
Clostridium difficile is a major enteropathogen of humans. It produces two main virulence factors, toxins A and B. A third, less well known toxin, C. difficile toxin (CDT), is a binary toxin composed of distinct enzymatic (CdtA) and cell binding/translocation (CdtB) proteins. We used a novel enzyme linked immunoassay (EIA) to detect CdtB protein in feces and culture fluids. Additionally, PCR was used to assay C. difficile isolates from fecal samples for the CDT locus (CdtLoc). Although the results from 80 isolates suggest no relationship between toxin concentrations in situ and in vitro, there is a good correlation between PCR detection of the cdtB gene and EIA detection of CdtB protein in vitro. Possible implications of the detection of CDT in patients are discussed.
Subject(s)
ADP Ribose Transferases/analysis , Bacterial Proteins/analysis , Bacterial Toxins/analysis , Clostridioides difficile/metabolism , Diarrhea/microbiology , ADP Ribose Transferases/genetics , Bacterial Proteins/genetics , Bacterial Toxins/genetics , Clostridioides difficile/genetics , Clostridioides difficile/isolation & purification , Feces/microbiology , Humans , Immunoenzyme TechniquesABSTRACT
We investigated the frequency of Clostridium perfringens in the normal fecal flora of healthy North Americans. About half of 43 subjects were colonized with C. perfringens at levels of approximately 10(6)cfu/g feces. Only type A strains were recovered. Spores sometimes outnumbered vegetative cells. Several genotypes were found. Some donors carried two genotypes, some only one. We found no alpha, beta2 or enterotoxin in the stools of any donors. Though some isolates carried toxin genes (e.g. cpe and cpb2) on plasmids, we saw no indication that healthy humans are the reservoir for the chromosomally-borne cpe recovered from cases of C. perfringens food poisoning.
Subject(s)
Bacterial Toxins/genetics , Clostridium perfringens/genetics , Calcium-Binding Proteins/genetics , Carrier State/microbiology , Clostridium perfringens/isolation & purification , Colony Count, Microbial , Enterotoxins/genetics , Feces/microbiology , Female , Genotype , Humans , Male , North America , Plasmids , Spores, Bacterial/isolation & purification , Type C Phospholipases/geneticsABSTRACT
Several different nomenclatures have been applied to the Clostridium difficile toxins and their associated genes. This paper summarizes the new nomenclature that has been agreed to by the research groups currently active in the field. The revised nomenclature includes C. difficile toxins and other related large clostridial toxins produced by Clostridium sordellii and Clostridium novyi, and corresponding toxin genes, as well as toxin production types of C. difficile strains.
Subject(s)
Bacterial Toxins/genetics , Clostridioides difficile/genetics , Terminology as Topic , Bacterial Proteins/genetics , Bacterial Toxins/isolation & purification , Enterotoxins/geneticsABSTRACT
RicinB, the non-toxic galactose/N-acetylgalactosamine-binding subunit of ricin, was fused to a model antigen, green fluorescent protein (GFP), and expressed in tobacco plants and hairy root cultures to test for utility in mucosal vaccine delivery/adjuvancy. The fusion protein retained both GFP fluorescence and galactose/galactosamine-binding activity. Intranasal immunization of mice with galactosamine-affinity purified ricinB:GFP recovered from tobacco root cultures triggered significant increases in GFP-specific serum IgGs. This strong humoral response was comparable to that observed following GFP immunization with cholera toxin adjuvant. GFP at the same concentrations but without an adjuvant was non-immunogenic. Induction of higher levels of IgG(1) than IgG(2a) following ricinB:GFP immunization suggested the presence of a Th2 response. Serum and fecal anti-GFP IgA were also induced by immunization with ricinB:GFP. Our data suggest that ricinB can be used as an adjuvant and antigen carrier to the mucosa and is efficient in eliciting systemic and mucosal immune responses.
Subject(s)
Adjuvants, Immunologic/administration & dosage , Ricin/administration & dosage , Vaccines/administration & dosage , Administration, Intranasal , Animals , Antibody Formation , Antigens/administration & dosage , Female , Green Fluorescent Proteins , Immunity, Mucosal , Luminescent Proteins/administration & dosage , Luminescent Proteins/genetics , Luminescent Proteins/immunology , Mice , Mice, Inbred ICR , Plants, Genetically Modified , Recombinant Proteins/administration & dosage , Recombinant Proteins/genetics , Recombinant Proteins/immunology , Ricin/genetics , Nicotiana/geneticsABSTRACT
Amebic colitis is an important worldwide parasitic disease for which there is not a well-established animal model. In this work we show that intracecal inoculation of Entamoeba histolytica trophozoites led to established infection in 60% of C3H mice, while C57BL/6 or BALB/c mice were resistant, including mice genetically deficient for IL-12, IFN-gamma, or inducible NO synthase. Infection was a chronic and nonhealing cecitis that pathologically mirrored human disease. Characterization of the inflammation by gene chip analysis revealed abundant mast cell activity. Parasite-specific Ab and cellular proliferative responses were robust and marked by IL-4 and IL-13 production. Depletion of CD4(+) cells significantly diminished both parasite burden and inflammation and correlated with decreased IL-4 and IL-13 production and loss of mast cell infiltration. This model reveals important immune factors that influence susceptibility to infection and demonstrates for the first time the pathologic contribution of the host immune response in amebiasis.