Biomedical subjects

K R Klimpel

Publications and source records attributed to K R Klimpel.

23 records · Page 2Linked to original sources

PubMed1992

Fusions of anthrax toxin lethal factor to the ADP-ribosylation domain of Pseudomonas exotoxin A are potent cytotoxins which are translocated to the cytosol of mammalian cells.

The lethal factor (LF) and edema factor (EF) components of anthrax toxin are toxic to animal cells only if internalized by interaction with the protective antigen (PA) component. PA binds to a cell surface receptor and is proteolytically cleaved to expose a binding site for LF and EF. To study how LF and EF are internalized and trafficked within cells, LF was fused to the translocation and ADP-ribosylation domains (domains II and III, respectively) of Pseudomonas exotoxin A. LF fusion proteins containing Pseudomonas exotoxin A domains II and III were less toxic than those containing only domain III. Fusion proteins with a functional endoplasmic reticulum retention sequence, REDLK, at the carboxyl terminus of domain III were less toxic than those with a nonfunctional sequence, LDER. The most potent fusion protein, FP33, had an EC50 = 2 pM on Chinese hamster ovary cells, exceeding that of native Pseudomonas exotoxin A (EC50 = 420 pM). Toxicity of all the fusion proteins required the presence of PA and was blocked by monensin. These data suggest that LF and LF fusion proteins are efficiently translocated from acidified endosomes directly to the cytosol without trafficking through other organelles, as is required for Pseudomonas exotoxin A. This system provides a potential vehicle for importing diverse proteins into the cytosol of mammalian cells.

ADP Ribose Transferases↗

PubMed1991

Functional mapping of anthrax toxin lethal factor by in-frame insertion mutagenesis.

Linker insertion mutagenesis was employed to create structural disruptions of the lethal factor (LF) protein of anthrax toxin to map functional domains. A dodecameric linker was inserted at 17 blunt end restriction enzyme sites throughout the gene. Paired MluI restriction sites within the linker allowed the inserts to be reduced from four to two amino acids. Shuttle vectors containing the mutated genes were transformed into the avirulent Bacillus anthracis UM23C1-1 for expression and secretion of the gene products. Mutations at five sites in the central one-third of the sequence made the protein unstable, and purified protein could not be obtained. Mutated LF proteins with insertions at the other sites were purified and assessed for toxic activity in a macrophage lysis assay and for their ability to bind to the protective antigen (PA) component of anthrax toxin, the receptor binding moiety. Most insertions located in the NH2-terminal one-third of the LF protein eliminated both toxicity and binding to PA, while all four insertions in the COOH-terminal one-third of the protein eliminated toxicity without affecting binding to PA. These data support the hypothesis that the NH2-terminal domain contains the structures required for binding to PA and the COOH-terminal domain contains the putative catalytic domain of LF.

Antigens, Bacterial↗

PubMed1991

The carboxyl-terminal end of protective antigen is required for receptor binding and anthrax toxin activity.

Anthrax toxin consists of three separate proteins produced by Bacillus anthracis: protective antigen (PA), lethal factor (LF), and edema factor (EF). Previous work showed that the process by which these proteins damage eukaryotic cells begins with binding of PA (83 kDa) to cell surface receptors. PA is then cleaved by a cell surface protease so as to expose a high-affinity binding site for LF or EF on the COOH-terminal, receptor-bound, 63-kilodalton fragment. In this report we more closely define a region of PA involved in receptor binding. The gene encoding PA was mutagenized so as to delete 3, 5, 7, 12, or 14 amino acids from the carboxyl terminus of the protein, and the truncated PA variants were purified from Bacillus subtilis or Escherichia coli. Deletion of 3, 5, or 7 amino acids reduced the binding of PA to cells and the subsequent toxicity of the PA.LF complex to J774A.1 cells and also the ability to cause EF binding to cells. Deletion of 12 or 14 amino acids completely eliminated all these activities. These results show that the carboxy terminus comprises or is part of the receptor-binding domain of PA.

Amino Acid Sequence↗

PubMed1988

Cell walls from avirulent variants of Histoplasma capsulatum lack alpha-(1,3)-glucan.

Cell wall composition of isogenic virulent-avirulent strain pairs of Histoplasma capsulatum varied markedly with respect to alpha-(1,3)-glucan content. When yeast cell walls were fractionated by standard techniques, the avirulent strains contained up to 1,000-fold less alpha-(1,3)-glucan than did their virulent parents. No alpha-(1,3)-glucan could be detected on the surface of the avirulent strain yeast cells if we used a mouse monoclonal antibody that recognized this polymer. A similar relationship between virulence and alpha-(1,3)-glucan has been described for Paracoccidioides brasiliensis. alpha-(1,3)-Glucan is also found in several other pathogenic fungi and may thus be an important common virulence determinant.

Antigens, Fungal↗

PubMed1987

Isolation and characterization of spontaneous avirulent variants of Histoplasma capsulatum.

A selection procedure was developed which allowed us to isolate spontaneous isogenic avirulent clones from virulent strains of Histoplasma capsulatum. The avirulent yeasts had a unique phenotype: they did not aggregate like the parental strains but grew as dispersed budded and unbudded single cells in liquid medium. On solid medium, the avirulent variant strains grew as smooth-textured colonies, whereas the virulent parental strains grew as rough convoluted colonies. Virulence testing in mice demonstrated that the smooth variants gave 50% lethal dose values similar to those of the avirulent Downs strain. Growth curves for the paired rough and smooth strains were similar. Furthermore, they had the same protein profiles when crude cell fractions were separated on one-dimensional polyacrylamide gels or when whole-cell extracts were separated by two-dimensional gel electrophoresis. Electrophoresis of culture supernatants, however, revealed a difference in a released low-molecular-weight peptide that may be related to virulence. In addition to their usefulness in comparative virulence studies, these avirulent strains should prove valuable for H. capsulatum genetic experiments because of the unique ability of these yeasts to grow without clumping.

Animals↗