Urease catalyzes the hydrolysis of urea to ammonia and carbamate and has been found to be an important pathogenic factor for certain bacteria. Cryptococcus neoformans is a significant human pathogenic fungus that produces large amounts of urease; thus we wanted to investigate the importance of urease in the pathogenesis of cryptococcosis. We cloned and sequenced the genomic locus containing the single-copy C. neoformans urease gene (URE1) and used this to disrupt the native URE1 in the serotype A strain H99. The ure1 mutant strains were found to have in vitro growth characteristics, phenoloxidase activity, and capsule size similar to those of the wild type. Comparison of a ure1 mutant with H99 after intracisternal inoculation into corticosteroid-treated rabbits revealed no significant differences in colony counts recovered from the cerebrospinal fluid. However, when these two strains were compared in both the murine intravenous and inhalational infection models, there were significant differences in survival. Mice infected with a ure1 strain lived longer than mice infected with H99 in both models. The ure1 strain was restored to urease positivity by complementation with URE1, and two resulting transformants were significantly more pathogenic than the ure1 strain. Our results suggest that urease activity is involved in the pathogenesis of cryptococcosis but that the importance may be species and/or infection site specific.Urease is a metalloenzyme that catalyzes the hydrolysis of urea to ammonia and carbamate. Under physiological conditions, this reaction can result in an increase in pH. Urease activity has been found in several bacteria, fungi, and plants and has been shown to be an important pathogenic factor for the bacteria Helicobacter pylori and Proteus mirabilis (12,14,33). H. pylori is the major cause of peptic ulcer disease in humans, and urease is essential for pathogenesis in experimental models. Urease-negative strains of H. pylori constructed using a variety of molecular and genetic techniques were unable to infect gastric mucosa (12,33). It has been postulated that the hydrolysis of urea and the resulting increase in pH allow H. pylori to survive within the gastric mucosa. It has also been postulated that much of the tissue damage induced by this organism is a result of ammonium hydroxide produced through the actions of urease (22,23), and there are data that the actions of urease may alter the function of white blood cells (18)(19)(20). Because of the importance of this enzyme in the pathogenesis of peptic ulcer disease, urease inhibitors and urease vaccines are currently being developed for clinical use.Many fungi pathogenic to humans have urease activity, among which are Cryptococcus neoformans, Coccidioides immitis, Histoplasma capsulatum, Sporothrix schenckii, and species of Trichosporon and Aspergillus. The first urease gene cloned from a human pathogenic fungus was that of C. immitis (35). The coccidioidal urease gene has been shown to be expressed in vivo, and there are suggestions that it p...
Silm~SL~Two immunoglobulin M monodonal antibodies (mAbs) derived from the same B cell recognize different epitopes on the capsular polysaccharide of the pathogenic yeast, Cryptococcus ,eoformans.Their respective epitopes are located in spatially distinct regions of the capsule. Passive administration of one mAb prolonged survival whereas the other mAb did not. The results indicate that specificity is an important determinant of antibody efficacy against C. ,eoformans and that somatic mutations occurring during the antibody response can affect the protective efficacy of antibodies to C. neoformans.
The antibody responses of BALB/c mice to serotype A Cryptococcus neoformans capsular polysaccharide (CNPS) were compared after cryptococcal infection and immunization with a serotype A glucuronoxylomannan-tetanus toxoid conjugate (GXM-TT). Infection rarely resulted in a rise of serum antibody titer to CNPS. In contrast, mice immunized with GXM-TT produced serum IgM and IgG to CNPS. Six IgM and one IgG1 monoclonal antibodies (MAbs) were generated from the spleen of one infected mouse. Nine IgM, 1 IgG3, 16 IgG1, and 7 IgA MAbs were generated from the spleen of one GXM-TT-immunized mouse. All MAbs generated from both mice bound to the GXM fraction of the capsular polysaccharide. For some MAbs, de-O-acetylation of serotype A GXM abolished or greatly reduced MAb binding compared with the native GXM. All MAbs reacted with CNPS from C. neoformans serotypes A-D. MAbs generated from the infected mouse competitively inhibited the binding of MAbs generated from the GXM-TT-immunized mouse. These results indicate that some antibodies elicited by infection with C. neoformans or by immunization with GXM-TT bind to the same antigenic determinant in the GXM.
Suml'naryThe molecular characteristics of the humoral immune response to a serotype A Cr~tococcus neoformans infection were compared with the response elicited by a cryptococcal ghicuronoxylomannan-tetanus toxoid (GXM-TT) conjugate. Anticryptococcal monoclonal antibodies (mAbs) isolated from both responses have previously been shown to recognize the same antigenic determinant of cryptococcal GXM. Southern blot and sequence analyses indicate that the hybridomas isolated from each response arose from only a few precursor B cells. All the mAbs generated from the infected and GXM-TT conjugate-immunized mice utilize the same VH7183 family member: J.2/J.4, v~5.1, and J~l; mAbs generated by different B cells had complementarity-determining region Ys (CDR3s) composed of seven amino acids with a common sequence motif. Thus, the molecular analysis of these anticryptococcal mAb-producing hybridomas indicated that the response to both cryptococcal infection and conjugate immunization was oligoclonal and highly restricted with regard to immunoglobulin gene utilization. The GXM-TT conjugate primarily stimulated isotype switching and clonal proliferation, and did not result in hybridomas expressing additional immunoglobulin repertoires. The mAbs from both responses had a number of replacement mutations at the 5' end of CDR2 that appear to be the result of antigen-driven selection. Somatic mutation also resulted in altered epitope specificity for one mAb, 13F1. Passive administration of representative mAbs from different clones generated in response to the GXM-TT conjugate prolonged survival of lethally infected mice.
Antitoxins are needed that can be produced economically with improved safety and shelf life compared to conventional antisera-based therapeutics. Here we report a practical strategy for development of simple antitoxin therapeutics with substantial advantages over currently available treatments. The therapeutic strategy employs a single recombinant ‘targeting agent’ that binds a toxin at two unique sites and a ‘clearing Ab’ that binds two epitopes present on each targeting agent. Co-administration of the targeting agent and the clearing Ab results in decoration of the toxin with up to four Abs to promote accelerated clearance. The therapeutic strategy was applied to two Botulinum neurotoxin (BoNT) serotypes and protected mice from lethality in two different intoxication models with an efficacy equivalent to conventional antitoxin serum. Targeting agents were a single recombinant protein consisting of a heterodimer of two camelid anti-BoNT heavy-chain-only Ab VH (VHH) binding domains and two E-tag epitopes. The clearing mAb was an anti-E-tag mAb. By comparing the in vivo efficacy of treatments that employed neutralizing vs. non-neutralizing agents or the presence vs. absence of clearing Ab permitted unprecedented insight into the roles of toxin neutralization and clearance in antitoxin efficacy. Surprisingly, when a post-intoxication treatment model was used, a toxin-neutralizing heterodimer agent fully protected mice from intoxication even in the absence of clearing Ab. Thus a single, easy-to-produce recombinant protein was as efficacious as polyclonal antiserum in a clinically-relevant mouse model of botulism. This strategy should have widespread application in antitoxin development and other therapies in which neutralization and/or accelerated clearance of a serum biomolecule can offer therapeutic benefit.
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