Jean Mukherjee

Urease as a Virulence Factor in Experimental Cryptococcosis

ABSTRACT 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 URE1in 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 aure1 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 withURE1, 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.

Monoclonal antibody based ELISAs for cryptococcal polysaccharide.

Mouse monoclonal antibody (MAb)-based enzyme-linked immunosorbent assays (ELISAs) have been developed to detect Cryptococcus neoformans capsular polysaccharide from the four serotypes A, B, C and D. The ELISAs avoid the problem of unreliable polysaccharide binding to polystyrene plates by using MAbs to capture and immobilize the polysaccharide antigen. The presence of polysaccharide is detected using MAbs of a different isotype from that of the capture MAb. The capturing MAbs are themselves immobilized on the plates using commercial goat anti-mouse polyclonal sera. The MAbs bind to the glucuronoxylomannan component of cryptococcal polysaccharide. The ELISAs can be used to measure the concentration of polysaccharide in biological fluids and are potentially useful tools for basic research and clinical studies.

Therapeutic efficacy of monoclonal antibodies to Cryptococcus neoformans glucuronoxylomannan alone and in combination with amphotericin B.

The therapeutic efficacy of the immunoglobulin G1 (IgG1) monoclonal antibody (MAb) 2H1 to the Cryptococcus neoformans capsular polysaccharide was studied with and without amphotericin B (AmB) in a murine model of intravenous (i.v.) infection. MAb and AmB were administered by intraperitoneal (i.p.) injection after i.v. infection with a C. neoformans serotype D strain. Intraperitoneal administration of MAb 2H1 resulted in rapid distribution to the intravascular compartment, and the half-lives of i.p. and i.v. administered MAb were similar. Administration of MAb 2H1 alone resulted in increased survival, decreased lung fungal burden, and reduced serum glucuronoxylomannan antigen levels when given 2 to 6 h but not 24 h after infection. In vivo, the combination of MAb 2H1 and AmB was more effective at prolonging survival than either agent alone. MAbs of IgM, IgG1, IgG3, and IgA isotypes given 1 day after infection were effective in reducing serum GXM-D levels, with their relative efficacy being IgG1 > IgG3 > IgM > IgA. In vitro, MAb 2H1 was a potent opsonin of C. neoformans and the combination of MAb 2H1 and AmB was more effective than either agent alone in decreasing C. neoformans colony counts in the presence of the murine macrophage cell line J774.16. The results confirm that capsule-binding MAbs can enhance the effect of AmB against C. neoformans and provide support for considering combined therapy in humans.

Antibody Efficacy in Murine Pulmonary Cryptococcus neoformans Infection: A Role for Nitric Oxide

We investigated the pathogenesis of pulmonary Cryptococcus neoformans infection and passive Ab efficacy in mice deficient in inducible NO synthase (NOS2−/−) and the parental strain. Parental mice lived significantly longer than NOS2−/− mice after intratracheal infection, despite having a higher lung fungal burden. Administration of Ab reduced lung CFU in both NOS2−/− and parental mice, but prolonged survival and increased the inflammatory response only in parental mice. Ab administration was associated with increased serum nitrite and reduced polysaccharide levels in parental mice. Eosinophils were present in greater numbers in the lung of infected NOS2−/− mice than parental mice, irrespective of Ab administration. C. neoformans infection in NOS2−/− mice resulted in significantly higher levels of IFN-γ, monocyte chemoattractant protein-1, and macrophage-inflammatory protein-1α than parental mice. Ab administration had different effects on infected NOS2−/− and parental mice with respect to IFN-γ, monocoyte chemoattractant protein-1, and macrophage-inflammatory protein-1α levels. Ab administration increased lung levels of IFN-γ in parental mice and reduced levels in NOS2−/− mice. The results indicate that NO is involved in the regulation of cytokine expression in response to cryptococcal pneumonia and is necessary for Ab efficacy against C. neoformans in mice. Our findings indicate a complex relationship between Ab efficacy against C. neoformans and cytokine expression, underscoring the interdependency of cellular and humoral defense mechanisms.

Mouse-human immunoglobulin G1 chimeric antibodies with activities against Cryptococcus neoformans.

Passive antibody administration is a potentially useful approach for the therapy of human Cryptococcus neoformans infections. To evaluate the efficacy of the human immunoglobulin G1 (IgG1) constant region against C. neoformans and to construct murine antibody derivatives with reduced immunogenicities and longer half-lives in humans, two mouse-human IgG1 chimeric antibodies were generated from the protective murine monoclonal antibodies 2D10 (IgM) and 18B7 (IgG1). The 2D10 mouse-human IgG1 chimeric antibody (ch2D10) had significantly lower binding affinity than its parent murine antibody (m2D10), presumably because of a loss of avidity contribution on switching from IgM to IgG. The 18B7 mouse-human IgG1 chimeric antibody (ch18B7) had higher affinity for cryptococcal polysaccharide antigen than its parent murine antibody (m18B7). ch18B7 and ch2D10 promoted phagocytosis of C. neoformans by primary human microglial cells and the murine J774.16 macrophage-like cell line. ch18B7 and m18B7 enhanced fungistatic or fungicidal activity of J774.16 cells and prolonged the survival of lethally infected mice. We conclude that the human IgG1 constant chain can be effective in mediating antifungal activity against C. neoformans. ch18B7 or similar antibodies are potential candidates for passive antibody therapy of human cryptococcosis.

Monoclonal antibodies to Cryptococcus neoformans glucuronoxylomannan enhance fluconazole efficacy.

Monoclonal antibody (MAb) 2H1, which binds to the capsular glucuronoxylomannan (GXM) of the fungus Cryptococcus neoformans, prolonged survival and decreased fungal burden in an experimental murine infection. Fluconazole (FLU) is a triazole antibiotic which is effective against C. neoformans. The efficacy of MAb 2H1 in combination with FLU was studied in vitro with the murine macrophage-like cell line J7741.16 and in vivo in mice infected intravenously. In vitro, the combination of MAb 2H1 and FLU was more effective than either agent alone in reducing the number of CFU of C. neoformans cocultured with J774.16 cells. In combination with FLU, GXM-binding MAbs of the immunoglobulin M (IgM), IgG1, IgG2a, IgG2b, IgG3, and IgA isotypes were effective in reducing the numbers of CFU in C. neoformans-J774.16 cocultures. For the in vivo experiments, A/JCr mice were infected intravenously with 5 x 10(5) organisms treated with MAb and FLU. The therapeutic effect of MAb 2H1 was primarily to reduce the number of CFU in the lung and the serum GXM level, whereas FLU was most effective in reducing the number of CFU in the brain. Mice receiving combination therapy had lower numbers of CFU in the lung and serum GXM levels than mice treated with FLU alone. Administration of MAb 2H1 with or without FLU had little or no effect on the number of CFU in the brain. The results provide support for combined therapy.