Stress tolerance and pathogenic potential of a mannitol mutant of Cryptococcus neoformans

Abstract

Cryptococcus neoformans produces large amounts of the acyclic hexitol mannitol in culture and infected animals, but the functional and pathogenic significance of mannitol production by this fungus is not known. We exposed C. neoformans H99 (Cn H99) to UV irradiation (1 x LD, , ) and screened survivors for mannitol production. A mutant, Cn MLP (Mannitol Low Producer), synthesized less mannitol from glucose (2.7 vs 8-2 nmol per lo8 cells min-' a t 37 "C) and contained less intracellular mannitol (1 vs 11 pmol per lo6 cells a t 37 "C) than did Cn H99. Cn MLP and Cn H99 were similar with respect to carbon assimilation patterns, rates of glucose consumption, growth rates a t 30 "C, urease and phenoloxidase activities, morphology, capsule formation, mating type, electrophoretic karyotype, rapid amplification of polymorphic DNA (RAPD) patterns and antifungal susceptibility. However, Cn MLP was more susceptible than was Cn H99 to growth inhibition and killing by heat and high NaCl concentrations. Also, the LD, , values in mice injected intravenously were 3.7 x lo6 c.f.u. for Cn MLP compared to 6 9 x lo2 c.f.u. for Cn H99. Moreover, 500 c.f.u. Cn H99 intravenously killed 12 of 12 mice by 60 d, whereas all mice given the same inoculum of Cn MLP survived. Classical genetic studies were undertaken to determine if these differences were due to a single mutation, but the basidiospores were nonviable. These results suggest that the abilities of C. neoformans to produce and accumulate mannitol may influence its tolerance to heat and osmotic stresses and its pathogenicity in mice.

Mannitol-1-phosphate dehydrogenase from Cryptococcus neoformans is a zinc-containing long-chain alcohol/polyol dehydrogenase The GenBank accession numbers for the nucleotide sequences for the C. neoformans mannitol-1-phosphate dehydrogenase cDNA and gene are AF175685 and AF186474, respectively.

“…Although this result implies that inability to express MPD mRNA was not responsible for mannitol underproduction in C. neoformans MLP, we have not ruled out a loss of function mutation in the gene encoding MPD in C. neoformans MLP. Lastly, because earlier studies suggested that mannitol may function in C. neoformans as an intracellular stress protectant (Chaturvedi et al, 1996a), we also examined MPD mRNA levels in C. neoformans cells exposed to temperature and osmotic stresses and we found that MPD mRNA levels increased when C. neoformans was cultured in YPD supplemented with 0n2 or 0n4 M NaCl (but not higher levels) and when the incubation temperature was increased from 25 mC to 30 mC or 37 mC.…”

“…C. neoformans strains H99 (wild-type, serotype A) and MLP (a UV-generated mutant of strain H99 that underproduced and underaccumulated mannitol) (Wong et al, 1990 ;Chaturvedi et al, 1996a) were cultured in YPD (1 % yeast extract, 2 % peptone, 2 % glucose) or synthetic complete medium (0n67 %, w\v, yeast nitrogen base with amino acids) containing either 2 % glucose or 2 % mannitol. Saccharomyces cerevisiae YPH252 (Matα ura3-52 lys2-801 ade2-101 trp1-1∆ his3-∆200) and YPH252 gpd1∆ ::leu2 (Eriksson et al, 1995) were cultured in YPD, and uracil prototrophs were cultured in synthetic complete (SC) medium containing 2 % (w\v) glucose but no uracil (SC glukura) or in SC medium containing 2 % (w\v) galactose but no uracil (SC galkura).…”

“…Specifically, we showed that a C. neoformans mutant that underproduced and underaccumulated mannitol was (i) hypersusceptible to heat and hyperosmolar stresses ; (ii) profoundly hypovirulent in mice (Chaturvedi et al, 1996a) ; and (iii) hypersusceptible to oxidative killing by human neutrophils and by oxidants generated by the iron-H # O # -halide system (Chaturvedi et al, 1996b). Furthermore, heterologous expression of bacterial mtlD [which encodes mannitol-1-phosphate dehydrogenase (MPD)] conferred on transgenic tobacco plants (Tarczynski et al, 1993) and on glycerol-underproducing S. cerevisiae gpd1 mutants (Chaturvedi et al, 1997) the abilities to synthesize mannitol and to tolerate hyperosmolar stresses.…”

31P and 13C nuclear magnetic resonance studies of metabolic pathways in Pasteurella multocida

“…The role of mannitol in fungi is unclear but it has recently been suggested [17] that mannitol is important to the pathogenicity of the fungus Cryptococcus neoformans in mice, aiding in the tolerance to heat and osmotic stress. In Eimeria, the most apparent role of this pathway is in the development and maturation of oocysts [15,16].…”

Does competition for existence pushed the evolution of the ‘once’ saprophytic fungi to parasitic life?

“…melanin and capsule production, mannitol utilization etc. (Chaturvedi et al, 1996) in C. neoformans are unique to certain geophilic group among the whole of the fungal community. Decoding of the 'adaptive ability' of these fungi would definitely provide a better management strategy for containing the diseases caused by them.…”