Figure 1.
The end product of purine metabolism varies from species to species.
While degradation of purines to uric acid is generally conserved among organisms, the end product of uric acid catabolism varies among taxa often due to the loss of functional catabolic enzymes in the pathway.
Figure 2.
Predicted pathway of uric acid degradation in C. neoformans.
While the metabolic pathway of purine degradation is present in bacteria, fungi, plants and animals, there is hidden complexity between species. In addition to species in which only part of the pathway is present, these variations include at least three examples of convergent evolution (conversion of xanthine to uric acid, allantoin to allantoate, and urea to ammonia), offshoots of the pathway that are only present in some species (allantoin racemase, ureidoglycine production and hydrolysis) as well as points at which spontaneous conversion occur without the need for enzyme activity (dashed arrows). Steps predicted to be absent from C. neoformans are opaque.
Figure 3.
Nitrogen assimilation phenotypes of the C. neoformans uric acid catabolic deletion mutants.
(A) Based on the enzymatic activity of homologs in other systems, inactivation of each respective catabolic enzyme (in red) is predicted to disrupt the ability of C. neoformans to utilize one or more pathway intermediates as the sole nitrogen source. (B) Ten-fold spot dilution assays on YNB supplemented with 10 mM uric acid or its derivatives revealed that the uro1Δ, uro2Δ, uro3Δ, dal1Δ, dal2,3,3Δ and ure1Δ mutants showed a correlation with the anticipated nitrogen utilization abilities.
Figure 4.
Uro1, Uro2, Uro3, Dal1, Dal2,3,3 and Ure1 are not required for C. neoformans initiation of mating.
Filamentation assays on V8, MS and pigeon guano (PG) medium showed that filament formation appeared indistinguishable between the crosses of H99 × KN99a and each of the uric acid mutants × KN99a. (A) Periphery of each individual colony. (B) Under higher magnification (×400).
Figure 5.
Uro1, Uro2, Uro3, Dal1, Dal2,3,3 and Ure1 are not required for expression of the three major virulence attributes.
(A) India ink cell staining under light microscopy revealed that the uric acid catabolic deletion mutants produced characteristic halos around its cells representing enlarged capsule that were similar to wild-type when cultured under serum-induced growth conditions. Scale bar, 10 µm. (B) Ten-fold spot dilution assays on L-DOPA medium at both 30 and 37°C showed that the catabolic deletion mutants melanized to the same extent as wild-type. C. Ten-fold spot dilution assays on YPD medium at human body temperature (37 and 39°C) demonstrated that the deletion mutants exhibited wild-type growth.
Figure 6.
HIU hydrolase plays a subtle role in C. neoformans-mediated killing of C. elegans.
∼50 nematode worms were transferred to a lawn of (A) wild-type H99, uro1Δ, uro1Δ + URO1, (B) uro2Δ, uro2Δ + URO2, (C) uro3Δ, uro3Δ + URO3, (D) dal1Δ, dal1Δ + DAL1, (E) dal2,3,3Δ, dal2,3,3Δ + DAL2,3,3, (F) ure1Δ or ure1Δ + URE1, as the sole food source on BHI medium and survival was monitored at 24-hr intervals. There was no observable difference in C. elegans killing by the uro1Δ, uro1Δ + URO1, uro2Δ + URO2, uro3Δ, uro3Δ + URO3, dal1Δ, dal1Δ + DAL1, dal2,3,3Δ, dal2,3,3Δ + DAL2,3,3, ure1Δ and ure1Δ + URE1 strains compared to wild-type. In contrast, the uro2Δ strain killed C. elegans slightly slower than wild-type. All experiments were repeated three times with similar results.
Figure 7.
Uro1, Uro2, Uro3, Dal1 and Dal2,3,3 are not required for infection of a murine host.
10 mice were each intranasally infected with either 5×105 cells of (A) wild-type H99, uro1Δ, uro1Δ +URO1, (B) uro2Δ, uro2Δ +URO2, (C) uro3Δ, uro3Δ + URO3, (D) dal1Δ, dal1Δ +DAL1, (E) dal2,3,3Δ or dal2,3,3Δ + DAL2,3,3, and survival was monitored daily. Mice infected with the uro1Δ, uro1Δ +URO1, uro2Δ, uro2Δ +URO2, uro3Δ, uro3Δ + URO3, dal1Δ, dal2,3,3Δ and dal2,3,3Δ + DAL2,3,3 strains progressed to morbidity as quickly as mice infected with the wild-type strain. Unexpectedly, the complemented dal1Δ +DAL1 strain appeared to be significantly less virulent than wild-type; this conundrum is likely caused by integration of DAL1 into a non-native, virulence-associated (possibly high temperature-associated) locus.