D-proline

Abstract

Two members of the Cryptococcus neoformans-gattii species complex, the etiologic agents of cryptococcosis, can be differentiated by biological, biochemical, serological and molecular typing techniques. Based on their differences in carbon and nitrogen utilization patterns, cost effective and very specific diagnostic tests using D-proline and canvanine-glycine-bromthymol blue (CGB) media have been formulated and are widely used for identification of the two species. However, these methods have yet to be tested for strains with confirmed molecular types to assess the degree of specificity for each molecular type in the two species. We collected global isolates of every major molecular type available and tested their patterns of nitrogen utilization. We confirmed specificity of the CGB test to be 100% regardless of molecular type while the D-proline test yielded 8–38% false negative results in three of the four C. gattii molecular types, VGI–VGIII. The utilization pattern of a new set of amino acids: D-alanine, L-tryptophan and L-phenylalanine, showed species specificity comparable to that of D-proline. We discovered that the transcription factor Gat1 (Are1) regulates the utilization of nitrogen differently between C. neoformans and C. gattii strains. Unlike in C. neoformans, expression of the genes encoding glycine decarboxylase complex in C. gatti was only partially suppressed by nitrogen catabolite repression in the presence of ammonium. GAT1 in C. neoformans controlled the induction of three of the four genes encoding the glycine decarboxylase complex when glycine was used as the sole nitrogen source while in C. gattii its regulation of these genes was less stringent. Moreover, while virulence of C. neoformans strains in mice was not affected by Gat1, the transcription factor positively influenced the virulence of C. gattii strain.

Discussion

The ability to utilize specific nitrogen and carbon sources is among the standard methods used for the identification of yeast species , as well as some filamentous fungi . Differentiation of the two species in Csc also has been effectively carried out by both nitrogen and carbon utilization tests , , , , . However, the efficacies of these biochemical tests were established long before molecular strain typing methods became available and the validity of these tests could have been compromised since the populations tested could contain strains of the same genotype. For example, previous studies have reported 100% and 95% specificity for D-proline utilization by C. gattii while we found a much lower specificity; only 80% of C. gattii strains yielded positive results in contrast to the 100% that were negative for C. neoformans. The CGB media, on the other hand, showed 100% specificity between the two species regardless of molecular type. Remarkably, the false negatives observed for D-proline utilization were found mainly among strains of the VGI and VGIII (5 negatives among 6 strains) molecular types. According to the phylogenetic analysis of global Csc strains based on multi gene sequence analyses, the VGI and VGIII molecular types formed a separate cluster with high bootstrap supports , . Our results underscore the biological variation between different molecular types within C. gattii.