Reactive oxygen species homeostasis and virulence of the fungal pathogen Cryptococcus neoformans requires an intact proline catabolism pathway

Abstract

Degradation of the multifunctional amino acid proline is associated with mitochondrial oxidative respiration. The two-step oxidation of proline is catalyzed by proline oxidase and Δ(1)-pyrroline-5-carboxylate (P5C) dehydrogenase, which produce P5C and glutamate, respectively. In animal and plant cells, impairment of P5C dehydrogenase activity results in P5C-proline cycling when exogenous proline is supplied via the actions of proline oxidase and P5C reductase (the enzyme that converts P5C to proline). This proline is oxidized by the proline oxidase-FAD complex that delivers electrons to the electron transport chain and to O2, leading to mitochondrial reactive oxygen species (ROS) overproduction. Coupled activity of proline oxidase and P5C dehydrogenase is therefore important for maintaining ROS homeostasis. In the genome of the fungal pathogen Cryptococcus neoformans, there are two paralogs (PUT1 and PUT5) that encode proline oxidases and a single ortholog (PUT2) that encodes P5C dehydrogenase. Transcription of all three catabolic genes is inducible by the presence of proline. However, through the creation of deletion mutants, only Put5 and Put2 were found to be required for proline utilization. The put2Δ mutant also generates excessive mitochondrial superoxide when exposed to proline. Intracellular accumulation of ROS is a critical feature of cell death; consistent with this fact, the put2Δ mutant exhibits a slight, general growth defect. Furthermore, Put2 is required for optimal production of the major cryptococcal virulence factors. During murine infection, the put2Δ mutant was discovered to be avirulent; this is the first report highlighting the importance of P5C dehydrogenase in enabling pathogenesis of a microorganism.

Keywords: Cryptococcus neoformans; P5C dehydrogenase; nitrogen; proline oxidase; virulence.

Figure 1

The C. neoformans genome contains two paralogs that encode proline oxidases and a single ortholog that encodes P5C dehydrogenase. (A) Scheme representing the proline degradation pathway. (B) Exon–intron structures of PUT1, PUT5, and PUT2 and their chromosomal locations.

Figure 2

Transcription of all the proline catabolic enzyme-encoding genes is regulated by pathway-specific induction. cDNA from wild-type H99 grown in YNB supplemented with proline, ammonium, glutamine, glutamate, asparagine, or uric acid (10 mM each) was amplified via qRT-PCR using primers for the proline catabolic genes PUT1, PUT5, and PUT2, normalized against the control gene ACT1. Expression of PUT1, PUT5, and PUT2 was induced in the presence of proline but not in the presence of the other nitrogen sources (*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001). Error bars represent standard errors across three biological replicates.

Figure 3

PUT5-encoded proline oxidase and PUT2-encoded P5C dehydrogenase are required for proline assimilation. Tenfold spot dilution assays for nitrogen or nitrogen and carbon source utilization showed that the put1Δ mutant exhibited wild-type growth on YNB supplemented with 10 mM proline (with or without 2% glucose as a carbon source). In contrast, the put5Δ, double put1Δ put5Δ, and put2Δ mutants were unable to proliferate on proline as the sole nitrogen or carbon source. The ammonium-supplemented YNB plates served as a control to demonstrate that, unlike proline, ammonium can be utilized by C. neoformans only as a nitrogen source but not as a carbon source.

Figure 4

The put5Δ and double put1Δ put5Δ mutants are less tolerant against the oxidative stressor tert-Butyl hydroperoxide and nitrosative stressor GSNO. Wild-type H99 and proline oxidase deletion mutant strains were grown in YNB [proline and glutamate (10 mM each)] supplemented with (A) 0–0.3 mM tert-Butyl hydroperoxide or (B) 0–9 mM GSNO. The put5Δ and double put1Δ put5Δ mutants exhibited a significantly lower growth percentage relative to the unstressed control when subjected to these individual stressors as compared to the wild-type or put1Δ strains (**P < 0.01, ***P < 0.001). Growth percentage relative to unstressed control is defined by dividing each stressed strain’s growth in OD₆₀₀ with the same strain’s growth in OD₆₀₀ when unstressed. Error bars represent standard errors across three biological replicates.

Figure 5

The put2Δ mutant generates excessive superoxide ions in the mitochondria when exposed to proline. Wild-type H99 and proline catabolic deletion mutant strains were briefly subjected to culture in YNB supplemented with 10 mM proline and stained with MitoSOX, and the accumulation of ROS in the mitochondria was assessed by fluorescence microscopy and flow cytometry. (A) Representative confocal images showing a subpopulation of put2Δ cells generate enhanced MitoSOX fluorescence compared to the wild-type or proline oxidase deletion mutant strains. Bar, 10 μm. (B) Representative histograms of flow cytometry showing two major distinct populations of put2Δ cells with one subpopulation exhibiting wild-type MitoSOX fluorescence and another subpopulation producing enhanced fluorescence. Overall, the put2Δ mutant generated a significant increase in mean fluorescence intensity of oxidized MitoSOX relative to the wild-type or complemented put2Δ + PUT2 strains (Figure S6). Unstained C. neoformans cells were used as a negative control.

Figure 6

P5C dehydrogenase is needed for optimal growth at high temperature and for optimal production of melanin. (A) Tenfold spot dilution assays on YPD medium demonstrated that the put2Δ mutant exhibited a slightly slower growth rate compared to wild type at 30°, and this growth defect was exacerbated at 37°. (B) Tenfold spot dilution assays on L-DOPA medium showed that the put2Δ mutant produced slightly less melanin than wild type at 30° and that this melanization defect was exacerbated at 37°.

Figure 7

The put2Δ mutant is avirulent during infection of a murine host. Ten mice were each intranasally infected with either 5 × 10⁵ cells of (A) wild-type H99, put1Δ, put1Δ +PUT1, (B) put5Δ, put5Δ +PUT5, (C) double put1Δ put5Δ, put1Δ put5Δ + PUT1 PUT5, and (D) put2Δ or put2Δ +PUT2, and survival was monitored daily. Mice infected with the put1Δ, put1Δ +PUT1, put5Δ, put5Δ +PUT5, double put1Δ put5Δ, put1Δ put5Δ + PUT1 PUT5, and put2Δ +PUT2 strains progressed to morbidity as quickly as mice infected with the wild-type strain. In contrast, mice infected with the put2Δ strain remained healthy throughout the experiment.