Role of alternative oxidase gene in pathogenesis of Cryptococcus neoformans

Abstract

We identified a homologue of the alternative oxidase gene in a screen to identify genes that are preferentially transcribed in response to a shift to 37 degrees C in the human-pathogenic yeast Cryptococcus neoformans. Alternative oxidases are nucleus-encoded mitochondrial proteins that have two putative roles: they can function in parallel with the classic cytochrome oxidative pathway to produce ATP, and they may counter oxidative stress within the mitochondria. The C. neoformans alternative oxidase gene (AOX1) was found to exist as a single copy in the genome, and it encodes a putative protein of 401 amino acids. An aox1 mutant strain was created using targeted gene disruption, and the mutant strain was reconstituted to wild type using a full-length AOX1. Compared to both the wild-type and reconstituted strains, the aox1 mutant strain was not temperature sensitive but did have significant impairment of both respiration and growth when treated with inhibitors of the classic cytochrome oxidative pathway. The aox1 mutant strain was also found to be more sensitive to the oxidative stressor tert-butyl hydroperoxide. The aox1 mutant strain was significantly less virulent than both the wild type and the reconstituted strain in the murine inhalational model, and it also had significantly impaired growth within a macrophage-like cell line. These data demonstrate that the alternative oxidase of C. neoformans can make a significant contribution to metabolism, has a role in the yeast's defense against exogenous oxidative stress, and contributes to the virulence composite of this organism, possibly by improving survival within phagocytic cells.

FIG. 1.

Schematic diagram of fungal mitochondrial electron transfer chain during respiration in the inner mitochondrial membrane. Electron transfer complexes (I to IV), alternative oxidase and sites of action of inhibitors, SHAM, myxothiazol, antimycin, and cyanide are shown.

FIG. 2.

(A) Disruption of C. neoformans AOX1 by homologous recombination. (I) Restriction map of the genomic fragment containing the alternative oxidase gene (AOX1). (II) A 1.9-kb URA5 gene is ligated into the XhoI site within AOX1. The solid arrows flanking the XhoI sites designate the sites of the PCR primers that were used to screen for disruption of the native gene. (III) Construct used to complement aox1 strain with AOX1. A nourseothricin resistance cassette (NAT) was ligated into the plasmid containing AOX1 and was used as a selectable marker. (B) PCR analysis using primers shown in panel A (I) gives a 700-bp fragment in H99. In aox1 strains (aox1-1 and aox1-2), this fragment is displaced by 2,600 bp due to insertion of URA5. The fragments from reconstituted strains (AOXRec1 to AOXRec3) demonstrate the disrupted native locus as well as that corresponding to the wild-type copy of AOX1. (C) Southern analysis of genomic DNA cut with XbaI confirms that AOX1 has been disrupted in the knockout mutant and that the reconstituted AOX1 strains show random ectopic insertion of AOX1.

FIG. 3.

C. neoformans has a functional cyanide-resistant respiratory pathway. Oxygen uptake was monitored in the presence of myxothiazol and SHAM. Data shown represent the means and standard errors of results from three independent experiments.

FIG. 4.

Growth patterns of C. neoformans (H99, aox1, and AOXRec1). A total of 10⁴ CFU/ml were treated with 1 mM KCN and 2 mM SHAM at 30°C for 24 h, and cells were plated for number of surviving colonies. (SHAM alone was not tested with aox1.)

FIG. 5.

Northern analysis of AOX1 expression. Total RNA obtained from aox1 grown at 37°C for 4 h, H99 grown at 30°C for 4 h, H99 grown at 37°C for 4 h, H99 grown at 30°C for 24 h, and H99 grown at 37°C for 24 h (lanes 1 to 5, respectively) was probed with AOX1 (top). Below are photos of gels showing rRNA bands for measurement of RNA loading (bottom).

FIG. 6.

Outcome of inhalational murine model. Shown is survival of mice inoculated with wild-type H99 (solid circles), AOXRec1 (solid inverted triangles), and the aox1 strain (open circles). Survival of mice infected with the aox1 mutant was significantly longer than that of mice infected with H99 (P = 0.001) and AOXRec1 (P = 0.01).