Redundant catalases detoxify phagocyte reactive oxygen and facilitate Histoplasma capsulatum pathogenesis

Abstract

Histoplasma capsulatum is a respiratory pathogen that infects phagocytic cells. The mechanisms allowing Histoplasma to overcome toxic reactive oxygen molecules produced by the innate immune system are an integral part of Histoplasma's ability to survive during infection. To probe the contribution of Histoplasma catalases in oxidative stress defense, we created and analyzed the virulence defects of mutants lacking CatB and CatP, which are responsible for extracellular and intracellular catalase activities, respectively. Both CatB and CatP protected Histoplasma from peroxide challenge in vitro and from antimicrobial reactive oxygen produced by human neutrophils and activated macrophages. Optimal protection required both catalases, as the survival of a double mutant lacking both CatB and CatP was lower than that of single-catalase-deficient cells. Although CatB contributed to reactive oxygen species defenses in vitro, CatB was dispensable for lung infection and extrapulmonary dissemination in vivo. Loss of CatB from a strain also lacking superoxide dismutase (Sod3) did not further reduce the survival of Histoplasma yeasts. Nevertheless, some catalase function was required for pathogenesis since simultaneous loss of both CatB and CatP attenuated Histoplasma virulence in vivo. These results demonstrate that Histoplasma's dual catalases comprise a system that enables Histoplasma to efficiently overcome the reactive oxygen produced by the innate immune system.

Fig 1

CATB and CATP are the major Histoplasma catalase genes expressed, but only CATB shows pathogenic-phase regulation. (A) Yeast- and mycelium-phase expression profiles for the extracellular catalase CATB gene and the CATP and CATA catalase genes. Relative expression of each catalase gene was determined by quantitative PCR of reverse-transcribed RNA from yeasts and mycelia after normalization to the level of expression of the RPS15 gene. Data represent the log (base 2) value of the fold change in expression of the CATB, CATP, and CATA genes in yeasts compared to mycelia. The fold change (yeast-phase compared to mycelium-phase expression) is indicated by the values above the respective columns. Error bars represent standard deviations of three biological replicate RNA samples. Asterisks represent significant differences between yeast and mycelial phases determined by Student's t test (ns, not significant; **, P < 0.01). (B) Estimation of the relative level of CATB, CATP, and CATA expression in yeasts. Expression levels were approximated by use of the RCN value, which was calculated from the RPS15-normalized cycle thresholds for each gene and compared to the value for RPS15-normalized actin (ACT1), set at 100%.

Fig 2

Deletion of the CATB and CATP genes specifically results in loss of extracellular CatB and intracellular CatP, respectively. (A) Analysis of extracellular CatB production. Total culture filtrate proteins from wild type, the catalase mutants, and their corresponding complemented strains were separated by SDS-PAGE, and proteins were visualized by silver staining to monitor production of the 90-kDa CatB protein. (B) Analysis of intracellular CatP production. Cellular lysates were prepared from wild type, the catalase mutants, and their corresponding complemented strains. Evidence of CatP protein in lysates was shown by immunoblotting of lysate proteins with a custom antibody to Histoplasma CatP. WT, wild type (OSU45); catBΔ, catB mutant (OSU16); catBΔ/CATB, complemented catB mutant (OSU51); catPΔ, catP mutant (OSU157); catPΔ/CATP, complemented catP mutant (OSU158); catBΔ catPΔ, catB catP double mutant (OSU159).

Fig 3

CatB and CatP are specifically responsible for extracellular and intracellular catalase activity, respectively. (A) Extracellular cell-free (soluble) catalase activity. Culture filtrates harvested from wild-type and catalase-deficient yeasts were tested for catalase activity by monitoring the ability of the culture filtrate proteins to destroy H₂O₂. Relative H₂O₂ destruction was measured as the decrease in absorbance (Abs) at 240 nm over time at 25°C. (B) Extracellular catalase activity associated with yeast cells. The ability of yeasts to eliminate H₂O₂ was monitored by incubation of washed yeast cells with H₂O₂ and quantitation of the H₂O₂ remaining by determination of the absorbance at 240 nm. At 1-min intervals, yeasts were removed by centrifugation through 0.45-μm-pore-size filters and the absorbance of the clarified solution was determined. The assay was performed at 4°C to slow the enzyme kinetics to allow experimental manipulations. (C) Intracellular catalase activity of yeast cells. Cellular lysates were tested for catalase activity by addition of total yeast lysates to 10 mM H₂O₂ and monitoring the decrease in H₂O₂ by determination of the absorbance at 240 nm over time at 25°C. Error bars represent standard deviations from three biological replicate samples. Asterisks represent significant differences from the wild type strain determined by Student's t test (ns, not significant; **, P < 0.01; ***, P < 0.001). WT, wild type (OSU45); catBΔ, catB mutant (OSU16); catBΔ/CATB, complemented catB mutant (OSU51); catPΔ, catP mutant (OSU157); catPΔ/CATP, complemented catP mutant (OSU158); catBΔ catPΔ, catB catP double mutant (OSU159).

Fig 4

CatB and CatP provide protection to yeasts from H₂O₂ in vitro. Resistance of wild-type and catalase-deficient yeasts to killing by H₂O₂. H₂O₂ killing of yeast was determined by a filter disk diffusion assay on solid medium using filters with 300 mM H₂O₂. (A) Quantitation of the sensitivity to H₂O₂. The zone of clearing around the disks was determined as the total area lacking yeast cell growth. Data presented are the average areas of clearing for three replicate tests, with error bars representing standard deviations. Asterisks represent significant differences from the wild-type strain determined by Student's t test (ns, not significant; *, P < 0.05; **, P < 0.01; ***, P < 0.001). (B) Images of Histoplasma yeast growth around H₂O₂-saturated disks showing the presence of microcolonies of surviving yeasts in zones of clearing for wild type and for CatB-deficient strains but not for strains lacking CatP (catPΔ or catBΔ catPΔ strains). WT, wild type (OSU45); catBΔ, catB mutant (OSU16); catBΔ/CATB, complemented catB mutant (OSU51); catPΔ, catP mutant (OSU157); catPΔ/CATP, complemented catP mutant (OSU158); catBΔ catPΔ, catB catP double mutant (OSU159).

Fig 5

CatP and CatB can provide protection to Histoplasma yeasts against phagocyte-produced reactive oxygen. (A) Survival of Histoplasma yeasts after coincubation with human PMNs. Wild-type and catalase- and/or superoxide dismutase-deficient mutant yeasts were added to PMNs isolated from peripheral human blood. Relative yeast survival (mean ± standard deviation) was determined by enumeration of the number of viable CFU after 4 h. In some experiments, DPI was added 24 h prior to yeast addition to inactivate the phagocyte NADPH oxidase. (B) Survival of Histoplasma yeast after infection of activated human macrophages. Wild-type, catalase, and superoxide dismutase (Sod3) mutants were added to IFN-γ-activated macrophages derived from human peripheral blood monocytes. Relative yeast survival (mean ± standard deviation) was determined by enumeration of viable CFU after 4 h. In some experiments, DPI was added 24 h prior to yeast addition to inactivate the phagocyte NADPH oxidase. Asterisks represent significant differences from survival of the wild-type strain determined by Student's t test of three replicates (ns, not significant; *, P < 0.05; **, P < 0.01; ***, P < 0.001). Additionally, statistical significance was determined for the Sod3-deficient mutant compared to the double mutant lacking both the extracellular superoxide dismutase and the extracellular catalase. WT, wild type (OSU45); catBΔ, catB mutant (OSU16); catBΔ/CATB, complemented catB mutant (OSU51); catPΔ, catP mutant (OSU157); catPΔ/CATP, complemented catP mutant (OSU158); catBΔ catPΔ, catB catP double mutant (OSU159); sod3Δ, sod3 mutant (OSU15); catBΔ sod3Δ, catB sod3 double mutant (OSU46).

Fig 6

Sod3 deficiency, but not CatB deficiency, attenuates Histoplasma virulence in mice. (A) Pulmonary fungal burden kinetics after sublethal lung infection by Histoplasma yeast. Wild-type C57BL/6 mice were infected intranasally with 2 × 10⁴ wild-type, CatB-deficient, Sod3-deficient, or doubly CatB- and Sod3-deficient yeasts. At 4-day intervals postinfection, fungal burdens were determined by quantitative plating of lung homogenates for determination of the number of Histoplasma CFU. The lower limit of detection was 10² CFU. Dashed horizontal lines indicate the actual number of CFU delivered in the inoculum. (B) Dissemination kinetics of Histoplasma yeasts after sublethal lung infection. Wild-type C57BL/6 mice were infected intranasally with 2 × 10⁴ wild-type, CatB-deficient, Sod3-deficient, or CatB- and Sod3-deficient yeasts. At 4-day intervals postinfection, splenic fungal burdens were determined by quantitative plating of spleen homogenates for determination of the number of Histoplasma CFU. The lower limit of detection was 60 CFU. No yeasts had disseminated from the lung to the spleen at day 0. Each data point represents the CFU counts per organ from an individual animal (n = 4). Horizontal bars represent the mean fungal burden. Asterisks indicate statistically significant differences from wild type determined by Student's t test (ns, not significant; *, P < 0.05; **, P < 0.01; ***, P < 0.001). WT, wild type (OSU45); catBΔ, catB mutant (OSU16); sod3Δ, sod3 mutant (OSU15); catBΔΔ sod3Δ, catB sod3 double mutant (OSU46).

Fig 7

CatB and CatP provide redundant catalase functions necessary for Histoplasma virulence in vivo. (A) Pulmonary fungal burden kinetics after sublethal lung infection by Histoplasma yeast. Wild-type C57BL/6 mice were infected intranasally with 2 × 10⁴ wild-type, CatB-deficient, CatP-deficient, or double mutant yeasts lacking both catalases. At 4-day intervals postinfection, fungal burdens were determined by quantitative plating of lung homogenates for determination of the number of Histoplasma CFU. The lower limit of detection was 10² CFU. Dashed horizontal lines indicate the actual number of CFU delivered in the inoculum. (B) Dissemination kinetics of Histoplasma yeast after sublethal lung infection. Wild-type C57BL/6 mice were infected intranasally with 2 × 10⁴ wild-type, CatB-deficient, CatP-deficient, or double catalase mutant yeasts. At 4-day intervals postinfection, splenic fungal burdens were determined by quantitative plating of spleen homogenates for determination of the number of Histoplasma CFU. The lower limit of detection was 60 CFU. No yeasts had disseminated from the lung to the spleen at day 0. Each data point represents CFU counts per organ from an individual animal (n = 4). Horizontal bars represent the mean fungal burden. Asterisks indicate statistically significant differences from wild type determined by Student's t test (ns, not significant; *, P < 0.05; **, P < 0.01). WT, wild type (OSU45); catBΔ, catB mutant (OSU16); catPΔ, catP mutant (OSU157); catBΔ catPΔ, catB catP double mutant (OSU159).