Cysteine and Related Compounds in the Growth of the Yeastlike Phase of Histoplasma Capsulatum

Salvin, S. B.

doi:10.1093/infdis/84.3.275

Cited by 95 publications

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“…Previous studies. showed that cysteine is required for maintenance of the yeast phase (2,4,15,16). Our results show that cysteine also may be required to activate mitochondrial respiration and that this requirement can be met by other reducing agents (2-mercaptoethanol and dithiothreitol).…”

Section: Discussionmentioning

confidence: 99%

“…Factors that affect morphogenesis and maintenance of one phase of the organism are of considerable interest because growth of the yeast phase appears to be required for pathogenicity (2). In culture, the transition from mycelial to yeast phase is triggered by a shift in temperature from 25°C to 37°C.…”

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“…In culture, the transition from mycelial to yeast phase is triggered by a shift in temperature from 25°C to 37°C. Growth and maintenance of the yeast phase in culture are also dependent upon a low oxidation-reduction potential (3) and on the presence of cysteine in the culture medium (2,4). Thus far, however, there has been little insight into the biochemical basis of any of these observations.…”

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Role of cysteine in regulating morphogenesis and mitochondrial activity in the dimorphic fungus Histoplasma capsulatum.

Maresca¹,

Lambowitz²,

Kumar³

et al. 1981

Proc. Natl. Acad. Sci. U.S.A.

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Three stages can be distinguished in the temperature-induced mycelial-to-yeast phase transition of Hwtoplasma capsulatum. Stage one is characterized by a progressive decrease in the respiration rate and in the intracellular concentrations of cysteine and other amino acids. By stage two, respiration has ceased completely and free cysteine has fallen to low levels. Exogenous cysteine is required during the second stage for activation of mitochondrial respiration (stage three) and completion of the morphological transition. Mitochondria isolated from cells in the second stage show no respiration with NADH, succinate, or other substrates unless they are first incubated with cysteine. In addition, a novel, cytosolic cysteine oxidase appears during the latter part of the second stage. In stage three, the respiration rate rises, intracellular concentrations offree cysteine and other amino acids increase to levels characteristic of yeast, and the morphological transition is completed. The results support the idea that alterations in cysteine metabolism play a key role in this differentiation process.Histoplasma capsulatum is a dimorphic pathogenic fungus that grows as a multicellular mycelium in soil and as a yeast in the tissues of infected humans and animals (1). Factors that affect morphogenesis and maintenance of one phase of the organism are of considerable interest because growth of the yeast phase appears to be required for pathogenicity (2). In culture, the transition from mycelial to yeast phase is triggered by a shift in temperature from 25°C to 37°C. Growth and maintenance of the yeast phase in culture are also dependent upon a low oxidation-reduction potential (3) and on the presence of cysteine in the culture medium (2, 4). Thus far, however, there has been little insight into the biochemical basis of any of these observations.We have shown that cysteine stimulates oxygen consumption in the yeast phase but not in the mycelial phase (5). We reasoned that this observation might provide a good starting point for the investigation of biochemical mechanisms in phase determination and morphogenesis ofH. capsulatum. In the present work, we show that the cysteine stimulation of oxygen consumption is due to a cytosolic cysteine oxidase activity, which is present only in yeast-phase cells. In addition, experiments monitoring cellular respiration, cysteine levels, and cysteine oxidase activity during the temperature-induced mycelial-to-yeast phase transition support the idea that alterations in cysteine metabolism play a key role in the differentiation process. They also demonstrate the occurrence of novel regulatory mechanisms involving the effect ofcysteine on the mitochondrial respiratory chain. On the basis of our results and work by others, we propose a model that outlines biochemical events at specific stages of the morphological transition.MATERIALS AND METHODS Growth of H. capsulatum. The wild-type Down's strain of H. capsulatum mating type (-) was used in this study. Cells were grown in the defined m...

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Section: Discussionmentioning

confidence: 99%

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confidence: 99%

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confidence: 99%

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Role of cysteine in regulating morphogenesis and mitochondrial activity in the dimorphic fungus Histoplasma capsulatum.

Maresca¹,

Lambowitz²,

Kumar³

et al. 1981

Proc. Natl. Acad. Sci. U.S.A.

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“…HMM, a medium optimized for growth of Histoplasma and macrophages, is based on Ham's F-12 cell culture medium and is supplemented with 1.8% glucose and 0.7 mM cystine (30). The increased cystine provides additional organic sulfur, since Histoplasma yeast cells and mycelia differ in their requirements for organic sulfur (31)(32)(33)(34)(35). Although both F-12 and RPMI have cysteine (0.2 mM and 0.4 mM, respectively), we tested if supplementation of F-12 or RPMI with cystine increased the yield of Histoplasma yeasts in microtiter plates.…”

Section: Methodsmentioning

confidence: 99%

Quantitative Microplate-Based Growth Assay for Determination of Antifungal Susceptibility of Histoplasma capsulatum Yeasts

2015

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bStandardized methodologies for determining the antifungal susceptibility of fungal pathogens is central to the clinical management of invasive fungal disease. Yeast-form fungi can be tested using broth macrodilution and microdilution assays. Reference procedures exist for Candida species and Cryptococcus yeasts; however, no standardized methods have been developed for testing the antifungal susceptibility of yeast forms of the dimorphic systemic fungal pathogens. For the dimorphic fungal pathogen Histoplasma capsulatum, susceptibility to echinocandins differs for the yeast and the filamentous forms, which highlights the need to employ Histoplasma yeasts, not hyphae, in antifungal susceptibility tests. To address this, we developed and optimized methodology for the 96-well microtiter plate-based measurement of Histoplasma yeast growth in vitro. Using optical density, the assay is quantitative for fungal growth with a dynamic range greater than 30-fold. Concentration and assay reaction time parameters were also optimized for colorimetric (MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] reduction) and fluorescent (resazurin reduction) indicators of fungal vitality. We employed this microtiter-based assay to determine the antifungal susceptibility patterns of multiple clinical isolates of Histoplasma representing different phylogenetic groups. This methodology fulfills a critical need for the ability to monitor the effectiveness of antifungals on Histoplasma yeasts, the morphological form present in mammalian hosts and, thus, the form most relevant to disease.T he increasing incidence of fungal disease necessitates adequate and timely assessment of antifungal susceptibility to guide the selection and implementation of antifungal therapies. Consequently, the Clinical and Laboratory Standards Institute (CLSI) and the European Committee on Antimicrobial Susceptibility Testing (EUCAST) have established reference test methods for susceptibility to the major classes of antifungals, which include the polyenes, azoles, and the recently developed echinocandins (1, 2). These standards utilize broth macrodilution or microdilution assays for testing yeasts (M27-A3 [3] and E.DEF 7.1 [4]) and filamentous fungi (M38-A2 [5] and E.DEF 9.1 [6]). Procedures, including inoculum preparation, media, assay duration, and endpoint definitions, have now been established for testing some of the more commonly encountered yeast-form pathogenic fungi (i.e., Candida species and Cryptococcus). However, notably missing from the M27-A3 and E.DEF 7.1 standards for testing of yeasts are methods for testing the yeast forms of the dimorphic fungi.The dimorphic fungi that cause systemic disease are characterized by distinct yeast and filamentous morphological states (7,8). Temperature is the principal morphology-determining factor, with yeast forms characterizing infections in mammals (9). Histoplasma capsulatum is the most common clinically encountered dimorphic fungal pathogen in the United States, with an estimated 10,000 to 20,000...

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“…Early studies of yeast growth in culture indicated that yeasts but not mycelia of most Histoplasma species are auxotrophic for cysteine due to temperature-dependent expression of sulfite reductase and the consequent inability to incorporate inorganic sulfate into cysteine (28)(29)(30)(31)(32). Organic sulfhydryls, such as cysteine, also reduce the redox potential, which contributes to yeast phase differentiation (33)(34)(35).…”

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Histoplasma capsulatum Depends on De Novo Vitamin Biosynthesis for Intraphagosomal Proliferation

2014

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During infection of the mammalian host, Histoplasma capsulatum yeasts survive and reside within macrophages of the immune system. Whereas some intracellular pathogens escape into the host cytosol, Histoplasma yeasts remain within the macrophage phagosome. This intracellular Histoplasma-containing compartment imposes nutritional challenges for yeast growth and replication. We identified and annotated vitamin synthesis pathways encoded in the Histoplasma genome and confirmed by growth in minimal medium that Histoplasma yeasts can synthesize all essential vitamins with the exception of thiamine. Riboflavin, pantothenate, and biotin auxotrophs of Histoplasma were generated to probe whether these vitamins are available to intracellular yeasts. Disruption of the RIB2 gene (riboflavin biosynthesis) prevented growth and proliferation of yeasts in macrophages and severely attenuated Histoplasma virulence in a murine model of respiratory histoplasmosis. Rib2-deficient yeasts were not cleared from lung tissue but persisted, consistent with functional survival mechanisms but inability to replicate in vivo. In addition, depletion of Pan6 (pantothenate biosynthesis) but not Bio2 function (biotin synthesis) also impaired Histoplasma virulence. These results indicate that the Histoplasma-containing phagosome is limiting for riboflavin and pantothenate and that Histoplasma virulence requires de novo synthesis of these cofactor precursors. Since mammalian hosts do not rely on vitamin synthesis but instead acquire essential vitamins through diet, vitamin synthesis pathways represent druggable targets for therapeutics.

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Cysteine and Related Compounds in the Growth of the Yeastlike Phase of Histoplasma Capsulatum

Cited by 95 publications

References 0 publications

Role of cysteine in regulating morphogenesis and mitochondrial activity in the dimorphic fungus Histoplasma capsulatum.

Role of cysteine in regulating morphogenesis and mitochondrial activity in the dimorphic fungus Histoplasma capsulatum.

Quantitative Microplate-Based Growth Assay for Determination of Antifungal Susceptibility of Histoplasma capsulatum Yeasts

Histoplasma capsulatum Depends on De Novo Vitamin Biosynthesis for Intraphagosomal Proliferation

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