Heat-shock proteins induced during the mycelial-to-yeast transitions of strains of Histoplasma capsulatum

Heat-shock proteins (hsp) were elicited when mycelia of the Downs strain and the more virulent G184A and G222B strains of Histoplasma capsulatum were shifted up to temperatures which induced the mycelial-to-yeast transition (34-40 degrees C). The classes of the major hsp which increased in synthesis in each strain were similar. However, the pattern of synthesis of these proteins at the different temperatures in Downs differed from those in the G184A and G222B strains: hsp synthesis in Downs peaked at 34 degrees C, whereas in G184A and G222B it was highest at 37 degrees C.     

Uptake of L-proline by Histoplasma capsulatum.

The uptake and incorporation of L-proline by yeast cells of the dimorphic zoopathogen Histoplasma capsulatum were studied. The amino acid was assimilated in at least two ways: by an active transport system with a Km of 1.7 X 10(-5) M and by simple diffusion. The active transport system was sterospecific and severely restricted to neutral aliphatic side-chain amino acids. Certain analogues inhibited L-proline uptake and prevented incorporation of the amino acid into cellular constituents. The inhibition of L-proline uptake by L-leucine was competitive. Since L-leucine and L-proline are seemingly transported by a system with similar characteristics, must be concluded, as originally postulated, that the buckled ring of L-proline, in solution, acts as an aliphatic side chain and that this cyclic amino acid is transported by a system more or less specific for amino acids with neutral aliphatic side chains.     

Regulation of dimorphism in Histoplasma capsulatum by cyclic adenosine 3',5'-monophosphate.

During temperature-induced transition of the dimorphic pathogenic fungus Histoplasma capsulatum from the single yeast cell form to the multicellular mycelial form, there was an increase in intracellular cyclic adenosine 3',5'-monophosphate (cAMP) levels as well as a striking accumulation of cAMP in the medium. cAMP levels also changed during the reverse mycelium-to-yeast transition.     

Selenocystine-resistant mutants of Histoplasma capsulatum

Cysteine metabolism has been thought to be important to the phenomenon of dimorphism inHistoplasma capsulatum. We sought mutants with genetic blocks in the metabolism of cysteine by selection of colonies resistant to the toxic analogue, selenocystine. The 22 resistant strains thus obtained were all deficient in uptake of cystine from the surrounding medium but were normally able to convert from mycelium to yeast and back again. Furthermore, they had normal quantities of NADH-dependent cystine reductase when this enzyme was measured. We conclude that mutants defective in cystine uptake can be readily obtained by selection of colonies resistant to selenocystine, and that a lesion in cystine-uptake does not appear to affect the phenomenon of dimorphism in this organism.Preliminary reports of this work were presented at the Second International Congress of Mycology, Tampa, 1977 and at the first International Conference on Histoplasmosis, Atlanta, 1978.     

Chemical composition of Histoplasma capsulatum

The chemical composition of yeast and mycelial cells of three strains ofHistoplasma capsulatum was analyzed and is expressed as per cent dry weight. Cultures were grown in a liquid synthetic medium, mycelial cells at 25°C and yeast at 37°C on gyrotory shakers. After 7 days, deoxyribonucleic acid (DNA), ribonucleic acid (RNA), and protein were higher in the yeast cells while mycelial cells contained more lipid and carbohydrate. The components of one strain were also studied at different stages of growth. The DNA in both yeast and mycelial cells remained relatively constant, but other components varied with the age of culture. In yeast cells the RNA level was 6.8 % at 2 days and then declined sharply remaining constant around 3.5 %. A protein content of 29 % on day 2 decreased gradually to 19 % on day 14. An initial lipid content of 21 % rose to 33 % on day 5 and then decreased. Similarly, an initial carbohydrate level of 17 % rose to 25 % on day 7 and then declined. The mycelial cells contained 4 % RNA up to 10 days followed by a slight decline to 3 % on day 14. A protein content of 20 % on day 5 increased to 24 % on day 10 and then decreased to 15 % on day 28. The lipid content of 33 % on day 5 rose to 38 % on day 7 and then decreased gradually. The carbohydrate level of 20 % at 5 days increased to 38 % on day 10 and declined gradually to 27 % after 28 days.

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Résumé La composition chimique des cellules levuriformes et mycéliennes de trois souches deHistoplasma capsulatum a été déterminée. Le champignon a été cultivé dans un milieu synthétique liquide secoué à 25° C pour la phase mycélienne et à 37° C pour la phase levuriforme. Après 7 jours d'incubation, les cellules levuriformes étaient plus riches en acides nucléiques et en protéines que les cellules mycéliennes qui étaient par contre plus riches en lipides et en hydrates de carbone. La composition d'une des souches fut étudiée à différentes étapes de la croissance. La teneur en ADN des deux phases resta relativement constante mais des variations furent observées dans le cas des autres constituants chimiques. Pour ce qui est des levures, l'ARN qui constituait 6,8 % du poids des cellules sèches à deux jours, tomba rapidement à 3,5 % et resta constant. Les proteines passèrent de 29 % au deuxième jour à 19 % au quatorzième jour. Au contraire, la teneur en lipides passa d'un valeur initiale de 21 % à 33 % au cinquième jour, pour diminuer de nouveau par la suite. De même, une teneur initiale en hydrates de carbone de 17 % passa à 25 % au septième jour puis diminua par la suite. Dans les cas des cellules mycéliennes contenaient 4 % de ARN jusqu'au dizième jours, puis cette valeur tomba légèrement jusqu'à 3 % au quatorzième jour. Les protéines qui représentaient 20 % au cinquième jour augmentèrent jusqu'à 24 % au dizième jour pour tomber à 15 % au vingthuitième jour. La teneur en lipides de 33 % au cinquième jour augmenta jusqu'à 38 % au septième jour pour diminuer graduellement. De même les taux en hydrates de carbones qui représentaient 20 % au cinquième jour augmentèrent jusqu'à 38 % au dixième jour et diminuèrent graduellement jusqu'à 27 % au vingt-huitième jour.

     

Electrophoretic analysis of Histoplasma capsulatum chromosomal DNA.

Seven chromosome-sized DNA molecules in the Downs strain of Histoplasma capsulatum were resolved by using chromosome-specific DNA probes in blot hybridizations of contour-clamped homogeneous electric field (CHEF) and field-inversion gel electrophoresis (FIGE) agarose gels. The sizes of the chromosomal DNA bands extended from that of the largest Saccharomyces cerevisiae chromosome to beyond that of the Schizosaccharomyces pombe chromosomes. Under our experimental conditions, the order of the five largest DNA bands was inverted in the FIGE gel relative to the CHEF gel, demonstrating a characteristic of FIGE whereby large DNA molecules may have greater rather than lesser mobility with increasing size. Comparison of the Downs strain with other H. capsulatum strains by CHEF and FIGE analysis revealed considerable variability in band mobility. The resolution of seven chromosome-sized DNA molecules in the Downs strain provides a minimum estimate of the chromosome number.     

Effect of thimerosal on the whole yeast phase antigen of Histoplasma capsulatum

Summary Thimerosal (merthiolate) and formalin treated whole-cell yeast phase antigens ofHistoplasma capsulatum were prepared and their reactivities with sera from cases of histoplasmosis, blastomycosis and coccidioidomycosis were compared. Thimerosal treated antigens often gave complement fixation titers with heterologous sera 2 to 8 fold lower than the titers obtained with formalin treated antigens. However, with certain anti-histoplasmosis sera, thimerosal killed antigens had less reactivity with homologous antisera also. In virtually all cases an equal or higher specificity ratio was obtained with thimerosal killed antigens. The effects of thimerosal and formalin were independent, indicating different sites of reactivity of these reagents. Uptake of thimerosal at several concentrations suggested two types of reactions with live yeast phase cells. Analyses of the cellular fractions for thimerosal showed it was present only in the soluble fractions from which it was readily removed by dialysis. Cellular fractions killed with thimerosal retained several of the same physical and antigenic characteristics of those fractions isolated from frozen and thawed cells.     

Cysteine-independent and cysteine-requiring yeast-strains of Histoplasma capsulatum

Recently we described a strain of Histoplasma capsulatum, designated H-35, which is able to grow as yeast on a minimal medium consisting of inorganic salts, glucose and a trace of biotin. Using this strain as a prototrophic wild type we sought auxotrophic mutants. Mutagenized yeast-cells were starved for inorganic sulfate in sulfur-free minimal medium. Sulfate was then added, and growing prototrophic cells were killed by addition of amphotericin B. After 24 hours non-growing auxotrophs were rescued by removal of amphotericin and addition of yeast extract. This mutant enrichment cycle was repeated two additional times, after which the cells were plated on blood agar and 800 yeast-colonies were picked. Seventeen of these yeast-strains required cysteine for growth, as compared with strain H-35, which grew as yeast on minimal medium.     

A prototrophic yeast-strain of Histoplasma capsulatum

A newly derived strain of Histoplasma capsulatum can be grown stably as yeast in a minimal medium containing glucose, biotin, tartrate and inorganic salts.     

The dimorphism and infectivity of Histoplasma capsulatum

Summary By examining mycelial cultures at regular intervals during incubation at 37° C in cysteine-enriched media, it was possible to detect differences in the conversion properties of six isolates ofHistoplasma capsulatum. The organisms varied according to conversion ability, rate and degree of transformation following yeast initiation, and cysteine sensitivity. These findings were unrelated to infectivity of the mycelial phase for mice as determined by percentage recovery of isolates from the cultured organs of inoculated animals.     

Digestion of Histoplasma capsulatum yeasts by human macrophages.

The strategies used by Histoplasma capsulatum yeasts to survive and multiply within human macrophages (M phi) are unknown. To better understand these strategies we studied the intracellular fate of viable vs heat-killed (HK) yeasts in human monocyte-derived M phi. Initial studies demonstrated that phagolysosome fusion was present in M phi ingesting either viable or HK yeasts. Viable yeasts multiplied within M phi phagolysosomes, whereas M phi completely digested intracellular FITC-labeled HK yeasts within 24 h after ingestion. This observation was confirmed by electron microscopy. M phi that had ingested colloidal gold-labeled HK yeasts contained gold particles but no visible yeasts at 24 h. Digestion of HK yeasts was evident as early as 4 h after phagocytosis, and was complete by 24 h. M phi digestion of HK yeasts was blocked completely when M phi were cultured for 24 h in the presence of chloroquine. In M phi simultaneously ingesting both viable and HK yeasts, viable yeasts multiplied, but HK yeasts were digested within the same cell. M phi that had ingested viable yeasts digested them completely when M phi were cultured for 24 h in the presence of cycloheximide or amphotericin B. Coculture of infected M phi with nystatin or ketoconazole resulted in inhibition of growth, but the yeasts were not digested. These data indicate that: 1), HK Hc yeasts are easily digested by preformed M phi lysosomal hydrolases; 2), viable Hc yeasts survive and multiply within M phi phagolysosomes, but the yeasts do not secrete a factor(s) that affects the ability of other phagolysosomes within the same M phi to digest killed yeasts; and 3), inhibition of yeast protein synthesis or cell wall biosynthesis is sufficient to render viable yeasts susceptible to digestion by human M phi.