Quantitative Studies of Total Lipids of Pathogenic Fungi

Ten species of dermatophytes and four of the systemic fungi were assayed for total lipids, acetone-soluble fraction, and phospholipid content in different types of cultures. The yeast phase of each of the systemic fungi grown on solid medium exhibited a higher total lipid content than did the mycelial growth in liquid medium, either shake or still. Shake cultures, in all the fungi tested, produced the least lipids. The yeasts were consistently higher also in the acetone-soluble fraction. Histoplasma duboisii in the yeast phase and Microsporum gypseum produced the greatest amount of phospholipid, and Blastomyces dermatitidis in the yeast phase and M. canis produced the largest acetone-soluble fraction among the systemic fungi and dermatophytes, respectively.     

The lipid content of cell walls obtained from juvenile,yeast-like and filamentous cells ofCandida albicans

Purified cell walls ofCandida albicans obtained from juvenile cells, mature yeast-like cells and filamentous cells were analyzed for their lipid components. Chloroform: methanol (2:1 v v) extraction of the acetone-treated dried cell walls indicated the total lipid content to be 2.1% of the dry weight of the juvenile cell walls, 1.8% of the mature yeast-like cell walls and 4.5% of the filamentous cell walls. Separation of the chloroform: methanol extractable fraction through a silicie acid column and quantitative determination of the fractions showed significant amounts of sterol esters, triglycerides, sterols, free fatty acids, and phospholipids in these extracts. Following acetone extraction sterols were shown to constitute a greater percentage of the cell wall of juvenile cells than mature cells. Thin-layer chromatography separated the acetone-extractable lipids into at least four components. Diethyl ether extracts of the cell walls indicated the presence of small amounts of glycerol phospholipids in the cell walls of juvenile and mature yeast cells. Boiling 95% ethanol also removed a small lipid fraction from the cell walls of both juvenile and mature yeast which could include sphingosine phosphatides or glycosides.     

Phospholipids from Bacillus stearothermophilus

The lipids of Bacillus stearothermophilus strain 2184 were extracted with chloroform-methanol and separated into neutral lipid and three phospholipid fractions by chromatography on silicic acid columns. The phospholipids were identified by specific staining reactions on silicic acid-impregnated paper, by chromatography of alkaline and acid hydrolysis products, and by determination of acyl ester:glycerol:nitrogen:phosphorus molar ratios. The total extractable lipid was 8% of the dry weight of whole cells and consisted of 30 to 40% neutral lipid and 60 to 70% phospholipid. The phospholipid consisted of diphosphatidyl glycerol (23 to 42%), phosphatidyl glycerol (22 to 39%), and phosphatidyl ethanolamine (21 to 32%). The concentrations of diphosphatidyl glycerol and phosphatidyl glycerol were lower in 2-hr cells than in 4- and 8-hr cells. Whole cells were fractionated by sonic treatment and differential centrifugation. The total lipid content, expressed in per cent of dry weight of each fraction was: whole protoplasts, 10%; membrane fraction, 18%; 30,000 x g particulate fraction, 22%; and 105,000 x g particulate fraction, 26%. The relative phospholipid concentrations in each fraction were about the same. As had been previously reported, none of the phospholipid was stable to alkaline hydrolysis.     

Preparation and study of an exoantigen of Histoplasma capsulatum for serological reactions.

The aim of this study was to determine the usefulness of a yeast-phase exo-antigen of Histoplasma capsulatum in standard serologic reactions. Three native strains of H.capsulatum which belong to Mycology Center collection were employed. They were maintained in their yeast-phase by weekly subcultures in 2% dextrose broth agar at 37 degrees C. After one week incubation yeast cells were suspended in distilled water containing thimerosal and phenylmethyl sulfonyl fluoride at a concentration of 1:5000. This suspension was left at room temperature for 72 h, then the supernatant was separated by centrifugation and it was lyophilized. Proteins and polysaccharides concentrations were determined. Immunodiffusion (ID) tests were carried out with an antigenic dilution containing 1.4 mg/ml of proteins. This exo-antigen was submitted to SDS-PAGE. Seven protein fractions were detected but only two of them showed antigenic activity against a pool of positive human sera; the molecular weights of these two proteins were 97 kDa and 66 kDa respectively. A metabolic antigen from the mycelial phase of H. capsulatum was used as control. A rabbit gammaglobulin anti-H. capsulatum was prepared and employed as positive control in serologic reactions. The antigenic capacity of ten batches of this exo-antigen was studied by ID and counterimmunoelectrophoresis (CIE) tests using serum samples of 20 hamsters experimentally infected by intracardiac inoculation of the yeast-phase of H. capsulatum. All tests presented positive results after three weeks of the infection. Fifty sera from patients suffering progressive histopasmosis were analyzed: ID, CIE and complement fixation (CF) tests were performed in all cases. HIV negative patients presented 7/7 (100%) positive reactions with the yeast-phase exoantigen and 5/7 (71.4%) with histoplasmin. In HIV positive patients CIE and CF were the most sensitive serologic tests, they gave positive results in 15/43 cases (34.8%) with the yeast-phase exo-antigen and in 7/43 cases (13.9%) with histoplasmin. Sera from 10 patients with paracoccidioidomycosis, aspergillosis and candidiasis respectively were studied by ID with the aim of detecting serologic cross reactions. No cross reaction was detected in these serum samples. This yeast-phase exo-antigen of H. capsulatum is more sensitive than and equally as specific as control histoplasmin.     

Monosaccharide and Chitin Content of Cell Walls of Histoplasma capsulatum and Blastomyces dermatitidis

Cell walls of Histoplasma capsulatum and Blastomyces dermatitidis, obtained by mechanical breakage of yeast- and mycelial-phase cultures, were lipid-extracted and then fractionated with ethylenediamine. Unextracted cell walls, lipid-extracted cell walls, and the three fractions resulting from ethylenediamine treatment were examined for monosaccharide and chitin content. The yeast-phase cell walls of five strains of H. capsulatum fell into two categories, designated chemotypes I and II, one of which, chemotype II, was similar to yeast-phase cell walls derived from three strains of B. dermatitidis. H. capsulatum chemotype I cell walls were characterized by lower content of material soluble in ethylenediamine, higher chitin content, and lower monosaccharide content than H. capsulatum chemotype II or B. dermatitidis cell walls. Approximately 80% of the monosaccharides of chemotype I cell walls was combined in forms susceptible to attack by mild acid hydrolysis, compared with about 50% of the monosaccharides of chemotype II and B. dermatitidis. H. capsulatum and B. dermatitidis yeast-phase cell walls could be distinguished, however, by their susceptibility to attack by a crude enzyme system derived from a Streptomyces sp. incubated with chitin as the only carbon source. Both glucose and acetylglucosamine were released from H. capsulatum cell walls, regardless of chemotype, during enzymatic hydrolysis, whereas only acetylglucosamine was released from B. dermatitidis yeast-phase cell walls. Mycelial-phase cell walls of H. capsulatum and B. dermatitidis were characterized by lower content of material soluble in ethylenediamine, higher proportions of mannose, and lower chitin content than their respective yeast phases. Glucose and acetylglucosamine were both released from all mycelial-phase cell walls, whether H. capsulatum or B. dermatitidis, by the crude enzyme system.     

Composition and fatty acid content of rat ventral prostate phospholipids

The major phospholipids of rat ventral prostate have been separated and examined using thin-layer chromatography, gas chromatography and mass spectrometry. The main phospholipid classes were choline and ethanolamine glycerophospholipids, accounting for 77.9% of total lipid phosphorus. The prostate also contained small amounts of serine glycerophospholipids and sphingomyelin. The relative proportions of fatty acids in the different phospholipid classes were also determined. Arachidonic acid in prostatic phospholipids is contributed primarily by ethanolamine glycerophospholipids. This fraction contained 65-69 mol% plasmalogens, whereas choline and serine glycerophospholipid fractions contained less than 5 mol% plasmalogens. Ethanolamine, choline and serine plasmalogens contained mainly vinyl ethers of palmitic and stearic aldehydes. Ethanolamine plasmalogens also contained the vinyl ether of oleic aldehyde.     

The lipids of the Golgi apparatus subfractions from rat liver.

Golgi apparatus were isolated from untreated rat liver and separated into three fractions. One consisted mainly of vesicles, a second of tubular particles (dictyosomes) and the third was a mixed fraction. Large differences between these fractions could be seen in the electron microscope and by enzyme analysis. The total lipid content of the vesicles was 3.5-times greater than that of the dictyosomes and the neutral lipid value was 7-times greater. The ratio of phospholipids to protein was approximately the same in the three fractions. However, the phospholipid patterns differed between the vesicle and dictyosome fractions.     

Structure of egg yolk very low density lipoprotein. Polydispersity of the very low density lipoprotein and the role of lipovitellenin in the structure

Egg yolk very low density lipoprotein contained on the average 75% of lipid which could be extracted by ether and 25% of a residual lipoprotein, the classical lipovitellenin. The ether-extracted lipid was composed of 75% triglycerides, 7% sterols, 2% mono- and diglycerides, and 16% phospholipids. Lipovitellenin contained 48% lipid composed of 87% phospholipids, 11% triglycerides, and 2% sterols. The protein vitellenin was composed for the most part of units of 74,000 and 270,000 daltons molecular weight.Egg yolk very low density lipoprotein is polydisperse. Preparative ultracentrifugation separated it into six fractions of different average molecular size, and gel chromatography separated it into five. The fractions of larger molecular size contained more lipid and triglyceride than did the fractions of smaller molecular size. The proteins of the fractions appeared to be similar.Egg yolk very low density lipoproteins appear to be a series of molecules composed of cores of lipid of varying sizes with each core surrounded by a layer of lipovitellenin, which is composed principally of glycoprotein and phospholipid.     

Histoplasma capsulatum pathogenesis: making a lifestyle switch

The dimorphism of Histoplasma reflects a developmental switch in morphology and lifestyle that is necessary for virulence. The dimorphism regulating kinase DRK1 and the Histoplasma WOR1 homolog RYP1 mediate the thermally induced transition to the pathogenic yeast-phase program. The genes expressed as part of this regulon influence the host-pathogen interaction to favor Histoplasma virulence. While surface localized HSP60 supports yeast attachment to host macrophages, yeast alpha-glucan polysaccharides conceal immunostimulatory cell wall beta-glucans from detection by macrophage receptors. Intramacrophage growth of yeast cells is facilitated by CBP a secreted, protease-resistant calcium-binding protein tailored to function within the phagolysosomal environment. In some Histoplasma strains, YPS3 promotes dissemination of yeast from pulmonary infection sites. The Histoplasma yeast-phase program includes additional cell surface and extracellular molecules that potentially function in further aspects of Histoplasma virulence.     

Antibodies in Histoplasmosis

Production of precipitating and complement-fixing antibody in rabbits and other animals was induced by immunization with live yeast-phase cells of Histoplasma capsulatum. Results of studies of polysaccharide antigens from three strains of H. capsulatum, by quantitative complement-fixation with human and rabbit antisera, strongly suggest the presence of type specificity. The variations of titer during 11 weeks in one patient with histoplasmosis and the variations of titer among a group of patients with histoplasmosis were studied by use of quantitative complement-fixation tests.     

Maintaining network security: how macromolecular structures cross the peptidoglycan layer

Histoplasma capsulatum is the leading cause of endemic mycosis in the world. Analyses of clinical isolates from different endemic regions show important diversity within the species. Recent molecular studies of two isolates, the Chemotype I NAm2 strain G217B and the Chemotype II Panamanian strain G186A, reveal significant genetic, structural, and molecular differences between these representative Histoplasma strains. Some of these variations have functional consequences, representing distinct molecular mechanisms that facilitate Histoplasma pathogenesis. The realization of Histoplasma strain diversity highlights the importance of characterizing Histoplasma virulence factors in the context of specific clinical strain isolates.     

Rapid synthesis and turnover of brain microsomal ether phospholipids in the adult rat.

The rates of synthesis, turnover, and half-lives were determined for brain microsomal ether phospholipids in the awake adult unanesthetized rat. A multicompartmental kinetic model of phospholipid metabolism, based on known pathways of synthesis, was applied to data generated by a 5 min intravenous infusion of [1,1-(3)H]hexadecanol. At 2 h post-infusion, 29%, 33%, and 31% of the total labeled brain phospholipid was found in the 1-O-alkyl-2-acyl-sn-glycero-3-phosphate, ethanolamine, and choline ether phospholipid fractions, respectively. Autoradiography and membrane fractionation showed that 3% of the net incorporated radiotracer was in myelin at 2 h, compared to 97% in gray matter microsomal and synaptosomal fractions. Based on evidence that ether phospholipid synthesis occurs in the microsomal membrane fraction, we calculated the synthesis rates of plasmanylcholine, plasmanylethanolamine, plasmenylethanolamine, and plasmenylcholine equal to 1.2, 9.3, 27.6, and 21.5 nmol. g(-1). min(-1), respectively. Therefore, 8% of the total brain ether phospholipids have half-lives of about 36.5, 26.7, 23.1, and 15.1 min, respectively. Furthermore, we clearly demonstrate that there are at least two pools of ether phospholipids in the adult rat brain. One is the static myelin pool with a slow rate of tracer incorporation and the other is a dynamic pool found in gray matter.The short half-lives of microsomal ether phospholipids and the rapid transfer to synaptosomes are consistent with evidence of the marked involvement of these lipids in brain signal transduction and synaptic function.     

Soluble Antigens for Immunofluorescence Detection of Histoplasma capsulatum Antibodies

Soluble antigens from Histoplasma capsulatum in the mycelial and yeast phase were purified by gel filtration, fixed onto paper discs, and employed in an indirect immunofluorescence procedure to detect antibody in sera from individuals infected with H. capsulatum. The elution patterns of crude histoplasmin passed through Sephadex G-200 revealed two minor peaks of protein showing immunofluorescence, complement fixing, and precipitating-antigen activity. A large peak containing the pigment and other low molecular weight materials showed no serological activity. A polysaccharide antigen obtained from fragmented, deproteinized yeast-phase cells was reactive in the fluorescent-antibody test but showed no antigen activity in complement fixation or precipitin tests. Although certain sera from culturally proven cases of blastomycosis, coccidioidomycosis, and cryptococcosis reacted with the purified Histoplasma antigens, preliminary evaluation indicated that the immunofluorescence technique may be of value as a screening procedure for the serodiagnosis of histoplasmosis.     

Studies on ether phospholipids. I. A new method of determination using phospholipase A1 from guinea pig pancreas: application to Krebs II ascites cells

A new method for ether phospholipid analysis has been devised, based on the selective destruction of diacyl phospholipids by guinea pig phospholipase A1 and of plasmalogens by acidolysis. The paper describes optimal conditions allowing a specific degradation of diacyl phospholipids by the enzyme(s). This requires the incubation of a total lipid extract in the presence of 2.4 mM sodium deoxycholate, at pH 8.0, at a temperature of 42 degrees C. As shown with various radioactive markers, all the diacyl phospholipids become degraded, whereas sphingomyelin and ether phospholipids remain refractory to phospholipase A1 attack. Phospholipids are then separated by a bidimensional thin-layer chromatography involving the exposure of the plates to HCl fumes between the two runs, in order to hydrolyse plasmalogens. Selectivity of both hydrolytic procedures is further demonstrated upon analysis of acetyl diacylglycerol derived from phospholipids. Various phospholipids can thus be determined by phosphorus measurement using sphingomyelin as an internal standard. By this way, it is shown that Krebs II cells present a very high content of ether phospholipid species (around 25% of total). Among these, about 50% are alkyl forms in ethanolamine phosphoglycerides, whereas this value reaches 70% in choline phosphoglycerides.     

Diabetes affects phospholipid content in the nuclei of the rat liver.

The aim of the present study was to examine the effect of acute streptozotocin diabetes on the content of different phospholipids and the incorporation of blood-borne 14C-palmitic acid into the phospholipid moieties in the rat liver nuclei. Diabetes was produced by intravenous administration of streptozotocin, and determinations were carried out two and seven days thereafter. Phospholipids were extracted from isolated nuclei and separated into the following fractions: sphingomyelin, phosphatidylcholine, phosphatidylserine, phosphatidylinositol, phosphatidylethanolamine and cardiolipin. Following that, they were quantified and radioactivity was measured. It was found that, in comparison to non-diabetic controls, two-day diabetes reduced the total content of phospholipids in the nuclei by 9.6%. The content of phospholipids in the nuclei by 9.6%. The content of phosphatidylcholine and phosphatidylserine was reduced and the content of the remaining phospholipids was stable. The specific activity of phosphatidylcholine, phosphatidylserine, phosphatidylethanolamine and cardiolipin, based on radioactivity incorporated from 14C-palmitic acid, was elevated. Seven-day diabetes resulted in a reduction of the total phospholipid content in the nuclei by 39.4%. This was accounted for by a reduction in the content of each phospholipid fraction with the exception of cardiolipin. The specific activity of each phospholipid fraction, was elevated in this group. It is concluded that insulin is involved in the regulation of the nuclear phospholipid content.     

The membrane lipids of Halobacterium halobium

The lipid content of the cell membrane of Halobacterium halobium increased from about 15% to 21% during exponential growth of the organism. Total lipid phosphorus more than doubled during the growth cycle. The mixture of membrane lipids from stationary-phase organisms was similar to lipid mixtures from whole cells of other halobacteria inasmuch as 80% of the lipid phosphorus occurred in a diether analogue of phosphatidylglycerophosphate and an additional 7.5% occurred in the ether analogue of phosphatidylglycerol. The lipid mixture was more complex than those reported for other halophils, however, 12 components being recognized in the acetone-insoluble fraction and 17 in the acetone-soluble fraction. There were major changes in the proportions of some minor components of the acetone-insoluble fraction during a growth cycle. Three nitrogenous lipids were recognized in the acetone-insoluble fraction, but all were present in relatively low proportion. One, which was not a phospholipid, contained a bound peptide. Of the 17 acetonesoluble compounds, 15 were pigments. The major carotenoids were alpha- and beta-bacteriorubrin. The carotenoid pigments occurred at maximal concentration after 6-7 days' growth.     

Growth of dimorphic human pathogenicfungi on media containing cycloheximide and chloramphenicol

Summary Studies of 24 strains ofBlastomyces dermatitidis confirmed previously published results that the yeast-phase of this fungus is more sensitive than the mycelial-phase to cycloheximide and chloramphenicol.Studies of 5 strains each ofHistoplasma capsulatum, Paracoccidioides brasiliensis andSporotrichum schenckii show that that these species also have a similar yeast-phase mycelial -phase sensitivity differential in regard to these antibiotics.A cycloheximide resistant strain ofB. dermatitidis was developed from a sensitive strain.The experimental results support the general practice of using 0.5 mg/ml cycloheximide and 0.05 mg/ml chloramphenicol in media for the isolation of the four fungi at 25° C. The results indicate, however, that some strains would not be recovered at 37° C with similar concentrations of these antibiotics.It is recommended that a concentration of not more than 0.2 mg/ml chloramphenicol should be used to preserve sputum which is subsequently to be cultured forB. dermatitidis, Histoplasma capsulatum, Paracoccidioides brasiliensis orS. schenckii.     

Proceedings: The phospholipid degradation and cellular death caused by freeze-thawing or freeze-drying of yeast

Freeze-thawing or freeeze-drying of yeast cells increased the amount of total lipid and phospholipid extractable to the level of the cell's total lipid contents. However, the amount of total lipid and phospholipid extractable from intact cells was usually less than half of these values.Phospholipase activity was apparent after freeze-thawing or freeze-drying of the cells, and phospholipids in the cells were decomposed to diglyceride and phosphoryl groups. Lipase activity was higher at pH 3–4.5, but at pH 6, practically no activity was noted.The cells incubated in medium at pH 6 after freeze-thawing or freeze-drying showed higher survivals than the cells incubated at pH 4.4 after the same treatments.     

Reduction of a Tetrazolium Salt in Determining Growth Activity of Yeast-Phase Histoplasma capsulatum

A colorimetric method using a tetrazolium compound, 3,4,5-dimethylthiozalil-(1 or 2),2,5-diphenyltetrazolium bromide (TTBr), was developed for studying the growth activity of yeast-phase Histoplasma capsulatum. Materials extracted in phosphate buffer, pH 7.0, from cells at different stages of growth reduced TTBr. Colorimetric changes were correlated with enzymatic activity. Under standardized conditions specified herein, the optical density of the reduced tetrazole was an index of the growth activity of the organism.     

Isolation of a New Soluble Antigen from the Yeast Phase of Histoplasma capsulatum

A method is described by which a soluble antigen was prepared from the yeast phase of Histoplasma capsulatum. This soluble preparation had a specificity greater than that of whole-cell yeast-phase antigens. In complement fixation tests with sera from human cases of histoplasmosis, blastomycosis, and coccidioidomycosis, the soluble antigen reacted in 12.1% of 141 tests with heterologous sera, whereas conventional whole-cell yeast antigens reacted in 47.3% of 91 tests with heterologous sera. The reactivities of the two types of antigens with homologous sera were essentially the same.