An homologue of the human 100-kDa protein (p100) is differentially expressed by Histoplasma capsulatum during infection of murine macrophages.

Using differential display reverse transcription-PCR (DDRT-PCR) we have identified several sequences that are specifically expressed by Histoplasma capsulatum during infection of murine macrophages (MPhi). Here, we report the characterization of a clone, pHc12, identified as a differentially expressed gene 1 hour after infection of MPhi. Screening of a cDNA library of H. capsulatum allowed us to isolate a clone, pHc12-E, that contains the complete coding sequence. We show that after infection the level of transcription of this gene increases about 5 fold. Analysis of its sequence revealed the presence of an open reading frame of 890 aa (ORF890) that shares respectively 30 and 33% identity with human and Caenorhabditis elegans p100 kD and rat p105 kD co-activator proteins. Using the two-dimensional Hydrophobic Cluster Analysis (HCA) method, we showed that H. capsulatum ORF890 and p100 kD co-activator proteins are clearly related. The H. capsulatum protein consists of a four-fold repeated module (domains I to IV) like the p100 kD co-activator proteins, whose three-dimensional (3D) structure is related to staphylococcal thermonuclease, followed by a modified fifth "hybrid" domain which partially resembles the structure of the tudor domain found in multiple copies in the Drosophila melanogaster tudor protein. These data strongly suggest that ORF890 is homologous to human p100 kD and that this protein, named Hcp100, may play an essential role during infection by co-activating the expression of specific genes.

Identification and isolation by DDRT-PCR of genes differentially expressed by Histoplasma capsulatum during macrophages infection.

Establishment of infection and disease implies modifications in the genetic programmes of the cell systems that are involved and the differential expression of genes in both parasite and host. In order to identify and isolate relevant genes of the fungus, Histoplasma capsulatum, in which expression is specifically induced during its interaction with murine macrophages (Mphi), we performed a comparative analysis of the pattern of gene expression of the fungus before and after exposure to, and internalization into Mphi by using differential display reverse transcriptase-PCR (DDRT-PCR). Using a limited set of primer combinations, six cDNA fragments of H. capsulatum were identified and isolated; five representing fungal genes in which expressions were enhanced during Mphi infection, whereas one mRNA fragment was down-regulated. Slot blots followed by Northern blot analyses confirmed that the transcripts detected with cDNA clones were over expressed after 1 h of Mphi infection, whereas no transcripts were detected with mRNA purified from H. capsulatum before infection. Sequence analyses and database searches revealed no significant homology to any known sequence for five of these clones. One of the clones showed homology to the rat p105 kD protein, and to the p100 kD co-activator proteins of human and Caenorhabditis elegans. To our knowledge, this is the first experimental evidence that specific genes are differentially expressed by a fungal pathogen when it is exposed to, and phagocytosed by Mphi. Furthermore, these results show that the DDRT-PCR procedure has adequate sensitivity to detect fungal genes induced during parasite-host interaction to identify potential new targets that can be used to develop new antifungal drugs.

The value of the Premier enzyme immunoassay for diagnosing Blastomyces dermatitidis infections.

One hundred and three sera drawn from 20 proven and 65 suspected cases of blastomycosis were examined concurrently with the enzymes immunoassay and microimmunodiffusion tests for the 'A' antibody specific for Blastomyces dermatitidis. Results indicated that all 20 proven sera were positive by both these tests. Thirteen of the 65 sera from suspected blastomycosis cases were positive by the enzyme immunoassay only, whereas none reacted positively in the micro-immunodiffusion test. Eighteen sera from apparently normal subjects, and patients with heterologous fungal and HIV infections were also tested by both tests. The sensitivity and specificity of the enzyme immunoassay test was 100% and 85.6%, respectively. The micro- immunodiffusion test was 100% sensitive and specific. In light of the fact that the enzyme immunoassay test is not entirely specific, a positive result should be confirmed by either a positive culture, histopathology or micro-immunodiffusion test.

Hsp70 in parasites: as an inducible protective protein and as an antigen.

The heat shock (HS) response is a general homeostatic mechanism that protects cells and the entire organism from the deleterious effects of environmental stresses. It has been demonstrated that heat shock proteins (HSP) play major roles in many cellular processes, and have a unique role in several areas of cell biology, from chronic degenerative diseases to immunology, from cancer research to interaction between host and parasites. This review deals with the hsp70 gene family and with its protein product, hsp70, as an antigen when pathogens infect humans. Members of HSP have been shown to be major antigens of many pathogenic organisms when they experience a major temperature shift upwards at the onset of infection and become targets for host B and T cells.

Comparative evaluation of the Premier enzyme immunoassay, micro-immunodiffusion and complement fixation tests for the detection of Histoplasma capsulatum var. capsulatum antibodies.

A total of 178 sera, including 68 from proven cases of histoplasmosis (65 positive for the presence of Histoplasma capsulatum var. capsulatum antibodies and three positive for antigen), 93 from patients with suspected histoplasmosis but with no laboratory evidence of H. capsulatum var. capsulatum infection, 14 from humans with heterologous fungal and non-fungal infections and three from normal individuals, were tested for IgG H. capsulatum antibodies and M or M and H precipitins by enzyme immunoassay (EIA) (Meridian Diagnostics, Cincinnati, OH, USA) and microimmunodiffusion (MID) respectively. Sixty-three of the 68 histoplasmosis case sera demonstrated IgG antibody, and 65 of 68 demonstrated the presence of specific precipitins in the MID test. Nine positive case sera, when tested with the Laboratory Branch complement fixation (LBCF) test, reacted positively to whole yeast and histoplasmin antigens (titres 1:8 to 1:512). Three histoplasmosis case sera repeatedly tested negative for IgG, specific precipitins and complement-fixing antibodies, whereas they were positive for Histoplasma antigen. Eighteen of 95 sera from patients without evidence of histoplasmosis demonstrated IgG antibody in the EIA only. Among these positive sera, three out of three cases of aspergillosis and three out of five cases of blastomycosis were confirmed. Sera from HIV-infected and healthy individuals did not show IgG or M and/or H antibodies to H. capsulatum. Ninety-three sera were negative by both EIA and MID. The EIA for IgG was less sensitive (97%) than MID (100%). The specificity of EIA and MID was 84% and 100% respectively.

Evaluation of a commercial enzyme immunoassay for the detection of cryptococcal antigen.

A total of 143 cerebrospinal and serum samples, from proven and suspected cases of cryptococcosis, were concurrently examined using a recently introduced enzyme immunoassay (EIA Premier, Meridian Diagnostics, Inc., Cincinnati, OH, USA) and three latex agglutination (LA) procedures (Immunomycologics, Inc., Norman, OK, USA; IBL, Inc., Cranbury, NJ, USA and a non-commercial LA test). Of these 143 specimens, 115 were negative for cryptococcal antigen (CrAg) with the EIA and LA tests. The remaining 28 specimens were evaluated by the LA tests, and all were positive for CrAg (with titres ranging from 1:2 to 1:8192). Of these 28 LA-positive specimens, 26 were also tested by the EIA. This procedure detected CrAg in 23 specimens (88.5%), with antigen levels ranging from 1:4 to 1:266,857. There were 3 LA-positive specimens (tires 1:4 to 1:32) which were negative by the EIA procedure (10.7%). One LA-negative specimen demonstrated CrAg (titre 1:30) by the EIA procedure. The sensitivity of the EIA and LA tests was 85.2 and 100%, respectively. The specificity of the LA test was 100%, whereas that of the EIA was 97%. The agreement among laboratories for testing the specimens with the three LA tests was 100%.

4-oxatetradecanoic acid is fungicidal for Cryptococcus neoformans and inhibits replication of human immunodeficiency virus I.

Candida albicans and Cryptococcus neoformans are major causes of systemic fungal infections, particularly in patients with acquired immunodeficiency syndrome. Metabolic labeling studies revealed that these organisms synthesize a small number of N-myristoylproteins, the most prominent being 20-kDa ADP-ribosylation factors (Arfs). C. albicans Arf has approximately 80% identity with the essential Arf1 and Arf2 proteins of Saccharomyces cerevisiae. [3H]Myristic acid analogs with oxygen for -CH2- substitutions at C4, C6, C11, and C13 are incorporated into cellular N-myristoylproteins, phospholipids, and neutral lipids produced by these three yeasts during exponential growth at 30 degrees C in complex media. Analog- and organism-specific differences in the efficiency of labeling of proteins and lipid classes were observed. The effects of oxatetradecanoic acids with oxygen for -CH2- substitutions at C3-C13 on C. neoformans, C. albicans, and S. cerevisiae were assessed during mid-log phase growth at 30 degrees C. A single dose of 3-oxa-, 4-oxa-, 5-oxa- or 6-oxatetradecanoic acid (O3-O6, final concentration = 300 microM) was able to inhibit growth of C. neoformans in the order O4 greater than O5 greater than O3 approximately O6. The other compounds were inactive. 4-Oxatetradecanoic acid was fungicidal, producing a 10,000-fold reduction in viable cell number 1 h after administration and continued suppression of cell growth for 7 h. A clear dose response was observed over a concentration range of 100-300 microM. 4-Oxatridecanoic acid was 100-fold less potent in reducing cell viability than 4-oxatetradecanoic acid but more potent than 5-oxatridecanoic acid. O4 produced approximately 10-100-fold reductions in the viability of C. albicans and S. cerevisiae at 300-500 microM, respectively, whereas O5 and O6 were less active. Since N-myristoylation of the Pr55gag polyprotein precursor produced by human immunodeficiency virus I (HIV-I) is essential for its assembly, we also assessed the antiviral effects of 4-oxatetradecanoic acid. O4 is able to produce a 50% reduction in the replication of HIV-I in acutely infected human T-lymphocyte cell lines at a concentration of 18 microM. Together, these data suggest that (i) the position of the oxygen for methylene substitution is a critical determinant of the fungicidal activity of O4 and (ii) NMT may be an attractive therapeutic target for treating opportunistic fungal infections in patients infected with HIV-I.

Mitochondrial activity and heat-shock response during morphogenesis in the pathogenic fungus Histoplasma capsulatum.

Changes in temperature and a variety of other stimuli coordinately induce transcription of a specific set of heat-shock genes in all organisms. In the human fungal pathogen Histoplasma capsulatum, a temperature shift from 25 to 37 degrees C acts not only as a signal that causes transcription of heat-shock genes, but also triggers a morphological mycelium- to yeast-phase transition. The temperature-induced morphological transition may be viewed as a heat-shock response followed by cellular adaptation to a higher temperature. We have found that by inducing thermotolerance, i.e., an initial incubation at 34 degrees C, the thermosensitive attenuated Downs strain of H. capsulatum can be made to resemble those of the more temperature-tolerant G222B strain with respect to mitochondrial ATPase activity and electron transport efficiency at elevated temperatures. Furthermore, if the heat-shock response is first elicited by preincubation at milder temperatures or stress, transcription of heat-shock mRNA in mycelial cells of Downs strain that shifted to 37 degrees C proceeds at rates comparable to those of the virulent strains.

Effects of Histoplasma capsulatum on murine macrophage functions: inhibition of macrophage priming, oxidative burst, and antifungal activities.

Histoplasma capsulatum yeast cells fail to trigger an oxidative burst response in normal murine macrophages. The results of this study, in which an in vitro assay of macrophage antifungal effects was used, extend these findings. During 18 h of incubation, unprimed elicited murine macrophages inhibited H. capsulatum growth only when macrophages were present in great excess. Gamma interferon (IFN-gamma)-primed macrophages showed enhanced fungal growth inhibition but a similar requirement for an excess of phagocytes. Macrophages containing heat-killed H. capsulatum exhibited diminished antifungal effects toward viable H. capsulatum and Saccharomyces cerevisiae cells. Parallel experiments showed no comparable effect of ingested latex particles on macrophage antifungal activity. Using chemiluminescence as a measure of the oxidative burst, we found that macrophages primed in vitro with IFN-gamma alone failed to exhibit a significant response to triggering by H. capsulatum yeast cells unless a second priming agent (tumor necrosis factor alpha or bacterial lipopolysaccharide) was added to IFN-gamma. Furthermore, macrophage priming with single agents was blocked by the prior ingestion of heat-killed H. capsulatum. These studies provide evidence that ingestion of H. capsulatum yeast cells can induce a prompt and enduring deactivation of murine macrophages.

Amphotericin B-resistant yeast infection in severely immunocompromised patients.

Systemic yeast infections are a major cause of morbidity and mortality in severely immunocompromised patients. The in vitro susceptibility to amphotericin B of 29 yeasts causing fungemia was examined in 26 patients undergoing allogeneic or autologous bone marrow transplantation and/or myelosuppressive chemotherapy. The minimal inhibitory concentrations (MICs) of amphotericin B observed with blood isolates from these patients were significantly higher than those observed with blood, sputum, or skin isolates from non-immunocompromised patients (p less than 0.01). All episodes (10 of 10) of bloodstream infection in immunocompromised patients caused by isolates with MICs greater than 0.8 micrograms/ml were fatal, versus eight of 17 episodes of bloodstream infection caused by yeasts with MICs of 0.8 micrograms/ml or less (p = 0.04). Although 15 of 26 patients received empiric treatment with amphotericin B before laboratory evidence of fungemia developed, the amphotericin B susceptibilities of their isolates were not significantly different from those of patients who had not received empiric amphotericin B treatment. It is concluded that yeast fungemia in severely immunocompromised patients is often caused by organisms resistant to the usual concentrations of amphotericin B obtainable in vivo, and that this finding is clinically significant.

Comparative in vitro activity of LY121019 and amphotericin B against clinical isolates of Candida species.

LY121019 and amphotericin B were equally active in vitro against most clinical isolates of Candida albicans and Candida tropicalis. Higher concentrations of LY121019 were required for inhibition of Candida glabrata. Other Candida species were inhibited by amphotericin B but not by LY121019.

Mycelial- to yeast-phase transitions of the dimorphic fungi Blastomyces dermatitidis and Paracoccidioides brasiliensis.

The physiological changes that occur during the mycelial- to yeast-phase transitions induced by a temperature shift from 25 to 37 degrees C of cultures of Blastomyces dermatitidis and Paracoccidioides brasiliensis can be divided into three stages. The triggering event is a heat-related insult induced by the temperature shift which results in partial uncoupling of oxidative phosphorylation and declines in cellular ATP levels, respiration rates, and concentrations of electron transport components (stage 1). The cells then enter a stage in which spontaneous respiration ceases (stage 2), and finally, there is a shift into a recovery phase during which transformation to yeast morphology occurs (stage 3). Cysteine is required during stage 2 for the operation of shunt pathways which permit electron transport to bypass blocked portions of the cytochrome system. The mycelial- to yeast-phase transitions of these two fungi are very similar to that of Histoplasma capsulatum. Therefore, these three dimorphic fungal pathogens have evolved parallel mechanisms to adjust to the temperature shifts which induce these mycelial- to yeast-phase transitions.

Modulation of the macrophage oxidative burst by Histoplasma capsulatum.

The production of reactive oxygen species by phagocytic cells is an important host defense against invading microorganisms. Because pathogens that achieve intracellular survival escape destruction by reactive oxidants, we investigated the relationship between the intracellular survival of H. capsulatum and the macrophage oxidative burst. H. capsulatum yeast failed to stimulate the release of reactive oxygen metabolites in unprimed murine macrophages despite extensive phagocytosis of the microorganisms. This effect was observed with live as well as heat-killed fungi over a wide range of yeast-to-macrophage ratios. Preincubation of murine macrophages with heat-killed H. capsulatum (but not with latex spheres), followed by incubation with unopsonized zymosan, resulted in inhibition of oxidative burst triggering without inhibition of zymosan phagocytosis. Ingestion of H. capsulatum yeast opsonized with the cognate mouse antibody resulted in significant oxidant release, suggesting that suppression of the respiratory burst may be circumvented through Fc-mediated phagocytosis.

Disseminated coccidioidomycosis in a patient with the acquired immune deficiency syndrome.

Disseminated coccidioidomycosis is a recognized but infrequent accompaniment of the acquired immune deficiency syndrome (AIDS). A patient with AIDS, Pneumocystis carinii pneumonia, and disseminated coccidioidomycosis occurring outside of an endemic area is described. Fungal infection presented atypically with progressive thoracic adenopathy and the development of cold soft tissue abscesses. As is often the case with AIDS, serologic testing proved to be unreliable and tissue biopsy the only means of accurate diagnosis.

Growth inhibition of Cryptococcus neoformans by cloned cultured murine macrophages.

Cloned and unselected bone marrow-derived macrophage cell lines were obtained from A/J, AKR/J, BIO.A(5R), CBA/J, DBA/2, HPC, NZW, and [NZB X NZW]F1 mice, and their interactions were studied in vitro with a lightly encapsulated natural serotype A isolate of Cryptococcus neoformans. Growth inhibition of C. neoformans was seen with all of the cell lines, as determined by enumeration of colony-forming units. Inhibition was enhanced by a high concentration (8%) of fresh mouse serum and was the same for serum obtained from AKR/J (C5 deficient) and BIO.A (C5 normal) mice. Macrophage incubation with fresh AKR/J serum which had been absorbed with heat-killed Cryptococcus cells also inhibited C. neoformans growth. Heat-inactivation, EDTA addition or anti-C3 antibody treatment of fresh serum abolished the opsonic activity for C. neoformans, while EGTA addition to fresh serum was without effect on opsonization. In addition, neither IgM nor IgG1 murine monoclonal antibodies specific for C. neoformans enhanced phagocytosis or killing of the yeast by macrophages. These findings are consistent with the interpretation that C3b is an important modulator of interactions between macrophages and C. neoformans.

Sulfhydryl induced respiratory "shunt" pathways and their role in morphogenesis in the fungus, Histoplasma capsulatum.

When the mycelial to yeast transition of the dimorphic fungus Histoplasma capsulatum is induced by a temperature shift from 25 to 37 degrees C, the activities of the cytochrome system and the alternate oxidase decrease in parallel over the first 24 to 40 h (stage 1 of the transition). The decrease in activity of the cytochrome system is correlated with extensive decreases in the amounts of cytochromes b, c, and aa3, assayed spectrophotometrically. After 40 h, the cells enter a dormant phase (stage 2 of the transition) and cysteine or other sulfhydryl-containing compounds are required to reactivate mitochondrial respiration. This reactivation is due to the establishment of shunt pathways which bypass blocked segments of the electron transport system. The "shunt" pathways operate normally in mycelia grown at 25 degrees C, but are shut down during the transition, possibly because of depletion of intracellular cysteine. The longstanding observation that cysteine is required to progress beyond the initial stages of the morphological transition may be due, at least in part, to the reactivation of these "shunt" pathways.

Possible relationship of morphogenesis in pathogenic fungus, Histoplasma capsulatum, to heat shock response.

Histoplasma capsulatum, like many other fungal pathogens, is dimorphic: it exists as mycelia in the soil and yeast in animal hosts. Because only the yeast phase is parasitic, factors which affect morphogenesis have been of interest for understanding and controlling pathogenicity. In culture, the mycelial to yeast transition of H. capsulatum is induced by a temperature shift from 25 to 37 degrees C (ref. 1). The transition occurs over several days and is accompanied by marked changes in metabolic processes, including respiration and cysteine metabolism. Here, we show that the triggering event for these morphological and biochemical changes is a rapid decline in intracellular ATP levels that follows uncoupling of oxidative phosphorylation when mycelia are shifted from 25 to 37 degrees C. We also show that respiration in the yeast phase is coupled at 37 degrees C and thus that the morphological transition may be viewed as a heat shock followed by cellular adaptation to higher temperature.

Purification and characterization of a cysteine dioxygenase from the yeast phase of Histoplasma capsulatum.

A cysteine dioxygenase, cysteine oxidase (EC 1.13.11.20), has been purified from the cytosolic fraction of yeast phase cells of the dimorphic fungus Histoplasma capsulatum. The cysteine oxidase is an iron-containing dioxygenase with a molecular weight of 10500 (+/- 1500) and is present only in the yeast phase of the fungus. The enzyme is highly specific for L-cysteine, with a Km of 2 X 10(-5) M in vitro. The product of cysteine oxidation is cysteinesulfinic acid, as analyzed by thin-layer chromatography and mass spectroscopy. To our knowledge, this is the first cysteine oxidase isolated from a fungus, and it probably plays an important role in the mycelial to yeast phase transition of H. capsulatum during which redox potential and cysteine levels are crucial factors.

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

Three stages can be distinguished in the temperature-induced mycelial-to-yeast phase transition of Histoplasma 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 of free 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.