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The development of strategies to reduce the load of unwanted bacteria is a fundamental challenge in industrial processing, environmental sciences and medical applications. Here, we report a new method to sequester motile bacteria from a liquid, based on passive, deployable micro-traps that confine bacteria using micro-funnels that open into trapping chambers. Even in low concentrations, micro-traps afford a 70% reduction in the amount of bacteria in a liquid sample, with a potential to reach >90% as shown by modelling improved geometries. This work introduces a new approach to contain the growth of bacteria without chemical means, an advantage of particular importance given the alarming growth of pan-drug-resistant bacteria.

We showed previously that insertion of Synechocystis Δ 12 -desaturase in salmonella's membrane alters membrane physical state (MPS), followed by the expression of stress genes causing inability to survive within murine macrophages (MΦ). Recently, we showed that expression of one membrane lipid domain (MLD) of Δ 12 -desaturase (ORF200) interferes with salmonella MPS, causing loss of virulence in mice and immunoprotection. Here, we postulate that an α-antimicrobial peptide (α-AMP) intercalates within membrane lipids, and depending on its amino acid sequence, it does so within specific key sensors of MLD. In this study, we choose as target for a putative synthetic AMP, PhoP/PhoQ, a sensor that responds to low Mg 2+ concentration. We synthesised a modified DNA fragment coding for an amino acid sequence (NUF) similar to that fragment and expressed it in salmonella typhimurium. We showed that the pattern of gene expression controlled by PhoP/PhoQ highlights dysregulation of pathways involving phospholipids biosynthesis, stress proteins and genes coding for antigens. RNA-Seq of strain expressing ORF200 showed that the pattern of those genes is also altered here. Accumulation of NUF conferred temporary immunoprotection. This represents a powerful procedure to address synthetic α-AMPs to a specific MLD generating live non-virulent bacterial strains., (© 2016 John Wiley & Sons Ltd.)

Obesity and dietary fats are well known risk factors for the pathogenesis of neurodegenerative diseases. The analysis of specific markers, whose brain level can be affected by diet, might contribute to unveil the intersection between inflammation/obesity and neurodegeneration. Haptoglobin (Hpt) is an acute phase protein, which acts as antioxidant by binding free haemoglobin (Hb), thus neutralizing its pro-oxidative action. We previously demonstrated that Hpt plays critical functions in brain, modulating cholesterol trafficking in neuroblastoma cell lines, beta-amyloid (Aβ) uptake by astrocyte, and limiting Aβ toxicity on these cells. A major aim of this study was to evaluate whether a long term (12 or 24 weeks) high-fat diet (HFD) influences Hpt and Hb expression in rat hippocampus. We also assessed the development of obesity-induced inflammation by measuring hippocampal level of TNF-alpha, and the extent of protein oxidation by titrating nitro-tyrosine (N-Tyr). Hpt concentration was lower (p < 0.001) in hippocampus of HFD rats than in control animals, both in the 12 and in the 24 weeks fed groups. HFD was also associated in hippocampus with the increase of Hb level (p < 0.01), inflammation and protein oxidative modification, as evidenced by the increase in the concentration of TNF-alpha and nitro-tyrosine. In fact, TNF-alpha concentration was higher in rats receiving HFD for 12 (p < 0.01) or 24 weeks (p < 0.001) compared to those receiving the control diet. N-Tyr concentration was more elevated in hippocampus of HFD than in control rats in both 12 weeks (p = 0.04) and 24 weeks groups (p = 0.01), and a positive correlation between Hb and N-Tyr concentration was found in each group. Finally, we found that the treatment of the human glioblastoma-astrocytoma cell line U-87 MG with cholesterol and fatty acids, such as palmitic and linoleic acid, significantly impairs (p < 0.001) Hpt secretion in the extracellular compartment. We hypothesize that the HFD-dependent decrease of Hpt in hippocampus, as associated with Hb increase, might enhance the oxidative stress induced by free Hb. Altogether our data, identifying Hpt as a molecule modulated in the brain by dietary fats, may represent one of the first steps in the comprehension of the molecular mechanisms underlying the diet-related effects in the nervous system.

Conventional approaches to create biomaterials rely on reverse engineering of biological structures, on biomimicking, and on bioinspiration. Plant nanobionics is a recent approach to engineer new materials combining plant organelles with synthetic nanoparticles to enhance, for example, photosynthesis. Biological structures often outperform man-made materials. For example, higher plants sense temperature changes with high responsivity. However, these properties do not persist after cell death. Here, we permanently stabilize the temperature response of isolated plant cells adding carbon nanotubes (CNTs). Interconnecting cells, we create materials with an effective temperature coefficient of electrical resistance (TCR) of -1,730% K(-1), ∼2 orders of magnitude higher than the best available sensors. This extreme temperature response is due to metal ions contained in the egg-box structure of the pectin backbone, lodged between cellulose microfibrils. The presence of a network of CNTs stabilizes the response of cells at high temperatures without decreasing the activation energy of the material. CNTs also increase the background conductivity, making these materials suitable elements for thermal and distance sensors.

We demonstrated previously that expression of a single trans-membrane region of the Δ(12) -desaturase gene of Synechocystis sp. PCC 6803 in Salmonella enterica serovar Typhimurium (Salmonella Typhimurium) altered the membrane physical state of this pathogen, induced a significant change in the pattern of mRNA transcription of major heat shock genes, and inhibited pathogen growth inside murine macrophages. In this study, we demonstrate that injection of the modified Salmonella strain [Stm(pBAD200)] into C57Bl6j mice is safe. Survival of mice was associated with bacterial clearance, an increased number of splenic leukocytes, and high levels of interleukin-12, interferon γ and tumor necrosis factor α in spleens as well as in sera. Furthermore, Stm(pBAD200)-injected mice developed a Salmonella-specific antibody and Th1-like responses. Mice challenged with Stm(pBAD200) are protected from systemic infection with Salmonella wild-type. Similarly, mice infected with Stm(pBAD200) by the oral route survived when challenged with an oral lethal dose of Salmonella wild-type. The avirulent Stm(pBAD200) phenotype is associated with a remarkable change in the expression of the hilC, hilD, hilA, invF and phoP genes, among others, whose products are required for invasion and replication of Salmonella inside phagocytic cells. These data demonstrate the use of trans-membrane peptides to generate attenuated strains, providing a potential novel strategy to develop vaccines for both animal and human use., (© 2014 FEBS.)

Beta-amyloid accumulation in brain is a driving force for Alzheimer's disease pathogenesis. Apolipoprotein E (ApoE) represents a critical player in beta-amyloid homeostasis, but its role in disease progression is controversial. We previously reported that the acute-phase protein haptoglobin binds ApoE and impairs its function in cholesterol homeostasis. The major aims of this study were to characterize the binding of haptoglobin to beta-amyloid, and to evaluate whether haptoglobin affects ApoE binding to beta-amyloid. Haptoglobin is here reported to form a complex with beta-amyloid as shown by immunoblotting experiments with purified proteins, or by its immunoprecipitation in brain tissues from patients with Alzheimer's disease. The interaction between ApoE and beta-amyloid was previously shown to be crucial for limiting beta-amyloid neurotoxicity and for promoting its clearance. We demonstrate that haptoglobin, rather than impairing ApoE binding to beta-amyloid, promotes to a different extent the formation of the complex between beta-amyloid and ApoE2 or ApoE3 or ApoE4. Our data suggest that haptoglobin and ApoE functions in brain should be evaluated taking into account their mutual interaction with beta-amyloid. Hence, the risk of developing Alzheimer's disease might not only be linked to the different ApoE isoforms, but also rely on the level of critical ligands, such as haptoglobin.

Alteration in cholesterol metabolism has been implicated in the pathogenesis of several neurodegenerative disorders. Apolipoprotein E (ApoE) is the major component of brain lipoproteins supporting cholesterol transport. We previously reported that the acute-phase protein Haptoglobin (Hpt) binds ApoE, and influences its function in blood cholesterol homeostasis. Major aim of this study was to investigate whether Hpt influences the mechanisms by which cholesterol is shuttled from astrocytes to neurons. In detail it was studied Hpt effect on ApoE-dependent cholesterol efflux from astrocytes and ApoE-mediated cholesterol incorporation in neurons. We report here that Hpt impairs ApoE-mediated cholesterol uptake in human neuroblastoma cell line SH-SY5Y, and limits the toxicity of a massive concentration of cholesterol for these cells, while it does not affect cholesterol efflux from the human glioblastoma-astrocytoma cell line U-87 MG. As aging is the most important non-genetic risk factor for various neurodegenerative disorders, and our results suggest that Hpt modulates ApoE functions, we evaluated the Hpt and ApoE expression profiles in cerebral cortex and hippocampus of adolescent (2 months), adult (5 and 8 months), and middle-aged (16 months) rats. Hpt mRNA level was higher in hippocampus of 8 and 16 month-old than in 2-month old rats (p < 0.05), and Hpt concentration increased with the age from adolescence to middle-age (p < 0.001). ApoE concentration, in hippocampus, was higher (p < 0.001) in 5 month-old rats compared to 2 month but did not further change with aging. No age-related changes of Hpt (protein and mRNA) were found in the cortex. Our results suggest that aging is associated with changes, particularly in the hippocampus, in the Hpt/ApoE ratio. Age-related changes in the concentration of Hpt were also found in human cerebrospinal fluids. The age-related changes might affect neuronal function and survival in brain, and have important implications in brain pathophysiology.

Intravascular ultrasound is a catheter-based imaging modality that was developed to investigate the condition of coronary arteries and assess the vulnerability of coronary atherosclerotic plaques in particular. Since its introduction in the clinic 20 years ago, use of intravascular ultrasound innovation has been relatively limited. Intravascular ultrasound remains a niche technology; its clinical practice did not vastly expand, except in Japan, where intravascular ultrasound is an appraised tool for guiding percutaneous coronary interventions. In this qualitative research study, we follow scholarship on the sociology of innovation in exploring both the current adoption practices and perspectives on the future of intravascular ultrasound. We conducted a survey of biomedical experts with experience in the technology, the practice, and the commercialization of intravascular ultrasound. The collected information enabled us to map intravascular ultrasound controversies as well as to outline the dynamics of the international network of experts that generates intravascular ultrasound innovations and uses intravascular ultrasound technologies. While the technology is praised for its capacity to measure coronary atherosclerotic plaque morphology and is steadily used in clinical research, the lack of demonstrated benefits of intravascular ultrasound guided coronary interventions emerges as the strongest factor that prevents its expansion. Furthermore, most of the controversies identified were external to intravascular ultrasound technology itself, meaning that decision making at the industrial, financial and regulatory levels are likely to determine the future of intravascular ultrasound. In light of opinions from the responding experts', a wider adoption of intravascular ultrasound as a stand-alone imaging modality seems rather uncertain, but the appeal for this technology may be renewed by improving image quality and through combination with complementary imaging modalities.

Improved diagnosis of psoriasis, by new biomarkers, is required for evaluating the progression rate of the disease and the response to treatment. Haptoglobin (Hpt), a glycoprotein secreted by hepatocytes and other types of cells including keratinocytes, was found with glycan changes in psoriasis and other diseases. We previously reported that Hpt isolated from plasma of psoriatic patients is more fucosylated than Hpt of healthy subjects. The aim of this study was to compare the glycosylation pattern of Hpt isolated from skin scales or plasma of patients with psoriasis with that of Hpt from cornified epidermal layer or plasma of healthy subjects. High performance liquid chromatography analysis of the glycans isolated from the protein backbone revealed that glycan patterns from skin and plasma of patients were similar, and mostly displayed quantitative rather than qualitative differences from normal pattern. Biotin-labeled lectins were used to evaluate quantitative differences in the glycoforms of Hpt from plasma and psoriatic skin scales. Hpt from skin and plasma of patients showed more fucosylated and branched glycans than Hpt from plasma of healthy subjects. Tryptic glycopeptides of Hpt were also analyzed by mass spectrometry, and a decreased amount of sialylated glycan chains was found in glycopeptides of skin Hpt, as compared with Hpt from plasma. High levels of glycans with fucosylated and tetra-antennary chains were detected on the peptide NLFLNHSENATAK from Hpt of psoriatic patients. Our data demonstrate that specific changes in glycan structures of Hpt, such as enhanced glycan branching and fucose content, are associated with psoriasis, and that differences between circulating and skin Hpt do exist. A lower extent of glycan fucosylation and branching was found in Hpt from plasma of patients in disease remission. Altered glycoforms might reflect changes of Hpt function in the skin, and could be used as markers of the disease.

Background and Purpose: The resistance of human colon adenocarcinoma cells to antineoplastic agents may be related to the high endogenous expression of stress proteins, including the family of heat shock proteins (HSPs). Recently, a quinone-based pentacyclic derivative, DTNQ-Pro, showed high cytotoxic activity in human colon carcinoma cell lines. The aim of the present study was to determine the precise cellular mechanisms of this cytotoxic action of DTNQ-Pro., Experimental Approach: Using human colorectal carcinoma-derived Caco-2 cells as a model, we studied the effects of DTNQ-Pro on cellular viability and oxidative stress; HSP70 and HSP27 accumulation; and cell cycle, differentiation and apoptosis., Key Results: Incubation of Caco-2 cells with DTNQ-Pro reduced cell growth and increased the levels of reactive oxygen species in mitochondria. After 48 h of treatment, cells surviving showed an increased expression of Mn-superoxide dismutase (SOD), nitric oxide production and membrane lipid peroxidation. Treatment with DTNQ-Pro decreased HSP70 expression, and redistributed HSP27 and vimentin within the cell. DTNQ-Pro down-regulated the expression of A and B cyclins with arrest of the cell cycle in S phase and increased cellular differentiation. A second treatment of Caco-2 cells with DTNQ-Pro induced cellular death by activation of the apoptotic pathway., Conclusions and Implications: DTNQ-Pro causes Caco-2 cell death by induction of apoptosis via inhibition of HSP70 accumulation and the intracellular redistribution of HSP27. These findings suggest the potential use of DTNQ-Pro in combination chemotherapy for colon cancer.

It is now recognized that membranes are not simple physical barriers but represent a complex and dynamic environment that affects membrane protein structures and their functions. Recent data emphasize the role of membranes in sensing temperature changes, and it has been shown that the physical state of the plasma membrane influences the expression of a variety of genes such as heat shock genes. It has been widely shown that minor alterations in lipid membranes are critically involved in the conversion of signals from the environment to the transcriptional activation of heat shock genes. Previously, we have proposed that the composition, molecular arrangement, and physical state of lipid membranes and their organization have crucial roles in cellular responses during stress caused by physical and chemical factors as well as in pathological states. Here, we show that transformation of Salmonella enterica serovar Typhimurium LT2 (Salmonella Typhimurium) with a heterologous Delta(12)-desaturase (or with its trans-membrane regions) causes major changes in the pathogen's membrane dynamic. In addition, this pathogen is strongly impaired in the synthesis of major stress proteins (heat shock proteins) under heat shock. These data support the hypothesis that the perception of temperature in Salmonella is strictly controlled by membrane order and by a specific membrane lipid/protein ratio that ultimately causes transcriptional activation of heat shock genes. These results represent a previously unrecognized mode of sensing temperature variation used by this pathogen at the onset of infection.

So far attenuation of pathogens has been mainly obtained by chemical or heat treatment of microbial pathogens. Recently, live attenuated strains have been produced by genetic modification. We have previously demonstrated that in several prokaryotes as well as in yeasts and mammalian cells the heat shock response is controlled by the membrane physical state (MPS). We have also shown that in Salmonella enterica serovar Typhimurium LT2 (Salmonella Typhimurium) overexpression of a Delta(12)-desaturase gene alters the MPS, inducing a sharp impairment of transcription of major heat shock genes and failure of the pathogen to grow inside macrophage (MPhi) (A. Porta et al., J. Bacteriol. 192:1988-1998, 2010). Here, we show that overexpression of a homologous Delta(9)-desaturase sequence in the highly virulent G217B strain of the human fungal pathogen Histoplasma capsulatum causes loss of its ability to survive and persist within murine MPhi along with the impairment of the heat shock response. When the attenuated strain of H. capsulatum was injected in a mouse model of infection, it did not cause disease. Further, treated mice were protected when challenged with the virulent fungal parental strain. Attenuation of virulence in MPhi of two evolutionarily distant pathogens was obtained by genetic modification of the MPS, suggesting that this is a new method that may be used to produce attenuation or loss of virulence in both other intracellular prokaryotic and eukaryotic pathogens. This new procedure to generate attenuated forms of pathogens may be used eventually to produce a novel class of vaccines based on the genetic manipulation of a pathogen's membrane fluid state and stress response.

Aim: To identify the altered gene expression patterns in squamous cell carcinoma of esophagus (ESCC) in relation to adjacent normal esophageal epithelium., Methods: Total RNA was extracted using SV total RNA isolation kit from snap frozen tissues of ESCC samples and normal esophageal epithelium far from the tumor. Radio-labeled cDNA were synthesized from equal quantities of total RNAs of tumor and normal tissues using combinations of 24 arbitrary 13-mer primers and three different anchoring oligo-dT primers and separated on sequencing gels. cDNA with considerable different amounts of signals in tumor and normal tissue were reamplified and cloned. Using southern blot, the clones of each band were controlled for false positive results caused by probable heterogeneity of cDNA population with the same size. Clones that confirmed differential expression by slot blot selected for sequencing and northern analysis. Corresponding full-length gene sequences was predicted using human genome project data, related transcripts were translated and used for various protein/motif searches to speculate their probable functions., Results: The 97 genes showed different levels of cDNA in tumor and normal tissues of esophagus. The expression of mal gene was remarkably down regulated in all 10 surveyed tumor tissues. Akr1c2, a member of the aldo-keto reductase 1C family, which is involved in metabolism of sex hormones and xenobiotics, was up-regulated in 8 out of 10 inspected ESCC samples. Rab11a, RPL7, and RPL28 showed moderate levels of differential expression. Many other cDNAs remained to further studies., Conclusion: The mal gene which is switched-off in all ESCC samples can be considered as a tumor suppressor gene that more studies in its regulation may lead to valuable explanations in ESCC development. Akr1c2 which is up-regulated in ESCC probably plays an important role in tumor development of esophagus and may be proposed as a potential molecular target in ESCC treatments. Differential display technique in spite of many disadvantages is still a valuable technique in gene function exploration studies to find new candidates for improved ones like gene chips.

The hydroxylamine derivative bimoclomol (BM) has been shown to activate natural cytoprotective homeostatic responses by enhancing the capability of cells to cope with various pathophysiological conditions. It exerts its effect in synergy with low levels of stress to induce the synthesis of members of major stress protein families. We show here that the presence of BM does not influence protein denaturation in the cells. BM and its derivatives selectively interact with acidic lipids and modulate their thermal and dynamic properties. BM acts as a membrane fluidizer at normal temperature, but it is a highly efficient membrane stabilizer, inhibiting the bilayer-nonbilayer phase transitions during severe heat shock. We suggest that BM and the related compounds modify those domains of membrane lipids where the thermally or chemically induced perturbation of lipid phase is sensed and transduced into a cellular signal, leading to enhanced activation of heat shock genes. BM may be a prototype for clinically safe membrane-interacting drug candidates that rebalance the level and composition of heat shock proteins.

Preservation of the chemical architecture of a cell or of an organism under changing and perhaps stressful conditions is termed homeostasis. An integral feature of homeostasis is the rapid expression of genes whose products are specifically dedicated to protect cellular functions against stress. One of the best known mechanisms protecting cells from various stresses is the heat-shock response which results in the induction of the synthesis of heat-shock proteins (HSPs or stress proteins). A large body of information supports that stress proteins--many of them molecular chaperones--are crucial for the maintenance of cell integrity during normal growth as well as during pathophysiological conditions, and thus can be considered "homeostatic proteins." Recently emphasis is being placed on the potential use of these proteins in preventing and/or treating diseases. Therefore, it would be of great therapeutic benefit to discover compounds that are clinically safe yet able to induce the accumulation of HSPs in patients with chronic disorders such as diabetes mellitus, heart disease or kidney failure. Here we show that a novel cytoprotective hydroxylamine derivative, [2-hydroxy-3-(1-piperidinyl) propoxy]-3-pyridinecarboximidoil-chloride maleate, Bimoclomol, facilitates the formation of chaperone molecules in eukaryotic cells by inducing or amplifying expression of heat-shock genes. The cytoprotective effects observed under several experimental conditions, including a murine model of ischemia and wound healing in the diabetic rat, are likely mediated by the coordinate expression of all major HSPs. This nontoxic drug, which is under Phase II clinical trials, has enormous potential therapeutic applications.

Heat shock protein (hsp) 70 from several microbes is antigenic in mammals. In this study we sequenced and expressed the gene encoding this protein from Histoplasma capsulatum to study its immunological activity. The deduced amino acid sequence of the gene demonstrated 71 and 76% identity to hsp7O from humans and Saccharomyces cerevisiae, respectively. A cDNA was synthesized by reverse transcription-PCR and was expressed in Escherichia coli. Recombinant protein reacted with a mouse monoclonal antibody raised against human hsp7O. Splenocytes from C57BL/6 mice immunized with recombinant hsp7O emulsified in adjuvant, but not yeast cells, reacted in vitro to the antigen. Recombinant hsp7O elicited a cutaneous delayed-type hypersensitivity response in mice immunized with protein or with viable yeast cells. Mice were injected with recombinant hsp7O and challenged intranasally with a sublethal inoculum of yeast cells. Vaccination did not confer protection in this model. Thus, recombinant hsp7O can induce a cell-mediated immune response but does not induce a protective response.

Addition of a saturated fatty acid (SFA) induced a strong increase in heat shock (HS) mRNA transcription when cells were heat-shocked at 37 degrees C, whereas treatment with an unsaturated fatty acid (UFA) reduced or eliminated the level of HS gene transcription at 37 degrees C. Transcription of the delta 9-desaturase gene (Ole1) of Histoplasma capsulatum, whose gene product is responsible for the synthesis of UFA, is up-regulated in a temperature-sensitive strain. We show that when the L8-14C mutant of Saccharomyces cerevisiae, which has a disrupted Ole1 gene, is complemented with its own Ole1 coding region under control of its own promoter or Ole1 promoters of H. capsulatum, the level of HS gene transcription depends on the activity of the promoters. Fluorescence anisotropy of mitochondrial membranes of completed strains corresponded to the different activity of the Ole1 promoter used. We propose that the SFA/UFA ratio and perturbation of membrane lipoprotein complexes are involved in the perception of rapid temperature changes and under HS conditions disturbance of the preexisting membrane physical state causes transduction of a signal that induces transcription of HS genes.

To understand the molecular mechanisms that control the reversible morphologic transition from mycelia to yeast in dimorphic fungi, we have isolated and characterized a cdc2 gene from Histoplasma capsulatum. This organism is a dimorphic pathogenic fungus that grows as a filamentous saprobic mold in soil and as a unicellular pathogenic yeast in human tissue. The cloned gene, whose protein product has a high degree of homology with other members of the cdc2 family, is split into four exons and three introns of 95, 52 and 85 nucleotides. Analyses of cDNA clones confirm the presence of the eukaryotic splice donor (GT) and acceptor (AG) sites. The spliced gene codes for a protein of 324 amino acids (aa) with a predicted molecular mass of 36.9 kDa. The H. capsulatum cdc2 product has 71% aa identity with Saccharomyces cerevisiae and 70% with Schizosaccharomyces pombe. The deduced protein contains the sequence, PSTAIRE, that is normally found in most p34cdc2 proteins. H. capsulatum cdc2 is transcriptionally regulated during the morphologic mycelium<==>yeast transitions and is more actively transcribed in the yeast than in the mycelial phase.

We have isolated and characterized a heat-inducible gene, hsp82, from the dimorphic pathogenic fungus Histoplasma capsulatum, which is a filamentous mold at 25 degrees C and a unicellular yeast at 37 degrees C. This gene, which has a high degree of homology with other members of the hsp82 gene family, is split into three exons and two introns of 122 and 86 nucleotides, respectively. Contrary to what has been demonstrated in Drosophila melanogaster, Saccharomyces cerevisiae, and other organisms, hsp82 mRNA in H. capsulatum is properly spliced during the severe heat conditions of 37 to 40 degrees C in the temperature-sensitive Downs strain. Splicing accuracy was also observed at 42 degrees C in the temperature-tolerant G222B strain, which showed no evidence of accumulation of primary transcripts. Furthermore, the intron containing the beta-tubulin gene is also properly spliced at the upper temperature range, suggesting that the lack of a block in splicing may be a general phenomenon in this organism.

We compared the mycelial to yeast transitions of the Downs strain of Histoplasma capsulatum (low level of virulence) with those of G184A and G222B, two more virulent strains having different levels of pathogenicity for mice. When the morphological transitions are initiated by a temperature shift from 25 degrees to 37 degrees C, all three strains undergo similar physiological changes, but these are less severe in G184A and G222B than in the Downs strain. The transitions from mycelial to yeast morphology in both of the more virulent strains are also one-third more rapid than in Downs. We also find that the differences in temperature sensitivity of the three strains can be correlated with the temperature required for complete uncoupling of oxidative phosphorylation. The differences in sensitivity to elevated temperatures extend to the growth of yeast cells of all three strains. Considered together, our results suggest that sensitivity to elevated temperatures may be a key factor accounting for differences in virulence and that uncoupling of oxidative phosphorylation may be the primary event in the morphological transition in all three strains.

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.

The response to heat shock has been examined in two strains of the dimorphic pathogenic fungus Histoplasma capsulatum, which differ considerably in thermotolerance and pathogenicity. The gene for the 70 kD heat shock protein (hsp70) was isolated using a Drosophila hsp70 gene to screen a cosmid library of the DNA from the temperature-sensitive Downs strain (low level of thermotolerance for mice). Using the cloned gene as a probe, we have measured the transcription of the endogenous hsp70 gene at 25 degrees C and in response to temperature shift to 34 degrees, 37 degrees and 40 degrees C, temperatures that trigger the mycelial to yeast phase transition in this fungus. The gene is constitutively transcribed at low levels, both in the yeast and the mycelial stages. Synthesis of hsp70 mRNA was transiently increased 1 to 3 h after the temperature shifts. By Northern analysis, peak levels of transcription were shown to occur at 34 degrees C in the Downs strain and at 37 degrees C in the more pathogenic G222B strain. Our results are consistent with reports in which it has been shown that heat shock gene expression is part of temperature adaptation and probably developmental processes. The low levels of transcription of the hsp70 gene in the Downs strain at 37 degrees C correlate with its greater temperature sensitivity and low level of virulence.

The transition from yeast to mycelia of Histoplasma capsulatum could be accomplished by shifting the temperature of incubation from 37 to 25 degrees C. It was accompanied by many changes in cellular metabolism, including changes in respiration, intracellular cyclic adenosine 3',5'-monophosphate (cAMP) levels, and activities of two enzymes specific for the yeast phase, cystine reductase (EC 1.6.4.1) and cysteine oxidase (EC 1.13.11.20). Even at 37 degrees C, the yeast to mycelial transition could be induced by cAMP and agents which raise the intracellular levels of cAMP (theophylline, acetylsalicylic acid, prostaglandin E1, and nerve growth factor). During this morphogenesis the same pattern of changes occurred as in the temperature-induced transition. Therefore, these changes were not simply dependent on a shift in temperature, but rather were part of the process of the phase transition.

Respiration in the yeast and mycelial phases of Histoplasma capsulatum proceeds via a cytochrome system and an alternate oxidase, both present constitutively. The mycelial cytochrome system is distinguished by an additional partial shunt around the antimycin-sensitive site.

Several fungi can assume either a filamentous or a unicellular morphology in response to changes in environmental conditions. This process, known as dimorphism, is a characteristic of several pathogenic fungi, e.g., Histoplasma capsulatum, Blastomyces dermatitidis, and Paracoccidioides brasiliensis, and appears to be directly related to adaptation from a saprobic to a parasitic existence. H. capsulatum is the most extensively studied of the dimorphic fungi, with a parasitic phase consisting of yeast cells and a saprobic mycelial phase. In culture, the transition of H. capsulatum from one phase to the other can be triggered reversibly by shifting the temperature of incubation between 25 degrees C (mycelia) and 37 degrees C (yeast phase). Mycelia are found in soil and never in infected tissue, in contrast to the yeast phase, which is the only form present in patients. The temperature-induced phase transition and the events in establishment of the disease state are very likely to be intimately related. Furthermore, the temperature-induced phase transition implies that each growth phase is an adaptation to two critically different environments. A fundamental question concerning dimorphism is the nature of the signal(s) that responds to temperature shifts. So far, both the responding cell component(s) and the mechanism(s) remain unclear. This review describes the work done in the last several years at the biochemical and molecular levels on the mechanisms involved in the mycelium to yeast phase transition and speculates on possible models of regulation of morphogenesis in dimorphic pathogenic fungi.

p-Chloromercuriphenylsulfonic acid (PCMS), a sulfhydryl inhibitor, prevented the mycelial-to-yeast transition of the dimorphic fungal pathogen, Histoplasma capsulatum. The effect of PCMS was specific for the mycelial-to-yeast transformation; it had no effect on growth of either the yeast or mycelial forms or on the yeast-to-mycelial transition. The failure of PCMS-treated mycelia to transform to yeast was permanent and irreversible. PCMS-treated mycelia could not infect mice but could stimulate resistance to infection by a pathogenic strain of Histoplasma capsulatum. These results suggest a new general strategy for vaccine development in diseases caused by dimorphic pathogens.

Organo-sulfur compounds favor the transition of mycelia of Histoplasma capsulatum to the yeast form (6, 8). Investigation of the role of cystine in the transition revealed that the two phases concentrated this amino acid at comparable rates and that mutants defective in the uptake of cystine were still able to undergo the transition normally. Uptake of cystine is therefore probably not a requirement for transition to or maintenance of the yeast phase. Both phases contained a reduced nicotinamide adenine dinucleotide phosphate-dependent glutathione reductase; but a reduced nicotinamide adenine dinucleotide-dependent cystine reductase was detectable only in the yeast phase. The cystine reductase appeared early in the transition of mycelium to yeast. Treatment of mycelia with p-chloromercuriphenylsulfonic acid, which prevented the transition to yeast, had no effect on cystine uptake but strongly inhibited the cystine reductase. These results suggest that cystine reductase may provide reduced sulfhydryl groups involved in the transition of mycelium to yeast.