Quantitative phosphoproteomic analysis of prion-infected neuronal cells

Following cultivation of distinct mesenchymal stem cell (MSC) populations derived from human umbilical cord under hypoxic conditions (between 1.5% to 5% oxygen (O₂)) revealed a 2- to 3-fold reduced oxygen consumption rate as compared to the same cultures at normoxic oxygen levels (21% O₂). A simultaneous measurement of dissolved oxygen within the culture media from 4 different MSC donors ranged from 15 μmol/L at 1.5% O₂ to 196 μmol/L at normoxic 21% O₂. The proliferative capacity of the different hypoxic MSC populations was elevated as compared to the normoxic culture. This effect was paralleled by a significantly reduced cell damage or cell death under hypoxic conditions as evaluated by the cellular release of LDH whereby the measurement of caspase3/7 activity revealed little if any differences in apoptotic cell death between the various cultures. The MSC culture under hypoxic conditions was associated with the induction of hypoxia-inducing factor-alpha (HIF-1α) and an elevated expression of energy metabolism-associated genes including GLUT-1, LDH and PDK1. Concomitantly, a significantly enhanced glucose consumption and a corresponding lactate production could be observed in the hypoxic MSC cultures suggesting an altered metabolism of these human stem cells within the hypoxic environment.     

Hypoxic adipocytes induce macrophages to release inflammatory cytokines that render skeletal muscle cells insulin resistant

Adipose tissue hypoxia occurs early in obesity and is associated with increased tissue macrophages and systemic inflammation that impacts muscle insulin responsiveness. We investigated how hypoxia interacted with adipocyte-macrophage crosstalk and inflammatory cytokine release, using co-culture and conditioned media (CM). Murine primary adipocytes from lean or obese mice were cultured under normoxic (21% O₂) or hypoxic (1% O₂) conditions. RAW264.7 macrophages were incubated under normoxic or hypoxic conditions with or without adipocyte conditioned media. Macrophage and adipocyte-macrophage co-culture CM were also collected. We found hypoxia did not elicit direct cytokine release from macrophages. However, adipocyte CM or adipocyte co-culture, synergistically stimulated TNFα and MCP-1 release from macrophages that was not further impacted by hypoxia. Exposure of muscle cells to elevated cytokines led to reduced insulin and muscle stress/inflammatory signaling. We conclude hypoxia or obesity induces release of inflammatory TNFα and MCP-1 from mice primary adipocytes but the two environmental conditions do not synergize to worsen macrophage signal transduction or insulin responsiveness.     

Dual factor delivery of CXCL12 and Exendin‐4 for improved survival and function of encapsulated beta cells under hypoxic conditions

A bioartifical pancreas (BAP) remains a promising approach for treating insulin‐dependent diabetes. Several obstacles to the clinical implementation of a BAP remain, including hypoxia following implantation. Within native pancreatic islets, CXCL12 and glucagon‐like peptide‐1 (GLP‐1) act in a paracrine fashion to promote the survival, function, and proliferation of β‐cells. This work sought to investigate if the presentation of CXCL12 and delivery of a GLP‐1 receptor analog, Exendin‐4 (Ex‐4), alone and in combination, conferred pro‐survival and insulinotropic effects on an encapsulated β‐cell line, βTC‐tet, cultured under hypoxic conditions of 7.6 mmHg O₂. Our findings indicate that presentation of CXCL12 in the encapsulation matrix completely abrogated apoptosis under hypoxic conditions. Delivery of Ex‐4 increased insulin secretion rate under both normoxic and hypoxic conditions, and additionally reduced apoptosis under hypoxic conditions. Furthermore, presentation of CXCL12 combined with Ex‐4 delivery significantly increased insulin secretion rate under hypoxic conditions compared to delivery of Ex‐4 alone. These findings demonstrate that the presentation of CXCL12 combined with the delivery of Ex‐4 may constitute a promising strategy for supporting β‐cell function and survival following transplantation. Biotechnol. Bioeng. 2013; 110: 2292–2300. © 2013 Wiley Periodicals, Inc.     

Growth of aortic vascular smooth muscle cells in lowered oxygen tension

Summary Primary cultures of vascular smooth muscle cells, isolated from rat aorta, were grown under normoxic (20% O₂) and mildly hypoxic (5 % O₂) conditions. Cells from both conditions were compared for growth characteristics, morphology, protein synthesis, lysosomal enzyme activity, and oxygen consumption. In no case was a consistently significant difference observed. These observations indicate that these cells can adapt or are adapted to mildly hypoxic conditions. Moreover, these results may indicate that the culture of vascular smooth muscle cells in mild hypoxia represents a closer approximation of in vivo growth conditions for these cells.Supported by HL19242     

Cartilage Tissue Engineering Using Dermis Isolated Adult Stem Cells: The Use of Hypoxia during Expansion versus Chondrogenic Differentiation

Dermis isolated adult stem (DIAS) cells, a subpopulation of dermis cells capable of chondrogenic differentiation in the presence of cartilage extracellular matrix, are a promising source of autologous cells for tissue engineering. Hypoxia, through known mechanisms, has profound effects on in vitro chondrogenesis of mesenchymal stem cells and could be used to improve the expansion and differentiation processes for DIAS cells. The objective of this study was to build upon the mechanistic knowledge of hypoxia and translate it to tissue engineering applications to enhance chondrogenic differentiation of DIAS cells through exposure to hypoxic conditions (5% O₂) during expansion and/or differentiation. DIAS cells were isolated and expanded in hypoxic (5% O₂) or normoxic (20% O₂) conditions, then differentiated for 2 weeks in micromass culture on chondroitin sulfate-coated surfaces in both environments. Monolayer cells were examined for proliferation rate and colony forming efficiency. Micromasses were assessed for cellular, biochemical, and histological properties. Differentiation in hypoxic conditions following normoxic expansion increased per cell production of collagen type II 2.3 fold and glycosaminoglycans 1.2 fold relative to continuous normoxic culture (p<0.0001). Groups expanded in hypoxia produced 51% more collagen and 23% more GAGs than those expanded in normoxia (p<0.0001). Hypoxia also limited cell proliferation in monolayer and in 3D culture. Collectively, these data show hypoxic differentiation following normoxic expansion significantly enhances chondrogenic differentiation of DIAS cells, improving the potential utility of these cells for cartilage engineering.     

Hyaluronic acid optimises therapeutic effects of hydrogen peroxide‐induced oxidative stress on breast cancer

Distinguishing the multiple effects of reactive oxygen species (ROS) on cancer cells is important to understand their role in tumour biology. On one side, ROS can be oncogenic by promoting hypoxic conditions, genomic instability and tumorigenesis. Conversely, elevated levels of ROS‐induced oxidative stress can induce cancer cell death. This is evidenced by the conflicting results of research using antioxidant therapy, which in some cases promoted tumour growth and metastasis. However, some antioxidative or ROS‐mediated oxidative therapies have also yielded beneficial effects. To better define the effects of oxidative stress, in vitro experiments were conducted on 4T1 and splenic mononuclear cells (MNCs) under hypoxic and normoxic conditions. Furthermore, hydrogen peroxide (H₂O₂; 10–1,000 μM) was used as an ROS source alone or in combination with hyaluronic acid (HA), which is frequently used as drug delivery vehicle. Our result indicated that the treatment of cancer cells with H₂O₂ + HA was significantly more effective than H₂O₂ alone. In addition, treatment with H₂O₂ + HA led to increased apoptosis, decreased proliferation, and multiphase cell cycle arrest in 4T1 cells in a dose‐dependent manner under normoxic or hypoxic conditions. As a result, migratory tendency and the messenger RNA levels of vascular endothelial growth factor, matrix metalloproteinase‐2 (MMP‐2), and MMP‐9 were significantly decreased in 4T1 cells. Of note, HA treatment combined with 100–1,000 μM H₂O₂ caused more damage to MNCs as compared to treatment with lower concentrations (10–50 μM). Based on these results, we propose to administer high‐dose H₂O₂ + HA (100–1000 μM) for intratumoural injection and low doses for systemic administration. Intratumoural route could have toxic and inhibitory effects not only on the tumour but also on residential myeloid cells defending it, whereas systemic treatment could stimulate peripheral immune responses against the tumour. More in vivo research is required to confirm this hypothesis.     

Protein profiling and angiogenic effect of hypoxia-cultured human umbilical cord blood-derived mesenchymal stem cells in hindlimb ischemia

The aim of the present study was to investigate protein profiles of human umbilical cord blood-derived mesenchymal stem cells (hUCB-MSCs) cultured in normoxic (21% O₂) and hypoxic (1% O₂) conditions, and evaluate oxygenation effects on angiogenesis in an ischemic hindlimb mouse model using a modified ischemic scoring system. Hypoxic conditions did not change the expression of phenotypic markers and increased adipogenesis and chondrogenesis. Epidermal growth factor (EGF), transforming growth factor alpha (TGF-α), TGF-β RII, and vascular endothelial growth factor (VEGF) were upregulated in the conditioned medium of hypoxic hUCB-MSCs, which are commonly related to angiogenesis and proliferation of biological processes by Gene Ontology. In the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway, significant enrichment of the phosphorylation of abelson murine leukemia viral oncogene homolog 1 (ABL1) (Phospho-Tyr204) and B-cell lymphoma-extra large (BCL-XL) (Phospho-Thr47) as anti-apoptotic pathways was observed in hypoxic hUCB-MSCs. Furthermore, hypoxic conditions induced proliferation and migration, and reduced apoptosis of hUCB-MSCs in vitro. Based on the results of protein antibody array, we evaluated the angiogenic effects of injecting normoxic or hypoxic hUCB-MSCs (1 × 10⁶) into the ischemic hindlimb muscles of mice. Ischemic scores and capillary generation were significantly greater in the hypoxic hUCB-MSC injection group than in the normoxic hUCB-MSC group. Our findings demonstrate that culturing hUCB-MSCs in hypoxic conditions not only significantly enriches phosphorylation in the anti-apoptosis pathway and enhances the secretion of several angiogenic proteins from cells, but also alleviates ischemic injury of hindlimb of mice.     

Mesenchymal stem cells cultured under hypoxia escape from senescence via down-regulation of p16 and extracellular signal regulated kinase

Hypoxia has been considered to affect the properties of tissue stem cells including mesenchymal stem cells (MSCs). Effects of long periods of exposure to hypoxia on human MSCs, however, have not been clearly demonstrated. MSCs cultured under normoxic conditions (20% pO₂) ceased to proliferate after 15-25 population doublings, while MSCs cultured under hypoxic conditions (1% pO₂) retained the ability to proliferate with an additional 8-20 population doublings. Most of the MSCs cultured under normoxic conditions were in a senescent state after 100 days, while few senescent cells were found in the hypoxic culture, which was associated with a down-regulation of p16 gene expression. MSCs cultured for 100 days under hypoxic conditions were superior to those cultured under normoxic conditions in the ability to differentiate into the chondro- and adipogenic, but not osteogenic, lineage. Among the molecules related to mitogen-activated protein kinase (MAPK) signaling pathways, extracellular signal regulated kinase (ERK) was significantly down-regulated by hypoxia, which helped to inhibit the up-regulation of p16 gene expression. Therefore, the hypoxic culture retained MSCs in an undifferentiated and senescence-free state through the down-regulation of p16 and ERK.     

O2 Level Controls Hematopoietic Circulating Progenitor Cells Differentiation into Endothelial or Smooth Muscle Cells

Background

Recent studies showed that progenitor cells could differentiate into mature vascular cells. The main physiological factors implicated in cell differentiation are specific growth factors. We hypothesized that simply by varying the oxygen content, progenitor cells can be differentiated either in mature endothelial cells (ECs) or contractile smooth muscle cells (SMCs) while keeping exactly the same culture medium.

Methodology/Principal Findings

Mononuclear cells were isolated by density gradient were cultivated under hypoxic (5% O₂) or normoxic (21% O₂) environment. Differentiated cells characterization was performed by confocal microscopy examination and flow cytometry analyses. The phenotype stability over a longer time period was also performed. The morphological examination of the confluent obtained cells after several weeks (between 2 and 4 weeks) showed two distinct morphologies: cobblestone shape in normoxia and a spindle like shape in hypoxia. The cell characterization showed that cobblestone cells were positive to ECs markers while spindle like shape cells were positive to contractile SMCs markers. Moreover, after several further amplification (until 3^rd passage) in hypoxic or normoxic conditions of the previously differentiated SMC, immunofluorescence studies showed that more than 80% cells continued to express SMCs markers whatever the cell environmental culture conditions with a higher contractile markers expression compared to control (aorta SMCs) signature of phenotype stability.

Conclusion/Significance

We demonstrate in this paper that in vitro culture of peripheral blood mononuclear cells with specific angiogenic growth factors under hypoxic conditions leads to SMCs differentiation into a contractile phenotype, signature of their physiological state. Moreover after amplification, the differentiated SMC did not reverse and keep their contractile phenotype after the 3^rd passage performed under hypoxic and normoxic conditions. These aspects are of the highest importance for tissue engineering strategies. These results highlight also the determinant role of the tissue environment in the differentiation process of vascular progenitor cells.     

Tradeoffs between respiration and feeding in Sacramento blackfish,Orthodon microlepidotus

Synopsis Suspension-feeding fishes use gill structures for both respiration (lamellae) and food capture (rakers). During hypoxic exposure in eutrophic lakes or poorly circulated sloughs, many fishes, including Sacramento blackfish, Orthodon microlepidotus, increase their gill water flows, in part by increasing ventilatory stroke volumes. Stroke volume increases could compromise particle sieving efficiency by spreading interdigitated gill rakers from adjacent gill arches, although blackfish capture food particles by raker-guided water flows to a sticky buccal root. Using van Dam-type respirometers, blackfish respiratory variables and feeding efficiency (Artemia nauplii) were measured under normoxia (> 130 torr PO₂) and hypoxia (60 torr PO₂). Compared with non-feeding, normoxic conditions, gill ventilation volume, frequency, stroke volume, and gape all increased, while O₂ uptake efficiency decreased, during hypoxia and during feeding. O₂ consumption increased during feeding treatments, and % uptake of nauplii showed no difference between normoxic and hypoxic groups. Thus, blackfish display respiratory adaptations, including increased ventilatory stroke volumes, to survive in hypoxic environments such as Clear Lake, California. Importantly, they have also evolved a particle capture mechanism that allows efficient suspension-feeding under both normoxic and hypoxic conditions.     

Reconstitution activity of hypoxic cultured human cord blood CD34-positive cells in NOG mice

Hematopoietic stem cells (HSCs) reside in hypoxic areas of the bone marrow. However, the role of hypoxia in the maintenance of HSCs has not been fully characterized. We performed xenotransplantation of human cord blood cells cultured in hypoxic or normoxic conditions into adult NOD/SCID/IL-2Rγ^null (NOG) mice. Hypoxic culture (1% O₂) for 6 days efficiently supported the maintenance of HSCs, although cell proliferation was suppressed compared to the normoxic culture. In contrast, hypoxia did not affect in vitro colony-forming ability. Upregulation of a cell cycle inhibitor, p21, was observed in hypoxic culture. Immunohistochemical analysis of recipient bone marrow revealed that engrafted CD34⁺CD38⁻ cord blood HSCs were hypoxic. Taken together, these results demonstrate the significance of hypoxia in the maintenance of quiescent human cord blood HSCs.     

Influence of Hypoxic Interval Training and Hyperoxic Recovery on Muscle Activation and Oxygenation in Connection with Double-Poling Exercise

Here, we evaluated the influence of breathing oxygen at different partial pressures during recovery from exercise on performance at sea-level and a simulated altitude of 1800 m, as reflected in activation of different upper body muscles, and oxygenation of the m. triceps brachii. Ten well-trained, male endurance athletes (25.3±4.1 yrs; 179.2±4.5 cm; 74.2±3.4 kg) performed four test trials, each involving three 3-min sessions on a double-poling ergometer with 3-min intervals of recovery. One trial was conducted entirely under normoxic (No) and another under hypoxic conditions (Ho; F_iO₂ = 0.165). In the third and fourth trials, the exercise was performed in normoxia and hypoxia, respectively, with hyperoxic recovery (HOX; F_iO₂ = 1.00) in both cases. Arterial hemoglobin saturation was higher under the two HOX conditions than without HOX (p<0.05). Integrated muscle electrical activity was not influenced by the oxygen content (best d = 0.51). Furthermore, the only difference in tissue saturation index measured via near-infrared spectroscopy observed was between the recovery periods during the NoNo and HoHOX interventions (P<0.05, d = 0.93). In the case of HoHo the athletes’ P_mean declined from the first to the third interval (P < 0.05), whereas P_mean was unaltered under the HoHOX, NoHOX and NoNo conditions. We conclude that the less pronounced decline in P_mean during 3 x 3-min double-poling sprints in normoxia and hypoxia with hyperoxic recovery is not related to changes in muscle activity or oxygenation. Moreover, we conclude that hyperoxia (F_iO₂ = 1.00) used in conjunction with hypoxic or normoxic work intervals may serve as an effective aid when inhaled during the subsequent recovery intervals.     

Attenuation of lipopolysaccharide-induced oxidative stress and apoptosis in fetal pulmonary artery endothelial cells by hypoxia

Pulmonary vascular endothelial injury resulting from lipopolysaccharide (LPS) and oxygen toxicity contributes to vascular simplification seen in the lungs of premature infants with bronchopulmonary dysplasia. Whether the severity of endotoxin-induced endothelial injury is modulated by ambient oxygen tension (hypoxic intrauterine environment vs. hyperoxic postnatal environment) remains unknown. We posited that ovine fetal pulmonary artery endothelial cells (FPAEC) will be more resistant to LPS toxicity under hypoxic conditions (20–25 Torr) mimicking the fetal milieu. LPS (10 μg/ml) inhibited FPAEC proliferation and induced apoptosis under normoxic conditions (21% O₂) in vitro. LPS-induced FPAEC apoptosis was attenuated in hypoxia (5% O₂) and exacerbated by hyperoxia (55% O₂). LPS increased intracellular superoxide formation, as measured by 2-hydroxyethidium (2-HE) formation, in FPAEC in normoxia and hypoxia. 2-HE formation in LPS-treated FPAEC increased in parallel with the severity of LPS-induced apoptosis in FPAEC, increasing from hypoxia to normoxia to hyperoxia. Differences in LPS-induced apoptosis between hypoxia and normoxia were abolished when LPS-treated FPAEC incubated in hypoxia were pretreated with menadione to increase superoxide production. Apocynin decreased 2-HE formation, and attenuated LPS-induced FPAEC apoptosis under normoxic conditions. We conclude that ambient oxygen concentration modulates the severity of LPS-mediated injury in FPAEC by regulating superoxide levels produced in response to LPS.     

New method for the detection of reactive oxygen species in anti-tumoural activity of adriamycin: A comparison between hypoxic and normoxic cells

Tumour hypoxia plays a role in chemoresistance in several human tumours. However, how hyperbaric oxygen leads to chemotherapeutic gain is unclear. This study investigates the relation of reactive oxygen species (ROS) generation with anti-tumoural effect of adriamycin (ADR) on CCRF-CEM cells under hypoxic (2% O₂) and normoxic (21% O₂) conditions. A new method was used to measure intracellular ROS variations through the fluorescence lifetime of 1-pyrenebutyric acid. At 24 h, ADR, probably via semiquinone radical, enhances ROS levels in normoxic cells compared to hypoxic cells. Long-term studies show that ROS are also generated by a second mechanism related to cell functions perturbation. ADR arrests the cell cycle progression both under hypoxia and normoxia, indicating that oxygen and ROS does not influence the DNA damaging activity of ADR. The findings reveal that moderate improvement of ADR cytotoxicity results from higher ROS formation in normoxic cells, leading to elevated induction of cell death.     

Characteristics of human lipoaspirate-isolated mesenchymal stromal cells cultivated under lower oxygen tension

The effect of reduced oxygen concentration in the gas phase on the proliferation, viability, and immunophenotype of human mesenchymal stromal cells isolated from lipoaspirate (lMSC) has been investigated. It was shown that the proliferation activity of cells under hypoxic conditions (5% O₂) was, on average, 2.9 times higher than those cultivated under routine (normoxic) (20% O₂) conditions. Decreased oxygen level in the culture medium did not cause any change in lMSC viability or immunophenotype. Thus, the permanent cultivation of lMSC in medium with a lower oxygen tension may be an efficient approach to obtaining a higher mass of cells that maintain their characteristics over a shorter period of time, which is a requirement for regenerative medicine.     

Aromatic Amino Acid Activation of Signaling Pathways in Bone Marrow Mesenchymal Stem Cells Depends on Oxygen Tension

The physiologic oxygen pressures inside the bone marrow environment are much lower than what is present in the peripheral circulation, ranging from 1–7%, compared to values as high as 10–13% in the arteries, lungs and liver. Thus, experiments done with bone marrow mesenchymal stem cells (BMMSCs) using standard culture conditions may not accurately reflect the true hypoxic bone marrow microenvironment. However, since aging is associated with an increased generation of reactive oxygen species, experiments done under 21%O₂ conditions may actually more closely resemble that of the aging bone marrow environment. Aromatic amino acids are known to be natural anti-oxidants. We have previously reported that aromatic amino acids are potent agonists for stimulating increases in intracellular calcium and phospho-c-Raf and in promoting BMMSC differentiation down the osteogenic pathway. Our previous experiments were performed under normoxic conditions. Thus, we next decided to compare a normoxic (21% O₂) vs. a hypoxic environment (3% O₂) alone or after treatment with aromatic amino acids. Reverse-phase protein arrays showed that 3% O₂ itself up-regulated proliferative pathways. Aromatic amino acids had no additional effect on signaling pathways under these conditions. However, under 21%O₂ conditions, aromatic amino acids could now significantly increase these proliferative pathways over this “normoxic” baseline. Pharmacologic studies are consistent with the aromatic amino acids activating the extracellular calcium-sensing receptor. The effects of aromatic amino acids on BMMSC function in the 21% O2 environment is consistent with a potential role for these amino acids in an aging environment as functional anti oxidants.     

Hypoxic culture enhances the expansion of rat bone marrow-derived mesenchymal stem cells via the regulatory pathways of cell division and apoptosis

This study aimed to examine the proliferative behavior and molecular mechanisms of rat bone marrow-derived MSCs (rBMSCs) cultured under three different oxygen concentrations. Passaged rBMSCs exhibited significantly greater proliferation rates at 1% O₂ and 5% O₂ than those at 18% O₂ and the cells exposed to 1% O₂ showed the highest proliferative potential, which was evidenced by the growth curves, colony-forming efficiencies, and CCK-8 absorbance values. The rBMSCs grown under hypoxic culture conditions (1% O₂ and 5% O₂) had the increased percentage of cells in S?+?G₂/M-phase and the decreased apoptotic index, compared with normoxia (18% O₂). It was revealed for the first time that there were more phosphohistone H3 (PHH3)-positive cells and higher expressions of proliferating cell nuclear antigen (PCNA) in the hypoxic cultures of rBMSCs than in the normoxic culture. Hypoxia upregulated the anti-apoptotic protein Bcl-2 and downregulated the pro-apoptotic proteins Bax and the cleaved caspase-3 in cultured rBMSCs. The levels of hypoxia-inducible factor-1α (HIF-1α) and phosphorylated extracellular signal-regulated kinase 1/2 (p-ERK1/2) were increased in the hypoxic-cultured rBMSCs. Nevertheless, no significant difference was observed in p53 level of rBMSCs between different oxygen concentrations. In conclusion, the hypoxia exerts a promoting effect on the in vitro expansion of rBMSCs via several signaling and molecular pathways involved in the control of cell cycle and apoptosis.     

Two Novel Class II Hydrophobins from Trichoderma spp. Stimulate Enzymatic Hydrolysis of Poly(Ethylene Terephthalate) when Expressed as Fusion Proteins

Poly(ethylene terephthalate) (PET) can be functionalized and/or recycled via hydrolysis by microbial cutinases. The rate of hydrolysis is however low. Here, we tested whether hydrophobins (HFBs), small secreted fungal proteins containing eight positionally conserved cysteine residues, are able to enhance the rate of enzymatic hydrolysis of PET. Species of the fungal genus Trichoderma have the most proliferated arsenal of class II hydrophobin-encoding genes among fungi. To this end, we studied two novel class II HFBs (HFB4 and HFB7) of Trichoderma. HFB4 and HFB7, produced in Escherichia coli as fusions to the C terminus of glutathione S-transferase, exhibited subtle structural differences reflected in hydrophobicity plots that correlated with unequal hydrophobicity and hydrophily, respectively, of particular amino acid residues. Both proteins exhibited a dosage-dependent stimulation effect on PET hydrolysis by cutinase from Humicola insolens, with HFB4 displaying an adsorption isotherm-like behavior, whereas HFB7 was active only at very low concentrations and was inhibitory at higher concentrations. We conclude that class II HFBs can stimulate the activity of cutinases on PET, but individual HFBs can display different properties. The present findings suggest that hydrophobins can be used in the enzymatic hydrolysis of aromatic-aliphatic polyesters such as PET.     

The pleiotropic functions of intracellular hydrophobins in aerial hyphae and fungal spores

Higher fungi can rapidly produce large numbers of spores suitable for aerial dispersal. The efficiency of the dispersal and spore resilience to abiotic stresses correlate with their hydrophobicity provided by the unique amphiphilic and superior surface-active proteins–hydrophobins (HFBs)–that self-assemble at hydrophobic/hydrophilic interfaces and thus modulate surface properties. Using the HFB-enriched mold Trichoderma (Hypocreales, Ascomycota) and the HFB-free yeast Pichia pastoris (Saccharomycetales, Ascomycota), we revealed that the rapid release of HFBs by aerial hyphae shortly prior to conidiation is associated with their intracellular accumulation in vacuoles and/or lipid-enriched organelles. The occasional internalization of the latter organelles in vacuoles can provide the hydrophobic/hydrophilic interface for the assembly of HFB layers and thus result in the formation of HFB-enriched vesicles and vacuolar multicisternal structures (VMSs) putatively lined up by HFBs. These HFB-enriched vesicles and VMSs can become fused in large tonoplast-like organelles or move to the periplasm for secretion. The tonoplast-like structures can contribute to the maintenance of turgor pressure in aerial hyphae supporting the erection of sporogenic structures (e.g., conidiophores) and provide intracellular force to squeeze out HFB-enriched vesicles and VMSs from the periplasm through the cell wall. We also show that the secretion of HFBs occurs prior to the conidiation and reveal that the even spore coating of HFBs deposited in the extracellular matrix requires microscopic water droplets that can be either guttated by the hyphae or obtained from the environment. Furthermore, we demonstrate that at least one HFB, HFB4 in T. guizhouense, is produced and secreted by wetted spores. We show that this protein possibly controls spore dormancy and contributes to the water sensing mechanism required for the detection of germination conditions. Thus, intracellular HFBs have a range of pleiotropic functions in aerial hyphae and spores and are essential for fungal development and fitness.