Respiratory syncytial virus (RSV) is an important cause of respiratory tract disease in infants and the elderly. Currently, no licensed vaccine against RSV is available. Here we describe the development of a safe and effective intranasal subunit vaccine that is based on recombinant fusion (F) protein bound to the surface of immunostimulatory bacterium-like particles (BLPs) derived from the food-grade bacterium Lactococcus lactis. Different variants of F were analyzed with respect to their conformation and reactivity with neutralizing antibodies, assuming that F proteins mimicking the metastable prefusion form of RSV F expose a more extensive and relevant epitope repertoire than F proteins corresponding to the postfusion structure. Our results indicate that the recombinant soluble ectodomain of RSV F readily adopts a postfusion conformation, generation of which cannot be prevented by C-terminal addition of a trimerization motif, but whose formation is prevented by mutation of the two furin cleavage sites in F. While the putative postfusion form of F is recognized well by the monoclonal antibody Palivizumab, this is much less so for the more potently neutralizing, prefusion-specific antibodies D25 and AM22. Both addition of the trimerization motif and mutation of the furin cleavage sites increased the reactivity of F with D25 and AM22, with the highest reactivity being observed for F proteins in which both these features were combined. Intranasal vaccination of mice or cotton rats with BLPs loaded with this latter prefusion-like F protein (BLP-F), resulted in the potent induction of F-specific immunoglobulins and in significantly decreased virus titers in the lungs upon RSV challenge. Moreover, and in contrast to animals vaccinated with formalin-inactivated RSV, animals that received BLP-F exhibited high levels of F-specific secretory IgA in the nose and RSV-neutralizing antibodies in sera, but did not show symptoms of enhanced disease after challenge with RSV.

Penicillium chrysogenum utilizes phenylacetic acid as a side chain precursor in penicillin G biosynthesis. During industrial production of penicillin G, phenylacetic acid is fed in small amounts to the medium to avoid toxic side effects. Phenylacetic acid is taken up from the medium and intracellularly coupled to 6-aminopenicillanic acid. To enter the fungal cell, phenylacetic acid has to pass the plasma membrane. The process via which phenylacetic acid crosses the plasma membrane was studied in mycelia and liposomes. Uptake of phenylacetic acid by mycelium was nonsaturable, and the initial velocity increased logarithmically with decreasing external pH. Studies with liposomes demonstrated a rapid passive flux of the protonated species through liposomal membranes. These results indicate that phenylacetic acid passes the plasma membrane via passive diffusion of the protonated species. The rate of phenylacetic acid uptake at an external concentration of 3 mM is at least 200-fold higher than the penicillin production rate in the Panlabs P2 strain. In this strain, uptake of phenylacetic acid is not the rate-limiting step in penicillin G biosynthesis.

Functional plasma membranes from the filamentous fungus Penicillium chrysogenum have been isolated with the objective of studying transport processes. The isolation procedure consists of three steps, namely homogenization of cells with a Braun MSK homogenizer, followed by Percoll gradient centrifugation and floatation of membranes in a three‐step Nycodenz gradient. This method can be applied to strains which differ significantly in morphology and penicillin‐production capacity. Plasma membranes were fused with liposomes containing the beef heart mitochondria1 cytochrome c oxidase. In the presence of reduced cytochrome c, the hybrid membranes maintained a high proton motive force that functions as a driving force for the uptake of the amino acids arginine and valine via distinct transport systems.

Transport studies with Penicillium chrysogenum plasma membranes fused with cytochrome c oxidase liposomes demonstrate that sulfate uptake is driven by the transmembrane pH gradient and not by the transmembrane electrical potential. Ca 2؉ and other divalent cations are not required. It is concluded that the sulfate transport system catalyzes the symport of two protons with one sulfate anion.One of the transport processes which plays an important role in the biosynthesis of penicillins by filamentous fungi is the uptake of sulfate. Current penicillin production processes yield final penicillin titers which are more than 500 times higher than those obtained with the parental Penicillium chrysogenum strain NRRL 1951 (9). Sulfate is the primary sulfur source in industrial production of penicillin and is the precursor of cysteine that together with valine forms the backbone of the penicillin molecule (9, 15). Therefore, the strongly increased demand for cysteine during penicillin production requires an elevated rate of sulfate uptake from the environment. Studies of the mechanism of sulfate uptake by P. chrysogenum, related filamentous fungi, and yeasts have led to the conclusion that this process occurs through active transport (1, 6, 8, 12-14, 16, 17). It has been suggested that in Penicillium notatum, sulfate is taken up in symport with one proton and one calcium ion (2). According to such a mechanism, the driving forces for sulfate uptake would be the transmembrane electrical potential (⌬⌿), the transmembrane pH gradient (⌬pH), and the chemical gradient of calcium ions (⌬ Ca 2ϩ/F). Although sulfate transport was shown to promote uptake of Ca 2ϩ , a stoichiometric coupling could not be demonstrated. It has been suggested that the sulfate transport system exchanges SO 4. Since these studies have been performed with metabolically active mycelium, the possibility of involvement of plasma membrane-localized Ca 2ϩ transport systems and Ca 2ϩ ATPases cannot be excluded. Moreover, sulfate appears to be sequestered in two distinct intracellular pools, one of which only slowly exchanges with external sulfate (7). The complex mycelial morphology and the presence of intracellular compartments make it impossible to relate the uptake of sulfate in a quantitative manner to the driving forces. We therefore reinvestigated the mechanism of sulfate uptake in P. chrysogenum using a well-defined model system, i.e., isolated plasma membranes fused with cytochrome c oxidase-containing liposomes (5).Sulfate transport in fused plasma membranes. P. chrysogenum shows a high level of sulfate uptake activity when cells are deprived of sulfur (19). P. chrysogenum Wisconsin 54-1255 (kindly supplied by Gist-brocades NV) was first grown for 70 h at 25ЊC on production medium (pH 6.3) (10) supplemented with 10 mM glutamate and 10% (mass/vol) glucose and subsequently incubated for 16 h in production medium in which all sulfate salts were replaced by equivalent chloride salts. Plasma membranes were isolated by a method previously described (5) and f...

The characteristics of the basic amino acid permease (system VI) of the filamentous fungus Penicillium chrysogenum were studied in plasma membranes fused with liposomes containing the beef heart mitochondrial cytochrome c oxidase. In the presence of reduced cytochrome c, the hybrid membranes accumulated the basic amino acids arginine and lysine. Inhibition studies with analogs revealed a narrow substrate specificity. Within the external pH range of 5.5 to 7.5, the transmembrane electrical potential (⌬) functions as the main driving force for uphill transport of arginine, although a low level of uptake was observed when only a transmembrane pH gradient was present. It is concluded that the basic amino acid permease is a H ؉ symporter. Quantitative analysis of the steady-state levels of arginine uptake in relation to the proton motive force suggests a H ؉ -arginine symport stoichiometry of one to one. Efflux studies demonstrated that the basic amino acid permease functions in a reversible manner.Amino acids are utilized by fungi as primary or secondary nitrogen sources or as building blocks for the synthesis of proteins and peptides. Systems involved in the translocation of amino acids across the plasma membrane have been studied in only a few filamentous fungi (5,8,25). Of these fungi, Neurospora crassa, Aspergillus nidulans, and, to a lesser extent, Penicillium chrysogenum are genetically and biochemically the most extensively characterized species. Two distinct classes of plasma membrane-located amino acid permeases are found in filamentous fungi: systems that catalyze the uptake of structurally related amino acids with a broad substrate specificity, such as the general amino acid permeases of plant and animal cells (2,15,18), and systems with a narrow substrate specificity, such as bacterial amino acid transporters (22).Fungi show a peculiar substrate specificity in their amino acid transport systems, and multiple transport mechanisms seem to exist for several amino acids. In N. crassa, five distinct transport systems have been identified, with specificity for aromatic and aliphatic amino acids (system I); aromatic, aliphatic, and basic amino acids (system II); basic amino acids (system III); acidic amino acids (system IV); and L-methionine (system V) (5,8,20,25). Studies with mycelium of P. chrysogenum indicate that this fungus possesses at least six distinct amino acid transport systems (1, 5, 8-10, 11, 24). Most of these systems are specific for one amino acid and analogs, except for system III, which is a general amino acid permease, and system IV, which transports acidic amino acids only (8, 10). Amino acid transporters of fungi are assumed to possess some typical properties: (i) they seem to function unidirectionally; i.e., only uphill transport is observed, while efflux or countertransport of the accumulated amino acids is not detected (8, 19); and (ii) their activity is regulated by transinhibition, i.e., a high internal concentration of an amino acid appears to lower the activity of the transport syst...