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The hygromycin B phosphotransferase gene from Escherichia coli and the pyrithiamine resistance gene from Aspergillus oryzae are two dominant selectable marker genes widely used to genetically manipulate several fungal species. Despite the recent development of CRISPR/Cas9 and marker-free systems, in vitro molecular tools to study Aspergillus&nbsp, fumigatus, which is a saprophytic fungus causing life-threatening diseases in immunocompromised hosts, still rely extensively on the use of dominant selectable markers. The limited number of drug selectable markers is already a critical aspect, but the possibility that their introduction into a microorganism could induce enhanced virulence or undesired effects on metabolic behavior constitutes another problem. In this context, here, we demonstrate that the use of ptrA in A. fumigatus leads to the secretion of a compound that allows the recovery of thiamine auxotrophy. In this study, we developed a simple modification of the two commonly used dominant markers in which the development of resistance can be controlled by the xylose-inducible promoter PxylP from Penicillium chrysogenum. This strategy provides an easy solution to avoid undesired side effects, since the marker expression can be readily silenced when not required.

β-Lactoglobulin (BLG) is a member of the lipocalin protein family and a major food-borne allergen in humans. Numerous in vitro studies have suggested a role for BLG in molecular transport processes; however, its physiological role remains enigmatic. A cellular receptor for BLG has been proposed, but has not yet been identified. Here we show that human LIMR, known to act as an endocytic receptor for lipocalin-1, also binds bovine BLG and mediates its cellular uptake. The specificity of this interaction is corroborated by a complete block of cellular uptake of BLG in the presence of LIMR antibodies or LIMR downregulation by antisense RNA. Furthermore, heterologous expression of human LIMR in insect cells mediates cellular internalization of FITC-BLG. Since LIMR is highly expressed in the human intestine, it might also function in the uptake of food-borne BLG.

Multiangle laser light scattering and fluorescence anisotropy decay measurements clarified the oligomeric states of native and recombinant tear lipocalin (lipocalin-1, TL). Native TL is monomeric. Recombinant TL (5-68 microM) with or without the histidine tag shows less than 7% dimer formation that is not in equilibrium with the monomeric form. Fluorescence anisotropy decay showed a correlation time of 9-10 ns for TL (10 microM-1 mM). Hydrodynamic calculations based on the crystallographic structure of a monomeric TL mutant closely concur with the observed correlation time. The solution properties calculated with HYDROPRO and SOLPRO programs from the available crystallographic structure of a monomeric TL mutant concur closely with the observed fluorescence anisotropy decay. The resulting model shows that protein topology is the major determinant of rotational correlation time and accounts for deviation from the Stokes-Einstein relation. The data challenge previous gel filtration studies to show that native TL exists predominantly as a monomer in solution rather than as a dimer. Delipidation of TL results in a formation of a complex oligomeric state (up to 25%). These findings are important as the dynamic processes in the tear film are limited by diffusional, translational as well as rotational, properties of the protein.

There is a lack of relevant methods to assess the colonization of textiles by skin bacteria because present methods are mainly culture-based procedures. Therefore, the goal of this study was to develop a fast and sensitive culture-independent procedure for the quantification of microbial colonization and growth on textiles. We have established a suitable protocol to use DNA quantification as a reliable method for in vitroand in vivoinvestigations of textiles. For DNA extraction, a two-step procedure comprising treatment of the textile with a solution containing Triton X-100 and lysozyme for 1 h and a successive treatment by SDS and proteinase K for 2 h turned out to be most efficient. DNA extracted from textiles and fabrics was than quantified with the highly sensitive PicoGreen fluorescent dye. In vitrochallenge tests demonstrated a strong correlation between numbers of bacteria on textiles and amount of DNA extracted from textiles. Therefore, this method was used to compare different materials after in vivotrials for assessment of their susceptibility for microbial colonization and growth.

Human tear lipocalin (TL; also known as Lcn1) is a secretory protein present in large amounts in fluids that cover epithelial surfaces such as tears and respiratory secretions. It is supposed to act as a physiological scavenger of hydrophobic, potentially harmful molecules, but there is evidence that it also inhibits bacterial growth. In the present study, we reconsidered the possibility that TL might interfere with microbial growth by scavenging of siderophores, as described for human neutrophil gelatinase-associated lipocalin (NGAL). Indeed, our experiments revealed that TL binds to microbial siderophores with high affinities. In contrast to NGAL, which was shown to have some specificity for bacterial catecholate-type siderophores, TL binds to a broad array of siderophores, including bacterial catecholate-type enterobactin and hydroxamate-type desferrioxamine B, and all major classes of fungal siderophores. By adding exogenous TL, bacterial and fungal growth could be inhibited under iron-limiting conditions. Thus, TL might be a novel member of the innate immune system especially involved in mucosal defense against fungal infections.

Human lipocalin-1 (Lcn-1, also called tear lipocalin), a member of the lipocalin structural superfamily, is produced by a number of glands and tissues and is known to bind an unusually large array of hydrophobic ligands. Apart from its specific function in stabilizing the lipid film of human tear fluid, it is suggested to act as a physiological scavenger of potentially harmful lipophilic compounds, in general. To characterize proteins involved in the reception, detoxification, or degradation of these ligands, a cDNA phage-display library from human pituitary gland was constructed and screened for proteins interacting with Lcn-1. Using this method an Lcn-1 interacting phage was isolated that expressed a novel human protein. Molecular cloning and analysis of the entire cDNA indicated that it encodes a 55-kDa protein, lipocalin-1 interacting membrane receptor (LIMR), with nine putative transmembrane domains. The cell membrane location of this protein was confirmed by immunocytochemistry and Western blot analysis of membrane fractions of human NT2 cells. Independent biochemical investigations using a recombinant N-terminal fragment of LIMR also demonstrated a specific interaction with Lcn-1 in vitro. Based on these data, we suggest LIMR to be a receptor of Lcn-1 ligands. These findings constitute the first report of cloning of a lipocalin interacting, plasma membrane-located receptor, in general. In addition, a sequence comparison supports the biological relevance of this novel membrane protein, because genes with significant nucleotide sequence similarity are present in Takifugu rubripes, Drosophila melanogaster, Caenorhabditis elegans, Mus musculus, Bos taurus, and Sus scrofa. According to data derived from the human genome sequencing project, the LIMR-encoding gene has to be mapped on human chromosome 12, and its intron/exon organization could be established. The entire LIMR-encoding gene consists of about 13.7 kilobases in length and contains 16 introns with a length between 91 and 3438 base pairs.

Northern analysis of human multiple tissue blots containing poly A+ RNA from spleen, thymus, prostate, testis, ovary, small intestine, colon and peripheral blood leukocytes revealed that a prostate specific transcript hybridizes to a tear lipocalin/von Ebner's gland protein (TL/VEGP) gene probe. To characterize this transcript, the corresponding cDNA was amplified by reverse transcription (RT)-PCR. Cloning and sequence analysis showed that it was identical to the tear lipocalin cDNA isolated from human lachrymal glands. Immunohistochemical analysis on thin layer sections of human prostate using a tear lipocalin specific antiserum confirmed the expression of this cDNA in prostate. Thus, our results clearly argue against a unique function of TL/VEGP in human tear fluid or saliva. The human cDNA was expressed in E. coli using the pQE system yielding a recombinant protein which shows biochemical properties identical to the native TL/VEGP.

Apolipoprotein D (apoD), a glycoprotein originally characterized as a component of the high density lipoprotein fraction of human plasma and known to be a member of the lipocalin protein superfamily, has been found in human tear fluid by Western blot analysis. Unlike serum it seems that in the tear fluid apoD exists mainly as a disulphide linked homodimer which is not associated with lecithin/cholesterol acyltransferase (LCAT) or apolipoprotein A-I (apo A-I). By reverse-transcription-PCR (RT-PCR) of mRNA extracted from a human lacrimal gland and use of specific primers we could demonstrate expression of the apoD gene in this tissue. The amplified cDNA was cloned and a subsequent sequence analysis confirmed the identity of apoD mRNA in the human lacrimal gland. These investigations indicate that the lacrimal gland is the site of synthesis of the tear fluid apoD. Although the physiological function of apoD is unknown, it has the ability to bind phospholipids, cholesterol and other small hydrophobic molecules. Therefore, this protein might interact with meibomian lipids present in human tear fluid and probably contribute to the surface spreading of these lipids or it may function as a clearance factor, protecting the cornea from harmful lipophilic molecules.

Human tear lipocalin (TL) is an unusual member of the lipocalin protein family, since it is known to bind a large variety of lipophilic ligands in vivo and acts as a cysteine proteinase inhibitor in vitro. It is suggested to function as a physiological protection factor by scavenging lipophilic potentially harmful compounds. Since protein-protein interaction or macromolecular complexation is a common feature of many lipocalins, we applied phage display technology to identify TL interacting proteins. By panning of a human prostate cDNA phagemid library against purified TL we isolated a thioredoxin (Trx) encoding phage clone. Biochemical analysis revealed that TL indeed interacts with Trx and is reduced by this redox protein. Reduction of the TL-specific disulfide bond is of functional relevance, since the reduced protein shows a nine-fold increase in ligand affinity when tested with retinoic acid as ligand.

Human complement component C8gamma is an unusual complement factor since it shows no homology to other complement proteins but is a member of the lipocalin superfamily. So far, it has been found exclusively in plasma, covalently linked to C8alpha by disulfide bridging. We have used dot blot and Northern blot analyses of a large number of different human tissues to survey systematically the expression pattern of C8gamma. Our experiments clearly showed that besides in liver, this gene is also expressed in fetal and adult kidney. Renal expression of C8gamma is not dependent on C8alpha expression, since we could not detect C8alpha expression in kidney. Thus its physiological function is not restricted to a specific action in association with complement components. As a prerequisite for further characterization of the structure and binding activities of the uncomplexed C8gamma, we have expressed the encoding cDNA in Escherichia coli. To increase the probability for proper folding of the characteristic intramolecular disulfide bridge the recombinant protein was produced by secretion to the periplasm.