Petra Merschak

Human Tear Lipocalin Exhibits Antimicrobial Activity by Scavenging Microbial Siderophores

ABSTRACT 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.

Expression of the gene for tear lipocalin/von Ebner's gland protein in human prostate.

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 Ebners 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.

The human lacrimal gland synthesizes apolipoprotein D mRNA in addition to tear prealbumin mRNA, both species encoding members of the lipocalin superfamily.

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.

Phage display reveals a novel interaction of human tear lipocalin and thioredoxin which is relevant for ligand binding

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.

Development of a Fast and Reliable Method for the Assessment of Microbial Colonization and Growth on Textiles by DNA Quantification

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.

Complement component C8γ is expressed in human fetal and adult kidney independent of C8α

Human complement component C8γ 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 C8α 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 C8γ. Our experiments clearly showed that besides in liver, this gene is also expressed in fetal and adult kidney. Renal expression of C8γ is not dependent on C8α expression, since we could not detect C8α 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 C8γ, 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.

Copper(I)oxide microparticles – synthesis and antimicrobial finishing of textiles

Copper containing particles are of high interest to provide antibacterial activity to textiles for medical products, hygiene application or where odor formation as result of bacterial activity has to be controlled. Cu(i)oxide microparticles with a rather uniform diameter between 1.5 and 2 μm can be prepared by controlled reduction of alkaline Cu(ii)-tartaric acid complexes. Such particles can be bound to textile surfaces by means of a pigment binder system used in pigment dyeing. By a simple pad-dry process textile fabrics with a Cu-content of 250-270 mg Cu per kg fabric could be prepared. The samples (fabrics) exhibited a reduction in viability of 100% for Staphylococcus aureus and 84% for Klebsiella pneumonia as estimated by the ASTM E2149 antimicrobial test. Simulated wash procedures led to a reduction in Cu-content to 60-50% of the initial value. Reduction in viability remained at 99% for Staphylococcus aureus and 78% for Klebsiella pneumoniae. The new process is of high value to impart antimicrobial properties to textile products because an antimicrobial product with good wash permanence can be delivered using rather simple processing and ordinary chemicals.

Mobilization of Metals from Pristine Mineral Soil by Nitrifying and Sulfur-Oxidizing Bacteria – The Leaching Potential of Indigenous Culture Enrichments

Indigenous chemolithotrophic nitrifying and sulfur-oxidizing culture enrichments mobilized metals from pristine mineral soil under conditions of ammonium or thiosulfate supplementation in a laboratory experiment carried out over a period of 40 days. The average mineralogical composition of the mineral soil was quartz (62%), feldspar (20%), muscovite (6%), chlorite (2%), hornblende (2%), dolomite (4%), and calcium carbonate (4%). The leaching efficiency of the nitrifying enrichment was calcium (27%), magnesium (15%), zinc (5.4%), manganese (0.6%), and cobalt (1.4%) after 40 days of incubation. In case of sulfur-oxidizing enrichment, leaching efficiency was calcium (56%), magnesium (36%), iron (0.8%), zinc (12%), manganese (2.1%), and cobalt (12%). The impact of these organisms on pristine mineral soil could be important in understanding primary colonization and the early stages of soil formation.