Characterization of Molecular Determinants of the Conformational Stability of Macrophage Migration Inhibitory Factor: Leucine 46 Hydrophobic Pocket

Macrophage Migration Inhibitory Factor (MIF) is a key mediator of inflammatory responses and innate immunity and has been implicated in the pathogenesis of several inflammatory and autoimmune diseases. The oligomerization of MIF, more specifically trimer formation, is essential for its keto-enol tautomerase activity and probably mediates several of its interactions and biological activities, including its binding to its receptor CD74 and activation of certain signaling pathways. Therefore, understanding the molecular factors governing the oligomerization of MIF and the role of quaternary structure in modulating its structural stability and multifunctional properties is crucial for understanding the function of MIF in health and disease. Herein, we describe highly conserved intersubunit interactions involving the hydrophobic packing of the side chain of Leu46 onto the β-strand β3 of one monomer within a hydrophobic pocket from the adjacent monomer constituted by residues Arg11, Val14, Phe18, Leu19, Val39, His40, Val41, Val42, and Pro43. To elucidate the structural significance of these intersubunit interactions and their relative contribution to MIF’s trimerization, structural stability and catalytic activity, we generated three point mutations where Leu46 was replaced by glycine (L46G), alanine (L46A) and phenylalanine (L46F), and their structural properties, stability, oligomerization state, and catalytic activity were characterized using a battery of biophysical methods and X-ray crystallography. Our findings provide new insights into the role of the Leu46 hydrophobic pocket in stabilizing the conformational state of MIF in solution. Disrupting the Leu46 hydrophobic interaction perturbs the secondary and tertiary structure of the protein but has no effect on its oligomerization state

5-ethyl-2'-deoxyuridine fragilizes Klebsiella pneumoniae outer wall and facilitates intracellular killing by phagocytic cells

Klebsiella pneumoniae is the causative agent of a variety of severe infections. Many K. pneumoniae strains are resistant to multiple antibiotics, and this situation creates a need for new antibacterial molecules. K. pneumoniae pathogenicity relies largely on its ability to escape phagocytosis and intracellular killing by phagocytic cells. Interfering with these escape mechanisms may allow to decrease bacterial virulence and to combat infections. In this study, we used Dictyostelium discoideum as a model phagocyte to screen a collection of 1,099 chemical compounds. Phg1A KO D. discoideum cells cannot feed upon K. pneumoniae bacteria, unless bacteria bear mutations decreasing their virulence. We identified 3 non-antibiotic compounds that restored growth of phg1A KO cells on K. pneumoniae, and we characterized the mode of action of one of them, 5-ethyl-2'-deoxyuridine (K2). K2-treated bacteria were more rapidly killed in D. discoideum phagosomes than non-treated bacteria. They were more sensitive to polymyxin and their outer membrane was more accessible to a hydrophobic fluorescent probe. These results suggest that K2 acts by rendering the membrane of K. pneumoniae accessible to antibacterial effectors. K2 was effective on three different K. pneumoniae strains, and acted at concentrations as low as 3 μM. K2 has previously been used to treat viral infections but its precise molecular mechanism of action in K. pneumoniae remains to be determined

Lipid components of olive oil from Tunisian cv. Sayali: characterization and authenticity

The analysis of the total lipid fraction from the Sayali variety of olive oil was accomplished in the present investigation. Glyceridic, unsaponifiable and flavour fractions of the oil were isolated and identified using several analytical methods. Chromatographic techniques have proven to be suitable for these determinations, especially capillary gas chromatography. Gas chromatography coupled to mass spectrometry was successfully used to identify sterols, triterpenes alcohols, 4-monomethylsterols, aliphatic alcohols and aroma compounds in our samples. Furthermore, solid phase microextraction was used to isolate volatiles from the total lipid fraction. Results from the quantitative characterization of Sayali olive oil showed that oleic acid (77.4%) and triolein (47.4%) were the dominant glyceridic components. However, the main compounds of the unsaponifiable fraction were b-sitosterol (147.5mg/100 g oil), 24-methylene cycloartenol (146.4mg/100 g oil) and hexacosanol (49.3 mg/100 g oil). Moreover, results showed that the aldehydic compounds were the major flavours present in Sayali olive oi

Morphology and chemical composition of Tunisian caper seeds: variability and population profiling

Caper, as a spontaneous plant, has a large natural distribution in the Mediterranean Sea basin. It is an interesting crop with an economic importance; especially the species Capparis spinosa. The morphology of seeds and their composition in lipid and protein were studied in 15 wild Tunisian caper populations: 9 populations represented the subspecies C. spinosa subsp. spinosa (thorny caper) and 6 populations represented the subspecies C. spinosa subsp. rupestris (inerm caper). Results show that seeds of C. spinosa are especially attractive because they can be grown to produce oil (ca. 30%) and protein (26%). Principal component analysis (PCA) did not show a geographic separation. A subspecies segregation between C. spinosa subsp. rupestris (as a homogeneous group) and C. spinosa subsp. spinosa (as a heterogeneous group) was clearly detected.Key words: Caper (Cappains spinosa), morphology; lipids, proteins, principal component analysis (PCA)

Exploring anti-bacterial compounds against intracellular Legionella.

Legionella pneumophila is a ubiquitous fresh-water bacterium which reproduces within its erstwhile predators, environmental amoeba, by subverting the normal pathway of phagocytosis and degradation. The molecular mechanisms which confer resistance to amoeba are apparently conserved and also allow replication within macrophages. Thus, L. pneumophila can act as an 'accidental' human pathogen and cause a severe pneumonia known as Legionnaires' disease. The intracellular localisation of L. pneumophila protects it from some antibiotics, and this fact must be taken into account to develop new anti-bacterial compounds. In addition, the intracellular lifestyle of L. pneumophila may render the bacteria susceptible to compounds diminishing bacterial virulence and decreasing intracellular survival and replication of this pathogen. The development of a single infection cycle intracellular replication assay using GFP-producing L. pneumophila and Acanthamoebacastellanii amoeba is reported here. This fluorescence-based assay allows for continuous monitoring of intracellular replication rates, revealing the effect of bacterial gene deletions or drug treatment. To examine how perturbations of the host cell affect L. pneumophila replication, several known host-targeting compounds were tested, including modulators of cytoskeletal dynamics, vesicle scission and Ras GTPase localisation. Our results reveal a hitherto unrealized potential antibiotic property of the β-lactone-based Ras depalmitoylation inhibitor palmostatin M, but not the closely related inhibitor palmostatin B. Further characterisation indicated that this compound caused specific growth inhibition of Legionella and Mycobacterium species, suggesting that it may act on a common bacterial target

BiodegChip : a DNA microarray to study the catabolic genes of aromatic "BTEX" pollutants in complex microbial communities

Different tools have been proposed to detect, characterize and quantify genes responsible for degradation of different pollutants in environmental samples such us PCR-SSCP DNA fingerprinting, PCR DNA clone libraries and microarrays. In the present work, we developed a custom array system termed BiodegChip to assess functional BTEX (Benzene, Toluene, Ethylbenzene, Xylene) catabolic gene diversity in contaminated environments. A custom CDS database was created including all catabolic genes encoding key activities in BTEX degradation pathways based on the Kyoto Encyclopedia of Genes and Genomes (KEGG) database, containing >15,000 genes in >30 functional groups (EC number). Resulting gene database were subjected to probe design (50 mer) using the software Oligoarrays 2.1 with a selection parameter of GC content between 35 and 50% and Tm range 80 and 88\ub0C. The verification of specificity of all probes was used using BLASTTN database. Printing and experimental procedures for application in environmental analyses are in progress

Inhibition of <i>M</i>. <i>marinum</i> sliding motility.

<p>A. Sliding motility of <i>M</i>. <i>marinum</i> was determined in the presence of compounds M39, M5 (0.3 μM each) or DMSO. Compounds were added in the center of each well containing 7H9 medium supplemented with 0.3% agarose, then mycobacteria were inoculated in the center and allowed to grow for 10 days at 30°C. The borders of the bacterial colony are indicated with black arrows. After 6 to 10 days, mycobacteria spread over the whole surface of the well. Spreading was inhibited by M39, but not by M5. Bar: 1 cm. B. Effect of each compound on sliding motility; (+) or (-) indicates if sliding motility occurred or not, respectively. For each compound, sliding motility could only be tested at concentrations where bacterial growth was not inhibited (NT: not tested).</p

Selected mycobacterial virulence inhibitors.

<p>Selected mycobacterial virulence inhibitors.</p