Mycobacterium and the coat of many lipids

Pathogenic Mycobacterium reside inside vacuoles in their host macrophages. These vacuoles fail to fuse with lysosomes yet interact with early endosomes. Glycoconjugates released by the intracellular bacilli traffic through the host cell and are released through exocytosis. These molecules represent both antigens for immune recognition and modulators of immune function. The molecules play key roles in the induction and maintenance of the granuloma, a tissue response that limits bacterial spread yet ensures persistence of the infection

Physico-chemical requirements and kinetics of membrane fusion of flavivirus-like particles.

Flaviviruses deliver their RNA genome into the host-cell cytoplasm by fusing their lipid envelope with a cellular membrane. Expression of the flavivirus pre-membrane and envelope glycoprotein genes in the absence of other viral genes results in the spontaneous assembly and secretion of virus-like particles (VLPs) with membrane fusion activity. Here, we examined the physico-chemical requirements for membrane fusion of VLPs from West Nile and Japanese encephalitis viruses. In a bulk fusion assay, optimal hemifusion (or lipid mixing) efficiencies were observed at 37 °C. Fusion efficiency increased with decreasing pH; half-maximal hemifusion was attained at pH 5.6. The anionic lipids bis(monoacylglycero)phosphate and phosphatidylinositol-3-phosphate, when present in the target membrane, significantly enhanced fusion efficiency, consistent with the emerging model that flaviviruses fuse with intermediate-to-late endosomal compartments, where these lipids are most abundant. In a single-particle fusion assay, VLPs catalysed membrane hemifusion, tracked as lipid mixing with the cellular membrane, on a timescale of 7-20 s after acidification. Lipid mixing kinetics suggest that hemifusion is a kinetically complex, multistep process.This work was supported by a Senior Research Fellowship from the Wellcome Trust, grant number 101908/Z/13/Z, to Y.M.; grant R01 GM102869 from the National Institutes of Health (NIH) to Y.M.; NIH grant NS079955 to E.R.; NIH grant T32 GM08283 to J.B.N.; and NIH grant T32 GM007223 to D.C.D. We thank Michel Ledizet, Martin Mattessich and Nathalie Bonafé (L2 Diagnostics, LLC) for helpful discussions.This is the author accepted manuscript. The final version is available from the Society for General Microbiology via http://dx.doi.org/10.1099/vir.0.00011

Conformational changes in Arp2/3 complex induced by ATP, WASp-VCA, and actin filaments

We used fluorescence spectroscopy and EM to determine how binding of ATP, nucleation-promoting factors, actin monomers, and actin filaments changes the conformation of Arp2/3 complex during the process that nucleates an actin filament branch. We mutated subunits of Schizosaccharomyces pombe Arp2/3 complex for labeling with fluorescent dyes at either the C termini of Arp2 and Arp3 or ArpC1 and ArpC3. We measured Förster resonance energy transfer (FRET) efficiency (ET_(eff)) between the dyes in the presence of the various ligands. We also computed class averages from electron micrographs of negatively stained specimens. ATP binding made small conformational changes of the nucleotide-binding cleft of the Arp2 subunit. WASp-VCA, WASp-CA, and WASp-actin-VCA changed the ET_(eff) between the dyes on the Arp2 and Arp3 subunits much more than between dyes on ArpC1 and ArpC3. Ensemble FRET detected an additional structural change that brought ArpC1 and ArpC3 closer together when Arp2/3 complex bound actin filaments. VCA binding to Arp2/3 complex causes a conformational change that favors binding to the side of an actin filament, which allows further changes required to nucleate a daughter filament

Structural Characterization of Phosphatidyl-myo-Inositol Mannosides from Mycobacterium bovis Bacillus Calmette Gúerin by Multiple-Stage Quadrupole Ion-Trap Mass Spectrometry with Electrospray Ionization. II. Monoacyl- and Diacyl-PIMs

The multiple-stage ion-trap mass spectrometric approaches towards to the structural characterization of the monoacyl-PIM (triacylated PIM) and the diacyl-PIM (tetracylated PIM), namely, the PIM (diacylated PIM) consisting of one or two additional fatty acid substituents attached to the glycoside, respectively, were described. While the assignment and confirmation of the fatty acid substituents on the glycerol backbone can be easily achieved by the methods described in the previous article, the identity of the glycoside moiety and its acylation state can be determined by the observation of a prominent acylglycoside ion arising from cleavage of the diacylglycerol moiety ([M − H − diacylglycerol]−) in the MS2-spectra of monoacyl-PIM and diacyl-PIM. The distinction of the fatty acid substituents on the 2-O-mannoside (i.e., R3CO2H) from that on the inositol (i.e., R4CO2H) is based on the findings that the MS3-spectrum of [M − H − diacylglycerol]− contains a prominent ion arising from further loss of the fatty acid at the 2-O-mannoside (i.e., the [M − H − diacylglycerol − R3CO2H]− ion), while the ion arising from loss of the fatty acid substituent at the inositol (i.e., the [M − H − diacylglycerol − R4CO2H]− ion) is of low abundance. The fatty acyl moiety on the inositol can also be identified by the product-ion spectrum from MS4 of the [M − H − diacylglycerol − R3CO2H]− ion, which gives rise to a prominent ion corresponding to loss of R4CO2H. An [M − H − acylmannose]− ion was also observed in the MS2-spectra and, thus, the identity of the fatty acid substituent attached to 2-O-mannoside can be confirmed. The combined information obtained from the multiple-stage product-ion spectra from MS2, MS3, and MS4 permit the assignment of the complex structures of monoacyl-PIMs and diacyl-PIMs in a mixture isolated from M. bovis Bacillus Calmette Guérin

Fibrinogen regulates the cytotoxicity of mycobacterial trehalose dimycolate, but is not required for cell recruitment, cytokine response, or control of mycobacterial infection

During inflammatory responses and wound healing, the conversion of soluble fibrinogen to fibrin, an insoluble extracellular matrix, long has been assumed to create a scaffold for the migration of leukocytes and fibroblasts. Previous studies concluded that fibrinogen is a necessary cofactor for mycobacterial trehalose 6,6-dimycolate-induced responses, because trehalose dimycolate-coated beads, to which fibrinogen was ad-sorbed, were more inflammatory than those to which other plasma proteins were adsorbed. Herein, we investigate roles for fibrin(ogen) in an in vivo model of mycobacterial granuloma formation and in infection with Mycobacterium tuberculosis, the causative agent of tuberculosis. In wild-type mice, the subcutaneous injection of trehalose dimycolate-coated polystyrene microspheres, suspended within Matrigel, elicited a pyogranulomatous response during the course of 12 days. In fibrinogen-deficient mice, neutrophils were recruited but a more suppurative lesion developed, with the marked degradation and disintegration of the matrix. Compared to that in wild-type mice, the early formation of granulation tissue in fibrinogen-deficient mice was edematous, hypocellular, and disorganized. These deficiencies were complemented by the addition of exogenous fibrinogen. The absence of fibrinogen had no effect on cell recruitment or cytokine production i

Conformational changes in Arp2/3 complex induced by ATP, WASp-VCA, and actin filaments

We used fluorescence spectroscopy and EM to determine how binding of ATP, nucleation-promoting factors, actin monomers, and actin filaments changes the conformation of Arp2/3 complex during the process that nucleates an actin filament branch. We mutated subunits of Schizosaccharomyces pombe Arp2/3 complex for labeling with fluorescent dyes at either the C termini of Arp2 and Arp3 or ArpC1 and ArpC3. We measured Förster resonance energy transfer (FRET) efficiency (ET_(eff)) between the dyes in the presence of the various ligands. We also computed class averages from electron micrographs of negatively stained specimens. ATP binding made small conformational changes of the nucleotide-binding cleft of the Arp2 subunit. WASp-VCA, WASp-CA, and WASp-actin-VCA changed the ET_(eff) between the dyes on the Arp2 and Arp3 subunits much more than between dyes on ArpC1 and ArpC3. Ensemble FRET detected an additional structural change that brought ArpC1 and ArpC3 closer together when Arp2/3 complex bound actin filaments. VCA binding to Arp2/3 complex causes a conformational change that favors binding to the side of an actin filament, which allows further changes required to nucleate a daughter filament

Spontaneous sharp bending of DNA: role of melting bubbles

The role of centrally located and distributed base pair mismatches (‘melting bubbles’) on localized bending and stiffness of short dsDNA fragments is evaluated using time-dependent fluorescence lifetime measurements. Distributed melting bubbles are found to induce larger bending angles and decreased levels of stiffness in DNA than centrally located ones of comparable overall size. Our results indicate that spontaneous local opening-up of the DNA duplex could facilitate sharp bending of short DNA strands even in the absence of DNA binding proteins. We also find that the occurrence of two closely spaced melting bubbles will generally be favored when a large energetic barrier must be overcome in forming the desired bent DNA structure