General N-and O-linked glycosylation of lipoproteins in mycoplasmas and role of exogenous oligosaccharide

The lack of a cell wall, flagella, fimbria, and other extracellular appendages and the possession of only a single membrane render the mycoplasmas structurally simplistic and ideal model organisms for the study of glycoconjugates. Most species have genomes of about 800 kb and code for few proteins predicted to have a role in glycobiology. The murine pathogens Mycoplasma arthritidis and Mycoplasma pulmonis have only a single gene annotated as coding for a glycosyltransferase but synthesize glycolipid, polysaccharide and glycoproteins. Previously, it was shown that M. arthritidis glycosylated surface lipoproteins through O-linkage. In the current study, O-linked glycoproteins were similarly found in M. pulmonis and both species of mycoplasma were found to also possess N-linked glycans at residues of asparagine and glutamine. Protein glycosylation occurred at numerous sites on surface-exposed lipoproteins with no apparent amino acid sequence specificity. The lipoproteins of Mycoplasma pneumoniae also are glycosylated. Glycosylation was dependent on the glycosidic linkages from host oligosaccharides. As far as we are aware, N-linked glycoproteins have not been previously described in Gram-positive bacteria, the organisms to which the mycoplasmas are phylogenetically related. The findings indicate that the mycoplasma cell surface is heavily glycosylated with implications for the modulation of mycoplasma-host interactions

Mycobacterium tuberculosis WhiB3 Maintains Redox Homeostasis by Regulating Virulence Lipid Anabolism to Modulate Macrophage Response

The metabolic events associated with maintaining redox homeostasis in Mycobacterium tuberculosis (Mtb) during infection are poorly understood. Here, we discovered a novel redox switching mechanism by which Mtb WhiB3 under defined oxidizing and reducing conditions differentially modulates the assimilation of propionate into the complex virulence polyketides polyacyltrehaloses (PAT), sulfolipids (SL-1), phthiocerol dimycocerosates (PDIM), and the storage lipid triacylglycerol (TAG) that is under control of the DosR/S/T dormancy system. We developed an in vivo radio-labeling technique and demonstrated for the first time the lipid profile changes of Mtb residing in macrophages, and identified WhiB3 as a physiological regulator of virulence lipid anabolism. Importantly, MtbΔwhiB3 shows enhanced growth on medium containing toxic levels of propionate, thereby implicating WhiB3 in detoxifying excess propionate. Strikingly, the accumulation of reducing equivalents in MtbΔwhiB3 isolated from macrophages suggests that WhiB3 maintains intracellular redox homeostasis upon infection, and that intrabacterial lipid anabolism functions as a reductant sink. MtbΔwhiB3 infected macrophages produce higher levels of pro- and anti-inflammatory cytokines, indicating that WhiB3-mediated regulation of lipids is required for controlling the innate immune response. Lastly, WhiB3 binds to pks2 and pks3 promoter DNA independent of the presence or redox state of its [4Fe-4S] cluster. Interestingly, reduction of the apo-WhiB3 Cys thiols abolished DNA binding, whereas oxidation stimulated DNA binding. These results confirmed that WhiB3 DNA binding is reversibly regulated by a thiol-disulfide redox switch. These results introduce a new paradigmatic mechanism that describes how WhiB3 facilitates metabolic switching to fatty acids by regulating Mtb lipid anabolism in response to oxido-reductive stress associated with infection, for maintaining redox balance. The link between the WhiB3 virulence pathway and DosR/S/T signaling pathway conceptually advances our understanding of the metabolic adaptation and redox-based signaling events exploited by Mtb to maintain long-term persistence

Tissue Localization and Solubilities of αA-crystallin and its Numerous C-terminal Truncation Products in Pre- and Postcataractous ICR/f Rat Lenses

The tissue localization of various αA-crystallin truncation products was discovered through the use of a new mass spectrometric technique, which revealed an interesting distribution pattern in a rat model of cataracts

LC MS/MS-CID showing hexosylation at Thr64 of the peptide Gt₆₄KDFLPIELQSLEVSK of MYPU_3230.

<p>The assigned b and y ions are shown in blue and red, respectively. Glycosylation of Q and S glycosites is absent in this spectrum as illustrated. The PEAKS peptide score (-10lgP) for this spectrum was 87. The charge state of the parental ion was <i>z</i> = 3.</p

LC MS/MS-CID showing hexosylation at Asn335 of the peptide STLEYTINNSQELQn₃₃₅ILKQTYEEFTK of MYPU_3200.

<p>The assigned b and y ions are shown in blue and red, respectively. Glycosylation of N, Q, T, S, and Y glycosites is absent in this spectrum as illustrated. The PEAKS peptide score (-10lgP) for this spectrum was 60. The charge state of the parental ion was <i>z</i> = 3.</p

Hexosylation of the peptide GTKDFLPIELQSLEVSK of MYPU_3230.

<p>Orbitrap MS showing the doubly and triply charged ions. The 81.0262 shift for <i>z</i> = 2 between the non-glycosylated and glycosylated peptides equates to a mass shift of 162.0524 Da, which corresponds to the addition of hexose (162.0528 Da) with a mass accuracy of 0.0004 Da. The 54.0169 shift for <i>z</i> = 3 between non-glycosylated and glycosylated forms equates to a mass shift of 162.0507 Da, which corresponds to hexosylation with a mass accuracy of 0.0021 Da. Monoisotopic values for the calculated theoretical and experimental masses of the peptide are given in bold. The images presented were obtained from an LC peak of MS scans and are expanded to show the charge states of each form.</p

Hexosylation of the peptide STLEYTINNSQELQNILKQTYEEFTK of MYPU_3200.

<p>Orbitrap MS showing the doubly and triply charged ions. The monoisotopic mass of the doubly charged species at 1648.8077 is consistent with the hexosylated peptide at <i>z</i> = 2 with a mass accuracy of 0.0012 Da. The 54.0175 shift for <i>z</i> = 3 between non-glycosylated and hexose forms equates to a mass shift of 162.0525 Da with a mass accuracy of 0.0003 Da. Monoisotopic values for the calculated theoretical and experimental masses of the peptide are given in bold. The images presented were obtained from an LC peak of MS scans and are expanded to show the charge states of each form.</p

Glycosites identified in this study.

<p>^a Accession numbers for MYPU_3200, MYPU_3230, MYPU_3460, and MARTH_403 are CAC13493, CAC13496, CAC13519, and YP_001999972, respectively.</p><p>Glycosites identified in this study.</p