Sugar Vinyl Sulfoxide Glycoconjugation of Peptides and Lysozynne: Abrogation of Proteolysis at the Lysine Sites

We describe a glycoconjugation strategy in which a sugar vinyl sulfoxide, acting as Michael donor, reacts efficiently with amine nucleophiles arising from the lysine side chain in peptides and proteins, at physiological pH and temperature. The method permits glycoconjugation of the lysine residues present in lysozyme with the sugar vinyl sulfoxide. The glycoconjugation of the protein abrogates the trypsin-mediated proteolysis at the lysine sites. The modified protein catalyzes digestion of the Gram-negative Escherichia coli cell wall and retains the same antimicrobial property as the native lysozyme

A staphylococcal anti-sigma factor possesses a single-domain, carries different denaturant-sensitive regions and unfolds via two intermediates.

RsbW, an anti-sigma factor possessing kinase activity, is expressed by many Gram-positive bacteria including Staphylococcus aureus. To obtain clues about the domain structure and the folding-unfolding mechanism of RsbW, we have elaborately studied rRsbW, a recombinant S. aureus RsbW. Sequence analysis of the protein fragments, generated by the limited proteolysis of rRsbW, has proposed it to be a single-domain protein. The unfolding of rRsbW in the presence of GdnCl or urea was completely reversible in nature and occurred through the formation of at least two intermediates. The structure, shape, and the surface hydrophobicity of no intermediate completely matches with those of other intermediates or the native rRsbW. Interestingly, one of the intermediates, formed in the presence of less GdnCl concentrations, has a molten globule-like structure. Conversely, all of the intermediates, like native rRsbW, exist as dimers in aqueous solution. The putative molten globule and the urea-generated intermediates also have retained some kinase activity. Additionally, the putative ATP binding site/catalytic site of rRsbW shows higher denaturant sensitivity than the tentative dimerization region of this enzyme

Mannopyranoside Glycolipids Inhibit Mycobacterial and Biofilm Growth and Potentiate Isoniazid Inhibition Activities in M. smegmatis

Lipomannan and lipoarabinomannan are integral components of the mycobacterial cell wall. Earlier studies demonstrated that synthetic arabinan and arabinomannan glycolipids acted as inhibitors of mycobacterial growth, in addition to exhibiting inhibitory activities of mycobacterial biofilm. Herein, it is demonstrated that synthetic mannan glycolipids are better inhibitors of mycobacterial growth, whereas lipoarabinomannan has a higher inhibition efficiency to biofilm. Syntheses of mannan glycolipids with a graded number of mannan moieties and an arabinomannan glycolipid are conducted by chemical methods and subsequent mycobacterial growth and biofilm inhibition studies are conducted on Mycobacterium smegmatis. Growth inhibition of (73 +/- 3) % is observed with a mannose trisaccharide containing a glycolipid, whereas this glycolipid did not promote biofilm inhibition activity better than that of arabinomannan glycolipid. The antibiotic supplementation activities of glycolipids on growth and biofilm inhibitions are evaluated. Increases in growth and biofilm inhibitions are observed if the antibiotic is supplemented with glycolipids, which leads to a significant reduction of inhibition concentrations of the antibiotic

Urea-induced unfolding of rRsbW.

<p>(A) The ellipticity values of rRsbW at 222 nm (θ₂₂₂), were derived from their far-UV CD spectra (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0195416#pone.0195416.s001" target="_blank">S1 Fig</a>), normalized (Nor), and plotted versus the corresponding urea concentrations. The ANS fluorescence intensity values at 480 nm (B) and the intrinsic Tyr fluorescence intensity values at 308 nm (C) were obtained from the corresponding spectra of rRsbW (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0195416#pone.0195416.s001" target="_blank">S1 Fig</a>) were plotted similarly.</p

Characterization of intermediates made by urea.

<p>(A) Analyses of proteolytic fragments of rRsbW by SDS-13.5% PAGE. Protein aliquots, pre-equilibrated with 0–7 M urea, were digested with (+)/without (-) trypsin for 8 min prior to gel analysis. Arrowheads indicate either new protein fragments or the fragments with a comparatively increased intensity at 1–4 M urea. (B) Near-UV CD spectra of equimolar concentrations of rRsbW at the indicated concentrations of urea. (C) Crosslinking of 0–7 M urea-treated rRsbW with (+)/without (-) glutaraldehyde (GCHO). The crosslinked molecules were separated by SDS-13.5% PAGE. ‘D’ indicates dimeric rRsbW. (D) The dimer-specific band intensity values were estimated from panel C and plotted versus the corresponding urea concentrations. (E) Gel filtration chromatography of rRsbW at 0–5 M urea. (F) Kinase assay. The assay was performed using the 0–7 M urea-exposed rRsbW, rRsbV, and 1 mM ATP for 10 min at room temperature. The reaction mixtures were analyzed by a 12% native PAGE. The protein bands corresponding to rRsbW, rRsbW-rRsbV complex, phosphorylated rRsbV, and non-phosphorylated rRsbV are indicated. The phosphorylated rRsbV bands were scanned to determine their intensity values at 0–7 M urea. Considering that the extent of rRsbV phosphorylation at 0 M urea corresponds to 100% kinase activity, kinase activities of rRsbW at 1–7 M urea were determined. After normalization, the kinase activity values were plotted against the corresponding urea concentrations (G).</p

Size and shape of rSarA in the presence/absence of GdnCl.

<p>(A) Analysis of the chemically cross-linked rSarA molecules by SDS-13.5% PAGE. Samples containing rSarA were pre-equilibrated with 0–5 M GdnCl before treating them with glutaraldehyde (GCHO). D and M indicate dimer- and monomer-specific rSarA. (B) Gel filtration chromatography of rSarA at the indicated concentrations of GdnCl. (C) DLS study of rSarA at the indicated concentrations of GdnCl.</p

Characterization of intermediates produced by GdnCl.

<p>(A) Near-UV CD spectra of rRsbW at 0–4 M GdnCl. (B) SDS-13.5% PAGE analysis of 0–3.5 M GdnCl-exposed rRsbW molecules crosslinked with (+)/without (-) glutaraldehyde (GCHO). (C) The plot of dimer-specific band intensity versus the matching GdnCl concentrations. The dimer-specific band intensity values were estimated from panel B as demonstrated above. (D) Gel filtration chromatography of rRsbW at 0–3 M GdnCl. (E) Kinase assay. The assay was carried out using a similar method as stated in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0195416#pone.0195416.g004" target="_blank">Fig 4F</a>. Only urea-treated rRsbW was replaced with the indicated GdnCl-treated rRsbW. (F) The plot of kinase activity versus equivalent GdnCl concentrations. The plot was made using the intensity of phosphorylated rRsbV bands as stated in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0195416#pone.0195416.g004" target="_blank">Fig 4F</a>.</p

A Surfactant-Induced Functional Modulation of a Global Virulence Regulator from <i>Staphylococcus aureus</i>

<div><p>Triton X-100 (TX-100), a useful non-ionic surfactant, reduced the methicillin resistance in <i>Staphylococcus</i> aureus significantly. Many <i>S</i>. <i>aureus</i> proteins were expressed in the presence of TX-100. SarA, one of the TX-100-induced proteins, acts as a global virulence regulator in <i>S</i>. <i>aureus</i>. To understand the effects of TX-100 on the structure, and function of SarA, a recombinant <i>S</i>. <i>aureus</i> SarA (rSarA) and its derivative (C9W) have been investigated in the presence of varying concentrations of this surfactant using various probes. Our data have revealed that both rSarA and C9W bind to the cognate DNA with nearly similar affinity in the absence of TX-100. Interestingly, their DNA binding activities have been significantly increased in the presence of pre-micellar concentration of TX-100. The increase of TX-100 concentrations to micellar or post-micellar concentration did not greatly enhance their activities further. TX-100 molecules have altered the secondary and tertiary structures of both proteins to some extents. Size of the rSarA-TX-100 complex appears to be intermediate to those of rSarA and TX-100. Additional analyses show a relatively moderate interaction between C9W and TX-100. Binding of TX-100 to C9W has, however, occurred by a cooperative pathway particularly at micellar and higher concentrations of this surfactant. Taken together, TX-100-induced structural alteration of rSarA and C9W might be responsible for their increased DNA binding activity. As TX-100 has stabilized the somewhat weaker SarA-DNA complex effectively, it could be used to study its structure in the future.</p></div

A schematic representation of the urea- and GdnCl-induced unfolding of rRsbW.

<p>Intermediates are indicated by I1 to I4.</p

Limited proteolysis of rSarA.

<p>(A) Amino acid sequence of SarA with the cleavage sites of proteinase K (Pk), chymotrypsin (Ch), and endoproteinase AspN (AspN). The cut sites of each enzyme in the SarA sequence were identified using the ‘PeptideCutter’ program in ExPasy server. Analyses of the proteinase K (B)-, chymotrypsin (C)-, and endoproteinase AspN (D)-cleaved rSarA fragments by SDS-13.5% PAGE. I–VIII indicates the major proteolytic fragments of rSarA. The rSarA-specific bands are denoted by an arrowhead. Western blotting analyses of the Pk (E)-, Ch (F)-, and AspN (G)-digested rSarA fragments using an anti-His antibody. The molecular masses (in kDa) of marker proteins are presented at the right side of the pictures.</p