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

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

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

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

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

Secondary and tertiary structures of C9W.

<p>Far-UV CD (A) and intrinsic Trp fluorescence (B) spectra of C9W in the presence of indicated concentrations of TX-100. One set of spectra are presented here.</p

Autoradiogram of DNase I footprinting assay.

<p>The bottom strand labelled (with ³²P) hla DNA was incubated with (+)/without (-) the saturating amount of rSarA followed by the digestion of the DNA with DNase I. The resulting DNA fragments, along with the G and A+G markers (made from the same labelled hla DNA by a standard method) were separated by a urea-8% PAGE. Sequence of the hla DNA protected by rSarA is shown at the right side of autoradiogram.</p