The N-Terminal GYPSY Motif Is Required for Pilin-Specific Sortase SrtC1 Functionality in Lactobacillus rhamnosus Strain GG

Predominantly identified in pathogenic Gram-positive bacteria, sortase-dependent pili are also found in commensal species, such as the probiotic-marketed strain Lactobacillus rhamnosus strain GG. Pili are typically associated with host colonization, immune signalling and biofilm formation. Comparative analysis of the N-terminal domains of pilin-specific sortases from various piliated Gram-positive bacteria identified a conserved motif, called GYPSY, within the signal sequence. We investigated the function and role of the GYPSY residues by directed mutagenesis in homologous (rod-shaped) and heterologous (coccoid-shaped) expression systems for pilus formation. Substitutions of some of the GYPSY residues, and more specifically the proline residue, were found to have a direct impact on the degree of piliation of Lb. rhamnosus GG. The present findings uncover a new signalling element involved in the functionality of pilin-specific sortases controlling the pilus biogenesis of Lb. rhamnosus GG and related piliated Gram-positive species.     

Aminothiazoles inhibit RANKL‐ and LPS‐mediated osteoclastogenesis and PGE2 production in RAW 264.7 cells

Periodontitis is characterized by chronic inflammation and osteoclast‐mediated bone loss regulated by the receptor activator of nuclear factor‐κB (RANK), RANK ligand (RANKL) and osteoprotegerin (OPG). The aim of this study was to investigate the effect of aminothiazoles targeting prostaglandin E synthase‐1 (mPGES‐1) on RANKL‐ and lipopolysaccharide (LPS)‐mediated osteoclastogenesis and prostaglandin E₂ (PGE₂) production in vitro using the osteoclast precursor RAW 264.7 cells. RAW 264.7 cells were treated with RANKL or LPS alone or in combination with the aminothiazoles 4‐([4‐(2‐naphthyl)‐1,3‐thiazol‐2‐yl]amino)phenol (TH‐848) or 4‐(3‐fluoro‐4‐methoxyphenyl)‐N‐(4‐phenoxyphenyl)‐1,3‐thiazol‐2‐amine (TH‐644). Aminothiazoles significantly decreased the number of multinucleated tartrate‐resistant acid phosphatase (TRAP)‐positive osteoclast‐like cells in cultures of RANKL‐ and LPS‐stimulated RAW 264.7 cells, as well as reduced the production of PGE₂ in culture supernatants. LPS‐treatment induced mPGES‐1 mRNA expression at 16 hrs and the subsequent PGE₂ production at 72 hrs. Conversely, RANKL did not affect PGE₂ secretion but markedly reduced mPGES‐1 at mRNA level. Furthermore, mRNA expression of TRAP and cathepsin K (CTSK) was reduced by aminothiazoles in RAW 264.7 cells activated by LPS, whereas RANK, OPG or tumour necrosis factor α mRNA expression was not significantly affected. In RANKL‐activated RAW 264.7 cells, TH‐848 and TH‐644 down‐regulated CTSK but not TRAP mRNA expression. Moreover, the inhibitory effect of aminothiazoles on PGE₂ production was also confirmed in LPS‐stimulated human peripheral blood mononuclear cell cultures. In conclusion, the aminothiazoles reduced both LPS‐ and RANKL‐mediated osteoclastogenesis and PGE₂ production in RAW 264.7 cells, suggesting these compounds as potential inhibitors for treatment of chronic inflammatory bone resorption, such as periodontitis.     

Aminothiazoles inhibit osteoclastogenesis and PGE2 production in LPS‐stimulated co‐cultures of periodontal ligament and RAW 264.7 cells,and RANKL‐mediated osteoclastogenesis and bone resorption in PBMCs

Inflammatory mediator prostaglandin E₂ (PGE₂) contributes to bone resorption in several inflammatory conditions including periodontitis. The terminal enzyme, microsomal prostaglandin E synthase‐1 (mPGES‐1) regulating PGE₂ synthesis is a promising therapeutic target to reduce inflammatory bone loss. The aim of this study was to investigate effects of mPGES‐1 inhibitors, aminothiazoles TH‐848 and TH‐644, on PGE₂ production and osteoclastogenesis in co‐cultures of periodontal ligament (PDL) and osteoclast progenitor cells RAW 264.7, stimulated by lipopolysaccharide (LPS), and bone resorption in RANKL‐mediated peripheral blood mononuclear cells (PBMCs). PDL and RAW 264.7 cells were cultured separately or co‐cultured and treated with LPS alone or in combination with aminothiazoles. Multinucleated cells stained positively for tartrate‐resistant acid phosphatase (TRAP) were scored as osteoclast‐like cells. Levels of PGE₂, osteoprotegerin (OPG) and interleukin‐6, as well as mRNA expression of mPGES‐1, OPG and RANKL were analysed in PDL cells. PBMCs were treated with RANKL alone or in combination with aminothiazoles. TRAP‐positive multinucleated cells were analysed and bone resorption was measured by the CTX‐I assay. Aminothiazoles reduced LPS‐stimulated osteoclast‐like cell formation both in co‐cultures and in RAW 264.7 cells. Additionally, aminothiazoles inhibited PGE₂ production in LPS‐stimulated cultures, but did not affect LPS‐induced mPGES‐1, OPG or RANKL mRNA expression in PDL cells. In PBMCs, inhibitors decreased both osteoclast differentiation and bone resorption. In conclusion, aminothiazoles reduced the formation of osteoclast‐like cells and decreased the production of PGE₂ in co‐cultures as well as single‐cell cultures. Furthermore, these compounds inhibited RANKL‐induced bone resorption and differentiation of PBMCs, suggesting these inhibitors for future treatment of inflammatory bone loss such as periodontitis.     

Virulence‐associated protein A from Rhodococcus equi is an intercompartmental pH‐neutralising virulence factor

Professional phagocytic cells such as macrophages are a central part of innate immune defence. They ingest microorganisms into membrane‐bound compartments (phagosomes), which acidify and eventually fuse with lysosomes, exposing their contents to a microbicidal environment. Gram‐positive Rhodococcus equi can cause pneumonia in young foals and in immunocompromised humans. The possession of a virulence plasmid allows them to subvert host defence mechanisms and to multiply in macrophages. Here, we show that the plasmid‐encoded and secreted virulence‐associated protein A (VapA) participates in exclusion of the proton‐pumping vacuolar‐ATPase complex from phagosomes and causes membrane permeabilisation, thus contributing to a pH‐neutral phagosome lumen. Using fluorescence and electron microscopy, we show that VapA is also transferred from phagosomes to lysosomes where it permeabilises the limiting membranes for small ions such as protons. This permeabilisation process is different from that of known membrane pore formers as revealed by experiments with artificial lipid bilayers. We demonstrate that, at 24 hr of infection, virulent R. equi is contained in a vacuole, which is enriched in lysosome material, yet possesses a pH of 7.2 whereas phagosomes containing a vapA deletion mutant have a pH of 5.8 and those with virulence plasmid‐less sister strains have a pH of 5.2. Experimentally neutralising the macrophage endocytic system allows avirulent R. equi to multiply. This observation is mirrored in the fact that virulent and avirulent R. equi multiply well in extracts of purified lysosomes at pH 7.2 but not at pH 5.1. Together these data indicate that the major function of VapA is to generate a pH‐neutral and hence growth‐promoting intracellular niche. VapA represents a new type of Gram‐positive virulence factor by trafficking from one subcellular compartment to another, affecting membrane permeability, excluding proton‐pumping ATPase, and consequently disarming host defences.     

Differences in variation of human immunodeficiency virus type 1 sequences from Henan and Shanghai regions of China

Illegally paid blood donation was a risk factor for HIV acquisition exclusively in Henan and Hubei Provinces of China, and not in Shanghai. Nucleotide sequences in the gag and env genes of HIV-1 were compared between isolates from Henan and Shanghai regions of China to test whether an expected higher degree of a common source of infections from this unique blood donation transmission risk would be evident as decreased variation among Henan isolates in an exploratory cross-sectional analysis. Among 38 isolates studied, 23 of 23 (100%) from Henan and 8 of 15 (54%) from Shanghai were subtype B. In addition, fewer sequence differences were found in gp41 of subtype B isolates from Henan than from Shanghai isolates. Further studies with additional controls are therefore warranted to confirm the role of the degree of a common source of infections in differences in HIV variation across populations. Fundation items: The Vanderbilt-Meharry Center for AIDS Research (P30 AI 54999); R.T.D (R01 AI 29193); Start Fund of Ministry of Education of China (for Hong-zhou LU, 2004BA719A10).     

Secondary structure and position of the cell-penetrating peptide transportan in SDS micelles as determined by NMR

Transportan is a 27-residue peptide (GWTLN SAGYL LGKIN LKALA ALAKK IL-amide) which has the ability to penetrate into living cells carrying a hydrophilic load. Transportan is a chimeric peptide constructed from the 12 N-terminal residues of galanin in the N-terminus with the 14-residue sequence of mastoparan in the C-terminus and a connecting lysine. Circular dichroism studies of transportan and mastoparan show that both peptides have close to random coil secondary structure in water. Sodium dodecyl sulfate (SDS) micelles induce 60% helix in transportan and 75% helix in mastoparan. The 600 MHz (1)H NMR studies of secondary structure in SDS micelles confirm the helix in mastoparan and show that in transportan the helix is localized to the mastoparan part. The less structured N-terminus of transportan has a secondary structure similar to that of the same sequence in galanin [Ohman, A., et al. (1998) Biochemistry 37, 9169-9178]. The position of mastoparan and transportan relative to the SDS micelle surface was studied by adding spin-labeled 5-doxyl- or 12-doxyl-stearic acid or Mn2+ to the peptide/micelle system. The combined results show that the peptides are for the most part buried in the SDS micelles. Only the C-terminal parts of both peptides and the central segment connecting the two parts of transportan are clearly surface exposed. For mastoparan, the secondary chemical shifts of the amide protons were found to vary periodically and display a pattern almost identical to those reported for mastoparan in phospholipid bicelles [Vold, R., et al. (1997) J. Biomol. NMR 9, 329-335], indicating similar structures and interactions in the two membrane-mimicking environments.