Role of Vertical Transmission of Shoot Endophytes in Root-Associated Microbiome Assembly and Heavy Metal Hyperaccumulation in Sedum alfredii.

The transmission mode of shoot-associated endophytes in hyperaccumulators and their roles in root microbiome assembly and heavy metal accumulation remain unclear. Using 16S rRNA gene profiling, we investigated the vertical transmission of shoot-associated endophytes in relation to growth and Cd/Zn accumulation of Sedum alfredii ( Crassulaceae). Endophytes were transmitted from shoot cuttings to the rhizocompartment of new plants in both sterilized (γ-irradiated) and native soils. Vertical transmission was far more efficient in the sterile soil, and the transmitted endophytes have become a dominant component of the newly established root-associated microbiome. Based on 16S rRNA genes, the vertically transmitted taxa were identified as the families of Streptomycetaceae, Nocardioidaceae, Pseudonocardiaceae, and Rhizobiaceae. Abundances of Streptomycetaceae, Nocardioidaceae, and Pseudonocardiaceae were strongly correlated with increased shoot biomass and total Cd/Zn accumulation. Inoculation of S. alfredii with the synthetic bacterial community sharing the same phylogenetic relatedness with the vertically transmitted endophytes resulted in significant improvements in plant biomass, root morphology, and Cd/Zn accumulation. Our results demonstrate that successful vertical transmission of endophytes from shoots of S. alfredii to its rhizocompartments is possible, particularly in soils with attenuated microbiomes. Furthermore, the endophyte-derived microbiome plays an important role in metal hyperaccumulation.

Three conserved MyD88-recruiting TLR residues exert different effects on the human TLR4 signaling pathway.

Stimulation of Toll-like receptor (TLR) 4 leads to the activation of both MyD88-dependent and MyD88-independent pathways through the recruitment of adaptors TIRAP/MyD88 and TRIF/TRAM, respectively. However, the molecular basis of the TLR4 Toll/interleukin-1 receptor (TIR) domain in recruiting these downstream adaptors is still not entirely clear. Here, we identify three amino acid residues (714P in the BB loop, 696L in the αA helix and 721N in the αB sheet) conserved in all MyD88-recruited TLRs, but not the TLR3 TIR domain, as being critical for TLR4 responsiveness to LPS. These results were based on the substitution of each residue with a residue of the opposite type (hydrophilic/hydrophobic). However, the responsiveness of the TLR4 mutants to LPS was only partially decreased when each residue was replaced with a residue having the same hydrophilicity/hydrophobicity. This result is likely associated with an alteration in the BB-loop conformation of each TLR4 mutant and its ability to recruit the downstream adaptor TRAM. Thus, we identified three amino acids essential for TLR4 signaling, and their replacement with a residue of the same or opposite hydrophilicity/hydrophobicity greatly affected TLR4 signaling. This study furthers our understanding of the molecular mechanism by which the TLR4 TIR domain modulates TLR4 signaling and also provides new insight for the design of antisepsis therapy.

Looped host defense peptide CLP-19 binds to microtubules and inhibits surface expression of TLR4 on mouse macrophages.

The looped host defense peptide CLP-19 is derived from a highly functional core region of the Limulus anti-LPS factor and exerts robust anti-LPS activity by directly interacting with LPS in the extracellular space. We previously showed that prophylactic administration of CLP-19 even 20 h prior to LPS challenge might significantly increase the survival rate in a lethal endotoxin shock mouse model. Such an effect may be associated with immune regulation of CLP-19. To investigate the underlying mechanisms, peptide affinity chromatography, immunofluorescence, and Western blotting procedures were used to identify α- and ß-tubulin as direct and specific binding partners of CLP-19 in the mouse macrophage cell line RAW 264.7. Bioinformatic analysis using the AutoDock Vina molecular docking and PyMOL molecular graphics system predicted that CLP-19 would bind to the functional residues of both α- and ß-tubulin and would be located within the groove of microtubules. Tubulin polymerization assay revealed that CLP-19 might induce polymerization of microtubules and prevent depolymerization. The immunoregulatory effect of CLP-19 involving microtubules was investigated by flow cytometry, immunofluorescence, and Western blotting, which showed that CLP-19 prophylactic treatment of RAW 264.7 cells significantly inhibited LPS-induced surface expression of TLR4. Taken together, these results suggest that CLP-19 binding to microtubules disrupts the dynamic equilibrium of microtubules, reducing the efficacy of microtubule-dependent vesicular transport that would otherwise translocate TLR4 from the endoplasmic reticulum to the cell surface.

Identification of a S100 calcium-binding protein expressed in HL-60 cells treated with all-trans retinoic acid by two-dimensional electrophoresis and mass spectrometry.

To characterize the alteration of protein expression during tumor cell differentiation induced by all-trans retinoic acid (ATRA) and to understand downstream signaling and molecular mechanism of ATRA action, we compared the protein expression profiles in HL-60 cells with ATRA treatment using two-dimensional electrophoresis (2-DE). Although many changes in protein expression were found in 2-DE maps, here we identified two protein spots remarkably expressed in the differentiated cells by nanoelectrospray ionization mass spectrometry and database searching. These two protein spots were found to be the same protein, namely S100 calcium-binding protein A9 (S100A9). Further study will be done to ascertain whether S100A9 plays a role in the regulation of differentiation or just a consequence of differentiation.