Ningsheng Shao

Hfq is a global regulator that controls the pathogenicity of Staphylococcus aureus.

The Hfq protein is reported to be an RNA chaperone, which is involved in the stress response and the virulence of several pathogens. In E. coli, Hfq can mediate the interaction between some sRNAs and their target mRNAs. But it is controversial whether Hfq plays an important role in S. aureus. In this study, we found that the deletion of hfq gene in S. aureus 8325-4 can increase the surface carotenoid pigments. The hfq mutant was more resistant to oxidative stress but the pathogenicity of the mutant was reduced. We reveal that the Hfq protein can be detected only in some S. aureus strains. Using microarray and qRT-PCR, we identified 116 genes in the hfq mutant which had differential expression from the wild type, most of which are related to the phenotype and virulence of S. aureus. Among the 116 genes, 49 mRNAs can specifically bind Hfq protein, which indicates that Hfq also acts as an RNA binding protein in S. aureus. Our data suggest that Hfq protein of S. aureus is a multifunctional regulator involved in stress and virulence.

Intronic miR-301 feedback regulates its host gene, ska2, in A549 cells by targeting MEOX2 to affect ERK/CREB pathways

miR-301 is localized in the first intron of ska2, whose function has not been clarified. Here, a new circuit model in which intronic miR-301 regulates the transcription and function of its host gene through a feedback mechanism has been described. Our results showed that blocking of miR-301 in A549 cells leads to a decrease in the expression of the host gene, ska2. Further analysis showed that miR-301 targets MEOX2 to affect the ERK/CREB pathway. CREB directly regulates the expression of the host gene, ska2. In addition, the inhibition of miR-301 or ska2 resulted in an increase of the mitotic index and a decrease in colony formation in soft agar, which may be related to lung tumorigenesis.

MicroRNA-200b regulates cyclin D1 expression and promotes S-phase entry by targeting RND3 in HeLa cells

MicroRNAs (miRNAs) are endogenous non-coding small RNAs that inhibit gene expression post-transcriptionally. By regulating their target genes, miRNAs play important roles in tumor generation and development. Recently, the mir-200 family was revealed to inhibit the epithelial-mesenchymal transition, which is viewed as an essential step in early tumor metastasis. Here, we used luciferase assays to demonstrate that mir-200b interacts with predicted target sites in the 3′ untranslated region of RND3. In HeLa cells, mir-200b directly reduced the expression of RND3 at the mRNA and protein levels, which thereby promoted expression of the downstream protein cyclin D1 and increased S-phase entry. In conclusion, our study demonstrates a novel role for mir-200b in cell cycle progression and identifies RND3 as a novel mir-200b target.

Inhibition of Staphylococcus aureus pathogenesis in vitro and in vivo by RAP-binding peptides

Staphylococcus aureus cause many diseases by producing toxins, whose synthesis is regulated by quorum-sensing mechanisms. S. aureus secretes a protein termed RNAIII activating protein (RAP) which autoinduces toxin production via the phosphorylation of is target protein TRAP. Mice vaccinated with RAP were protected from S. aureus infection, suggesting that RAP is an useful target for selecting potential therapeutic molecules to inhibit S. aureus pathogenesis. We show here that RAP (native and recombinant) was used to select RAP-binding peptides (RBPs) from a random 12-mer phage-displayed peptide library. Two RBPs were shown to inhibit RNAIII production in vitro (used a marker for pathogenesis). The peptide WPFAHWPWQYPR, which had the strongest inhibitory activity, was chemically synthesized and also expressed in Escherichia coli as a GST-fusion. Both synthetic peptide and GST-fusion peptide decreased RNAIII levels in a dose-dependent manner. The GST-fusion peptide was also shown to protect mice from a S. aureus infection in vivo (tested in a murine cutaneous S. aureus infection model). Our results suggest the potential use of RAP-binding proteins in treating clinical S. aureus infections.

Selection of Novel Nickel‐binding Peptides from Flagella Displayed Secondary Peptide Library

Nickel (Ni) performs its biological or toxic functions in nickel–protein coordination form. Novel Ni‐binding peptides were isolated from a random dodecapeptide library displayed on the flagella of Escherichia coli against immobilized ions. On the basis of isolated sequences rich in histidine residues, two secondary libraries were constructed respectively. By consequent selection, more Ni‐chelating peptides were identified and the consensus motif RHXHR (where X was always H) was deduced. The result suggested that not only histidine, but also arginine, play an important role in Ni‐binding. Furthermore, two selected clones (1035 and 2022) were chosen for further identification. They exhibited similar relative binding affinity, which was about nine times that of the original library derived clones and statistically much more significant than the positive control with polyhistidine insert. Free nickel ions could almost completely inhibit the binding of the clones 1035 and 2022 to immobilized nickel, implicating that the peptides were able to chelate nickel ions. These studies reveal that bacterial surface displayed peptide libraries may have promising future potential for the development of metal bioadsorbents. Furthermore, novel Ni‐binding peptides may provide lead molecules for Ni‐chelation and applications thereof.

SsrA (tmRNA) acts as an antisense RNA to regulate Staphylococcus aureus pigment synthesis by base pairing with crtMN mRNA

SsrA RNA (small stable RNA A), also known as tmRNA and 10Sa RNA, functions both as tRNA and mRNA through its unique structure. The carotenoid pigment is the eponymous feature of human pathogen Staphylococcus aureus. Here, we found that the pigment of the mutant strain with ssrA deletion was increased. Furthermore, it was demonstrated that ssrA could act as an antisense RNA aside from its well‐known biological function, and crtMN, encoding two essential enzymes for the pigment synthesis, was identified as target of ssrA. Further investigation showed ssrA could specifically base pair with the RBS (ribosomal binding site) region of the crtMN mRNA. Our results revealed a new mechanism by which ssrA regulated the biosynthesis of pigment in S. aureus.

MiR‐152 reduces human umbilical vein endothelial cell proliferation and migration by targeting ADAM17

As a cleavage enzyme of precursor TNF‐α, the high expression level of ADAM17 in endothelial cells is an important factor in atherosclerosis. In this study, we demonstrate that ADAM17 is the target of miR‐152. We found that miR‐152 could reduce TNF precursor cleavage and inhibit cell proliferation and migration by targeting ADAM17 in human umbilical vein endothelial cells (HUVECs). Furthermore, the expression pattern of miR‐152 and corresponding target ADAM17 was opposite in HUVECs under hypoxic conditions. The levels of circulating miR‐152 in AS patient sera were lower than those detected in the sera of normal individuals. Our results indicate that miR‐152 may be involved in the development of human atherosclerosis and could be used as diagnostic biomarker or therapeutic target in atherosclerosis.

Inhibiting platelets aggregation could aggravate the acute infection caused by Staphylococcus aureus

Several fibrinogen binding proteins (Fibs) play important roles in the pathogenesis of Staphylococcus aureus (S. aureus). Most Fibs can promote the aggregation of platelets during infection, but the extracellular fibrinogen-binding protein (Efb) is an exception. It is reported that Efb can specifically bind fibrinogen and inhibit the aggregation of platelet with its N terminal. However, the biological significance of platelet aggregation inhibition in the infection caused by S. aureus is unclear until now. Here, we demonstrated that the persistence and aggregation of platelets were important for killing S. aureus in whole blood. It was found that the N terminal of Efb (EfbN) and platelets inhibitors could increase the survival of S. aureus in whole blood. The study in vivo also showed that EfbN and platelets inhibitors could reduce the killing of S. aureus and increase the lethality rate of S. aureus in the acute infection mouse model.

Exosomal transfer of vasorin expressed in hepatocellular carcinoma cells promotes migration of human umbilical vein endothelial cells.

Vasorin (VASN) is a type I transmembrane protein that plays important roles in tumor development and vasculogenesis. In this paper, we showed that VASN could be a key mediator of communication between tumor cells and endothelial cells. We confirmed for the first time that HepG2-derived VASN can be transferred to human umbilical vein endothelial cells (HUVECs) via receptor mediated endocytosis of exosomes, at least in part through HSPGs. The HepG2-derived VASN containing exosomes promote migration of recipient HUVECs cells. Our results identify a novel pathway by which a functional protein expressed in tumor cells affects the biological fate of endothelial cells via exosomes.

The Production of Extracellular Proteins Is Regulated by Ribonuclease III via Two Different Pathways in Staphylococcus aureus

Staphylococcus aureus ribonuclease III belongs to the enzyme family known to degrade double-stranded RNAs. It has previously been reported that RNase III cannot influence cell growth but regulates virulence gene expression in S. aureus. Here we constructed an RNase III inactivation mutant (Δrnc) from S. aureus 8325-4. It was found that the extracellular proteins of Δrnc were decreased. Furthermore, we explored how RNase III regulated the production of the extracellular proteins in S. aureus. We found during the lag phase of the bacterial growth cycle RNase III could influence the extracellular protein secretion via regulating the expression of secY2, one component of accessory secretory (sec) pathway. After S. aureus cells grew to exponential phase, RNase III can regulate the expression of extracellular proteins by affecting the level of RNAIII. Further investigation showed that the mRNA stability of secY2 and RNAIII was affected by RNase III. Our results suggest that RNase III could regulate the pathogenicity of S. aureus by influencing the level of extracellular proteins via two different ways respectively at different growth phases.