TolC promotes ExPEC biofilm formation and curli production in response to medium osmolarity.

While a high osmolarity medium activates Cpx signaling and causes CpxR to repress csgD expression, and efflux protein TolC protein plays an important role in biofilm formation in Escherichia coli, whether TolC also responds to an osmolarity change to regulate biofilm formation in extraintestinal pathogenic E. coli (ExPEC) remains unknown. In this study, we constructed ΔtolC mutant and complement ExPEC strains to investigate the role of TolC in the retention of biofilm formation and curli production capability under different osmotic conditions. The ΔtolC mutant showed significantly decreased biofilm formation and lost the ability to produce curli fimbriae compared to its parent ExPEC strain PPECC42 when cultured in M9 medium or 1/2 M9 medium of increased osmolarity with NaCl or sucrose at 28°C. However, biofilm formation and curli production levels were restored to wild-type levels in the ΔtolC mutant in 1/2 M9 medium. We propose for the first time that TolC protein is able to form biofilm even under high osmotic stress. Our findings reveal an interplay between the role of TolC in ExPEC biofilm formation and the osmolarity of the surrounding environment, thus providing guidance for the development of a treatment for ExPEC biofilm formation.

Screening of differentially expressed genes in the growth plate of broiler chickens with tibial dyschondroplasia by microarray analysis.

BACKGROUND: Tibial dyschondroplasia (TD) is a common skeletal disorder in broiler chickens. It is characterized by the presence of a non-vascularized and unmineralized cartilage in the growth plate. Previous studies have investigated differential expression of genes related to cartilage development during latter stages of TD. The aim of our study was to identify differentially expressed genes (DEGs) in the growth plate of broiler chickens, which were associated with early stage TD. We induced TD using tetramethylthiuram disulfide (thiram) for 1, 2, and 6 days and determined DEGs with chicken Affymetrix GeneChip assays. The identified DEGs were verified by quantitative polymerase chain reaction (qPCR) assays. RESULTS: We identified 1630 DEGs, with 82, 1385, and 429 exhibiting at least 2.0-fold changes (P < 0.05) at days 1, 2, and 6, respectively. These DEGs participate in a variety of biological processes, including cytokine production, oxidation reduction, and cell surface receptor linked signal transduction on day 1; lipid biosynthesis, regulation of growth, cell cycle, positive and negative gene regulation, transcription and transcription regulation, and anti-apoptosis on day 2; and regulation of cell proliferation, transcription, dephosphorylation, catabolism, proteolysis, and immune responses on day 6. The identified DEGs were associated with the following pathways: neuroactive ligand-receptor interaction on day 1; synthesis and degradation of ketone bodies, terpenoid backbone biosynthesis, ether lipid metabolism, JAK-STAT, GnRH signaling pathway, ubiquitin mediated proteolysis, TGF-ß signaling, focal adhesion, and Wnt signaling on day 2; and arachidonic acid metabolism, mitogen-activated protein kinase (MAPK) signaling, JAK-STAT, insulin signaling, and glycolysis on day 6. We validated seven DEGs by qPCR. CONCLUSIONS: Our findings demonstrate previously unrecognized changes in gene transcription associated with early stage TD. The DEGs we identified by microarray analysis will be used in future studies to clarify the molecular pathogenic mechanisms of TD. From these findings, potential pathways involved in early stage TD warrant further investigation.

[Genetic analysis of the nonstructural gene (NS1) of H9N2 avian influenza viruses isolated in China].

The nonstructural genes (NS1) of nine H9N2 subtype avian influenza viruses isolated from diseased chickens on different farms during 1998-2005 were amplified by RT-PCR and completely sequenced. The nucleotide and deduced amino acid sequences of NS1 genes of these isolates were compared. The results showed that NS1 genes of all H9N2 isolates contained 654 bp and encoded 217 amino acids. The homologies of the nucleotide and deduced amino sequences of the isolates were 95.4%-99.8% and 93.6%-100%, respectively. Comparison of the amino acid sequences of NS1 proteins of these isolates with other H9N2 viruses demonstrated that NS1 proteins of the nine strains had a deletion of 13 amino acid residues at the carboxyl terminus, which may be the molecule mark of the isolates in mainland China. Phylogenetic analysis revealed that the NS1 genes of these isolates fell into the same lineage and belonged to allele A. Eight out of nine isolates belonged to the CK/SH/F/98-like lineage while only Ck/HN/A3/98 strain belonged to the Ck/HK/Y280/97-like linease. All the isolates were derived from Ck/BJ/1/94 strain which was the first isolate is mainland China in 1994. The results indicated that H9N2 subtype AIV appeared differentiation following the passage of time and the viruses belonging to Ck/SH/F/98-like acquired an epidemic spread advantage in chicken population in mainland China.

[Study progress of NS1 protein of influenza A virus].

NS1 protein is the only non-structural protein of influenza A virus, which is encoded by the collinear mRNA transcribed from the viral RNA segment 8. It is a RNA-banding protein which has important posttranscriptional regulatory activity. It accumulates in the nucleus of the infected cell at early times but is also present in the cytoplasm later in the infection, Two functional domains have been identified in the NS1 protein: a RNA-binding domain at the N-terminus, and an effector domain at the C-terminus, which are associated with the inhibiting host celluar protein synthesis, inducing apoptosis and resisting the production of interferon-alpha/beta. In addition, NS1 protein could provide a means for the distinction between vaccinated and virus-infected poultry and act as a vaccine vector because it can tolerate large inserts of foreign sequences. Moreover, it might be an attractive target for antiviral drug design. All of these demonstrate that NS1 protein have the potential application benefits.

[Display of avian influenza virus nucleoprotein on Bacillus thuringiensis cell surface].

S-layer protein CTC surface display system of Bacillus thuringiensis (Bt) was used to test the possibility of displaying avian influenza virus nucleoprotein (NP) on Bt cell surface. Four recombinant plasmids were constructed by replacing 3'-terminal or central part below the surface anchor sequence slh of S-layer protein gene ctc with full length of np gene or part np gene (npp). The four resulting plasmids were pSNP (harboring fusion gene ctc-np), pCSA-SNP (harboring fusion gene csa-ctc-up, csa represents csaAB operon which is very important to the anchoring of S-layer protein on the bacterial cell surface), pCTC-NPP (harboring fusion gene ctc-npp) and pCSNPP (harboring fusion gene csa-ctc-npp). Five recombinant Bt strains were constructed by electro-transferring recombinant plasmids to Bt plasmid-free derivative strain BMB171. The resulting strains were BN (harboring pSNP), BCN (harboring pSNP as well as the plasmid pMIL-CSA which carried csaAB operon), C-S (harboring pCSA-SNP), BCCN (harboring pCTC-NPP and pMIL-CSA) and CN (harboring pCSNPP). The vegetative cells of five recombinant strains were used as agglutinogens of slide agglutination assay. Slide agglutination assay showed recombinant NP proteins were successfully displayed on the surface of five recombinant strains, respectively. After immunizing mice with vegetative cells of five recombinant strains respectively, five recombinant strains all elicited humoral respones to NP and exhibited immunogenicity as assayed by enzyme-linked immunosorbent assay (ELISA). Meanwhile these assays showed recombinant strain CN (harboring fusion gene csa-ctc-npp) exhibited the highest immunogenicity among five recombinant strains. That means the best way of constructing S-layer fusion gene is csa-ctc-* (* denotes heterologous antigen gene) which means the central part of S-layer protein gene ctc replaced by the heterologous antigen gene and csaAB operon located on the upstream of fusion gene. The strategy developed in this study gives a possibility to generate heat stable,oral, veterinary vaccine with Bt S-layer protein CTC surface display system.