Mapping Causal Variants with Single-Nucleotide Resolution Reveals Biochemical Drivers of Phenotypic Change

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金黄色葡萄球菌CHIPS基因克隆及原核表达

趋化抑制蛋白(Chemotaxis Inhibitory Protein ofStaphylococcus aureus,CHIPS)是由金黄色葡萄球菌分泌到菌体外的一种蛋白。在感染早期,它能特异性地与中性粒细胞和单核细胞上的C5a受体(C5aR)和fMLP受体(FPR)结合,从而阻止中性粒细胞和单核细胞对C5a和fMLP的结合作用,导致对病原吞噬作用的延迟。人们可以利用CHIPS对C5a-C5aR的阻止作用来研制治疗由C5a诱发的炎症性疾病的药物。将CHIPS作为免疫原防治疾病也将成为新的研究课题。实验成功构建了CHIPS蛋白的原核表达系统,为CHIPS免疫原性研究及蛋白的其他功能研究奠定了基础。     

金黄色葡萄球菌甲氧西林耐药辅助基因femB的克隆和原核表达

金黄色葡萄球菌femB基因与甲氧西林高水平耐药密切相关,可能成为开发抗MRSA药物的新靶位.以金葡菌基因组DNA为模板,PCR扩增femB全长基因,所得片段与pGM-T载体连接并转化感受态大肠杆菌DH5α,阳性克隆以PCR、双酶切及测序鉴定.将鉴定正确的目的片段定向克隆到pGEX-4T-1表达载体中,转化至大肠杆菌BL21后经IPTG诱导表达GST/FemB融合蛋白;采用SDS-PAGE及Western blot对融合蛋白进行鉴定.结果显示,重组质粒在宿主菌中获得了高效表达,融合蛋白相对分子质量为75 kD,该融合蛋白可与抗GST-tag抗体特异结合;表明femB基因的原核表达系统构建成功,为进一步研究其生物学功能奠定了基础.     

Differential Gene Expression in Ovaries of Qira Black Sheep and Hetian Sheep Using RNA-Seq Technique

The Qira black sheep and the Hetian sheep are two local breeds in the Northwest of China, which are characterized by high-fecundity and low-fecundity breed respectively. The elucidation of mRNA expression profiles in the ovaries among different sheep breeds representing fecundity extremes will helpful for identification and utilization of major prolificacy genes in sheep. In the present study, we performed RNA-seq technology to compare the difference in ovarian mRNA expression profiles between Qira black sheep and Hetian sheep. From the Qira black sheep and the Hetian sheep libraries, we obtained a total of 11,747,582 and 11,879,968 sequencing reads, respectively. After aligning to the reference sequences, the two libraries included 16,763 and 16,814 genes respectively. A total of 1,252 genes were significantly differentially expressed at Hetian sheep compared with Qira black sheep. Eight differentially expressed genes were randomly selected for validation by real-time RT-PCR. This study provides a basic data for future research of the sheep reproduction.     

Novel Inhibitors of Staphylococcus aureus Virulence Gene Expression and Biofilm Formation

Staphylococcus aureus is a major human pathogen and one of the more prominent pathogens causing biofilm related infections in clinic. Antibiotic resistance in S. aureus such as methicillin resistance is approaching an epidemic level. Antibiotic resistance is widespread among major human pathogens and poses a serious problem for public health. Conventional antibiotics are either bacteriostatic or bacteriocidal, leading to strong selection for antibiotic resistant pathogens. An alternative approach of inhibiting pathogen virulence without inhibiting bacterial growth may minimize the selection pressure for resistance. In previous studies, we identified a chemical series of low molecular weight compounds capable of inhibiting group A streptococcus virulence following this alternative anti-microbial approach. In the current study, we demonstrated that two analogs of this class of novel anti-virulence compounds also inhibited virulence gene expression of S. aureus and exhibited an inhibitory effect on S. aureus biofilm formation. This class of anti-virulence compounds could be a starting point for development of novel anti-microbial agents against S. aureus.     

Characterization of the Humoral Immune Response during Staphylococcus aureus Bacteremia and Global Gene Expression by Staphylococcus aureus in Human Blood

Attempts to develop an efficient anti-staphylococcal vaccine in humans have so far been unsuccessful. Therefore, more knowledge of the antigens that are expressed by Staphylococcus aureus in human blood and induce an immune response in patients is required. In this study we further characterize the serial levels of IgG and IgA antibodies against 56 staphylococcal antigens in multiple serum samples of 21 patients with a S. aureus bacteremia, compare peak IgG levels between patients and 30 non-infected controls, and analyze the expression of 3626 genes by two genetically distinct isolates in human blood. The serum antibody levels were measured using a bead-based flow cytometry technique (xMAP®, Luminex corporation). Gene expression levels were analyzed using a microarray (BµG@s microarray). The initial levels and time taken to reach peak IgG and IgA antibody levels were heterogeneous in bacteremia patients. The antigen SA0688 was associated with the highest median initial-to-peak antibody fold-increase for IgG (5.05-fold) and the second highest increase for IgA (2.07-fold). Peak IgG levels against 27 antigens, including the antigen SA0688, were significantly elevated in bacteremia patients versus controls (P≤0.05). Expression of diverse genes, including SA0688, was ubiquitously high in both isolates at all time points during incubation in blood. However, only a limited number of genes were specifically up- or downregulated in both isolates when cultured in blood, compared to the start of incubation in blood or during incubation in BHI broth. In conclusion, most staphylococcal antigens tested in this study, including many known virulence factors, do not induce uniform increases in the antibody levels in bacteremia patients. In addition, the expression of these antigens by S. aureus is not significantly altered by incubation in human blood over time. One immunogenic and ubiquitously expressed antigen is the putative iron-regulated ABC transporter SA0688.     

Model-Based Deconvolution of Cell Cycle Time-Series Data Reveals Gene Expression Details at High Resolution

In both prokaryotic and eukaryotic cells, gene expression is regulated across the cell cycle to ensure “just-in-time” assembly of select cellular structures and molecular machines. However, present in all time-series gene expression measurements is variability that arises from both systematic error in the cell synchrony process and variance in the timing of cell division at the level of the single cell. Thus, gene or protein expression data collected from a population of synchronized cells is an inaccurate measure of what occurs in the average single-cell across a cell cycle. Here, we present a general computational method to extract “single-cell”-like information from population-level time-series expression data. This method removes the effects of 1) variance in growth rate and 2) variance in the physiological and developmental state of the cell. Moreover, this method represents an advance in the deconvolution of molecular expression data in its flexibility, minimal assumptions, and the use of a cross-validation analysis to determine the appropriate level of regularization. Applying our deconvolution algorithm to cell cycle gene expression data from the dimorphic bacterium Caulobacter crescentus, we recovered critical features of cell cycle regulation in essential genes, including ctrA and ftsZ, that were obscured in population-based measurements. In doing so, we highlight the problem with using population data alone to decipher cellular regulatory mechanisms and demonstrate how our deconvolution algorithm can be applied to produce a more realistic picture of temporal regulation in a cell.     

Golden Pigment Production and Virulence Gene Expression Are Affected by Metabolisms in Staphylococcus aureus

The pathogenesis of staphylococcal infections is multifactorial. Golden pigment is an eponymous feature of the human pathogen Staphylococcus aureus that shields the microbe from oxidation-based clearance, an innate host immune response to infection. Here, we screened a collection of S. aureus transposon mutants for pigment production variants. A total of 15 previously unidentified genes were discovered. Notably, disrupting metabolic pathways such as the tricarboxylic acid cycle, purine biosynthesis, and oxidative phosphorylation yields mutants with enhanced pigmentation. The dramatic effect on pigment production seems to correlate with altered expression of virulence determinants. Microarray analysis further indicates that purine biosynthesis impacts the expression of ∼400 genes involved in a broad spectrum of functions including virulence. The purine biosynthesis mutant and oxidative phosphorylation mutant strains exhibit significantly attenuated virulence in a murine abscess model of infection. Inhibition of purine biosynthesis with a known small-molecule inhibitor results in altered virulence gene expression and virulence attenuation during infection. Taken together, these results suggest an intimate link between metabolic processes and virulence gene expression in S. aureus. This study also establishes the importance of purine biosynthesis and oxidative phosphorylation for in vivo survival.Staphylococcus aureus causes a variety of infections in humans, ranging from minor skin and wound infections to life-threatening diseases (31). The pathogenesis of staphylococcal infections is a multifactorial process that depends on the expression of different virulence factors controlled by multiple regulatory systems in conjunction with environmental and nutritional signals (46). The high degree of variability in the expression of virulence genes is modulated by a complex network regulated by factors such as the agr locus (RNAIII), SarA, and SigB (5, 9), which allows the bacterium to adapt to changing environmental conditions for survival and developing infection.The species epithet of S. aureus reflects its characteristic surface pigmentation (aureus, meaning “golden” in Latin) (43). The yellowish-orange (golden) pigment produced by S. aureus has been linked to virulence, owing to its antioxidant property (29, 30). The golden pigmentation of S. aureus is the product of a C₃₀ triterpenoid carotenoid biosynthesis pathway, and the carotenoid pigment biosynthesis genes are organized in an operon crtOPQMN controlled by the alternative sigma factor SigB (3, 39). Since many virulence genes are coordinately regulated in S. aureus (5, 9, 31), we hypothesized that genes affecting pigmentation may also influence the production of virulence determinants and have an impact on the pathogenesis of S. aureus.Herein, we present an analysis of S. aureus golden pigment biosynthesis and regulatory pathways at the genomic level by screening the Phoenix (ΦNΞ) library, a collection of defined transposon insertions into 1,812 open reading frames of S. aureus strain Newman (1). This study indicates an intimate link between metabolic processes and virulence gene expression. It demonstrates the importance of purine biosynthesis and oxidative phosphorylation for in vivo survival and pathogenesis of S. aureus. Our results show that targeting purine biosynthesis is a promising strategy to develop anti-S. aureus therapies.