The Zur of Xanthomonas campestris functions as a repressor and an activator of putative zinc homeostasis genes via recognizing two distinct sequences within its target promoters

It has been long considered that zinc homeostasis in bacteria is maintained by export systems and uptake systems, which are separately controlled by their own regulators and the uptake systems are negatively regulated by Zur which binds to an about 30-bp AT-rich sequence known as Zur-box present in its target promoters to block the entry of RNA polymerase. Here, we demonstrated in vivo and in vitro that in addition to act as a repressor of putative Zn(2+)-uptake systems, the Zur of the bacterial phytopathogen Xanthomonas campestris pathovar campestris (Xcc) acts as an activator of a Zn(2+) efflux pump. The Xcc Zur binds to a similar Zur-box with approximately 30-bp AT-rich sequence in the promoters of the genes encoding putative Zn(2+)-uptake systems but a 59-bp GC-rich sequence with a 20-bp inverted repeat overlapping the promoter's -35 to -10 sequence of the gene encoding a Zn(2+)-export system. Mutagenesis of the inverted repeat sequence resulted in abolishment of the in vitro binding and the in vivo and in vitro activation of the export gene's promoter by Zur. These results reveal that the Xcc Zur functions as a repressor and an activator of putative zinc homeostasis genes via recognizing two distinct sequences within its target promoters.     

Identification of candidate susceptibility and resistance genes of mice infected with Streptococcus suis type 2

Streptococcus suis type 2 (SS2) is an important swine pathogen and zoonosis agent. A/J mice are significantly more susceptible than C57BL/6 (B6) mice to SS2 infection, but the genetic basis is largely unknown. Here, alterations in gene expression in SS2 (strain HA9801)-infected mice were identified using Illumina mouse BeadChips. Microarray analysis revealed 3,692 genes differentially expressed in peritoneal macrophages between A/J and B6 mice due to SS2 infection. Between SS2-infected A/J and control A/J mice, 2646 genes were differentially expressed (1469 upregulated; 1177 downregulated). Between SS2-infected B6 and control B6 mice, 1449 genes were differentially expressed (778 upregulated; 671 downregulated). These genes were analyzed for significant Gene Ontology (GO) categories and signaling pathways using the Kyoto Encylopedia of Genes and Genomes (KEGG) database to generate a signaling network. Upregulated genes in A/J and B6 mice were related to response to bacteria, immune response, positive regulation of B cell receptor signaling pathway, type I interferon biosynthesis, defense and inflammatory responses. Additionally, upregulated genes in SS2-infected B6 mice were involved in antigen processing and presentation of exogenous peptides, peptide antigen stabilization, lymphocyte differentiation regulation, positive regulation of monocyte differentiation, antigen receptor-mediated signaling pathway and positive regulation of phagocytosis. Downregulated genes in SS2-infected B6 mice played roles in glycolysis, carbohydrate metabolic process, amino acid metabolism, behavior and muscle regulation. Microarray results were verified by quantitative real-time PCR (qRT-PCR) of 14 representative deregulated genes. Four genes differentially expressed between SS2-infected A/J and B6 mice, toll-like receptor 2 (Tlr2), tumor necrosis factor (Tnf), matrix metalloproteinase 9 (Mmp9) and pentraxin 3 (Ptx3), were previously implicated in the response to S. suis infection. This study identified candidate genes that may influence susceptibility or resistance to SS2 infection in A/J and B6 mice, providing further validation of these models and contributing to understanding of S. suis pathogenic mechanisms.     

猪链球菌2型强毒株05ZYH33转录调控因子Rgg基因敲除突变体与野生株的基因表达差异分析

目的:为了解猪链球菌2型强毒株05Z33转录调控因子Rgg的调控作用,用基因芯片方法分析野生株与rgg基因敲除突变体之间的差异表达基因。方法:用猪链球菌2型全基因组序列点样制备芯片,将芯片运用于rgg敲除株与野生株的基因表达差异研究,采用定量real-time PCR(qRT-PCR)验证表达谱结果。结果:在突变体中共发现45个基因表达量变化在2倍以上,其中19个基因表达上调,26个基因表达下调。这些基因在细菌毒力、免疫抗原、DNA合成和修复、基础代谢和ABC转运系统等方面起着重要作用。结论:转录调控因子Rgg是一个全局调控因子,但rgg敲除后并不影响猪链球菌的毒力。     

The zinc-responsive regulator Zur and its control of the znu gene cluster encoding the ZnuABC zinc uptake system in Escherichia coli

The synthesis of the Escherichia coli zinc transporter, encoded by the znuACB gene cluster, is regulated in response to the intracellular zinc concentration by the zur gene product. Inactivation of the zur gene demonstrated that Zur acts as a repressor when binding Zn(2+). Eight chromosomal mutant zur alleles were sequenced to correlate the loss of Zur function with individual mutations. Wild-type Zur and ZurDelta46-91 formed homo- and heterodimers. Dimerization was independent of metal ions since it also occurred in the presence of metal chelators. Using an in vivo titration assay, the znu operator was narrowed down to a 31-base pair region overlapping the translational start site of znuA. This location was confirmed by footprinting assays. Zur directly binds to a single region comprising a nearly perfect palindrome. Zinc chelators completely inhibited and Zn(2+) in low concentrations enhanced DNA binding of Zur. No evidence for autoregulation of Zur was found. Zur binds at least 2 zinc ions/dimer specifically. Although most of the mutant Zur proteins bound to the znu operator in vitro, no protection was observed in in vivo footprinting experiments. Analysis of the mutant Zur proteins suggested an amino-terminal DNA contact domain around residue 65 and a dimerization and Zn(2+)-binding domain toward the carboxyl-terminal end.     

猪链球菌2型89K毒力岛研究进展

高致病性猪链球菌2型的致病机制仍是未解之谜.毒力岛不仅赋予病原菌特殊的致病能力,而且在细菌的适应性进化过程中扮演重要角色.对猪链球菌2型89K毒力岛功能性基因的深入剖析有助于更全面地掌握病原菌的致病特性.综述了猪链球菌2型89K毒力岛的结构与进化过程,以及国内外对毒力岛中二元信号转导系统、Ⅳ型分泌系统、ABC转运蛋白、毒素-抗毒素系统等重要基因的研究进展,力图从基因水平为猪链球菌2型的致病机制寻找突破口.     

Biochemical characterization of the histidine triad protein PhtD as a cell surface zinc-binding protein of pneumococcus

Zinc homeostasis is critical for pathogen host colonization. Indeed, during invasion, Streptococcus pneumoniae has to finely regulate zinc transport to cope with a wide range of Zn(2+) concentrations within the various host niches. AdcAII was identified as a pneumococcal Zn(2+)-binding protein; its gene is present in an operon together with the phtD gene. PhtD belongs to the histidine triad protein family, but to date, its function has not been clarified. Using several complementary biochemical methods, we provide evidence that like AdcAII, PhtD is a metal-binding protein specific for zinc. When Zn(2+) binds (K(d) = 131 ± 10 nM), the protein displays substantial thermal stabilization. We also present the first direct evidence of a joint function of AdcAII and PhtD by demonstrating that their expression is corepressed by Zn(2+), that they interact directly in vitro, and that they are colocalized at the bacterial surface. These results suggest the common involvement of the AdcAII-PhtD system in pneumococcal zinc homeostasis.     

猪链球菌2型菌毛骨架蛋白编码基因SSU2101敲除突变株的构建及其生物学功能

利用同源重组基因敲除方法构建猪链球菌2型(Streptococcus suis serotype2,S.suis2)中国强毒株05ZYH33菌毛骨架蛋白(Backbone protein,BP)编码基因SSU2101敲除突变株。采用引物特异性PCR分析、Southern杂交及RT-PCR等方法鉴定,证实成功构建了BP基因缺失突变株。生物学特性显示,突变株的菌落形态、溶血活性以及染色特性方面与野生株之间均无明显差异。小鼠致病性试验结果显示,突变株的毒力比野生株显著减弱。研究结果提示菌毛在S.suis2感染致病过程中起重要作用,为系统研究S.suis2菌毛分子装配机制及其生物学功能奠定了基础。     

The zinc uptake regulator Zur is essential for the full virulence of Xanthomonas campestris pv. campestris

Zur is a regulator of the high-affinity zinc uptake system in many bacteria. In Xanthomonas campestris pv. campestris 8004, a putative protein encoded by the open reading frame designated as XC1430 shows 42% amino acid similarity with the Zur of Escherichia coli. An XC1430-disrupted mutant 1430nk was constructed by homologous suicide plasmid integration. 1430nk failed to grow in rich medium supplemented with Zn2+ at a concentration of 400 microM and in nonrich medium supplemented with Zn2+ at a concentration of 110 microM, whereas the wild-type strain grew well in the same conditions. In rich medium with 400 microM Zn2+, 1430nk accumulated significantly more Zn2+ than the wild-type strain. 1430nk showed a reduction in virulence on the host plant Chinese radish (Raphanus sativus L. var. radiculus Pers.) and produced less extracellular polysaccharide (EPS) than did the wild-type strain in the absence of added zinc. These results revealed that XC1430 is a functional member of the Zur regulator family that controls zinc homeostasis, EPS production, and virulence in X. campestris pv. campestris.     

Characterization and functional analysis of atl, a novel gene encoding autolysin in Streptococcus suis

Streptococcus suis serotype 2 (S. suis 2) is an important swine and human pathogen responsible for septicemia and meningitis. A novel gene, designated atl and encoding a major autolysin of S. suis 2 virulent strain HA9801, was identified and characterized in this study. The Atl protein contains 1,025 amino acids with a predicted molecular mass of 113 kDa and has a conserved N-acetylmuramoyl-l-alanine amidase domain. Recombinant Atl was expressed in Escherichia coli, and its bacteriolytic and fibronectin-binding activities were confirmed by zymography and Western affinity blotting. Two bacteriolytic bands were shown in the sodium dodecyl sulfate extracts of HA9801, while both were absent from the atl inactivated mutant. Cell chains of the mutant strain became longer than that of the parental strain. In the autolysis assay, HA9801 decreased to 20% of the initial optical density (OD) value, while the mutant strain had almost no autolytic activity. The biofilm capacity of the atl mutant was reduced ～30% compared to the parental strain. In the zebrafish infection model, the 50% lethal dose of the mutant strain was increased up to 5-fold. Furthermore, the adherence to HEp-2 cells of the atl mutant was 50% less than that of the parental strain. Based on the functional analysis of the recombinant Atl and observed effects of atl inactivation on HA9801, we conclude that Atl is a major autolysin of HA9801. It takes part in cell autolysis, separation of daughter cells, biofilm formation, fibronectin-binding activity, cell adhesion, and pathogenesis of HA9801.     

Promotion of Zn<Superscript>2+</Superscript> Uptake by Al<Superscript>3+</Superscript> in a <Emphasis Type="Italic">Saccharomyces Cerevisiae</Emphasis> Mutant that Lacks the <Emphasis Type="Italic">ZRT1</Emphasis> Gene Encoding a High-Affinity Zn Transporter

A Saccharomyces cerevisiae mutant (zrt1Delta) lacking the ZRT1 gene, which encodes a high-affinity Zn(2+) transporter, scarcely thrived in a low-pH, low-phosphate medium because of Zn(2+) deficiency. Supplementation of the medium with Al(3+) restored growth to a level comparable to that of a wild-type strain. A metal determination study clearly demonstrated that Al(3+) induced the incorporation of Zn(2+) into zrt1Delta cells, probably through the low-affinity Zn(2+) transporter Zrt2p, given that the zrt1Deltazrt2Delta double mutant did not show Al-induced growth enhancement. Al(3+) may have altered the speciation of Zn(2+) in the medium, resulting in enhanced levels of free Zn(2+). Alternatively, it might be that Zrt2p was degraded by endocytosis in the absence of Al(3+) and Al(3+) interfered with this process, resulting in enhanced Zn(2+) accumulation.     

Identification and proteome analysis of the two-component VirR/VirS system in epidemic Streptococcus suis serotype 2

Streptococcus suis serotype 2 (SS2) is an important zoonotic pathogen that infects pigs and sporadically causes serious infections in humans. Two recent large-scale outbreaks of human streptococcal toxic-shock-like syndrome with high mortality occurred in China, posing new challenges for global public health. However, the global regulation of the virulence of epidemic SS2 isolates lacks a systematic understanding. In this study, we performed a mutational and functional analysis of an SS2 two-component system that is orthologous to the VirR/VirS regulatory system of Clostridium perfringens. An isogenic knockout mutant of VirR/VirS (ΔvirRS) was found to exhibit marked phenotypic changes, including the formation of shorter chains and thinner capsular walls, more easily cleared in whole blood, and decreased oxidative stress tolerance. Furthermore, the ΔvirRS mutant was greatly attenuated in a mouse model. Comparative proteome analysis of the expression profiles of the wild-type strain with the ΔvirRS mutant allowed us to identify 72 proteins that are differentially expressed in the absence of the VirR/VirS system and that are directly responsible for the pleiotropic phenotype of the ΔvirRS mutant.     

Microarray-based gene expression profiles in rabbit retina due to negative pressure suction

We investigated a possible molecular pathogenesis involving retinal ganglion cell apoptosis following transient high intraocular pressure. Changes in the gene expression profiles of the retina were detected via gene chip methodology. Twelve New Zealand white rabbits were randomly assigned to control and 3-min negative pressure suction groups. The control group was treated only with a laser, and the experimental group was also treated with suction for 3 min, using a negative pressure generator. Total RNA was then extracted from the retinal tissue at different recovery stages to analyze gene expression profiles using the Agilent rabbit one-way gene chip. The groups were then compared. Immediately after negative pressure suction induction, 704 genes were differentially expressed. Among these, 485 genes were upregulated, and 219 were downregulated. Expression of the genes encoding CRYAA, CRYAB, and TLR3 genes, which are involved in apoptosis, was elevated. The KRT18 gene, which is involved in apoptosis, had reduced expression. Seven days after negative pressure suction, 482 genes were differentially expressed. Among these, 178 genes were upregulated, and 304 were downregulated. Expression of the genes encoding CRYAB, IL1-BETA and IL1R1, which are involved in apoptosis, was upregulated. Ten days after negative pressure suction, 402 genes were differentially expressed. Of these, 213 genes were upregulated, and 189 were downregulated. Apoptosis genes CRYAB, CRYBA3, CRYBB2, IL1- BETA, and IL1R1 showed higher expression levels. We concluded that negative pressure suction for long periods of time (for example, 3 min) results in changes in gene expression. Genes with higher fold changes help protect retinal ganglion cells from apoptosis. We suggest that promoting the expression of these genes should be considered as a new means for treating ischemic-hypoxic retinopathy.