Global Regulator MorA Affects Virulence-Associated Protease Secretion in Pseudomonas aeruginosa PAO1

Bacterial invasion plays a critical role in the establishment of Pseudomonas aeruginosa infection and is aided by two major virulence factors – surface appendages and secreted proteases. The second messenger cyclic diguanylate (c-di-GMP) is known to affect bacterial attachment to surfaces, biofilm formation and related virulence phenomena. Here we report that MorA, a global regulator with GGDEF and EAL domains that was previously reported to affect virulence factors, negatively regulates protease secretion via the type II secretion system (T2SS) in P. aeruginosa PAO1. Infection assays with mutant strains carrying gene deletion and domain mutants show that host cell invasion is dependent on the active domain function of MorA. Further investigations suggest that the MorA-mediated c-di-GMP signaling affects protease secretion largely at a post-translational level. We thus report c-di-GMP second messenger system as a novel regulator of T2SS function in P. aeruginosa. Given that T2SS is a central and constitutive pump, and the secreted proteases are involved in interactions with the microbial surroundings, our data broadens the significance of c-di-GMP signaling in P. aeruginosa pathogenesis and ecological fitness.     

Structural and Functional Characterization of Pseudomonas aeruginosa AlgX: ROLE OF AlgX IN ALGINATE ACETYLATION*

The exopolysaccharide alginate, produced by mucoid Pseudomonas aeruginosa in the lungs of cystic fibrosis patients, undergoes two different chemical modifications as it is synthesized that alter the properties of the polymer and hence the biofilm. One modification, acetylation, causes the cells in the biofilm to adhere better to lung epithelium, form microcolonies, and resist the effects of the host immune system and/or antibiotics. Alginate biosynthesis requires 12 proteins encoded by the algD operon, including AlgX, and although this protein is essential for polymer production, its exact role is unknown. In this study, we present the X-ray crystal structure of AlgX at 2.15 Å resolution. The structure reveals that AlgX is a two-domain protein, with an N-terminal domain with structural homology to members of the SGNH hydrolase superfamily and a C-terminal carbohydrate-binding module. A number of residues in the carbohydrate-binding module form a substrate recognition “pinch point” that we propose aids in alginate binding and orientation. Although the topology of the N-terminal domain deviates from canonical SGNH hydrolases, the residues that constitute the Ser-His-Asp catalytic triad characteristic of this family are structurally conserved. In vivo studies reveal that site-specific mutation of these residues results in non-acetylated alginate. This catalytic triad is also required for acetylesterase activity in vitro. Our data suggest that not only does AlgX protect the polymer as it passages through the periplasm but that it also plays a role in alginate acetylation. Our results provide the first structural insight for a wide group of closely related bacterial polysaccharide acetyltransferases.     

Structural and Functional Studies of the Pseudomonas aeruginosa Minor Pilin,PilE

Many bacterial pathogens, including Pseudomonas aeruginosa, use type IVa pili (T4aP) for attachment and twitching motility. T4aP are composed primarily of major pilin subunits, which are repeatedly assembled and disassembled to mediate function. A group of pilin-like proteins, the minor pilins FimU and PilVWXE, prime pilus assembly and are incorporated into the pilus. We showed previously that minor pilin PilE depends on the putative priming subcomplex PilVWX and the non-pilin protein PilY1 for incorporation into pili, and that with FimU, PilE may couple the priming subcomplex to the major pilin PilA, allowing for efficient pilus assembly. Here we provide further support for this model, showing interaction of PilE with other minor pilins and the major pilin. A 1.25 Å crystal structure of PilE_Δ1–28 shows a typical type IV pilin fold, demonstrating how it may be incorporated into the pilus. Despite limited sequence identity, PilE is structurally similar to Neisseria meningitidis minor pilins PilX_Nm and PilV_Nm, recently suggested via characterization of mCherry fusions to modulate pilus assembly from within the periplasm. A P. aeruginosa PilE-mCherry fusion failed to complement twitching motility or piliation of a pilE mutant. However, in a retraction-deficient strain where surface piliation depends solely on PilE, the fusion construct restored some surface piliation. PilE-mCherry was present in sheared surface fractions, suggesting that it was incorporated into pili. Together, these data provide evidence that PilE, the sole P. aeruginosa equivalent of PilX_Nm and PilV_Nm, likely connects a priming subcomplex to the major pilin, promoting efficient assembly of T4aP.     

施氏假单胞菌A1501铁吸收调节蛋白Fur的表达特性与功能鉴定

铁是细胞生理代谢的重要金属元素,是多种氧化还原酶类、过氧化物酶类的重要组成部分。革兰氏阴性菌中,胞内铁转运、储存和利用主要由铁吸收调节蛋白Fur（ferric uptake regulator）调控。水稻根际联合固氮菌——施氏假单胞菌（Pseudomonas stutzeri）A1501基因组中含有一个fur同源基因,但其具体功能尚不清楚。比较了A1501中fur基因及其编码蛋白的序列特征,并通过实时荧光定量PCR（real-time quantitative,qRT-PCR）和Western blotting杂交方法测定了fur基因和Fur蛋白的表达特性,结果发现fur基因表达水平与环境中铁浓度呈负相关,在铁限制条件下高表达,铁过量条件下表达受抑制。构建了fur基因突变株和功能回补株,表型测定发现fur突变影响了A1501的碳源代谢谱,并导致A1501对过氧化氢胁迫敏感。胞内铁含量测定表明,fur突变株内的铁含量为野生型的1.3倍,进一步qRT-PCR测定表明fur突变影响了铁离子转运系统及过氧化物酶编码基因的表达。研究结果为解析根际微生物胞内铁平衡调节及根际环境适应机制奠定了理论基础。     

Crystal Structure of BamB from Pseudomonas aeruginosa and Functional Evaluation of Its Conserved Structural Features

The assembly of β-barrel Outer Membrane Proteins (OMPs) in the outer membrane is essential for Gram-negative bacteria. The process requires the β-Barrel Assembly Machine (BAM), a multiprotein complex that, in E. coli, is composed of the OMP BamA and four lipoproteins BamB-E. Whereas BamA and BamD are essential, deletion of BamB, C or E produce membrane permeability defects. Here we present the high-resolution structure of BamB from Pseudomonas aeruginosa. This protein can complement the deletion of bamB in E. coli indicating that they are functionally equivalent. Conserved structural features include an eight-bladed β-propeller fold stabilized by tryptophan docking motifs with a central pore about 8 Å in diameter at the narrowest point. This pore distinguishes BamB from related β-propellers, such as quinoprotein dehydrogenases. However, a double mutation designed to block this pore was fully functional indicating that the opening is not essential. Two loops protruding from the bottom of the propeller are conserved and mediate binding to BamA. Conversely, an additional loop only present in E. coli BamB is not required for function. A cluster of highly conserved residues in a groove between blades 6 and 7 is crucial for proper BamB folding or biogenesis. It has been proposed that BamB may bind nascent OMPs by β-augmentation to its propeller outer strands, or recognize the aromatic residue signature at the C-terminus of OMPs. However, Isothermal Titration Calorimetry experiments and structural analysis do not support these proposals. The structural and mutagenesis analysis suggests that the main function of BamB is to bind and modulate BamA, rather than directly interact with nascent OMPs.     

Characterization of Pseudomonas aeruginosa Bacteriophage 95

A new strain of bacteriophage, phage 95, specific to Pseudomonas aeruginosa and Ps. schuylkilliensis was isolated. It has been shown that the phage induces a lytic enzyme which hydrolyzes peptide bond between l-alanine and d-glutamic acid in the peptidoglycan of cell wall.

Here the characteristics of phage 95 are described. It possesses a hexagonal head of 650Å, a contractile tail of 1150Å and spike with fibers. Its DNA has a GC content of 48%, a density of 1.700 g · cm^?3 and Tm of 87.7°C. Photoreactivation was observed. Latent period is 20 min and burst size is 50. The phage is stable between pH 5 and 7.5, and unstable above 55°C. Culture condition and 300 liter scale cultivation are also presented.     

Structural Insights into the Regulatory Mechanism of the Response Regulator RocR from Pseudomonas aeruginosa in Cyclic Di-GMP Signaling

The nucleotide messenger cyclic di-GMP (c-di-GMP) plays a central role in the regulation of motility, virulence, and biofilm formation in many pathogenic bacteria. EAL domain-containing phosphodiesterases are the major signaling proteins responsible for the degradation of c-di-GMP and maintenance of its cellular level. We determined the crystal structure of a single mutant (R286W) of the response regulator RocR from Pseudomonas aeruginosa to show that RocR exhibits a highly unusual tetrameric structure arranged around a single dyad, with the four subunits adopting two distinctly different conformations. Subunits A and B adopt a conformation with the REC domain located above the c-di-GMP binding pocket, whereas subunits C and D adopt an open conformation with the REC domain swung to the side of the EAL domain. Remarkably, the access to the substrate-binding pockets of the EAL domains of the open subunits C and D are blocked in trans by the REC domains of subunits A and B, indicating that only two of the four active sites are engaged in the degradation of c-di-GMP. In conjunction with biochemical and biophysical data, we propose that the structural changes within the REC domains triggered by the phosphorylation are transmitted to the EAL domain active sites through a pathway that traverses the dimerization interfaces composed of a conserved regulatory loop and the neighboring motifs. This exquisite mechanism reinforces the crucial role of the regulatory loop and suggests that similar regulatory mechanisms may be operational in many EAL domain proteins, considering the preservation of the dimerization interface and the spatial arrangement of the regulatory domains.     

Structural and functional characterization of Pseudomonas aeruginosa CupB chaperones

Pseudomonas aeruginosa, an important human pathogen, is estimated to be responsible for ∼10% of nosocomial infections worldwide. The pathogenesis of P. aeruginosa starts from its colonization in the damaged tissue or medical devices (e.g. catheters, prothesis and implanted heart valve etc.) facilitated by several extracellular adhesive factors including fimbrial pili. Several clusters containing fimbrial genes have been previously identified on the P. aeruginosa chromosome and named cup [1]. The assembly of the CupB pili is thought to be coordinated by two chaperones, CupB2 and CupB4. However, due to the lack of structural and biochemical data, their chaperone activities remain speculative. In this study, we report the 2.5 Å crystal structure of P. aeruginosa CupB2. Based on the structure, we further tested the binding specificity of CupB2 and CupB4 towards CupB1 (the presumed major pilus subunit) and CupB6 (the putative adhesin) using limited trypsin digestion and strep-tactin pull-down assay. The structural and biochemical data suggest that CupB2 and CupB4 might play different, but not redundant, roles in CupB secretion. CupB2 is likely to be the chaperone of CupB1, and CupB4 could be the chaperone of CupB4:CupB5:CupB6, in which the interaction of CupB4 and CupB6 might be mediated via CupB5.     

Characterization of Pseudomonas aeruginosa derepressed R-plasmids.

A genetic study of conjugal transmissibility of two R-plasmids was undertaken in Pseudomonas aeruginosa. Conjugally derepressed mutants of the R-plasmids were isolated, and examination of 11 independent mutants revealed that 10 were recessive to the wild-type transfer repressor, whereas 1 mutant was cis dominant. Cross-repression was observed between the two R-plasmids, suggesting that they have functionally equivalent systems for regulating the expression of tra loci. The derepressed R-plasmid mutants exhibited several characteristics, in addition to derepressed transfer, that were not expressed by the parental plasmids. These included sensitivity to certain donor-specific phages, inhibition of multiplication of a transducing phage, and, in the one case examined, a high degree of entry exclusion. The coexpression of these different functions suggests that their respective genetic loci are controlled by the same regulatory system as that of tra, or else that they are part of the tra complex.     

Characterization of guanosine diphospho-D-mannose dehydrogenase from Pseudomonas aeruginosa. Structural analysis by limited proteolysis.

Alginate is believed to be a major virulence factor in the pathogenicity of Pseudomonas aeruginosa in the lungs of patients suffering from cystic fibrosis. Guanosine diphospho-D-mannose dehydrogenase (GDPmannose dehydrogenase, EC 1.1.1.132) is a key enzyme in the alginate biosynthetic pathway which catalyzes the oxidation of guanosine diphospho-D-mannose (GDP-D-mannose) to GDP-D-mannuronic acid. In this paper, we report the structural analysis of GMD by limited proteolysis using three different proteases, trypsin, submaxillary Arg-C protease, and chymotrypsin. Treatment of GMD with these proteases indicated that the amino-terminal part of this enzyme may fold into a structural domain with an apparent molecular mass of 25-26 kDa. Multiple proteolytic cleavage sites existed at the carboxyl-terminal end of this domain, indicating that this segment may represent an exposed region of the protein. Initial proteolysis also generated a carboxyl-terminal fragment with an apparent molecular mass of 16-17 kDa which was further digested into smaller fragments by trypsin and chymotrypsin. The proteolytic cleavage sites were localized by partial amino-terminal sequencing of the peptide fragments. Arg-295 was identified as the initial cleavage site for trypsin and Tyr-278 for chymotrypsin. Catalytic activity of GMD was totally abolished by the initial cleavage. However, binding of the substrate, GDP-D-mannose, increased stability toward proteolysis and inhibited the loss of enzyme activity. GMP and GDP (guanosine 5'-mono- and diphosphates) also blocked the initial cleavage, but NAD and mannose showed no effect. These results suggest that binding of the guanosine moiety at the catalytic site of GMD may induce a conformational change that reduces the accessibility of the cleavage sites to proteases. Binding of [14C]GDP-D-mannose to the amino-terminal domain was not affected by the removal of the carboxyl-terminal 16-kDa fragment. Furthermore, photoaffinity labeling of GMD with [32P]arylazido-beta-alanine-NAD followed by proteolysis demonstrated that the radioactive NAD was covalently linked to the amino-terminal domain. These observations imply that the amino-terminal domain (25-26 kDa) contains both the substrate and cofactor binding sites. However, the carboxyl-terminal fragment (16-17 kDa) may possess amino acid residues essential for catalysis. Thus, proteolysis had little effect on substrate binding, but totally eliminated catalysis. These biochemical data are in complete agreement with amino acid sequence analysis for the existence of substrate and cofactor sites of GMD. A linear peptide map of GMD was constructed for future structure/functional studies.     

双链RNA依赖性蛋白激酶的结构与作用

dsRNA-依赖的蛋白激酶(PKR)在信号转导、细胞生长、分化和凋亡的控制中起重要作用。最近证实PKR是一个重要的凋亡效应物,是许多不同刺激物诱导凋亡的一个转导物,所以一些研究者开始关注是否PKR的调节可以作为一种新的抗肿瘤策略。由于PKR强大的生长抑制功能和促凋亡功能,使之可能成为又一个肿瘤基因治疗的靶点。该文介绍PKR的结构、活性调节、促凋亡活性及应用于肿瘤治疗的潜力。     

桩蛋白的结构与功能

桩蛋白(paxillin)是近年来发现的一种信号蛋白,主要定位于黏着斑,包含LD模体、LIM结构域、SH2和SH3结合结构域,在整个分子中还散在着多种磷酸化位点,共同构成了桩蛋白的多结构域性结构。桩蛋白分子本身的酶活性尚不清楚,但很可能作为细胞内的一种接头蛋白与多种功能蛋白质结合。另外．作为黏着斑的重要组成部分,桩蛋白不仅参与了整合蛋白介导的信号转导和黏着斑的组装,在细胞黏附和迁移过程中也发挥了重要作用。     

Characterization of the GO system of Pseudomonas aeruginosa

The mutT, mutM, and mutY genes of the GO system of the Pseudomonas aeruginosa PAO1 strain have been characterized by cloning, sequencing, and complementation analysis. The three genes, when cloned in a plasmid, were able to complement the high mutation frequency of the corresponding Escherichia coli deficient strains. Our results demonstrate that the putative mutT, mutM, and mutY gene products from P. aeruginosa are able to perform the expected activity. In addition, the sequence of the P. aeruginosa mutT gene strongly suggested that the product of this gene has a bifunctional activity in P. aeruginosa, being the C-terminal part 40% identical to a consensus sequence of thiamine monophosphate synthases. Our results also demonstrated that the N-terminal part of the protein is necessary and sufficient for the 8-oxodGTP hydrolase activity.     

Characterization of polyagglutinating and surface antigens in Pseudomonas aeruginosa

Mutants of Pseudomonas aeruginosa PAC1R (serotype O:3) which were resistant to bacteriophage D were isolated and shown to react with O:5d, O:9 and O:13 antisera as well as O:3. Antisera to the parent strain and to the three polyagglutinating (PA) mutants also showed cross-reactions. The mutants differed from the parent strain in their lipopolysaccharide (LPS) composition. The LPS from two of the three mutants yielded high molecular weight polysaccharide fractions. Although the high molecular weight fraction from one of the mutants contained the amino sugars and other components characteristic of the O:3 serotype strains, its mobility on Sephadex G75 was different from that of the parent strain. The high molecular weight material from the second mutant lacked the O-antigenic determinants but these were present in a semi-rough LPS fraction. The third mutant appeared rough and completely lacked the O-antigenic components. These three mutants were compared with the parent strain and with a non-agglutinating LPS-defective mutant which lacked both O-antigenic side chains and all neutral sugars in the outer core. Agglutination with absorbed sera and haemagglutination using purified LPS and ELISA detection suggested that wall components other than LPS were responsible for some of the cross-reactions observed. The components responsible were detected after SDS-PAGE of crude outer membrane fractions by a combination of Coomassie blue and silver-staining and antigenic components were detected by immunoelectrophoresis and ELISA-linked immunoblotting of the gels. The main antigenic determinants detected by antiserum to the parent strain were in the high molecular weight O-polysaccharide fractions and in the semirough fractions of the LPS, with some activity due to the H protein of the outer membrane. O:5d antisera reacted with unidentified high molecular weight polysaccharide fractions. Cross-reactions with the O:9 antiserum appeared to be due mainly to the F porin and, to a lesser extent, to the G and E proteins of the outer membrane. O:13 antiserum reacted with high molecular weight polysaccharide fractions but also with the rough core and F and H protein. Cross-reactivity of the other three mutant antisera could largely be interpreted in terms of the outer membrane components exposed in each strain. One reacted strongly with the F porin and the rough core, while the others reacted with a number of protein and LPS-derived fractions.(ABSTRACT TRUNCATED AT 400 WORDS)