Pathogenic characteristics of Pseudomonas aeruginosa

P. aeruginosa strains isolated from clinical material have been found capable of inducing various types of lesions in agricultural plants: tissue growth, soft and dry rot, changes in the color of plant tissue. In connection with the capacity of P. aeruginosa for adaptation to various conditions of existence, plants may be one of the reservoirs of infection. The authors suggest the P. aeruginosa is not an opportunistic, but a complete pathogen with a high degree of adaptability to the environment.     

应用代表性差异分析法筛选人癌候选抑癌基因

运用cDNA代表性差异分析法(cDNA representational difference analysis,cDNA RDA),以正常人鼻咽上皮细胞及鼻咽癌HNE1细胞作为比较的样品来源,分离了四个在鼻咽癌中缺失的cDNA片段.以此四个片段作探针,分别进行DNA杂交、RNA杂交,结果显示,这些差异性的cDNA序列确实来自正常人鼻咽上皮且只在其中表达和/或在鼻咽癌HNE1中表达降低,并在鼻咽癌病人中存在不同程度的缺失.序列分析结果表明这些差异性表达的基因为具有相当抑癌基因功能的已知基因和可能与鼻咽癌相关的抑癌基因的新基因.从而说明cDNA RDA是一种高效、敏感、假阳性低的克隆抑癌基因的有效方法.     

Identification of Genes Involved in Pseudomonas aeruginosa Biofilm-Specific Resistance to Antibiotics

Pseudomonas aeruginosa is a key opportunistic pathogen characterized by its biofilm formation ability and high-level multiple antibiotic resistance. By screening a library of random transposon insertion mutants with an increased biofilm-specifc antibiotic susceptibility, we previously identified 3 genes or operons of P. aeruginosa UCBPP-PA14 (ndvB, PA1875–1877 and tssC1) that do not affect biofilm formation but are involved in biofilm-specific antibiotic resistance. In this study, we demonstrate that PA0756–0757 (encoding a putative two-component regulatory system), PA2070 and PA5033 (encoding hypothetical proteins of unknown function) display increased expression in biofilm cells and also have a role in biofilm-specific antibiotic resistance. Furthermore, deletion of each of PA0756, PA2070 and PA5033 resulted in a significant reduction of lethality in Caenorhabditis elegans, indicating a role for these genes in both biofilm-specific antibiotic resistance and persistence in vivo. Together, these data suggest that these genes are potential targets for antimicrobial agents.     

Differentiation in Quinolone Resistance by Virulence Genotype in Pseudomonas aeruginosa

Pseudomonas aeruginosa is a leading pathogen that has become increasingly resistant to the fluoroquinolone antibiotics due to widespread prescribing. Adverse outcomes have been shown for patients infected with fluoroquinolone-resistant strains. The type III secretion system (TTSS) is a major virulence determinant during acute infections through the injection of effector toxins into host cells. Most strains exhibit a unique TTSS virulence genotype defined by the presence of either exoS or exoU gene encoding two of the effector toxins, ExoS and ExoU, respectively. Specific TTSS effector genotype has been shown previously to differentially impact virulence in pneumonia. In this study, we examined the relationship between TTSS effector genotype and fluoroquinolone resistance mechanisms in a collection of 270 respiratory isolates. We found that a higher proportion of exoU+ strains were fluoroquinolone-resistant compared to exoS+ strains (63% vs 49%, p = 0.03) despite its lower overall prevalence (38% exoU+ vs 56% exoS+). Results from sequencing the quinolone resistance determining regions (QRDRs) of the 4 target genes (gyrA, gyrB, parC, parE) indicated that strains containing the exoU gene were more likely to acquire ≥2 mutations than exoS+ strains at MICs ≤8 µg/ml (13% vs none) and twice as likely to have mutations in both gyrA and parC than exoS+ strains (48% vs 24% p = 0.0439). Our findings indicate that P. aeruginosa strains differentially develop resistance-conferring mutations that correlate with TTSS effector genotype and the more virulent exoU+ subpopulation. Differences in mutational processes by virulence genotype that were observed suggest co-evolution of resistance and virulence traits favoring a more virulent genotype in the quinolone-rich clinical environment.     

Identification of a chitin-binding protein secreted by Pseudomonas aeruginosa

One of the major proteins secreted by Pseudomonas aeruginosa is a 43-kDa protein, which is cleaved by elastase into smaller fragments, including a 30-kDa and a 23-kDa fragment. The N-terminal 23-kDa fragment was previously suggested as corresponding to a staphylolytic protease and was designated LasD (S. Park and D. R. Galloway, Mol. Microbiol. 16:263-270, 1995). However, the sequence of the gene encoding this 43-kDa protein revealed that the N-terminal half of the protein is homologous to the chitin-binding proteins CHB1 of Streptomyces olivaceoviridis and CBP21 of Serratia marcescens and to the cellulose-binding protein p40 of Streptomyces halstedii. Furthermore, a short C-terminal fragment shows homology to a part of chitinase A of Vibrio harveyi. The full-length 43-kDa protein could bind chitin and was thereby protected against the proteolytic activity of elastase, whereas the degradation products did not bind chitin. The purified 43-kDa chitin-binding protein had no staphylolytic activity, and comparison of the enzymatic activities in the extracellular medium of a wild-type strain and a chitin-binding protein-deficient mutant indicated that the 43-kDa protein supports neither chitinolytic nor staphylolytic activity. We conclude that the 43-kDa protein, which was found to be produced by many clinical isolates of P. aeruginosa, is a chitin-binding protein, and we propose to name it CbpD (chitin-binding protein D).     

Biosynthesis of Pyocyanine by a Paraffin Hydrocarbon-oxidizing Strain of Pseudomonas aeruginosa

A paraffin-oxidizing bacterium, designated as Pseudomonas aeruginosa ATS-14, was isolated from soil samples obtained from the Athabasca "tar sands." This strain utilized kerosene as the only carbon source of energy and produced a high concentration of pyocyanine in the culture medium. Aromatic carbons were not attacked, but C(10) to C(17)n-alkanes were readily oxidized by the pseudomonad and formed pyocyanine. The highest yield of the pigment was obtained from hexadecane and heptadecane.     

Reduction and Acetylation of 2,4-Dinitrotoluene by a Pseudomonas aeruginosa Strain

Aerobic and anoxic biotransformation of 2,4-dinitrotoluene (DNT) was examined by using a Pseudomonas aeruginosa strain isolated from a plant treating propellant manufacturing wastewater. DNT biotransformation in the presence and absence of oxygen was mostly reductive and was representative of the type of cometabolic transformations that occur when a high concentration of an easily degradable carbon source is present. P. aeruginosa reduced both nitro groups on DNT, with the formation of mainly 4-amino-2-nitrotoluene and 2-amino-4-nitrotoluene and small quantities of 2,4-diaminotoluene. Acetylation of the arylamines was a significant reaction. 4-Acetamide-2-nitrotoluene and the novel compounds 2-acetamide-4-nitrotoluene, 4-acetamide-2-aminotoluene, and 2,4-diacetamidetoluene were identified as DNT metabolites. The biotransformation of 2,4-diaminotoluene to 4-acetamide-2-aminotoluene was 24 times faster than abiotic transformation. 2-Nitrotoluene and 4-nitrotoluene were also reduced to their corresponding toluidines and then acetylated. However, the yield of 4-acetamidetoluene was much higher than that of 2-acetamidetoluene, demonstrating that acetylation at the position para to the methyl group was favored.     

光声光谱技术用于绿脓杆菌的鉴定

【目的】探索一种无创、快速、可靠、经济地鉴定绿脓杆菌的新方法。【方法】利用光声光谱技术分别对大肠杆菌、绿脓杆菌在35°C下培养24 h后产生的挥发性代谢产物(Bacterial volatile compounds,BVCs)进行连续检测,获得各细菌挥发性气体的光声光谱图谱,并用“逆向思维”的方法对其检测结果进行分析。【结果】利用光声光谱技术对绿脓杆菌、大肠杆菌的挥发性代谢产物进行检测分析,发现绿脓杆菌产生了较高浓度的氰化氢(HCN),而大肠杆菌并未检测出HCN,据此可以对绿脓杆菌进行初步鉴定。【结论】光声光谱技术为绿脓杆菌的鉴定提供了一种简单、快速、经济的方法。为加速其在临床中的应用,提出了“三步走”方案:建立“大数据”、完善“比对算法”、创建“自动检测-比对-校对-鉴定”系统。     

The Multiple Signaling Systems Regulating Virulence in Pseudomonas aeruginosa

Summary: Cell-to-cell communication is a major process that allows bacteria to sense and coordinately react to the fluctuating conditions of the surrounding environment. In several pathogens, this process triggers the production of virulence factors and/or a switch in bacterial lifestyle that is a major determining factor in the outcome and severity of the infection. Understanding how bacteria control these signaling systems is crucial to the development of novel antimicrobial agents capable of reducing virulence while allowing the immune system of the host to clear bacterial infection, an approach likely to reduce the selective pressures for development of resistance. We provide here an up-to-date overview of the molecular basis and physiological implications of cell-to-cell signaling systems in Gram-negative bacteria, focusing on the well-studied bacterium Pseudomonas aeruginosa. All of the known cell-to-cell signaling systems in this bacterium are described, from the most-studied systems, i.e., N-acyl homoserine lactones (AHLs), the 4-quinolones, the global activator of antibiotic and cyanide synthesis (GAC), the cyclic di-GMP (c-di-GMP) and cyclic AMP (cAMP) systems, and the alarmones guanosine tetraphosphate (ppGpp) and guanosine pentaphosphate (pppGpp), to less-well-studied signaling molecules, including diketopiperazines, fatty acids (diffusible signal factor [DSF]-like factors), pyoverdine, and pyocyanin. This overview clearly illustrates that bacterial communication is far more complex than initially thought and delivers a clear distinction between signals that are quorum sensing dependent and those relying on alternative factors for their production.     

Identification,Characterization, and Molecular Application of a Virulence-Associated Autotransporter from a Pathogenic Pseudomonas fluorescens Strain

A gene, pfa1, encoding an autotransporter was cloned from a pathogenic Pseudomonas fluorescens strain, TSS, isolated from diseased fish. The expression of pfa1 is enhanced during infection and is regulated by growth phase and growth conditions. Mutation of pfa1 significantly attenuates the overall bacterial virulence of TSS and impairs the abilities of TSS in biofilm production, interaction with host cells, modulation of host immune responses, and dissemination in host blood. The putative protein encoded by pfa1 is 1,242 amino acids in length and characterized by the presence of three functional domains that are typical for autotransporters. The passenger domain of PfaI contains a putative serine protease (Pap) that exhibits apparent proteolytic activity when expressed in and purified from Escherichia coli as a recombinant protein. Consistent with the important role played by PfaI in bacterial virulence, purified recombinant Pap has a profound cytotoxic effect on cultured fish cells. Enzymatic analysis showed that recombinant Pap is relatively heat stable and has an optimal temperature and pH of 50°C and pH 8.0. The domains of PfaI that are essential to autotransporting activity were localized, and on the basis of this, a PfaI-based autodisplay system (named AT1) was engineered to facilitate the insertion and transport of heterologous proteins. When expressed in E. coli, AT1 was able to deliver an integrated Edwardsiella tarda immunogen (Et18) onto the surface of bacterial cells. Compared to purified recombinant Et18, Et18 displayed by E. coli via AT1 induced significantly enhanced immunoprotection.Protein secretion plays important roles in bacterial life, as many of the secreted proteins are involved in biological processes that are fundamental to the survival and environmental adaptations of the cells. Gram-negative bacteria have evolved a number of secretion systems that utilize different secretion apparatus and mechanisms (9). The classical autotransporter secretion pathway belongs to the type V secretion system (8, 12, 13). Compared to other types of secretion mechanisms, the autotransporter system is unique in that all the components that are required for protein translocation are contained within a single polypeptide. Structurally, autotransporters are characterized by three domains: (i) an N-terminal signal sequence that is recognized by the Sec translocon; (ii) a central passenger domain (or α-domain) that contains the effector molecule and is highly variable; and (iii) a C-terminal translocation domain (or β-/autotransporter domain) that is conserved in length (250 to 300 amino acids) but varies in primary structure (7, 12, 55). In most cases, the β-domain contains 12 antiparallel strands of 9 to 12 residues that, upon integration into the outer membrane, form a β-barrel conformation (27, 57). Another conserved feature of the β-domain is the presence at the C terminus of a sequence motif, (Y/V/I/F/W)-X-(F/W), that is characterized by alternating hydrophobic and hydrophilic residues and ends with either a tryptophan or a phenylalanine (12, 16, 27). The integrity of this end motif seems to be required for protein translocation, as deletion of certain residues in this sequence impairs protein secretion. The secretion process of the autotransporter is initiated by the signal sequence, which directs the translocation of the protein precursor across the inner membrane into the periplasmic space via the Sec system. Once inside the periplasm, the β-domain inserts into the outer membrane and adopts the structure of a β-barrel through which the passenger domain is translocated to the cell surface, where it may exist as a membrane-anchored protein covalently linked to the β-domain or be cleaved from the β-domain as a result of proteolysis (7).Since the discovery of the gonococcal immunoglobulin A1 protease (35), the first autotransporter, and especially with the advent of genome sequencing technology, autotransporters have been identified in many bacterial species (18, 19, 28, 33). Functions assigned to autotransporters are mostly associated with bacterial pathogenicity, which includes adhesion and invasion into host cells, biofilm formation, and cytotoxicity (11, 54). In the present study, we identified and analyzed an autotransporter, PfaI, from a pathogenic Pseudomonas fluorescens strain isolated from diseased fish. We found that, like many of the autotransporters identified in other pathogens, PfaI is a virulence factor that is involved in interactions with host cells and modulation of host immune responses via a protease effector. In addition, we found that the autotransporter property of PfaI could be exploited for the delivery and surface display of an immunoprotective antigen.     

Identification of Biofilm-Associated Cluster (bac) in Pseudomonas aeruginosa Involved in Biofilm Formation and Virulence

Biofilms are prevalent in diseases caused by Pseudomonas aeruginosa, an opportunistic and nosocomial pathogen. By a proteomic approach, we previously identified a hypothetical protein of P. aeruginosa (coded by the gene pA3731) that was accumulated by biofilm cells. We report here that a ΔpA3731 mutant is highly biofilm-defective as compared with the wild-type strain. Using a mouse model of lung infection, we show that the mutation also induces a defect in bacterial growth during the acute phase of infection and an attenuation of the virulence. The pA3731 gene is found to control positively the ability to swarm and to produce extracellular rhamnolipids, and belongs to a cluster of 4 genes (pA3729–pA3732) not previously described in P. aeruginosa. Though the protein PA3731 has a predicted secondary structure similar to that of the Phage Shock Protein, some obvious differences are observed compared to already described psp systems, e.g., this unknown cluster is monocistronic and no homology is found between the other proteins constituting this locus and psp proteins. As E. coli PspA, the amount of the protein PA3731 is enlarged by an osmotic shock, however, not affected by a heat shock. We consequently named this locus bac for biofilm-associated cluster.     

Clustering of Functionally Related Genes in Pseudomonas aeruginosa

Genes for the mandelate and benzoate pathways in Pseudomonas aeruginosa are clustered to a greater degree than that predicted on the basis of the induction pattern.     

铜绿假单胞杆菌QS相关基因差异表达的研究

选取100个与铜绿假单胞杆菌（Pseudomonas aeruginosa）群感效应（quorum-sensing,QS）相关的基因,克隆这些基因片段于pMD-18T载体,测序鉴定,点样制备cDNA基因芯片。制备cy3-dCTP／cy5-dCTP标记的探针,与芯片杂交。初步研究了处于不同生长期的铜绿假单胞杆菌基因的表达差异。指数中期和平台初期相比,有9个QS基因表达量最著增加,有6个基因表达量显著下降。利用芯片做针对铜绿菌假单胞杆菌药物的筛选：妥布霉素（Tobramycin）给药后细菌基因发生差异表达。证明了该cDNA芯片用于药物筛选的可行性。在国内首次研制开发了QS相关基因的cDNA芯片。应用基因芯片技术建立的铜绿假单胞杆菌QS相关基因研究平台,为找到能较好抑制铜绿假单胞杆菌正常生长的药物研究提出新的解决方法。