Pseudomonas aeruginosa proteases and corneal virulence

Pseudomonas aeruginosa is a common cause of corneal infections, particularly among users of soft contact lenses. Previous studies with chemically induced mutants deficient in alkaline protease (AP) or elastase (LasB) suggested that these proteases contributed to the rapid liquifactive stromal necrosis characteristic of P. aeruginosa corneal infections. Because these mutants might harbor other chromosomal changes that could affect virulence, the role of these proteases in the pathogenesis of corneal disease (as well as a second elastase, LasA protease) was reexamined by constructing isogenic mutants deficient only in these enzymes. Allelic exchange was used to construct mutants of P. aeruginosa PAO1-V deficient in AP (PAO1-V AP[ - ]), LasB and LasA protease (PDO801 LasB[ - ]), or all three proteases (PDO801 TM). These mutants were then evaluated for virulence using mouse scratch and rabbit intrastromal injection models of corneal disease. Loss of AP significantly increased disease scores in the rabbit (P < 0.030) but not the mouse (P > 0.060) model of infection. Loss of both elastases had no effect on ocular virulence in either animal model of corneal disease (P > 0.100). The loss of all three proteases significantly decreased disease scores in the rabbit (P < 0.035), but not in the mouse (P > 0.110). Taken together, these data suggest that AP, LasB, and LasA protease are not essential for initiating or maintaining a corneal infection. Furthermore, AP appears to be an important mediator of pathology depending on the location of the organism within the cornea and whether or not concomitant elastolytic activity is present.     

Flagella and pili-mediated near-surface single-cell motility mechanisms in P. aeruginosa

Bacterial biofilms are structured multicellular communities that are responsible for a broad range of infections. Knowing how free-swimming bacteria adapt their motility mechanisms near a surface is crucial for understanding the transition from the planktonic to the biofilm phenotype. By translating microscopy movies into searchable databases of bacterial behavior and developing image-based search engines, we were able to identify fundamental appendage-specific mechanisms for the surface motility of Pseudomonas aeruginosa. Type IV pili mediate two surface motility mechanisms: horizontally oriented crawling, by which the bacterium moves lengthwise with high directional persistence, and vertically oriented walking, by which the bacterium moves with low directional persistence and high instantaneous velocity, allowing it to rapidly explore microenvironments. The flagellum mediates two additional motility mechanisms: near-surface swimming and surface-anchored spinning, which often precedes detachment from a surface. Flagella and pili interact cooperatively in a launch sequence whereby bacteria change orientation from horizontal to vertical and then detach. Vertical orientation facilitates detachment from surfaces and thereby influences biofilm morphology.     

Siderophore-mediated cooperation and virulence in Pseudomonas aeruginosa

Why should organisms cooperate with each other? Helping close relatives that are likely to share the same genes (kin selection) is one important explanation that is likely to apply across taxa. The production of metabolically costly extracellular iron-scavenging molecules (siderophores) by microorganisms is a cooperative behaviour because it benefits nearby conspecifics. We review experiments focusing on the production of the primary siderophore (pyoverdin) of the opportunistic bacterial pathogen, Pseudomonas aeruginosa, which test kin selection theories that seek to explain the evolution of cooperation. First, cooperation is indeed favoured when individuals interact with their close relatives and when there is competition between groups of cooperators and noncooperators, such that the benefit of cooperation can be realized. Second, the relative success of cheats and cooperators is a function of their frequencies within populations. Third, elevated mutation rates can confer a selective disadvantage under conditions when cooperation is beneficial, because high mutation rates reduce how closely bacteria are related to each other. Fourth, cooperative pyoverdin production is also shown to be favoured by kin selection in vivo (caterpillars), and results in more virulent infections. Finally, we briefly outline ongoing and future work using this experimental system.     

Overexpressed recombinant quorum quenching lactonase reduces the virulence,motility and biofilm formation of multidrug-resistant Pseudomonas aeruginosa clinical isolates

The increasing occurrence of resistance among Pseudomonas aeruginosa clinical isolates necessitates finding alternatives to antibiotics for controlling the infection of such pathogenic bacteria. In this study, lactonase gene ahl-1 from Bacillus weihenstephanensis isolate-P65 was successfully cloned and expressed in Escherichia coli BL21 (DE3) under the control of T7 promoter for utilizing its quorum quenching activity against three multidrug-resistant (MDR) P. aeruginosa clinical isolates. The biological activity of the overexpressed lactonase enzyme (Ahl-1), tested using a synthetic signal and Chromobacterium violaceum CV026 as a biosensor, displayed good catalytic activity using hexanoyl homoserine lactone (HHL) as a substrate and Chromobacterium violaceum (CV026) as a biosensor (77.2 and 133 nm min⁻¹ for the crude and the purified Ahl-lactonase enzymes, respectively). Upon challenging its ability to inhibit the virulence of three MDR P. aeruginosa clinical isolates, recombinant Ahl-1 successfully prevented the accumulation of acylhomoserine lactone signals resulting in a significant reduction in the investigated virulence determinants; protease (from 40 up to 75.5%), pyocyanin (48–75.9%), and rhamnolipids (52.7–63.4%) (P value < 0.05). Ahl-1 also displayed significant inhibitory activities on the swarming motility and biofilm formation of the three tested MDR P. aeruginosa clinical isolates (P value < 0.05). Consequently, Ahl-1 lactonase enzyme in this study is considered a promising therapeutic agent to inhibit P. aeruginosa pathogenicity with no fear of emergence of resistance.

     

Cooperation and virulence in acute Pseudomonas aeruginosa infections

Background  

Efficient host exploitation by parasites is frequently likely to depend on cooperative behaviour. Under these conditions, mixed-strain infections are predicted to show lower virulence (host mortality) than are single-clone infections, due to competition favouring non-contributing social 'cheats' whose presence will reduce within-host growth. We tested this hypothesis using the cooperative production of iron-scavenging siderophores by the pathogenic bacterium Pseudomonas aeruginosa in an insect host.     

Pseudomonas aeruginosa exoproducts as pulmonary virulence factors

An experimental animal model of chronic pseudomonas pneumonia was used to document the production of potential virulence factors by Pseudomonas aeruginosa during the infection. The production of exotoxin A, proteolytic enzymes, and the serotype-specific lipopolysaccharide and slime-layer antigens during the infection was examined by solid-phase radioimmunoassay of serum from infected rats and by indirect immunofluorescence tests of their lung tissue. Rats inoculated intratracheally with purified bacterial exoproducts, delivered alone or in combination, developed pulmonary histopathology similar to that induced by the experimental infection. The results indicate that these exoproducts are produced during the course of the pulmonary infection and suggest that they are involved in the observed lung pathology.     

Quorum sensing regulates exopolysaccharide production, motility, and virulence in Pseudomonas syringae

The N-acyl homoserine lactone (AHL)-mediated quorum-sensing system in the phytopathogen Pseudomonas syringae pv. syringae requires the AHL synthase AhlI and the regulator AhlR, and is additionally subject to regulation by AefR. The contribution of quorum sensing to the expression of a variety of traits expected to be involved in epiphytic fitness and virulence of P syringae were examined. Both an aefR- mutant and an ahlI- ahlR- double mutant, deficient in AHL production, were significantly impaired in alginate production and had an increased susceptibility to hydrogen peroxide compared with the wild-type strain. These mutants were hypermotile in culture, invaded leaves more rapidly, and caused an increased incidence of brown spot lesions on bean leaves after a 48-h moist incubation. Interestingly, an aefR- mutant was both the most motile and virulent. Like the wild-type strain, the AHL-deficient mutant strains incited water-soaked lesions on bean pods. However, lesions caused by an ahlI- ahlR- double mutant were larger, whereas those incited by an aefR- mutant were smaller. In contrast, tissue maceration of pods, which occurs at a later stage of infection, was completely abolished in the AHL-deficient mutants. Both the incidence of disease and in planta growth of P syringae pv. tabaci were greatly reduced in transgenic tobacco plants that produced AHL compared with wild-type plants. These results demonstrate that quorum sensing in E syringae regulates traits that contribute to epiphytic fitness as well as to distinct stages of disease development during plant infection.     

Rhamnolipids modulate swarming motility patterns of Pseudomonas aeruginosa

Pseudomonas aeruginosa is capable of twitching, swimming, and swarming motility. The latter form of translocation occurs on semisolid surfaces, requires functional flagella and biosurfactant production, and results in complex motility patterns. From the point of inoculation, bacteria migrate as defined groups, referred to as tendrils, moving in a coordinated manner capable of sensing and responding to other groups of cells. We were able to show that P. aeruginosa produces extracellular factors capable of modulating tendril movement, and genetic analysis revealed that modulation of these movements was dependent on rhamnolipid biosynthesis. An rhlB mutant (deficient in mono- and dirhamnolipid production) and an rhlC mutant (deficient in dirhamnolipid production) exhibited altered swarming patterns characterized by irregularly shaped tendrils. In addition, agar supplemented with rhamnolipid-containing spent supernatant inhibited wild-type (WT) swarming, whereas agar supplemented with spent supernatant from mutants that do not make rhamnolipids had no effect on WT P. aeruginosa swarming. Addition of purified rhamnolipids to swarming medium also inhibited swarming motility of the WT strain. We also show that a sadB mutant does not sense and/or respond to other groups of swarming cells and this mutant was capable of swarming on media supplemented with rhamnolipid-containing spent supernatant or purified rhamnolipids. The abilities to produce and respond to rhamnolipids in the context of group behavior are discussed.     

The contribution of exoproducts to virulence of Pseudomonas aeruginosa

Pseudomonas aeruginosa produces a large number of extracellular products which may contribute to its virulence. We have employed a genetic approach to determine the contribution of toxin A, exoenzyme S, elastase and alkaline protease to the pathogenesis of P. aeruginosa. Mutations have been introduced with chemicals or transposons. Mutants have been identified using immunological, chemical, or toxicity assays. Mutants were extensively characterized in vitro to ascertain that they were identical to their parent strain except for the production of the desired product. Appropriate mutants were compared with their parent strains in several animal models: the burned mouse model, the mouse corneal infection model, and a rat model of chronic lung infection. The data indicate that virulence of P. aeruginosa is multifactorial. Further, the relative contribution of a given P. aeruginosa product may vary with the type of infection.     

The response of Pseudomonas aeruginosa to iron: genetics, biochemistry and virulence

During the past decade significant progress has been made towards identifying some of the schemes that Pseudomonas aeruginosa uses to obtain iron and towards cataloguing and characterizing many of the genes and gene products that are likely to play a role in these processes. This review will largely recount what we have learned in the past few years about how P. aeruginosa regulates its acquisition, intake and, to some extent, trafficking of iron, and the role of iron acquisition systems in the virulence of this remarkable opportunistic pathogen. More specifically, the genetics, biochemistry and biology of an essential regulator (Ferric uptake regulator - Fur) and a Fur-regulated alternative sigma factor (PvdS), which are central to these processes, will be discussed. These regulatory proteins directly or indirectly regulate a substantial number of other genes encoding proteins with remarkably diverse functions. These genes include: (i) other regulatory genes, (ii) genes involved in basic metabolic processes (e.g. Krebs cycle), (iii) genes required to survive oxidative stress (e.g. superoxide dismutase), (iv) genes necessary for scavenging iron (e.g. siderophores and their cognate receptors) or genes that contribute to the virulence (e.g. exotoxin A) of this opportunistic pathogen. Despite this recent expansion of knowledge about the response of P. aeruginosa to iron, many significant biological issues surrounding iron acquisition still need to be addressed. Virtually nothing is known about which of the distinct iron acquisition mechanisms P. aeruginosa brings to bear on these questions outside the laboratory, whether it be in soil, in a pipeline, on plants or in the lungs of cystic fibrosis patients.     

Effects of ginseng on Pseudomonas aeruginosa motility and biofilm formation

Biofilm-associated chronic Pseudomonas aeruginosa lung infections in patients with cystic fibrosis are virtually impossible to eradicate with antibiotics because biofilm-growing bacteria are highly tolerant to antibiotics and host defense mechanisms. Previously, we found that ginseng treatments protected animal models from developing chronic lung infection by P. aeruginosa. In the present study, the effects of ginseng on the formation of P. aeruginosa biofilms were further investigated in vitro and in vivo. Ginseng aqueous extract at concentrations of 0.5-2.0% did not inhibit the growth of P. aeruginosa, but significantly prevented P. aeruginosa from forming biofilm. Exposure to 0.5% ginseng aqueous extract for 24 h destroyed most 7-day-old mature biofilms formed by both mucoid and nonmucoid P. aeruginosa strains. Ginseng treatment enhanced swimming and twitching motility, but reduced swarming of P. aeruginosa at concentrations as low as 0.25%. Oral administration of ginseng extracts in mice promoted phagocytosis of P. aeruginosa PAO1 by airway phagocytes, but did not affect phagocytosis of a PAO1-filM mutant. Our study suggests that ginseng treatment may help to eradicate the biofilm-associated chronic infections caused by P. aeruginosa.     

铜绿假单胞菌蹭行运动单细胞分析方法的建立及应用

蹭行运动在生物被膜形成过程中对细菌适应表面环境以及后续生物被膜三维结构的形成起重要作用。因此,对蹭行运动的原位表征、量化是生物被膜研究中的重要科学问题之一。我们通过高通量数据采集、自动化图像处理、数据库建立以及图形化输出等技术手段,建立了一整套基于单细菌的统计分析方法。利用这一方法对蹭行运动中的行走、弹射过程进行了详细分析,发现弹射运动过程中存在以0.9 s为周期的周期性弛豫。并定量比较了群体感知信号分子对蹭行运动的影响,发现加入信号分子后蹭行运动在高速区明显增强。该方法的建立为后续蹭行运动分子机制以及调节方式的研究奠定了基础。     

Adenylate kinase as a virulence factor of Pseudomonas aeruginosa

Adenylate kinase (AK; ATP:AMP phosphotransferase, EC 2.7.4.3) is a ubiquitous enzyme that contributes to the homeostasis of adenine nucleotides in eukaryotic and prokaryotic cells. AK catalyzes the reversible reaction Mg. ATP + AMP <--> Mg. ADP + ADP. In this study we show that AK secreted by the pathogenic strains of Pseudomonas aeruginosa appears to play an important role in macrophage cell death. We purified and characterized AK from the growth medium of a cystic fibrosis isolate strain of P. aeruginosa 8821 and hyperproduced it as a fusion protein with glutathione S-transferase. We demonstrated enhanced macrophage cell death in the presence of both the secreted and recombinant purified AK and its substrates AMP plus ATP or ADP. These data suggested that AK converts its substrates to a mixture of AMP, ADP, and ATP, which are potentially more cytotoxic than ATP alone. In addition, we observed increased macrophage killing in the presence of AK and ATP alone. Since the presence of ATPase activity on the macrophages was confirmed in the present work, external macrophage-effluxed ATP is converted to ADP, which in turn can be transformed by AK into a cytotoxic mixture of three adenine nucleotides. Evidence is presented in this study that secreted AK was detected in macrophages during infection with P. aeruginosa. Thus, the possible role of secreted AK as a virulence factor is in producing and keeping an intact pool of toxic mixtures of AMP, ADP, and ATP, which allows P. aeruginosa to exert its full virulence.     

Ralstonia solanacearum needs motility for invasive virulence on tomato

Ralstonia solanacearum, a widely distributed and economically important plant pathogen, invades the roots of diverse plant hosts from the soil and aggressively colonizes the xylem vessels, causing a lethal wilting known as bacterial wilt disease. By examining bacteria from the xylem vessels of infected plants, we found that R. solanacearum is essentially nonmotile in planta, although it can be highly motile in culture. To determine the role of pathogen motility in this disease, we cloned, characterized, and mutated two genes in the R. solanacearum flagellar biosynthetic pathway. The genes for flagellin, the subunit of the flagellar filament (fliC), and for the flagellar motor switch protein (fliM) were isolated based on their resemblance to these proteins in other bacteria. As is typical for flagellins, the predicted FliC protein had well-conserved N- and C-terminal regions, separated by a divergent central domain. The predicted R. solanacearum FliM closely resembled motor switch proteins from other proteobacteria. Chromosomal mutants lacking fliC or fliM were created by replacing the genes with marked interrupted constructs. Since fliM is embedded in the fliLMNOPQR operon, the aphA cassette was used to make a nonpolar fliM mutation. Both mutants were completely nonmotile on soft agar plates, in minimal broth, and in tomato plants. The fliC mutant lacked flagella altogether; moreover, sheared-cell protein preparations from the fliC mutant lacked a 30-kDa band corresponding to flagellin. The fliM mutant was usually aflagellate, but about 10% of cells had abnormal truncated flagella. In a biologically representative soil-soak inoculation virulence assay, both nonmotile mutants were significantly reduced in the ability to cause disease on tomato plants. However, the fliC mutant had wild-type virulence when it was inoculated directly onto cut tomato petioles, an inoculation method that did not require bacteria to enter the intact host from the soil. These results suggest that swimming motility makes its most important contribution to bacterial wilt virulence in the early stages of host plant invasion and colonization.