Proteome analysis reveals adaptation of Pseudomonas aeruginosa to the cystic fibrosis lung environment

Pseudomonas aeruginosa is known for the chronic lung colonization of cystic fibrosis (CF) patients in addition to eye, ear and urinary tract infections. With the underlying disease CF patients are predisposed to P. aeruginosa chronic lung infection, which leads to morbidity and mortality. In this study, we compared the protein expression profile of a CF lung-adapted P. aeruginosa strain C with that of the burn-wound isolate PAO. Differentially expressed proteins from the whole-cell, membrane, periplasmic as well as extracellular fraction were identified. The whole-cell proteome of strain C showed down-regulation of several proteins involved in amino acid metabolism, fatty acid metabolism, energy metabolism and adaptation leading to a highly distinct proteome pattern for strain C in comparison to PAO. Analysis of secreted proteins by strain C compared to PAO revealed differential expression of virulence factors under non-inducing conditions. The membrane proteome of strain C showed modulation of the expression of porins involved in nutrient and antibiotic influx. The proteome of the periplasmic space of strain C showed retention of elastase despite that the equal amounts were secreted by strain C and PAO. Altogether, our results elucidate adaptive strategies of P. aeruginosa towards the nutrient-rich CF lung habitat during the course of chronic colonization.     

Outer membrane antigens of mucoid Pseudomonas aeruginosa isolated directly from the sputum of a cystic fibrosis patient

Abstract The antigenicity of the outer membrane components of mucoid Pseudomonas aeruginosa directly isolated from the sputum of a cystic fibrosis patient and those of the same isolate cultivated under iron-depleted conditions in the presence of sub-in-hibitory concentrations of piperacillin and/or tobramycin was investigated by immunoblotting using the patient's own serum. The results indicated that iron-regulated membrane proteins as well as other major outer membrane proteins were antigenic and recognised by the patient's serum. The antibiotics used profoundly influenced the surface antigen pattern.     

The puzzling construction of the conidial outer layer of Aspergillus fumigatus

If the mycelium of Aspergillus fumigatus is very short‐lived in the laboratory, conidia can survive for years. This survival capacity and extreme resistance to environmental insults is a major biological characteristic of this fungal species. Moreover, conidia, which easily reach the host alveola, are the infective propagules. Earlier studies have shown the role of some molecules of the outer conidial layer in protecting the fungus against the host defense. The outer layer of the conidial cell wall, directly in contact with the host cells, consists of α‐(1,3)‐glucan, melanin, and proteinaceous rodlets. This study is focused on the global importance of this outer layer. Single and multiple mutants without one to three major components of the outer layer were constructed and studied. The results showed that the absence of the target molecules resulting from multiple gene deletions led to unexpected phenotypes without any logical additivity. Unexpected compensatory cell wall surface modifications were indeed observed, such as the synthesis of the mycelial virulence factor galactosaminogalactan, the increase in chitin and glycoprotein concentration or particular changes in permeability. However, sensitivity of the multiple mutants to killing by phagocytic host cells confirmed the major importance of melanin in protecting conidia.     

烟曲霉细胞壁的组分、组装及其功能概述

环境中普遍存在的腐生丝状真菌烟曲霉Aspergillus fumigatus在免疫功能低下或缺陷的人群中可引起多种急慢性疾病,包括致死率很高的侵袭性曲霉病。细胞壁作为真菌的细胞外骨架结构不仅起维持细胞形状、保护细胞抵抗外界压力等作用,在病原真菌极性生长、入侵新的生态域、启动宿主免疫反应中也起重要作用。细胞壁组分还是真菌感染的分子诊断基础和开发抗真菌药物的理想靶标。近几十年来烟曲霉细胞壁的遗传、生物化学及免疫学方向的研究使其成为研究真菌细胞壁的模式真菌。本文主要概述烟曲霉细胞壁的组分、分子组装机制及其在真菌生存和感染中的作用,并对未来研究方向提出了展望。     

Molecular arrangement between multivalent glycocluster and Pseudomonas aeruginosa LecA (PA‐IL) by atomic force microscopy: influence of the glycocluster concentration

New therapeutics strategy against cystic fibrosis seeks to prevent the adhesion of the bacterium Pseudomonas aeruginosa (PA) on the epithelial cells in the lungs. One of the factors that induces the adhesion is the interaction between natural glycocluster present on the cells and lectins such as the PA lectin LecA (PA‐IL) present on the bacterium. By introducing synthetic glycoclusters with a great affinity with the lectin PA‐IL, the adhesion can be prevented. In this study, we characterized, by atomic force microscopy, the interaction between a tetra‐galactosylated glycocluster and the PA‐IL lectin for high concentration of lectins (2.5 μM).We showed that the strong lectin/lectin interaction is reduced even for low concentration of glycoclusters (1 for 20 000 lectins). We assumed that it is due to the tensioactive behavior of the glycoclusters. It was shown that the arrangement of the created complexes induced different structures evolving from one‐dimensional elongated aggregates to two‐dimensional compact islands when increasing the glycocluster concentration. This evolution can be interpreted as the predominance of the glycocluster/lectin interaction. Copyright © 2013 John Wiley & Sons, Ltd.     

Bacterial cis-2-unsaturated fatty acids found in the cystic fibrosis airway modulate virulence and persistence of Pseudomonas aeruginosa

There is an increasing appreciation of the polymicrobial nature of many bacterial infections such as those associated with cystic fibrosis (CF) and of the potentially important role for interspecies interactions in influencing both bacterial virulence and response to therapy. Patients with CF are often co-infected with Pseudomonas aeruginosa and other pathogens including Burkholderia cenocepacia and Stenotrophomonas maltophilia. These latter bacteria produce signal molecules of the diffusible signal factor (DSF) family, which are cis-2-unsaturated fatty acids. We have previously shown by in vitro studies that DSF from S. maltophilia leads to altered biofilm formation and increased resistance to antibiotics by P. aeruginosa; these responses of P. aeruginosa require the sensor kinase PA1396. Here we show that DSF signals are present in sputum taken from patients with CF. Presence of these DSF signals was correlated with patient colonization by S. maltophilia and/or B. cenocepacia. Analysis of 50 clinical isolates of P. aeruginosa showed that each responded to the presence of synthetic DSF by increased antibiotic resistance and these strains demonstrated little sequence variation in the PA1396 gene. In animal experiments using CF transmembrane conductance regulator knockout mice, the presence of DSF promoted P. aeruginosa persistence. Furthermore, antibiotic resistance of P. aeruginosa biofilms grown on human airway epithelial cells was enhanced in the presence of DSF. Taken together, these data provide substantial evidence that interspecies DSF-mediated bacterial interactions occur in the CF lung and may influence the efficacy of antibiotic treatment, particularly for chronic infections involving persistence of bacteria.     

青蒿琥酯抗蜡螟烟曲霉感染自噬机制初步研究

目的 探究青蒿琥酯在蜡螟感染烟曲霉后,对蜡螟的自噬相关蛋白的表达影响。方法 用一定量的烟曲霉的活化孢子感染蜡螟,经过1 h,用青蒿琥酯注射一组蜡螟,两性霉素B注射一组蜡螟,剩余的作为感染组。12 h后,取各组蜡螟进行病理切片染色,观察各组的病理情况;取各组的蜡螟的淋巴液,收集各组的孢子,用真菌活性检测试剂盒检测孢子活性并将淋巴细胞分离、裂解,离心取上清,用Western-blot法检测上清液中的Dectin-1、ROS、LC3Ⅱ的表达水平。结果 青蒿琥酯注射组的蜡螟病理切片中的孢子比感染组数量少且聚集在一起,未长出菌丝,而体外分离的真菌孢子,青蒿琥酯组的活性明显受到抑制。经Western-blot显示青蒿琥酯能增强淋巴细胞Dectin-1、ROS、LC3Ⅱ的表达。结论 青蒿琥酯可通过抑制烟曲霉的活性和增强蜡螟淋巴细胞的自噬水平,对抗蜡螟烟曲霉感染。     

Evidence for genetic differentiation and variable recombination rates among Dutch populations of the opportunistic human pathogen Aspergillus fumigatus

As the frequency of antifungal drug resistance continues to increase, understanding the genetic structure of fungal populations, where resistant isolates have emerged and spread, is of major importance. Aspergillus fumigatus is an ubiquitously distributed fungus and the primary causative agent of invasive aspergillosis (IA), a potentially lethal infection in immunocompromised individuals. In the last few years, an increasing number of A. fumigatus isolates has evolved resistance to triazoles, the primary drugs for treating IA infections. In most isolates, this multiple-triazole-resistance (MTR) phenotype is caused by mutations in the cyp51A gene, which encodes the protein targeted by the triazoles. We investigated the genetic differentiation and reproductive mode of A. fumigatus in the Netherlands, the country where the MTR phenotype probably originated, to determine their role in facilitating the emergence and distribution of resistance genotypes. Using 20 genome-wide neutral markers, we genotyped 255 Dutch isolates including 25 isolates with the MTR phenotype. In contrast to previous reports, our results show that Dutch A. fumigatus genotypes are genetically differentiated into five distinct populations. Four of the five populations show significant linkage disequilibrium, indicative of an asexual reproductive mode, whereas the fifth population is in linkage equilibrium, indicative of a sexual reproductive mode. Notably, the observed genetic differentiation among Dutch isolates does not correlate with geography, although all isolates with the MTR phenotype nest within a single, predominantly asexual, population. These results suggest that both reproductive mode and genetic differentiation contribute to the structure of Dutch A. fumigatus populations and are probably shaping the evolutionary dynamics of drug resistance in this potentially deadly pathogen.     

Adhesion of Pseudomonas aeruginosa,Achromobacter xylosoxidans,Delftia acidovorans,Stenotrophomonas maltophilia to contact lenses under the influence of an artificial tear solution

Abstract

Corneal infection is a devastating sight-threatening complication that is associated with contact lens (CL) wear, commonly caused by Pseudomonas aeruginosa. Lately, Achromobacter xylosoxidans, Delftia acidovorans, and Stenotrophomonas maltophilia have been associated with corneal infection. This study investigated the adhesion of these emerging pathogens to CLs, under the influence of an artificial tear solution (ATS) containing a variety of components commonly found in human tears. Two different CL materials, etafilcon A and senofilcon A, either soaked in an ATS or phosphate buffered saline, were exposed to the bacteria. Bacterial adhesion was investigated using a radio-labeling technique (total counts) and plate count method (viable counts). The findings from this study revealed that in addition to P. aeruginosa, among the emerging pathogens evaluated, A. xylosoxidans showed an increased propensity for adherence to both CL materials and S. maltophilia showed lower viability. ATS influenced the viable counts more than the total counts on CLs.     

Use of artificial sputum medium to test antibiotic efficacy against Pseudomonas aeruginosa in conditions more relevant to the cystic fibrosis lung

There is growing concern about the relevance of in vitro antimicrobial susceptibility tests when applied to isolates of P. aeruginosa from cystic fibrosis (CF) patients. Existing methods rely on single or a few isolates grown aerobically and planktonically. Predetermined cut-offs are used to define whether the bacteria are sensitive or resistant to any given antibiotic. However, during chronic lung infections in CF, P. aeruginosa populations exist in biofilms and there is evidence that the environment is largely microaerophilic. The stark difference in conditions between bacteria in the lung and those during diagnostic testing has called into question the reliability and even relevance of these tests. Artificial sputum medium (ASM) is a culture medium containing the components of CF patient sputum, including amino acids, mucin and free DNA. P. aeruginosa growth in ASM mimics growth during CF infections, with the formation of self-aggregating biofilm structures and population divergence. The aim of this study was to develop a microtitre-plate assay to study antimicrobial susceptibility of P. aeruginosa based on growth in ASM, which is applicable to both microaerophilic and aerobic conditions. An ASM assay was developed in a microtitre plate format. P. aeruginosa biofilms were allowed to develop for 3 days prior to incubation with antimicrobial agents at different concentrations for 24 hours. After biofilm disruption, cell viability was measured by staining with resazurin. This assay was used to ascertain the sessile cell minimum inhibitory concentration (SMIC) of tobramycin for 15 different P. aeruginosa isolates under aerobic and microaerophilic conditions and SMIC values were compared to those obtained with standard broth growth. Whilst there was some evidence for increased MIC values for isolates grown in ASM when compared to their planktonic counterparts, the biggest differences were found with bacteria tested in microaerophilic conditions, which showed a much increased resistance up to a > 128 fold, towards tobramycin in the ASM system when compared to assays carried out in aerobic conditions. The lack of association between current susceptibility testing methods and clinical outcome has questioned the validity of current methods. Several in vitro models have been used previously to study P. aeruginosa biofilms. However, these methods rely on surface attached biofilms, whereas the ASM biofilms resemble those observed in the CF lung. In addition, reduced oxygen concentration in the mucus has been shown to alter the behavior of P. aeruginosa and affect antibiotic susceptibility. Therefore using ASM under microaerophilic conditions may provide a more realistic environment in which to study antimicrobial susceptibility.     

Susceptibility of compound 48/80-sensitized Pseudomonas aeruginosa to the hydrophobic biocide triclosan

Pseudomonas aeruginosa is intrinsically resistant to the hydrophobic biocide triclosan, and yet it can be sensitized to low concentrations by permeabilization of the outer membrane using compound 48/80. A selective plating assay revealed that compound 48/80-permeabilized YM64, a triclosan-recognizing efflux pump-deficient variant, was unable to initiate growth on a medium containing triclosan. Macrobroth dilution assay data revealed that treatment with compound 48/80 synergistically decreased minimal inhibitory concentrations of the hydrophobic antibacterial agents rifamycin SV and chloramphenicol for all cell envelope variant strains examined. A low concentration of triclosan exerted a transient bactericidal effect on permeabilized wild-type strain PAO1, after which exponential growth resumed within 4 h. Permeabilized strain YM64 was unable to overcome the inhibition; yet, both strains remained susceptible to chloramphenicol for as long as 6 h, thereby suggesting that the outer membrane remained permeable to nonpolar compounds. These data support the notion that the transitory nature of compound 48/80 sensitization to triclosan in P. aeruginosa does not involve obviation of the hydrophobic diffusion pathway through the outer membrane. The inability of strain YM64 to overcome the synergistic effect of compound 48/80 and triclosan strongly suggests that triclosan-recognizing efflux pumps are involved in maintaining viability in wild-type cells whose outer membranes are otherwise compromised.     

In vitro lung delivery of bacteriophages KS4‐M and ΦKZ using dry powder inhalers for treatment of Burkholderia cepacia complex and Pseudomonas aeruginosa infections in cystic fibrosis

Aims: To determine the feasibility of formulating and aerosolizing powders containing bacteriophages KS4‐M and ΦKZ for lung delivery and treatment of pulmonary Burkholderia cepacia complex and Pseudomonas aeruginosa infections. Methods and Results: Endotoxin‐removed bacteriophages KS4‐M and ΦKZ were lyophilized in lactose/lactoferrin 60 : 40 w/w matrix and deagglomerated in a mixer mill (without beads) to formulate respirable powders. The powders were then aerosolized using an Aerolizer^® capsule inhaler. Mass median aerodynamic diameter (MMAD) of this inhalable aerosol was determined using Andersen cascade impactor at 60 l min^?1. Measured MMAD for both types of powders was 3·4 μm, and geometric standard deviation was 1·9–2·0. Viability of bacteriophages delivered distal to an idealized mouth‐throat replica was determined from bioassays of samples collected on filters placed after the idealized replica. As a percentage of inhaler load, amount of powder delivered distal to the mouth‐throat replica, which is a measure of lung delivery, was 33·7 ± 0·3% for KS4‐M and 32·7 ± 0·9% for ΦKZ. Titres collected downstream of the mouth throat were (3·4 ± 2·5) × 10⁶ PFU for KS4‐M with an Aerolizer capsule load of (9·8 ± 4·8) × 10⁶ and (1·9 ± 0·6) × 10⁷ for ΦKZ with an Aerolizer capsule load of (6·5 ± 1·9) × 10⁷. Conclusions: Bacteriophages KS4‐M and ΦKZ can be lyophilized without significant loss of viability in a lactose/lactoferrin 60 : 40 w/w matrix. The resulting powders can be aerosolized to deliver viable bacteriophages to the lungs. Significance and Impact of the Study: Development of lactoferrin‐based bacteriophage aerosol powders solidifies the ground for future research on developing novel formulations as an alternative to inhaled antibiotic therapy in patients with cystic fibrosis.     

Heterotypic communication as the promoter of phenotypic plasticity of cancer cells: The role of cancer secretomes

Cellular communication relies on signaling circuits whose statuses are mainly modulated by soluble biomolecules such as carbohydrates, lipids, proteins, and metabolites as well as extracellular vesicles (EVs). Therefore, the active secretion of such biomolecules is critical for both cell homeostasis and proper pathophysiological responses in a timely fashion. In this context, proteins are among the main modulators of such biological responses. Hence, profiling cell line secretomes may be an opportunity for the identification of “signatures” of specific cell types (i.e., stromal or metastatic cells) with important prognostic/therapeutic value. This review will focus on the biological implications of cell secretomes in the context of cancer, as well as their functional roles in shaping the tumoral microenvironment (TME) and communication status of participating cells.     

The influence of flow cell geometry related shear stresses on the distribution,structure and susceptibility of Pseudomonas aeruginosa 01 biofilms

The effects of non-uniform hydrodynamic conditions resulting from flow cell geometry (square and rectangular cross-section) on Pseudomonas aeruginosa 01 (PAO1) biofilm formation, location, and structure were investigated for nominally similar flow conditions using a combination of confocal scanning laser microscope (CSLM) and computational fluid dynamics (CFD). The thickness and surface coverage of PAO1 biofilms were observed to vary depending on the location in the flow cell and thus also the local wall shear stress. The biofilm structure in a 5:1 (width to height) aspect ratio rectangular flow cell was observed to consist mainly of a layer of bacterial cells with thicker biofilm formation observed in the flow cell corners. For square cross-section (1:1 aspect ratio) flow cells, generally thicker and more uniform surface coverage biofilms were observed. Mushroom shaped structures with hollow centers and wall breaks, indicative of ‘seeding’ dispersal structures, were found exclusively in the square cross-section tubes. Exposure of PAO1 biofilms grown in the flow cells to gentamicin revealed a difference in susceptibility. Biofilms grown in the rectangular flow cell overall exhibited a greater susceptibility to gentamicin compared to those grown in square flow cells. However, even within a given flow cell, differences in susceptibility were observed depending on location. This study demonstrates that the spanwise shear stress distribution within the flow cells has an important impact on the location of colonization and structure of the resultant biofilm. These differences in biofilm structure have a significant impact on the susceptibility of the biofilms grown within flow channels. The impact of flow modification due to flow cell geometry should be considered when designing flow cells for laboratory investigation of bacterial biofilms.     

The role of nitric oxide in the regulation of ion channels in airway epithelium: Implications for diseases of the lung

The human respiratory tract is covered with airway surface liquid (ASL) that is essential for lung defense and normal airway function. The quantity and composition of ASL is regulated by active ion transport across the airway epithelium. Abnormal electrolyte transport produces changes in ASL volume and composition, inhibits mucociliary clearance and leads to chronic infection of airway surfaces, as is evident in cystic fibrosis. Agonists that induce intracellular increases in cAMP or Ca²⁺ are generally associated with electrolyte secretion. While these mechanisms have been studied in detail for many years, modulation of ion channels by nitric oxide (NO) has emerged only recently as a significant determinant of ion channel function. NO is a physiological regulator of transepithelial ion movement and alterations of its generation and action may play an important role in the pathogenesis of lung disorders characterized by hypersecretion of ASL. This review presents the current understanding of regulation of airway epithelial ion channels by NO and attempts to highlight the importance of this regulation for lung defense.     

烟曲霉sho1、pbs2基因额外拷贝对几种应激能力的影响

目的构建烟曲霉额外拷贝菌株,了解额外拷贝烟曲霉sho 1、pbs 2基因能否增强菌株对高渗透压、过氧化氢(H2O2)、碱性pH、刚果红应激的抵抗能力,探讨HOG通路(high osmolarity glycerol pathway)参与的应激反应。方法用原生质体法构建分别含有烟曲霉sho 1、pbs 2基因的额外拷贝菌株,采用Real-time PCR方法检测额外拷贝株中sho 1、pbs 2的表达情况。观察并比较缺陷株、额外拷贝株对NaCl(1 mol/L)、H2O2(5 mmol/L)、刚果红(400 mg/L)及碱性pH(10.0)应激的反应。结果获得了含有烟曲霉sho 1、pbs 2基因的额外拷贝菌株MCsho1、MCpbs2,和含空白质粒的对照株Empty。额外拷贝株sho 1、pbs 2的表达水平增高,对NaCl(1 mol/L)、H2O2(5 mmol/L)、刚果红(400 mg/L)、碱性pH(10.0)应激的抵抗强于Empty。MCpbs2对这些应激的抵抗较MCsho1更显著。烟曲霉缺陷株△sho 1、△pbs 2对NaCl(1 mol/L)、H2O2(5mmol/L)、碱性pH(10.0)的敏感性高于野生株AF293。△sho 1对刚果红(400 mg/L)的敏感性高于野生株,△pbs 2对刚果红的敏感性与野生株比,无显著差别。结论额外拷贝烟曲霉sho 1或pbs 2基因能增强菌株对高渗透压、氧化压力、刚果红、碱性pH应激的抵抗能力。