铜绿假单胞菌生物膜悬液和藻酸盐对小鼠巨噬细胞吞噬功能的影响

目的探讨铜绿假单胞菌（PA）生物膜和藻酸盐成分对小鼠巨噬细胞吞噬功能的影响。方法用具有生物膜成分的PA悬液分别感染肺部巨噬细胞缺乏小鼠和正常小鼠,比较组织中的细菌数量。提取小鼠腹腔巨噬细胞,藻酸盐作用后加入PA悬液,测定巨噬细胞对细菌的吞噬率。巨噬细胞经不同浓度的藻酸盐作用后,中性红法检测巨噬细胞吞噬能力。结果巨噬细胞缺乏组和对照组肺部组织的细菌数量分别为（4.16±3.36）×10^5/ml和（5.15±1.92）×10^5/ml,t=0.7211,P=0.483。生物膜细菌组的巨噬细胞吞噬率与对照组的吞噬率分别为（13.82±4.71）%和（42.73±11.00）%,Q=12.3231,P〈0.01。表明生物膜细菌组比对照组更能抵抗巨噬细胞的吞噬。加藻酸盐组的巨噬细胞吞噬率与对照组的吞噬率分别为（22.91±6.20）%和（42.73±11.00）%,Q=8.4465,P〈0.01。表明加藻酸盐组比对照组更能抵抗巨噬细胞的吞噬。当藻酸盐浓度为0、25、50、75、100、125、150μg/ml时,以吸光度A（540nm）值表示其巨噬细胞吞噬中性红的能力分别为：0.271±0.044、0.456±0.062、0.445±0.061、0.551±0.065、0.210±0.053、0.186±0.026、0.195±0.025。当藻酸盐≤75μg/ml时巨噬细胞吞噬中性红的能力增强,与0μg/ml组相比P〈0.05;当藻酸盐〉75μg/ml时巨噬细胞吞噬中性红的能力降低,与0μg/ml组相比P〈0.05。结论巨噬细胞有阻止PA入侵的作用。PA生物膜可以抑制巨噬细胞的吞噬。PA生物膜藻酸盐成分在〈75μg/ml时促进巨噬细胞的吞噬,而在较大剂量时抑制巨噬细胞的吞噬功能。     

Cholesterol-rich membrane rafts and Lyn are involved in phagocytosis during Pseudomonas aeruginosa infection

The mechanism of phagocytosis of pathogens remains to be fully characterized. We report a novel phagocytosis pathway for Pseudomonas aeruginosa, which is initiated by cholesterol-rich membrane rafts and is dependent on Lyn, primarily an immune regulator with both positive and negative roles. Blocking of Lyn or blocking of cholesterol synthesis significantly inhibited phagocytosis by alveolar macrophages. We found that Lyn, via Src homology 2 and 3 domains, bound to and then activated PI3K and Akt to regulate intracellular routing of the engulfed P. aeruginosa. Further analysis indicates that Lyn and raft components entered in phagosomes and late lysosomes. Finally, respiratory burst was dependent on Lyn and membrane rafts, as confirmed by small interfering RNA and dominant-negative strategies. Our investigations demonstrate that Lyn along with membrane rafts plays a fundamental role in phagocytosis by alveolar macrophages during infection.     

Contribution of macrophage secretory products to urovirulence of Pseudomonas aeruginosa

Macrophages form one of the first lines of defense on mucosal surfaces like urinary tract, providing protection against pathogens. These cells pour their secretory products, which include a cocktail of biomolecules, at the site of infection. In the present investigation, the effect of macrophage secretory products (MSPs) obtained after interaction of macrophages with Pseudomonas aeruginosa on the virulence of this organism in planktonic and biofilm cell mode was assessed employing a mouse model of ascending pyelonephritis. When urinary tract infection (UTI) was established with P. aeruginosa grown in the presence of 30% MSPs, the extent of pyelonephritis was enhanced. Of the two cell forms, biofilm cells had an edge over the planktonic cells with respect to in vivo virulence. The enhanced virulence of MSP-grown P. aeruginosa may be attributed to increased production of quorum-sensing systems as well as increased adherence to uroepithelial cells and evasion of phagocytosis. The results of the present study reveal that macrophages can play a key role during the course of UTI, not only through their phagocytic activity, but also through effects mediated by their secretory products. Utilization of MSPs by P. aeruginosa can have far-reaching consequences, including chronicity and recurrence of infections caused by this pathogen.     

Internalization and degradation of macrophage Fc receptors during receptor-mediated phagocytosis

Macrophage receptors for the Fc domain of immunoglobulin G (IgG) can mediate the efficient binding and phagocytosis of IgG-coated particles. After internalization, phagocytic vacuoles fuse with lysosomes, initiating the degradation of their contents. Using specific monoclonal and polyclonal antireceptor antibodies, we have now analyzed the internalization and fate of Fc receptors during the uptake of IgG- coated erythrocytes and erythrocyte ghosts by mouse peritoneal macrophages. Receptor-mediated phagocytosis led to the selective and largely irreversible removal of Fc receptors (greater than 50%) from the macrophage plasma membrane. The expression of several other plasma membrane proteins (including a receptor for complement), recognized by a series of antimacrophage monoclonal antibodies, was affected only slightly. Interiorized Fc receptors were rapidly and selectively degraded. This was demonstrated by a series of turnover studies in which Fc receptor was immunoprecipitated from lysates of 125I-labeled macrophages. These experiments were made possible by the development of a polyclonal rabbit antiserum, raised against isolated Fc receptor, which recognized the receptor even in the presence of bound ligand. In control cells, the receptor turned over with a t1/2 of approximately 10 h; after phagocytosis, greater than 50% of the receptors were degraded with a t1/2 of less than 2 h. The turnover of other unrelated plasma membrane proteins was unaffected (t1/2 of 18-23 h) under these conditions.     

Incorporation of bacterial peptidoglycan constituents into macrophage lipids during phagocytosis

It has previously been established that several glycopeptides of peptidoglycan origin are formed as a result of processing of Bacillus subtilis cell walls by the macrophage-like cell line RAW264. Although the formation of these glycopeptides could account for the humoral immune responses characteristic of bacterial peptidoglycans, their formation does not account for the cellular-mediated immune responses observed for water-in-oil emulsions of peptidoglycan or for lipophilic derivatives of glycopeptide fragments thereof. Therefore, the processing of peptidoglycan by macrophages was reexamined to establish whether the lipophilic derivative of any peptidoglycan-derived glycopeptide was formed. The experiments were performed by incubating B. subtilis cell walls radiolabeled in muramic acid, glucosamine, alanine, glutamic acid, and diaminopimelic acid residues in the presence of the macrophage-like cell line RAW264. The crude lipid fraction derived from the macrophages was further fractionated and analyzed, revealing the presence of two lipophilic glycopeptides that contained glucosamine, muramic acid, and alanine of bacterial origin.     

The Pseudomonas aeruginosa proteome during anaerobic growth

Isotope-coded affinity tag analysis and two-dimensional gel electrophoresis followed by tandem mass spectrometry were used to identify Pseudomonas aeruginosa proteins expressed during anaerobic growth. Out of the 617 proteins identified, 158 were changed in abundance during anaerobic growth compared to during aerobic growth, including proteins whose increased expression was expected based on their role in anaerobic metabolism. These results form the basis for future analyses of alterations in bacterial protein content during growth in various environments, including the cystic fibrosis airway.     

Defective phagocytosis and clearance of Pseudomonas aeruginosa in the lung following bone marrow transplantation

Bone marrow transplantation (BMT) is an important therapeutic option for a variety of malignant and nonmalignant disorders. Unfortunately, BMT recipients are at increased risk of infection, and in particular, pulmonary complications occur frequently. Although the risk of infection is greatest during the neutropenic period immediately following transplant, patients are still vulnerable to pulmonary infections even after neutrophil engraftment. We evaluated the risk of infection in this postengraftment period by using a well-established mouse BMT model. Seven days after syngeneic BMT, B6D2F(1) mice are no longer neutropenic, and by 3 wk, they demonstrate complete reconstitution of the peripheral blood. However, these mice remain more susceptible throughout 8 wk to infection after intratracheal administration of Pseudomonas aeruginosa; increased mortality in the P. aeruginosa-infected BMT mice correlates with increased bacterial burden in the lungs as well as increased systemic dissemination. This heightened susceptibility to infection was not secondary to a defect in inflammatory cell recruitment to the lung. The inability to clear P. aeruginosa in the lung correlated with reduced phagocytosis of the bacteria by alveolar macrophages (AMs), but not neutrophils, decreased production of TNF-alpha by AMs, and decreased levels of TNF-alpha and IFN-gamma in the bronchoalveolar lavage fluid following infection. Expression of the beta(2) integrins CD11a and CD11c was reduced on AMs from BMT mice compared with wild-type mice. Thus, despite restoration of peripheral blood count, phagocytic defects in the AMs of BMT mice persist and may contribute to the increased risk of infection seen in the postengraftment period.     

The exopolysaccharide alginate protects Pseudomonas aeruginosa biofilm bacteria from IFN-gamma-mediated macrophage killing

The ability of Pseudomonas aeruginosa to form biofilms and cause chronic infections in the lungs of cystic fibrosis patients is well documented. Numerous studies have revealed that P. aeruginosa biofilms are highly refractory to antibiotics. However, dramatically fewer studies have addressed P. aeruginosa biofilm resistance to the host's immune system. In planktonic, unattached (nonbiofilm) P. aeruginosa, the exopolysaccharide alginate provides protection against a variety of host factors yet the role of alginate in protection of biofilm bacteria is unclear. To address this issue, we tested wild-type strains PAO1, PA14, the mucoid cystic fibrosis isolate, FRD1 (mucA22+), and the respective isogenic mutants which lacked the ability to produce alginate, for their susceptibility to human leukocytes in the presence and absence of IFN-gamma. Human leukocytes, in the presence of recombinant human IFN-gamma, killed biofilm bacteria lacking alginate after a 4-h challenge at 37 degrees C. Bacterial killing was dependent on the presence of IFN-gamma. Killing of the alginate-negative biofilm bacteria was mediated through mononuclear cell phagocytosis since treatment with cytochalasin B, which prevents actin polymerization, inhibited leukocyte-specific bacterial killing. By direct microscopic observation, phagocytosis of alginate-negative biofilm bacteria was significantly increased in the presence of IFN-gamma vs all other treatments. Addition of exogenous, purified alginate to the alginate-negative biofilms restored resistance to human leukocyte killing. Our results suggest that although alginate may not play a significant role in bacterial attachment, biofilm development, and formation, it may play an important role in protecting mucoid P. aeruginosa biofilm bacteria from the human immune system.     

铜绿假单胞菌及L型诱导巨噬细胞凋亡的实验研究

目的研究铜绿假单胞菌（PA）及L型诱导巨噬细胞凋亡的能力,比较二者的差异。方法用生物素断端标记（TUNEL）法检测PA及L型感染巨噬细胞2、4、8、12、16和20h后各时间段的细胞凋亡率,Giemsa染色观察细胞凋亡情况,硝酸还原酶法检测培养液中一氧化氮（NO）的浓度变化。结果 PA及L型能诱导巨噬细胞发生凋亡,与对照组比较差异有统计学意义（P〈0.05）;L型诱导细胞的凋亡率弱于原菌（P〈0.05）;PA及L型感染组培养液NO浓度较对照组明显升高（P〈0.05）。结论 PA及L型可诱导巨噬细胞发生凋亡,L型较其原型诱导细胞凋亡的能力弱,NO可能在巨噬细胞凋亡中发挥一定作用。PA及L型可通过诱导巨噬细胞凋亡,发挥致病作用。     

Pseudomonas aeruginosa elastase provides an escape from phagocytosis by degrading the pulmonary surfactant protein-A

Pseudomonas aeruginosa is an opportunistic pathogen that causes both acute pneumonitis in immunocompromised patients and chronic lung infections in individuals with cystic fibrosis and other bronchiectasis. Over 75% of clinical isolates of P. aeruginosa secrete elastase B (LasB), an elastolytic metalloproteinase that is encoded by the lasB gene. Previously, in vitro studies have demonstrated that LasB degrades a number of components in both the innate and adaptive immune systems. These include surfactant proteins, antibacterial peptides, cytokines, chemokines and immunoglobulins. However, the contribution of LasB to lung infection by P. aeruginosa and to inactivation of pulmonary innate immunity in vivo needs more clarification. In this study, we examined the mechanisms underlying enhanced clearance of the ΔlasB mutant in mouse lungs. The ΔlasB mutant was attenuated in virulence when compared to the wild-type strain PAO1 during lung infection in SP-A^+/+ mice. However, the ΔlasB mutant was as virulent as PAO1 in the lungs of SP-A^-/- mice. Detailed analysis showed that the ΔlasB mutant was more susceptible to SP-A-mediated opsonization but not membrane permeabilization. In vitro and in vivo phagocytosis experiments revealed that SP-A augmented the phagocytosis of ΔlasB mutant bacteria more efficiently than the isogenic wild-type PAO1. The ΔlasB mutant was found to have a severely reduced ability to degrade SP-A, consequently making it unable to evade opsonization by the collectin during phagocytosis. These results suggest that P. aeruginosa LasB protects against SP-A-mediated opsonization by degrading the collectin.     

Flagellation of Pseudomonas aeruginosa during the cell division cycle

Flagellation of Pseudomonas aeruginosa during the cell division cycle was examined by scanning electron microscopy. A new flagellum grows on an old polar end located at the opposite position of the parental flagellum in the late stage of the cell cycle.     

2014—2016年某院铜绿假单胞菌耐药性监测

摘要：目的 分析铜绿假单胞菌的分布和耐药性变化,为临床防治铜绿假单胞菌感染提供依据。方法 收集成都大学附属医院2014—2016年所分离的铜绿假单胞菌,采用VITEK 2-Compact全自动细菌鉴定系统进行鉴定和药敏试验,采用WHONET 5.6软件对数据进行分析。结果 3年间共分离出1 945株铜绿假单胞菌,多重耐药铜绿假单胞菌分离率为34.2%（666/1945）。标本来源以呼吸道为主,占82.2%（1598/1945）。科室分布以呼吸内科最多,ICU其次。铜绿假单胞菌对头孢曲松和头孢噻肟的耐药率最高,均＞57.0%;对阿米卡星耐药率最低,为2.0%。3年来铜绿假单胞菌对17种抗生素的耐药率呈整体上升的趋势。结论 铜绿假单胞菌对头孢曲松、头孢噻肟、亚胺培南耐药率较高,对阿米卡星耐药率较低。铜绿假单胞菌的耐药率呈整体上升的趋势,应重视细菌耐药性的监测,以延缓耐药性产生、促进临床合理用药。     

Pseudomonas aeruginosa Psl polysaccharide reduces neutrophil phagocytosis and the oxidative response by limiting complement-mediated opsonization

Pseudomonas aeruginosa causes chronic lung infections in the airways of cystic fibrosis (CF) patients. Psl is an extracellular polysaccharide expressed by non-mucoid P. aeruginosa strains, which are believed to be initial colonizers. We hypothesized that Psl protects P. aeruginosa from host defences within the CF lung prior to their conversion to the mucoid phenotype. We discovered that serum opsonization significantly increased the production of reactive oxygen species (ROS) by neutrophils exposed to a psl-deficient mutant, compared with wild-type (WT) and Psl overexpressing strains (Psl(++)). Psl-deficient P. aeruginosa were internalized and killed by neutrophils and macrophages more efficiently than WT and Psl(++) variants. Deposition of complement components C3, C5 and C7 was significantly higher on psl-deficient strains compared with WT and Psl(++) bacteria. In an in vivo pulmonary competition assay, there was a 4.5-fold fitness advantage for WT over psl-deficient P. aeruginosa. Together, these data show that Psl inhibits efficient opsonization, resulting in reduced neutrophil ROS production, and decreased killing by phagocytes. This provides a survival advantage in vivo. Since phagocytes are critical in early recognition and control of infection, therapies aimed at Psl could improve the quality of life for patients colonized with P. aeruginosa.     

Interleukin-1 deficiency in combination with macrophage depletion increases susceptibility to Pseudomonas aeruginosa bacteremia

We evaluated the role of IL-1 during Pseudomonas aeruginosa bacteremia by intravenously injecting P. aeruginosa strain D4 into IL-1-deficient and WT mice. The two strains showed equivalent mortality rates. However, when the mice were pretreated with cyclophosphamide, bacteremia-induced mortality was significantly greater in the IL-1-deficient mice than in the WT mice ( P < 0.01). We then investigated the role of neutrophils and macrophages in protecting IL-1-deficient mice from bacteremia by administering anti-Gr-1 antibody or liposomes containing dichloromethylene diphosphonate, respectively. After P. aeruginosa inoculation survival was significantly lower in the macrophage-depleted IL-1-deficient mice than in the WT mice. In contrast, neutrophil depletion did not have this effect. Compared to the macrophage-depleted WT mice, the macrophage-depleted IL-1-deficient bacteremic mice had higher bacterial counts in various organs 48 and 72 hr post-infection. They also had lower TNF-α, IL-6, and INF-γ concentrations in their livers during the early phase of sepsis. Thus, IL-1 deficiency becomes disadvantageous during P. aeruginosa bacteremia when it is accompanied by immunosuppression, particularly when macrophage functions are seriously impaired.     

Pseudomonas aeruginosa exotoxin

Different P. aeruginosa strains have been found to differ in exotoxin synthesis. The strain isolated at the Mechnikov Research Institute for Vaccines and Sera (Moscow) and newly isolated cultures obtained from patients with the severe course of the infectious process have been found to possess the highest toxigenic activity and to synthesize exotoxins with the most complete set of pathogenically important antigens. The technological scheme for the production of stable exotoxin which can be used for the development of diagnostic, therapeutic and prophylactic preparations against Pseudomonas infections is proposed.     

Pseudomonas aeruginosa toxoid

P. aeruginosa adsorbed toxoid has been obtained. The stabilization of exotoxins and the content of proteases, hemolysin, lecithinase in their structure have been found to enhance the immunogenic potency of preparations which protect test animals from death caused by the experimental injections of toxins, homologous and heterologous to bacterial strains of different O-serogroups, into these animals. Antibodies neutralizing the lethal action of P. aeruginosa exotoxin have been detected in the blood sera of immunized animals.