Specific and non-specific mouse protection induced by different chemotypes of the Pseudomonas aeruginosa lipopolysaccharides

Abstract Lipopolysaccharides (LPS) of Pseudomonas aeruginosa were studied by the mouse active, cross-protection test. The primary structure of O-specific polysaccharides (O-repeating units) of different chemotypes was determined and their cross-protective activity demonstrated. Low doses of LPS (0.1–1 μg) stimulated chemotype-specific protection against P. aeruginosa in mice. This immunity was associated with the primary structure of the LPS and it lasted for 14 days after the first or second immunization. High doses of LPS (10–100 μg) induced cross-protection against P. aeruginosa in mice. The cross-protective capacity was caused evidently by the secondary structure or conformation of LPS molecule, i.e. by the common conformational protective determinant. This cross-protection lasted for only 5 days after the first or second immunization.     

Characterization of two Pseudomonas aeruginosa mutants with defective secretion of extracellular proteins and comparison with other mutants

We have isolated 2 new pleiotropic mutants of Pseudomonas aeruginosa strain PAO with defective secretion of extracellular proteins (Xcp mutants). One of these mutants was compared to 2 different, previously isolated secretion mutants. All had similar phenotypes and were unable to release at least 4 exoproteins (lipase, elastase, alkaline phosphatase, and phospholipase C), whilst alkaline protease was still secreted. The exoproteins appeared to be blocked in the periplasmic space. No difference in molecular weight was detected between cell-bound forms of elastase and alkaline phosphatase in the different mutants and the corresponding extracellular forms from the wild-type strain. Genetic mapping showed that the mutations were located in the 55′ region of the chromosome.     

Structure of a highly phosphorylated lipopolysaccharide core in the Delta algC mutants derived from Pseudomonas aeruginosa wild-type strains PAO1 (serogroup O5) and PAC1R (serogroup O3)

Lipopolysaccharides (LPS) were isolated from rough-type mutant strains of Pseudomonas aeruginosa (Delta algC) derived from wild-type strains PAO1 (serogroup O5) and PAC1R (serogroup O3). Structural studies of the LPS core region with a special focus on the phosphorylation pattern were performed by 2D NMR spectroscopy, including a 1H,(31)P HMQC-TOCSY experiment, MALDI-TOF MS, and Fourier-transform ion cyclotron resonance ESIMS using the capillary skimmer dissociation technique. Both LPS were found to contain two residues each of 3-deoxy-D-manno-oct-2-ulosonic acid (Kdo) and L-glycero-D-manno-heptose (Hep), one residue of N-(L-alanyl)-D-galactosamine and one O-carbamoyl group (Cm) on the distal Hep residue. The following structures of a tetrasaccharide trisphosphate from strain PAC1R Delta algC and that carrying an additional ethanolamine phosphate group (PEtN) from strain PAO1 Delta algC were elucidated: [carbohydrate structre: see text] where R=P in PAC1R Delta algC and PPEtN in PAO1 Delta algC. To our knowledge, in this work the presence of ethanolamine diphosphate is unambiguously confirmed and its position established for the first time in the LPS core of a rough-type strain of P. aeruginosa. In addition, the structure of the complete LPS core of wild-type strain P. aeruginosa PAO1 was reinvestigated and the position of the phosphorylation sites was revised.     

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。     

Characteristics of sheep-rumen isolates of Pseudomonas aeruginosa inhibitory to the growth of Escherichia coli O157

Screening facultative sheep-rumen bacteria which inhibit growth of Escherichia coli produced 11 strains of Pseudomonas aeruginosa. The isolates showed three different pulsed-field gel electrophoresis patterns and strains from different sheep produced pyocins that varied in strain specificity. Representative strains were resistant to ampicillin, methicillin, erythromycin, fusidic acid and augmentin, but not to tetracycline or nalidixic acid. Tested strains attached in large numbers to cultured rumen epithelial cells, potentially providing a means of survival in this ecosystem.     

Pseudomonas aeruginosa B-band lipopolysaccharide genes wbpA and wbpI and their Escherichia coli homologues wecC and wecB are not functionally interchangeable

The O antigen unit of Pseudomonas aeruginosa serotype O5 is a complex trisaccharide containing 2-acetamido-3-acetiminido-2, 3-dideoxy-beta-D-mannuronic acid, 2-acetimido-3-acetimido-2, 3-dideoxy-beta-D-mannuronic acid, and 2-acetimido-2, 6-deoxy-beta-D-galactosamine. Specific knockout mutations in the putative UDP-D-N-acetylglucosamine (UDP-D-GlcNAc) epimerase gene, wbpI, or the putative UDP-D-N-acetylmannosamine dehydrogenase gene, wbpA, resulted in strains that no longer produced B-band lipopolysaccharide, confirming the essential roles of these genes in B-band O antigen synthesis. Despite approximately 50% similarity of wbpI and wbpA to the Escherichia coli genes wecB (rffE) and wecC (rffD) involved in enterobacterial common antigen synthesis, cross-complementation experiments were not successful. These results imply that the P. aeruginosa UDP-D-GlcNAc precursor may be di-N-acetylated prior to further modification, preventing the E. coli enzymes from recognizing it as a substrate.     

Polycrylamide gel electrophoresis analysis of lipopolysaccharide from Pseudomonas aeruginosa growing planktonically and as biofilm

Abstract Lipopolysaccharide (LPS, endotoxin) was extracted from biofilm and planktonically grown monoagglutinable (1118) and polyagglutinable (258 and 15703) strains of Pseudomonas aeruginosa isolated from cystic fibrosis patients with chronic pulmonary infections. Analysis by polyacrylamide gel electrophoresis (PAGE) followed by immune-detection of LPS fractions showed an S-form appearance of strain 1118 and 258 with three distinct clusters of high molecular weight bands, whereas 15703 appeared semi-rough. LPS of semi-rough cells grown planktonically and as biofilm showed a very similar PAGE pattern; however, the core/lipid A R-LPS fraction was more prominent in biofilm-LPS than in planktonic-LPS extracted from the S-form bacteria (1118 and 258). The apparent change in LPS sub-unit components of the bacteria when grown as biofilm may reflect changes in the outer membrane structure that contribute to the altered physico-chemical properties of biofilm bacteria in foreign-device associated infections and chronic P. aeruginosa lung infection in cystic fibrosis patients.     

The antimicrobial mechanism of electrochemically activated water against Pseudomonas aeruginosa and Escherichia coli as determined by SDS-PAGE analysis

Aims:  To expose bacteria to anolyte and subsequently investigate the effect of anolyte on the protein profiles of treated bacteria.
Methods and Results:  Proteins were extracted from bacteria treated with different concentrations of anolyte and analysed using SDS-PAGE. Fewer and more faint protein bands were observed for concentrated halide anolyte treated bacteria when compared to untreated bacteria while extra protein bands were observed for bacteria exposed to dilute concentrations.
Conclusions:  The undiluted and the 10⁻¹ dilution of halide derived anolyte was effective in killing the test bacteria. Anolyte caused bacterial death by complete destruction of proteins or by causing oxidative stress which resulted in protein fragmentation.
Significance and Impact of the Study:  The results of this study provide information on the antimicrobial mechanism of anolyte on other bacteria for which the information is currently unavailable.     

Site-directed mutagenesis reveals key residue for O antigen chain length regulation and protein stability in Pseudomonas aeruginosa Wzz2

The production of preferred lipopolysaccharide O antigen chain lengths is important for the survival of pathogenic Gram-negative bacteria in different environments, yet how Wzz proteins regulate these lengths is not well understood. The Wzz2 proteins from two different serotype O11 Pseudomonas aeruginosa strains are responsible for the expression of different very long chain lengths despite high sequence homology. Site-directed mutagenesis was performed to determine whether a specific amino acid was responsible for this difference in chain length; the residue present in position 321 within the second predicted coiled-coil region was able to determine which chain length was produced. A panel of site-directed mutants introducing different amino acids at this position implicated that the charge of the amino acid affected chain length, with positively charged residues associated with shorter chain lengths. Expression data also suggested this site was important for overall stability of the protein because mutants predicted to disrupt proper folding of the α helix led to lower protein levels. Cross-linking studies found that Wzz2 proteins producing shorter chain lengths had more stable higher-order oligomers. Mapping residue 321 onto the solved Escherichia coli Wzz FepE crystal structure predicted it to be located within α helix 8, which participates in intermonomeric interactions. These data further support the observation that Wzz oligomerization is necessary for chain length regulating activity but also provide evidence that differences in complex stability or changes in the conformation of the oligomer can lead to shifts in the length of the O antigen side chain.     

Structural relationship between the polyagglutinable antigen and the core polysaccharide of Pseudomonas aeruginosa

Abstract It has been observed that each strain of the Pseudomonas aeruginosa species harbours the so-called polyagglutinable antigen (PA). Some strains may produce it in a form which is linked to the core moiety of lipopolysaccharide (LPS) and this type of PA can thus be detected by passive haemagglutination using the isolated LPS as coating antigen. Other strains synthesize PA exclusively in a free form, which is also coextractable with LPS, its presence can, however, be demonstrated by the haemagglutination inhibition test. From a polyagglutinable strain of P. aeruginosa an R-type LPS was isolated having the core-linked PA. This LPS preparation was highly immunogenic with regard to its PA moiety. The core-bound PA seems to exert an immunosuppression on the core region, hence, the polyagglutinable strains isolated from cystic fibrosis patients only engender anti-PA antibodies, whereas antibodies against both, side chain and core region of LPS, are not engendered. The mucoid exopolysacharides also contains the PA which could possibly play an important role in the patient by protecting P. aeruginosa cells against anti-PA antibodies.     

Quorum-sensing and siderophore biosynthesis in Pseudomonas aeruginosa: lasRllasI mutants exhibit reduced pyoverdine biosynthesis

Cell density-dependent gene expression in Pseudomonas aeruginosa is controlled, in part, by the quorum-sensing regulator LasR. lasR null mutants exhibited a reproducible 2-fold decrease in production of the catecholate-hydroxamate siderophore pyoverdine during grown under iron-limiting conditions. Similarly, lasI mutants defective in the biosynthesis of the autoinducer PAI-1 also exhibited a 2-fold decrease in pyoverdine production which could be largely restored upon addition of exogenous PAI-1. lasR mutants were not altered with respect to expression of the pvdD gene involved in the synthesis of the peptide portion of pyoverdine, indicating that some other pyoverdine biosynthetic gene(s) were affected by the LasRI status of the cell. This represents the first report of quorum-sensing regulation of siderophore production in bacteria and highlights the fact that cell density, while not an essential signal for pyoverdine expression, does enhance production of this siderophore.     

铜绿假单胞菌lasI,rhlI基因功能缺陷株的构建和生物学功能验证

构建铜绿假单胞菌lasI,rhlI基因功能缺陷株,为进一步阐明氦氧饱和高气压暴露条件诱导lasI,rhlI基因介导铜绿假单胞菌毒力调节的分子机制研究奠定基础。用双亲株接合转移法删除lasI,rhlI基因ORF编码区,通过RT-PCR方法验证目标基因编码序列mRNA的缺失;通过对细菌生长增殖能力、弹性蛋白酶代谢活性和细菌绿脓菌素分泌能力等表型的测定,验证目标基因编码序列缺失后的基因调节功能的缺陷。结果表明成功构建铜绿假单胞菌lasI,rhlI基因功能缺陷株,可作为进一步研究的基因工程菌。     

Actin cytoskeleton disruption by ExoY and its effects on Pseudomonas aeruginosa invasion

Three of the Type III-secreted effectors of Pseudomonas aeruginosa (ExoS, ExoT, and ExoY) each alter mammalian cell morphology in culture without causing a loss of cell viability. For ExoS and ExoT this property involves RhoGAP activity, and leads to actin cytoskeleton disruption and a reduced capacity for internalizing bacteria. ExoY does not possess RhoGAP activity. Instead, cell rounding depends upon its adenylate cyclase catalytic region. Since anti-phagocytic activities of ExoS and ExoT are associated with cell rounding and cytoskeleton disruption, we hypothesized that ExoY would also inhibit P. aeruginosa invasion of epithelial cells coinciding with adenylate cyclase-mediated cytoskeleton disruption. The results showed actin disruption of epithelial cells at 2 h post-infection associated with both adenylate cyclase-active ExoY and its catalytic mutant form ExoYK81M, and which coincided with inhibition of bacterial invasion (76% inhibition by ExoY, and 37% by ExoYK81M). Surprisingly, at 4h post-infection, neither form of ExoY inhibited invasion despite extensive actin disruption. These data suggest that ExoY, like ExoS and ExoT, contains more than one active domain affecting mammalian cell function. The data also suggest that cytoskeleton disruption does not necessarily predict invasion inhibitory activity, supporting the recently proposed model that P. aeruginosa internalization can proceed through more than one pathway.     

Salmonella Typhimurium strain expressing OprF‐OprI protects mice against fatal infection by Pseudomonas aeruginosa

Pseudomonas aeruginosa poses a major threat to human health and to the mink industry. Thus, development of vaccines that elicit robust humoral and cellular immunity against P. aeruginosa is greatly needed. In this study, a recombinant attenuated Salmonella vaccine (RASV) that expresses the outer membrane proteins fusion OprF_190–342‐OprI_21–83 (F1I2) from P. aeruginosa was constructed and the potency of this vaccine candidate assessed by measuring F1I2‐specific humoral immune responses upon vaccination through s.c. or oral routes. S.C. administration achieved higher serum IgG titers and IgA titers in the intestine and induced stronger F1I2‐specific IgG and IgA titers in lung homogenate than did oral administration, which resulted in low IgG titers and no local IgA production. High titers of IFN‐γ, IL‐4, and T‐lymphocyte subsets induced a mixed Th1/Th2 response in mice immunized s.c., indicating elicitation of cellular immunity. Importantly, when immunized mice were challenged with P. aeruginosa by the intranasal route 30 days after the initial immunization, s.c. vaccination achieved 77.78% protection, in contrast to 41.18% via oral administration and 66.67% via Escherichia coli‐expressed F1I2 (His‐F1I2) vaccination. These results indicate that s.c. vaccination provides a better protective response against P. aeruginosa infection than do oral administration and the His‐F1I2 vaccine.     

Purification and characterization of an alkaline protease from Pseudomonas aeruginosa MN1

An alkaline protease produced by Pseudomonas aeruginosa MN1, isolated from an alkaline tannery waste water, was purified and characterized. The enzyme was purified 25-fold by gel filtration and ion exchange chromatography to a specific activity of 82350 U mg⁻¹. The molecular weight of the enzyme was estimated to be 32000 daltons. The optimum pH and temperature for the proteolytic activity were pH 8.00 and 60°C, respectively. Enzyme activity was inhibited by EDTA suggesting that the preparation contains a metalloprotease. Enzyme activity was strongly inhibited by Zn²⁺, Cu²⁺ and Hg²⁺(5 mM), while Ca²⁺ and Mn²⁺ resulted in partial inhibition. The enzyme is different from other Pseudomonas aeruginosa alkaline proteases in its stability at high temperature; it retained more than 90% and 66% of the initial activity after 15 and 120 min incubation at 60°C. Journal of Industrial Microbiology & Biotechnology (2000) 24, 291–295. Received 09 June 1999/ Accepted in revised form 24 January 2000     

Effects of oxygen and nitrate on growth of Escherichia coli and Pseudomonas aeruginosa in the presence of organic solvents

Escherichia coli and Pseudomonas aeruginosa grown in the presence of certain harmful organic solvents become susceptible to these solvents during the cultivation. This susceptibility is conspicuous in the stationary phase of growth. The organic solvent tolerance levels of these microorganisms were maintained when the oxygen concentration was kept high. The tolerance levels were maintained also when these organisms were grown with nitrate present under anaerobic respiratory conditions. Received: 21 March, 1997 / Accepted: July 20, 1997