Type 1 and type 2 responses to Leishmania major

Pseudomonas aeruginosa and Burkholderia cepacia cause destructive lung disease in cystic fibrosis (CF) patients. Both pathogens employ 'quorum sensing', i.e. cell-to-cell communication, via diffusible N-acyl-L-homoserine lactone (AHL) signal molecules, to regulate the production of a number of virulence determinants in vitro. However, to date, evidence that quorum sensing systems are functional and play a role in vivo is lacking. This study presents the first direct evidence for the presence of AHLs in CF sputum. A total of 42 samples from 25 CF patients were analysed using lux-based Escherichia coli AHL biosensors. AHLs were detected in sputum from patients colonised by P. aeruginosa or B. cepacia but not Staphylococcus aureus. Furthermore, using liquid chromatography-mass spectrometry and thin layer chromatography, we confirmed the presence of N-hexanoylhomoserine lactone and N-(3-oxododecanoyl)homoserine lactone respectively in sputum samples from patients colonised by P. aeruginosa.     

Induction of ornithine decarboxylase by treatment of guinea pig lymphocytes with phospholipase C

Treatment of guinea pig lymphocytes with Clostridium perfringens phospholipase C but not with Naja naja snake venom phospholipase A2 increased ornithine decarboxylase activity. The increase in ornithine decarboxylase activity was suppressed by actinomycin D or cycloheximide, suggesting that de novo syntheses of RNA and protein are necessary for the increase in the enzyme activity. These results suggest that the activation of phospholipase C rather than that of phospholipase A2 is responsible for induction of ornithine decarboxylase during lymphocyte transformation.     

铜绿假单胞菌中鼠李糖脂生物合成的研究进展*

鼠李糖脂因其具有环境友好和卓越的物理化学特性,而有望成为化学合成表面活性剂的替代物。近年来鼠李糖脂得到了广泛的研究,其目的是利用低价的可再生资源进行大规模生产,但目前的研究成果仍不足以选育出更具商业竞争力的鼠李糖脂过量合成菌株。为此,进一步理解鼠李糖脂生物合成的复杂基因调控网络,探索降低生产成本的发酵工艺势在必行。综述了铜绿假单胞菌中鼠李糖脂的生物合成途径、群体感应对主要基因的调控、鼠李糖脂在生物膜形成中所发挥的作用,以及发酵优化对鼠李糖脂产量的影响。有助于加深对鼠李糖脂生物合成的认识,为提高鼠李糖脂产量提供重要参考信息。     

Production of N-acyl-L-homoserine lactones by P. aeruginosa isolates from chronic lung infections associated with cystic fibrosis

The N-acyl-L-homoserine lactones (AHLs) produced by sequential Pseudomonas aeruginosa isolates from chronically infected patients with cystic fibrosis were analyzed by thin-layer chromatography. It is demonstrated that both the amounts and the types of molecules synthesized by isolates from patients who were monitored over periods of up to 11 years do not change significantly during chronic colonization. However, in the case of a patient who became co-infected with an AHL-producing Burkholderia cepacia strain a dramatic reduction in the amounts of AHLs produced by the co-residing P. aeruginosa isolates was observed.     

N-hexanoyl-L-homoserine lactone, a mediator of bacterial quorum-sensing regulation, exhibits plant-dependent stability and may be inactivated by germinating Lotus corniculatus seedlings

The half-life of N-hexanoyl-l-homoserine lactone (C6-HSL) was determined under various pH and temperature conditions, and in several plant environments. C6-HSL was sensitive to alkaline pH, a process that was also temperature-dependent. In addition, C6-HSL disappeared from plant environments, i.e. axenic monocot and dicot plants cultivated under gnotobiotic, hydroponic conditions, albeit with variable kinetics. The disappearance was rapid at the root system of legume plants such as clover or Lotus, and slow or non-existent at the root system of monocots such as wheat or corn. These variable kinetics were not dependent upon pH changes that may have affected the growth media of the plants. Furthermore, C6-HSL did not accumulate in the plant, and the plant did not produce inhibitors of the C6-HSL signal. HPLC analyses revealed that C6-HSL disappeared from the media, and hence, Lotus exhibited a natural C6-HSL inactivating ability. This ability was not specific for C6-HSL and allowed the degradation of other N-acyl-homoserine lactones such as 3-oxo-C6-HSL, 3-oxo-octanoyl-HSL and 3-oxo-decanoyl-HSL. Preliminary investigation revealed that the inactivating ability is temperature-dependant and possibly of enzymatic origin.     

Genotypic and phenotypic characterization of a biofilm-forming Serratia plymuthica isolate from a raw vegetable processing line

Recently, we isolated from a raw vegetable processing line a Serratia strain with strong biofilm-forming capacity and which produced N-acyl-L-homoserine lactones (AHLs). Within the Enterobacteriaceae, strains of the genus Serratia are a frequent cause of human nosocomial infections; in addition, biofilm formation is often associated with persistent infections. In the current report, we describe the detailed characterization of the isolate using a variety of genotypic and phenotypic criteria. Although the strain was identified as Serratia plymuthica on the basis of its small subunit ribosomal RNA (16S rRNA) gene sequence, it differed from the S. plymuthica type strain in production of pigment and antibacterial compounds, and in AHL production profile. Nevertheless, the identification as S. plymuthica could be confirmed by gyrB phylogeny and DNA:DNA hybridization.     

GeneChip expression analysis of the VqsR regulon of Pseudomonas aeruginosa TB

Two interlinked quorum sensing circuits, las and rhl, which control pathogenesis of Pseudomonas aeruginosa are further modulated by numerous regulators, including VqsR (virulence and quorum sensing regulator). High-density oligonucleotide microarrays were used to compare the global expression profile of a wild-type and VqsR mutant in ABC minimal medium. The expression of a large group of metabolic genes, ECF sigma factors as well as of many quorum-sensing genes previously not assigned to VqsR-regulon was found to be affected by the disruption of vqsR.     

Galectin-3 interacts with membrane lipids and penetrates the lipid bilayer

The precise mechanism by which galectin-3 and other cytosolic proteins that lack signal peptides are secreted is yet to be elucidated. In the present analyses, we determined that galectin-3, a beta-galactoside binding protein, can interact directly with membrane lipids in solid phase binding assays. More interestingly, we determined by spectrophotometric methods that it can spontaneously penetrate the lipid bilayer of liposomes in either direction. These findings suggest that galectin-3 on its own has the capacity to traverse the lipid bilayer. Whereas the situation is rather simplified in liposomes, the interaction of galectin-3 with the plasma membrane may involve cholesterol-rich membrane domains where galectin-3 can be concentrated and form multimers or interact covalently with other proteins.     

Microbial metabolomics: replacing trial-and-error by the unbiased selection and ranking of targets

Microbial production strains are currently improved using a combination of random and targeted approaches. In the case of a targeted approach, potential bottlenecks, feed-back inhibition, and side-routes are removed, and other processes of interest are targeted by overexpressing or knocking-out the gene(s) of interest. To date, the selection of these targets has been based at its best on expert knowledge, but to a large extent also on educated guesses and gut feeling. Therefore, time and thus money is wasted on targets that later prove to be irrelevant or only result in a very minor improvement. Moreover, in current approaches, biological processes that are not known to be involved in the formation of a specific product are overlooked and it is impossible to rank the relative importance of the different targets postulated. Metabolomics, a technology that involves the non-targeted, holistic analysis of the changes in the complete set of metabolites in the cell in response to environmental or cellular changes, in combination with multivariate data analysis (MVDA) tools like principal component discriminant analysis and partial least squares, allow the replacement of current empirical approaches by a scientific approach towards the selection and ranking of targets. In this review, we describe the technological challenges in setting up the novel metabolomics technology and the principle of MVDA algorithms in analyzing biomolecular data sets. In addition to strain improvement, the combined metabolomics and MVDA approach can also be applied to growth medium optimization, predicting the effect of quality differences of different batches of complex media on productivity, the identification of bioactives in complex mixtures, the characterization of mutant strains, the exploration of the production potential of strains, the assignment of functions to orphan genes, the identification of metabolite-dependent regulatory interactions, and many more microbiological issues.