Type III Secretion System and Virulence Markers Highlight Similarities and Differences between Human- and Plant-Associated Pseudomonads Related to Pseudomonas fluorescens and P. putida

Pseudomonas fluorescens is commonly considered a saprophytic rhizobacterium devoid of pathogenic potential. Nevertheless, the recurrent isolation of strains from clinical human cases could indicate the emergence of novel strains originating from the rhizosphere reservoir, which could be particularly resistant to the immune system and clinical treatment. The importance of type three secretion systems (T3SSs) in the related Pseudomonas aeruginosa nosocomial species and the occurrence of this secretion system in plant-associated P. fluorescens raise the question of whether clinical isolates may also harbor T3SSs. In this study, isolates associated with clinical infections and identified in hospitals as belonging to P. fluorescens were compared with fluorescent pseudomonads harboring T3SSs isolated from plants. Bacterial isolates were tested for (i) their genetic relationships based on their 16S rRNA phylogeny, (ii) the presence of T3SS genes by PCR, and (iii) their infectious potential on animals and plants under environmental or physiological temperature conditions. Two groups of bacteria were delineated among the clinical isolates. The first group encompassed thermotolerant (41°C) isolates from patients suffering from blood infections; these isolates were finally found to not belong to P. fluorescens but were closely related and harbored highly conserved T3SS genes belonging to the Ysc-T3SS family, like the T3SSs from P. aeruginosa. The second group encompassed isolates from patients suffering from cystic fibrosis; these isolates belonged to P. fluorescens and harbored T3SS genes belonging to the Hrp1-T3SS family found commonly in plant-associated P. fluorescens.     

Metabolisme lipidique chez l'Aspergillus versicolor (Vuill.) tiraboschi. Relations avec la biogenese de la sterig matocystine

Résumé L' Aspergillus versicolor est cultivé sur un milieu synthétique pendant 22 jours. Les productions de lipides et de stérigmatocystine sont comparées. Les acides gras des fractions neutres et polaires sont essentiellement: C 160, C 180, C 181 C 182, et C 183. Les quantités maximales de mycélium sec, de lipides neutres et de stérigmatocystine apparaissent respectivement au 4e, 7e et 20e jours. Une diminution de la teneur en lipides précède la phase de concentration maximale en métabolites secondaires du type polycéto-acide. Il semble que les lipides, et tout particulièrement l'acide palmitique, participent à la biogenèse de ces dérivés.

---

Summary Aspergillus versicolor is cultivated in a synthetic medium for 22 days. Bioproduction of lipids and sterigmatocystin are compared. The fatty acids of the neutral lipid and polar lipids fractions are mainly: C 160, C 180, C 181, C 182, C 183. Maximal yields of dry weight, neutral lipids and sterigmatocystin occur, respectively, on the 4th, the 7th and the 20th days. These results and their comparison with other works emphasize that a fall of concentration in lipids precedes the phase of highest concentration in secondary metabolites of polyketide type; it appears that fats and particularly palmitic acid are present in biogenesis of these derivatives.

     

Angiotensinase activity and angiotensin receptor binding in guinea pig aorta.

The angiotensinase (EC 3.4.99.3) activity of the subcellular fractions of guinea pig aorta has been studied in relation to their [14C]angiotensin binding capacity. The enzyme activity occurs in the following decreasing order: supernatant greater than plasma membrane fraction greater than 105 000 X g pellet greater than mitochondrial fraction. The specific binding of [14C]angiotensin to these fractions follows the same pattern. Pretreatment of the subcellular fractions at 47 degrees C for 20 min was performed in an attempt to differentiate binding of angiotensin to the pharmacological receptor from binding to the destroying enzymes. This procedure decreased the angiotensinase activity in the plasma membrane fraction only whereas the specific binding of [14C]angiotensin to this fraction was not significantly decreased, suggesting that the plasma membrane angiotensinase is a thermolabile enzyme.     

青海藏区B型沙眼衣原体的分离培养与鉴定

【目的】对青海藏区沙眼患者标本进行沙眼衣原体分离培养与鉴定。【方法】分别采集患者的单眼结膜和结膜囊拭子标本至1 mL样本保护培养基中。取50μL样本采用离心法感染BGM细胞,37°C培养72 h,连续传代3次,相差显微镜观察衣原体包涵体。对临床样本和分离株分别进行主要外膜蛋白基因ompA序列分析。【结果】共采集了45例活动性沙眼患者的115份临床样本,其中54份样本为ompA PCR阳性,15份样本为沙眼衣原体培养阳性。ompA分析发现,青海藏区沙眼衣原体有3个不同的同源ompA变异株,均为基因B型,都包含有一个泌尿生殖道型沙眼衣原体特有密码子。分离株QH111L和QH111R分别来自编号111患者的左眼和右眼样本,它们ompA基因的可变区有一个非同义碱基差异。该碱基变异仅存在于111号患者的左眼样本中,说明QH111L可能是新出现的ompA突变体。【结论】青海藏区的眼型沙眼衣原体流行株为基因B型,至少存在3个不同的ompA变异株。从青海藏区分离培养了15株眼型沙眼衣原体,发现同一患者的左右眼样本中的沙眼衣原体有不同ompA。本研究为研制沙眼疫苗和诊断试剂奠定了基础,也将有助于理解沙眼的进化和传播。     

Low tumor cell density environment yields survival advantage of tumor cells exposed to MTX in vitro

Stable resistance to methotrexate has been well characterized after prolonged treatment of the HT-29 colon cancer cell line, but the mechanism of cell survival at the early stages of the drug resistance process still remains unclear. Here, we demonstrate that human cancer cells in vitro are sensitive to methotrexate only above a critical cell culture density, which specifically coincides with their ability to deplete the extracellular nucleosides from a fully supplemented culture medium. At lower cell densities, extracellular nucleosides remain intact and allow salvage nucleotide synthesis that renders cells insensitive to the drug. Consistently, medium conditioned by cells seeded at standard cell densities sensitizes low cell density cultures. Extracellular nucleosides are the determinants of sensitivity because the latter effect can be mimicked with the use of inhibitors of nucleoside cellular import and reversed by supplying exogenous thymidine and hypoxanthine. Interestingly, treatment at a sensitizing cell density does not preclude the survival of less than 1% of the cells--which have no intrinsic resistance--owing to the inability of the dying cell population to condition the culture medium; this population thus survives indefinitely to continuous treatment by keeping adapted to a low cell number. This cell density-dependent adaptive process accounts for the initial steps of in vitro resistance to methotrexate (MTX) and provides a novel mechanistic insight into the cell population dynamics of cell survival and cell death during drug treatment.     

Crystal structures and proposed structural/functional classification of three protozoan proteins from the isochorismatase superfamily

We have determined the crystal structures of three homologous proteins from the pathogenic protozoans Leishmania donovani, Leishmania major, and Trypanosoma cruzi. We propose that these proteins represent a new subfamily within the isochorismatase superfamily (CDD classification cd004310). Their overall fold and key active site residues are structurally homologous both to the biochemically well-characterized N-carbamoylsarcosine-amidohydrolase, a cysteine hydrolase, and to the phenazine biosynthesis protein PHZD (isochorismase), an aspartyl hydrolase. All three proteins are annotated as mitochondrial-associated ribonuclease Mar1, based on a previous characterization of the homologous protein from L. tarentolae. This would constitute a new enzymatic activity for this structural superfamily, but this is not strongly supported by the observed structures. In these protozoan proteins, the extended active site is formed by inter-subunit association within a tetramer, which implies a distinct evolutionary history and substrate specificity from the previously characterized members of the isochorismatase superfamily. The characterization of the active site is supported crystallographically by the presence of an unidentified ligand bound at the active site cysteine of the T. cruzi structure.     

Lack of chemokine receptor CCR5 promotes murine fulminant liver failure by preventing the apoptosis of activated CD1d-restricted NKT cells

Fulminant liver failure (FLF) consists of a cascade of events beginning with a presumed uncontrolled systemic activation of the immune system. The etiology of FLF remains undefined. In this study, we demonstrate that CCR5 deficiency promotes the development of acute FLF in mice following Con A administration by preventing activated hepatic CD1d-restricted NKT cells (but not conventional T cells) from dying from activation-induced apoptosis. The resistance of CCR5-deficient NKT cells from activation-induced apoptosis following Con A administration is not due to a defective Fas-driven death pathway. Moreover, FLF in CCR5-deficient mice also correlated with hepatic CCR5-deficient NKT cells, producing more IL-4, but not IFN-gamma, relative to wild-type NKT cells. Furthermore, FLF in these mice was abolished by IL-4 mAb or NK1.1 mAb treatment. We propose that CCR5 deficiency may predispose individuals to the development of FLF by preventing hepatic NKT cell apoptosis and by regulating NKT cell function, establishing a novel role for CCR5 in the development of this catastrophic liver disease that is independent of leukocyte recruitment.     

Proteome signatures for stress and starvation in Bacillus subtilis as revealed by a 2-D gel image color coding approach

In this paper we have defined proteome signatures of Bacillus subtilis in response to heat, salt, peroxide, and superoxide stress as well as after starvation for ammonium, tryptophan, glucose, and phosphate using the 2-D gel-based approach. In total, 79 stress-induced and 155 starvation-induced marker proteins were identified including 50% that are not expressed in the vegetative proteome. Fused proteome maps and a color coding approach have been used to define stress-specific regulons that are involved in specific adaptative functions (HrcA for heat, PerR and Fur for oxidative stress, RecA for peroxide, CymR and S-box for superoxide stress). In addition, starvation-specific regulons are defined that are involved in the uptake or utilization of alternative nutrient sources (TnrA, sigmaL/BkdR for ammonium; tryptophan-activated RNA-binding attenuation protein for tryptophan; CcpA, CcpN, sigmaL/AcoR for glucose; PhoPR for phosphate starvation). The general stress or starvation proteome signatures include the CtsR, Spx, sigmaL/RocR, sigmaB, sigmaH, CodY, sigmaF, and sigmaE regulons. Among these, the Spx-dependent oxidase NfrA was induced by all stress conditions indicating stress-induced protein damages. Finally, a subset of sigmaH-dependent proteins (sporulation response regulator, YvyD, YtxH, YisK, YuxI, YpiB) and the CodY-dependent aspartyl phosphatase RapA were defined as general starvation proteins that indicate the transition to stationary phase caused by starvation.