Zhang X et al. (2014) World J Microbiol Biotechnol "High-level soluble expression of one model olfactory receptor (ODR-10) in Escherichia coli cell-free system."

Huang L, Xu Z, Cen P, Zhang X, Cai J, Du L, Sheng J

[World J Microbiol Biotechnol, 2014]

High-level production of G protein-coupled receptors (GPCRs) is usually difficult to achieve in heterologous cell systems. The inherent hydrophobicity of these receptors could cause aggregation and possible cytotoxicity. Cell-free (CF) expression has become a highly promising tool for the fast and efficient production of integral membrane proteins. Here we reported the CF production of an olfactory receptor from Caenorhabditis elegans, odorant response abnormal protein 10 (ODR-10), a member of GPCRs, using the Escherichia coli extracts. Different expression vectors were investigated and 175g/ml total ODR-10 was achieved with pIVEX2.4c. To obtain soluble ODR-10, different detergents and liposome with varied concentrations were respectively added into the CF system. High-level expression of soluble ODR-10 (150g/ml) was attained with the addition of 1.5% polyoxyethylene-(20)-cetyl-ether (Brij58) into the CF system. Furthermore, the yield of total ODR-10 was improved to 350g/ml by supplementing liposomes into the CF system, and the maximal concentration of the soluble receptor (102g/ml) was achieved in this liposome-assisted CF system. Both strategies produced ODR-10 efficiently by using CF system, and the direct reconstitution of the in vitro expressed receptor into liposomes will be preferred for its potential applications in many areas.

Penesyan A et al. (2015) PLoS One "Genetically and Phenotypically Distinct Pseudomonas aeruginosa Cystic Fibrosis Isolates Share a Core Proteomic Signature."

Kamath K, Penesyan A, Shathili AM, Venkatakrishnan V, Krisp C, Packer NH, Kumar SS, Paulsen IT, Molloy MP

[PLoS One, 2015]

The opportunistic pathogen Pseudomonas aeruginosa is among the main colonizers of the lungs of cystic fibrosis (CF) patients. We have isolated and sequenced several P. aeruginosa isolates from the sputum of CF patients and compared them with each other and with the model strain PAO1. Phenotypic analysis of CF isolates showed significant variability in colonization and virulence-related traits suggesting different strategies for adaptation to the CF lung. Genomic analysis indicated these strains shared a large set of core genes with the standard laboratory strain PAO1, and identified the genetic basis for some of the observed phenotypic differences. Proteomics revealed that in a conventional laboratory medium PAO1 expressed 827 proteins that were absent in the CF isolates while the CF isolates shared a distinctive signature set of 703 proteins not detected in PAO1. PAO1 expressed many transporters for the uptake of organic nutrients and relatively few biosynthetic pathways. Conversely, the CF isolates expressed a narrower range of transporters and a broader set of metabolic pathways for the biosynthesis of amino acids, carbohydrates, nucleotides and polyamines. The proteomic data suggests that in a common laboratory medium PAO1 may transport a diverse set of "ready-made" nutrients from the rich medium, whereas the CF isolates may only utilize a limited number of nutrients from the medium relying mainly on their own metabolism for synthesis of essential nutrients. These variations indicate significant differences between the metabolism and physiology of P. aeruginosa CF isolates and PAO1 that cannot be detected at the genome level alone. The widening gap between the increasing genomic data and the lack of phenotypic data means that researchers are increasingly reliant on extrapolating from genomic comparisons using experimentally characterized model organisms such as PAO1. While comparative genomics can provide valuable information, our data suggests that such extrapolations may be fraught with peril.

Carter ME et al. (2010) J Infect Dis "A subtype of a Pseudomonas aeruginosa cystic fibrosis epidemic strain exhibits enhanced virulence in a murine model of acute respiratory infection."

Winstanley C, Kadioglu A, Fothergill JL, Rajakumar K, Carter ME, Walshaw MJ

[J Infect Dis, 2010]

BACKGROUND: The Liverpool epidemic strain (LES) of Pseudomonas aeruginosa is a particularly successful cystic fibrosis (CF) pathogen associated with transmissibility, increased patient morbidity, and, unusually, infection of the non-CF parents of a patient with CF. METHODS: Using assays for virulence-associated exoproducts, biofilm formation, Caenorhabditis elegans killing, and a murine model of acute respiratory infection, we compared the pathogenic behavior of representatives of 4 subtypes of the LES, including LES431, an isolate associated with the infection of a parent without CF. RESULTS: The quorum-sensing-defective lasR mutant LES400 produced less exoproduct and had less C. elegans killing activity than the other LES subtypes, which were represented by LES431, LESB58, and LESB65. LES431 was deficient in biofilm formation, compared with the other LES sub-types. The LES subtypes displayed a range of virulence in the mouse model, with LES431 being by far the most virulent. The genome-sequenced isolate LESB58, effective at establishing infections in a rat model of chronic infection, was the least virulent subtype in the murine acute infection model. CONCLUSIONS: LES isolates display widely variable pathogenic characteristics. LES431, associated with transmission to the non-CF parent of a CF patient, represents a "hypervirulent" subtype more adapted to acute infections than chronic infections.

Lore NI et al. (2012) PLoS One "Cystic fibrosis-niche adaptation of Pseudomonas aeruginosa reduces virulence in multiple infection hosts."

Eberl L, Cigana C, Lore NI, Juhas M, Bragonzi A, De Fino I, Riva C, Schwager S

[PLoS One, 2012]

The opportunistic pathogen Pseudomonas aeruginosa is able to thrive in diverse ecological niches and to cause serious human infection. P. aeruginosa environmental strains are producing various virulence factors that are required for establishing acute infections in several host organisms; however, the P. aeruginosa phenotypic variants favour long-term persistence in the cystic fibrosis (CF) airways. Whether P. aeruginosa strains, which have adapted to the CF-niche, have lost their competitive fitness in the other environment remains to be investigated. In this paper, three P. aeruginosa clonal lineages, including early strains isolated at the onset of infection, and late strains, isolated after several years of chronic lung infection from patients with CF, were analysed in multi-host model systems of acute infection. P. aeruginosa early isolates caused lethality in the three non-mammalian hosts, namely Caenorhabditis elegans, Galleria mellonella, and Drosophila melanogaster, while late adapted clonal isolates were attenuated in acute virulence. When two different mouse genetic background strains, namely C57Bl/6NCrl and Balb/cAnNCrl, were used as acute infection models, early P. aeruginosa CF isolates were lethal, while late isolates exhibited reduced or abolished acute virulence. Severe histopathological lesions, including high leukocytes recruitment and bacterial load, were detected in the lungs of mice infected with P. aeruginosa CF early isolates, while late isolates were progressively cleared. In addition, systemic bacterial spread and invasion of epithelial cells, which were detected for P. aeruginosa CF early strains, were not observed with late strains. Our findings indicate that niche-specific selection in P. aeruginosa reduced its ability to cause acute infections across a broad range of hosts while maintaining the capacity for chronic infection in the CF host.

Harmer C et al. (2013) Microbiology "Modulation of gene expression by Pseudomonas aeruginosa during chronic infection in the adult cystic fibrosis lung."

Alnassafi K, Cordwell S, Rose B, Elkins M, Manos J, Hu H, Harmer C, Harbour C, Bye P, Triccas JA

[Microbiology, 2013]

Chronic Pseudomonas aeruginosa infection is the leading cause of morbidity and mortality in cystic fibrosis (CF) patients. P. aeruginosa isolates undergo significant transcriptomic and proteomic modulation as they adapt to the niche environment of the CF lung and the host defences. This study characterized the in vitro virulence of isogenic strain pairs of P. aeruginosa epidemic or frequent clonal complexes (FCCs) and non-epidemic or infrequent clonal complexes (IFCCs) that were collected 5-8 years apart from five chronically infected adult CF patients. Strains showed a significant decrease in virulence over the course of chronic infection using a Caenorhabditis elegans slow-killing assay and in phenotypic tests for important virulence factors. This decrease in virulence correlated with numerous differentially expressed genes such as oprG, lasB, rsaL and lecB. Microarray analysis identified a large genomic island deletion in the IFCC strain pair that included type three secretion system effector and fimbrial subunit genes. This study presents novel in vitro data to examine the transcriptomic profiles of sequentially collected P. aeruginosa from CF adults. The genes with virulence-related functions identified here present potential targets for new therapies and vaccines against FCCs and IFCCs.

Hilbi H et al. (2011) Environ Microbiol Rep "Legionella spp. outdoors: colonization, communication and persistence."

Harrison CF, Hoffmann C, Hilbi H

[Environ Microbiol Rep, 2011]

Bacteria of the genus Legionella persist in a wide range of environmental habitats, including biofilms, protozoa and nematodes. Legionellaceae are 'accidental' human pathogens that upon inhalation cause a severe pneumonia termed 'Legionnaires' disease'. The interactions of L. pneumophila with eukaryotic hosts are governed by the Icm/Dot type IV secretion system (T4SS) and more than 150 'effector proteins', which subvert signal transduction pathways and promote the formation of the replication-permissive 'Legionella-containing vacuole'. The Icm/Dot T4SS is essential to infect free-living protozoa, such as the amoeba Dictyostelium discoideum, as well as the nematode Caenorhabditis elegans, or mammalian macrophages. To adapt to different niches, L. pneumophila not only responds to exogenous cues, but also to endogenous signals, such as the -hydroxyketone compound LAI-1 (Legionella autoinducer-1). The long-term adaptation of Legionella spp. is based on extensive horizontal DNA transfer. In fact, Legionella spp. have acquired canonical 'genomic islands' of prokaryotic origin, but also a number of eukaryotic genes. Since many aspects of Legionella virulence against environmental predators and immune phagocytes are similar, an understanding of Legionella ecology provides valuable insights into the pathogenesis of legionellaceae for humans.

Chen T et al. (2023) Int J Biol Macromol "Antioxidant potential evaluation of polysaccharides from Camellia oleifera Abel in vitro and in vivo."

Ding CB, Liu L, Tang M, Cao XH, Chen T, Yang HY, Huang Y, Zhao XR, Zhou LJ, Feng SL

[Int J Biol Macromol, 2023]

The extraction process, structural characterization and free radical scavenging ability of polysaccharides from Camellia oleifera have already been widely studied. However, the antioxidant activities are still lack of systematic experiments. In this study, we used Hep G2 cells and Caenorhabditis elegans to evaluate the antioxidant potential of polysaccharides that from C. oleifera flowers (P-CF), leaves (P-CL), seed cakes (P-CC) and fruit shells (P-CS). The results showed all these polysaccharides could protect cells from oxidative damage induced by t-BHP. The highest cell viabilities were 66.46 ± 1.36 % (P-CF), 55.2 ± 2.93 % (P-CL), 54.49 ± 1.29 % (P-CC) and 61.45 ± 1.67 % (P-CS), respectively. Studies have shown that four polysaccharides may protect cells from apoptosis by reducing ROS levels and maintaining MMP balance. Moreover, P-CF, P-CL, P-CC and P-CS increased the survival rate of C. elegans under thermal stress, which reduced the production of ROS by 56.1 ± 0.67 %, 59.37 ± 1.79 %, 16.63 ± 2.51 % and 27.55 ± 2.62 %, respectively. P-CF and P-CL showed stronger protective effects on C. elegans by increasing the nuclear entry rate of DAF-16 and stimulating the expression of SOD-3. Our study suggested that C. oleifera polysaccharides have the potential to develop into a natural supplement agent.

Dupont C et al. (2017) Front Microbiol "Highly Diversified Pandoraea pulmonicola Population during Chronic Colonization in Cystic Fibrosis."

Aujoulat F, Condom P, Jumas-Bilak E, Chiron R, Dupont C, Marchandin H

[Front Microbiol, 2017]

Several environmental bacteria are considered as opportunistic pathogens in cystic fibrosis (CF) and are able to persistently colonize the CF respiratory tract (CFRT). Beside Pseudomonas aeruginosa and Burkholderia cepacia complex, Pandoraea spp. are defined as pathogenic. During chronic colonization, adaptive evolution and diversified population have been demonstrated, notably for P. aeruginosa. However, the persistence of Pandoraea in the CFRT remains largely unexplored. We studied genomic and phenotypic traits of Pandoraea pulmonicola isolates successively recovered from the airways of a single CF patient and relate the results to qualitative and quantitative evolution of other cultivable pathogens and to patient clinical status. A total of 31 isolates recovered from 18 sputum samples over a 7-year period in a single CF patient were studied. Genome dynamics was assessed by pulsed-field gel electrophoresis, ERIC-PCR fingerprinting and 16S rRNA gene PCR-temporal temperature gel electrophoresis. Phenotypic features included antimicrobial susceptibility, motility, biofilm production, and virulence in Caenorhabditis elegans model. Variability was observed for all the characteristics studied leading to highly diversified patterns (24 patterns) for the 31 clonally related isolates. Some of these modifications, mainly genomic events were concomitantly observed with CFRT microbiota composition shifts and with severe exacerbations. The diversity of P. pulmonicola population studied, observed for isolates recovered from successive samples but also within a sample suggested that existence of a diversified population may represent a patho-adaptive strategy for host persistence in the heterogeneous and fluctuating CFRT environment.

Gimenez-Pardo C et al. (1999) Journal of Helminthology "Proteolytic activity in Caenorhabditis elegans: Soluble and insoluble fractions."

Rodriquez-Caabeiro F, Gimenez-Pardo C, de Armas-Serra C, Vazquez-Lopez C

[Journal of Helminthology, 1999]

Proteolytic activities of soluble and insoluble fractions of the free-living soil nematode Caenorhabditis elegans were measured across a range of substrates, temperatures and pH conditions. Several protease inhibitors were also tested under these conditions. Results of these studies indicate that proteolytic activity is present in cytosolic (CF) and non-cytosolic (NCF) fractions of C. elegans extracts at every condition of pH, temperature and buffer assayed. On the other hand, the use of different protease inhibitors demonstrated the existence of exo and endoproteases types in CF as well as NCF. Moreover our results show that the use of two protease inhibitor mixed types proposed by several authors are not enough to avoid this lytic activity when homogenates of this nematode are employed in biochemical assays.

Bird AF (1979) Journal of Nematology "A method of distinguishing between living and dead nematodes by enzymatically induced fluorescence."

Bird AF

[Journal of Nematology, 1979]

Since it is sometimes difficult to distinguish between living and dead nematodes, dyes are used, such as New Blue R, Chrysoidin, Eosin-Y, and several fluorochromes, with varied success. A method is described here that is rapid (results in 15 min) and has a mechanism of staining that is understood. The technique was described first by Rotman and Papermaster, working with living mammalian cells, and later by Heslop-Harrison and Heslop-Harrison, working with plant material. It takes advantage of the presence of esterases which hydrolyse nonfluorescent fatty acid esters of fluorescein to yield fluorescein, which accumulates and is detectable by its fluorescence. Since esterases are known to be present in quantity in nematodes, concentrated principally in the nervous system, male spicules, and gut, this technique seemed worth testing as a rapid means of distinguishing between living and dead nematodes.