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.

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.

Moreira AS et al. (2016) Virulence "Variation of Burkholderia cenocepacia virulence potential during cystic fibrosis chronic lung infection."

Ramos CG, Pinto-de-Oliveira A, Fialho AM, Moreira AS, Leitao JH, Coutinho CP, Dos Santos SC, Sa-Correia I, Sousa SA, Mil-Homens D

[Virulence, 2016]

During long-term lung infection in cystic fibrosis (CF) patients, Burkholderia cenocepacia faces multiple selective pressures in this highly stressful and fluctuating environment. As a consequence, the initial infecting strain undergoes genetic changes that result in the diversification of genotypes and phenotypes. Whether this clonal expansion influences the pathogenic potential is unclear. The virulence potential of 39 sequential B. cenocepacia (recA lineage IIIA) isolates, corresponding to three different clones retrieved from three chronically infected CF patients was compared in this study using the non-mammalian infection hosts Galleria mellonella and Caenorhabditis elegans. The isolates used in this retrospective study were picked randomly from selective agar plates as part of a CF Center routine, from the onset of infection until patients' death after 3.5 and 7.5years or the more recent isolation date after 12.5years of chronic infection. The infection models proved useful to assess virulence potential diversification, but for some isolates the relative values diverged in C. elegans and G. mellonella. Results also reinforce the concept of the occurrence of clonal diversification and co-existence of multiple phenotypes within the CF lungs, also with respect to pathogenicity. No clear trend of decrease (or increase) of the virulence potential throughout long-term infection was found but there is an apparent tendency for a clone/patient-dependent decrease of virulence when the G. mellonella model was used. The sole avirulent variant in both infection hosts was found to lack the small third replicon previously associated to virulence. Although possible, the in vivo loss of this nonessential megaplasmid was found to be a rare event (1 among a total of 64 isolates examined).

Kang, Donghoon et al. (2017) International Worm Meeting "Siderophore-mediated mitochondrial damage and host immune response."

Kang, Donghoon, Kirienko , Natalia, Kirienko, Daniel

[International Worm Meeting, 2017]

Pseudomonas aeruginosa is a re-emerging, multi-drug resistant pathogen responsible for a large number of hospital-acquired infections each year. Virulence in this organism largely requires siderophore pyoverdine, which plays a central role in growth and regulation of a number of toxins and other pathogenic determinants. Here, we show that pyoverdine also directly damages host tissues by removing iron, disrupting normal metabolic functions. We demonstrate that pyoverdine can translocate into C. elegans cells, even in the absence of live pathogen. Once inside, pyoverdine binds intracellular iron, making it unavailable to the host. Within a short period of time, iron-bound pyoverdine exits the cell, significantly depleting host iron. Since mitochondria are a rich source of iron within the host, we assayed mitochondrial health in C. elegans exposed to pyoverdine. Using mitochondrially-targeted GFP, we demonstrate that pyoverdine causes extensive mitochondrial disruption. Such damage is sufficient to severely compromise host ATP production. Finally, a PINK-1-GFP reporter demonstrated that pyoverdine-mediated damage activates mitophagy. Using transcriptional profiling, we investigated the host immune response to pyoverdine. Microarray data indicates that pyoverdine exposure triggers a SKN-1 independent detoxification response that resembles a response to hypoxia. In contrast, C. elegans genes upregulated by pyoverdine bear little resemblance to those activated by P. aeruginosa in slow kill assays or infection with Yersinia pestis and Candida albicans. Cystic fibrosis (CF) patients are particularly sensitive to P. aeruginosa infections, and infection strongly correlates with pronounced downturn in patient prognosis., Despite the general importance of pyoverdine in P. aeruginosa virulence, its role in CF pathology is controversial. We used a pair of humanized pgp-3/CFTR alleles (one intact, one missing F508, which recapitulates the most common genetic lesion causing CF) to assess the impact of this allele on pyoverdine sensitivity. Surprisingly, the CF allele of pgp-3/CFTR dramatically increases sensitivity to pyoverdine damage. Our study demonstrates a mechanism for pyoverdine toxicity for the first time. We show that pyoverdine causes substantial mitochondrial damage, and that host activates a specific immune response to this damage. Finally, our data suggest that CF patients may have a previously unappreciated hypersensitivity to pyoverdine damage.