Molecular regulation of iron uptake in pseudomonas aeruginosa

The microbial demand for iron is often met by the elaboration of siderophores into the surrounding medium and expression of cognate outer membrane receptors for the ferric siderophore complexes. Conditions of iron limitation, such as those encountered in vivo, cause Pseudomonas aeruginosa to express two high-affinity iron-uptake systems based on pyoverdin and pyochelin. These systems will operate both in the organism's natural habitat, soil and water, where the solubility of iron at neutral pH is extremely low, and in the human host where the availability of free iron is too low to sustain bacterial growth due to the iron-binding glycoproteins transferrin and lactoferrin. Cross-feeding and radiolabelled iron uptake experiments demonstrated that pyoverdin biosynthesis and uptake were highly heterogeneous amongst P.aeruginosa strains, that growth either in the presence of pyoverdin or pyochelin resulted in induction of specific IROMPs, and that induction of iron uptake is siderophore-specific. The P.aeruginosa Tn5 mutant PH1 is deficient in ferripyoverdin uptake and resistant to pyocin Sa, suggesting that the site of interaction of pyocin Sa is a ferripyoverdin receptor. Additional Tn5 mutants appeared to exploit different strategies to achieve pyocin Sa-resistance, involving modifications in expression of pyoverdin-mediated iron uptake, indicating that complex regulatory systems exist to enable these organisms to compete effectively for iron. Modulation of expression of IROMPs prompted a study of the mechanism of uptake of a semi-synthetic C(7) α-formamido substituted cephalosporin BRL 41897A. Sensitivity to this agent correlated with expression of the 75 kDa ferri-pyochelin receptor and demonstrated the potential of high-affinity iron uptake systems for targeting of novel antibiotics. Studies with ferri-pyoverdin uptake-deficient mutant PH1 indicated that expression of outer membrane protein G (OprG), which is usually expressed under iron-rich conditions and repressed under iron-deficient conditions, was perturbed. Attempts were made to clone the oprG gene using a degenerate probe based on the N-terminal amino acid sequence. A strongly hybridising HindIll restriction fragment was cloned and sequenced, but failed to reveal an open reading frame correspondmg to OprG. However, there appears to be good evidence that a part of the gene codmg for the hydrophilic membrane-associated ATP-binding component of a hitherto uncharacterised periplasmic- binding-protein-dependent transport system has been isolated. The full organisation and sequence of the operon, and substrate for this putative transport system, are yet: to be elucidated,

Influence of iron on bacterial infections in leukaemia

The influence of iron metabolism, both on the invading bacterial pathogen and in the host is widespread and often appears to be crucial in determining the outcome of an infection. This study involved the investigation of leukaemia, a clinical disease where abnormal availability of iron may play a part in predisposing patients to bacterial infection. The iron status throughout a Gram-negative septicaemia and in 20 random, newly diagnosed leukaemic patients was assessed. The results revealed that the majority of the patients exhibited high serum iron levels and serum transferrin saturation often at 100%, with an inability to reduce the latter to within normal values during an infection episode. The antibody response to P.aeruginosa, E.coli and K.pneumoniae outer membrane protein (OMP) antigens were investigated by immunoblotting with sequential serum samples during infection in the leukaemic host. Antibodies to all the major OMPs, were observed, although recognition of iron-regulated membrane proteins (IRMPs) was in many cases weak. Results from the enzyme-linked immunosorbent assay indicated that in all patients antibody titre in response to infection was poor. Sub-MICs of mitomycin C significantly altered the surface characteristics of P.aeruginosa. The silver-stained SDS-PAGE gels of proteinase K digested whole cell lysates of strains PAO1, 6750, M7 and PAJ indicated that core LPS was affected in the presence of mitomycin C. In contrast, the rough strain AK1012 showed no observable differences. Results obtained using quantitative gas-liquid chromatographic analysis showed the amount of LPS fatty acids to be unaffected, however, the KDO and carbohydrate content in strains PAO1, 6750 and M7 under Fe+ and Fe- growth conditions were decreased by up to 4-fold in the presence of mitomycin C, indicating perturbed expression of LPS. The cell surface became significantly more hydrophobic in the P.aeruginosa strains, except AK1012 which was comparatively unaffected. The induction of protein G (OprG) in P.aeruginosa was found to be a sensitive indicator of media iron. The data indicated that expression of OprG can be modulated by growth rate/phase, availability of iron and by the presence of ciprofloxacin in the growth medium.

An emerging consensus on aquaporin translocation as a regulatory mechanism

Water passes through cell membranes relatively slowly by diffusion. In order to maintain water homeostasis, the rapid and specific regulation of cellular water flow is mediated by the aquaporin (AQP) family of membrane protein water channels. The wide range of tissues that are known to express AQPs is reflected by their involvement in many physiological processes and diseases; thirteen human AQPs have been identified to date and the majority are highly specific for water while others show selectivity for water, glycerol and other small solutes. Receptor mediated translocation, via hormone activation, is an established method of AQP regulation, especially for AQP2. There is now an emerging consensus that the rapid and reversible translocation of other AQPs from intracellular vesicles to the plasma membrane, triggered by a range of stimuli, confers altered membrane permeability thereby acting as a regulatory mechanism. This review examines the molecular components that may enable such AQP regulation; these include cytoskeletal proteins, kinases, calcium and retention or localization signals. Current knowledge on the dynamic regulation of sub-cellular AQP translocation in response to a specific trigger is explored in the context of the regulation of cellular water flow. © 2013 Informa UK, Ltd.

Oxidized phospholipids:their properties and interactions with proteins

Lipids are a highly diverse class of biomolecules, with an average eukaryotic cell estimated as containing at least 100,000 different species. The significance of this diversity is still poorly understood, yet it has become clear that lipids have critical regulatory as well as structural roles, varying from signaling (e.g. phosphatidylinositols, prostaglandins, platelet activating factor, ceramide) to the control of permeability properties of skin, for instance. An unprecedented discovery from recent efforts in lipidomics, aimed at the elucidation of the functional roles of lipids in cells, was the key role for lipid oxidation in cell behavior and pathology. More specifically, oxidized phospholipids (oxPL) have been shown to increase significantly in apoptosis as well as in inflammation and to be involved in several pathological conditions, such as atherosclerosis, cancer, inflammation, Alzheimer's and Parkinson's disease, as well as type 2 diabetes, with the detailed mechanisms remaining to be established. However, a coherent overall view of the causalities and mechanisms has been lacking, mainly because of insufficient understanding of the cellular as well as molecular level mechanisms. This Special Issue represents a focused, integrated interdisciplinary approach summarizing very recent leading edge developments in this emerging field with emphasis on lipid–protein interactions. The data now becoming available are paving the way to the development of improved diagnostics, therapies and preventive measures to combat the above diseases.

Joining S100 proteins and migration:for better or for worse, in sickness and in health

The vast diversity of S100 proteins has demonstrated a multitude of biological correlations with cell growth, cell differentiation and cell survival in numerous physiological and pathological conditions in all cells of the body. This review summarises some of the reported regulatory functions of S100 proteins (namely S100A1, S100A2, S100A4, S100A6, S100A7, S100A8/S100A9, S100A10, S100A11, S100A12, S100B and S100P) on cellular migration and invasion, established in both culture and animal model systems and the possible mechanisms that have been proposed to be responsible. These mechanisms involve intracellular events and components of the cytoskeletal organisation (actin/myosin filaments, intermediate filaments and microtubules) as well as extracellular signalling at different cell surface receptors (RAGE and integrins). Finally, we shall attempt to demonstrate how aberrant expression of the S100 proteins may lead to pathological events and human disorders and furthermore provide a rationale to possibly explain why the expression of some of the S100 proteins (mainly S100A4 and S100P) has led to conflicting results on motility, depending on the cells used. © 2013 Springer Basel.

A short regulatory domain restricts glycerol transport through yeast Fps1p

The controlled export of solutes is crucial for cellular adaptation to hypotonic conditions. In the yeast Saccharomyces cerevisiae glycerol export is mediated by Fpslp, a member of the major intrinsic protein (MIP) family ]of channel proteins. Here we describe a short regulatory domain that restricts glycerol transport through Fpslp. This domain is required for retention of cellular glycerol under hypertonic stress and hence acquisition of osmotolerance. It is located in the N-terminal cytoplasmic extension close to the first transmembrane domain. Several residues within that domain and its precise position are critical for channel control while the proximal residues 13-215 of the N-terminal extension are not required. The sequence of the regulatory domain and its position are perfectly conserved in orthologs from other yeast species. The regulatory domain has an amphiphilic character, and structural predictions indicate that it could fold back into the membrane bilayer. Remarkably, this domain has structural similarity to the channel forming loops B and E of Fpslp and other glycerol facilitators. Intragenic second-site suppressor mutations of the sensitivity to high osmolarity conferred by truncation of the regulatory domain caused diminished glycerol transport, confirming that elevated channel activity is the cause of the osmosensitive phenotype.

Cross-linking of cellular proteins by tissue transglutaminase during necrotic cell death:a mechanism for maintaining tissue integrity

Tissue transglutaminase (tTG) is a Ca2+-dependent enzyme which cross-links proteins via e(g-glutamyl)lysine bridges. There is increasing evidence that tTG is involved in wound repair and tissue stabilization, as well as in physiological mechanisms leading to cell death. To investigate the role of this enzyme in tissue wounding leading to loss of Ca2+ homoeostasis, we initially used a model involving electroporation to reproduce cell wounding under controlled conditions. Two cell models were used whereby tTG expression is regulated either by antisense silencing in ECV 304 cells or by using transfected Swiss 3T3 cells in which tTG expression is under the control of the tet regulatory system. Using these cells, loss of Ca2+ homoeostasis following electroporation led to a tTG-dependent formation of highly cross-linked proteinaceous shells from intracellular proteins. Formation of these structures is dependent on elevated intracellular Ca2+, but it is independent of intracellular proteases and is near maximal after only 20min post-wounding. Using labelled primary amines as an indicator of tTG activity within these 'wounded cells', we demonstrate that tTG modifies a wide range of proteins that are present in both the perinuclear and intranuclear spaces. The demonstration of entrapped DNA within these shell structures, which showed limited fragmentation, provides evidence that the high degree of transglutaminase cross-linking results in the prevention of DNA release, which may serve to dampen any subsequent inflammatory response. Comparable observations were shown when monolayers of cells were mechanically wounded by scratching. In this second model of cell wounding, redistribution of tTG activity to the extracellular matrix was also demonstrated, an effect which may serve to stabilize tissues post-trauma, and thus contribute to the maintenance of tissue integrity.

Role of Pseudomonas aeruginosa PA4157in Quorum Sensing and Iron Transport Regulation

Pseudomonas aeruginosa, a Gram-negative opportunistic pathogen, is a pnmary contributing factor responsible for the morbidity and mortality in patients with cystic fibrosis. One of the trademarks of P. aeruginosa is its ability to resist antibiotics. P. aeruginosa does so in part through the LysR-type transcription factor, AmpR. To identify additional members of the AmpR regulon, a new algorithm called iterative enhancement of motifs was used to identify putative AmpR binding sites upstream of open reading frames in the P. aeruginosa genome. The surprising primary hit of this analysis was the promoter of an uncharacterized open reading frame, P A 415 7. P A 415 7 is located upstream ofthefep operon, which is known to be involved in iron acquisition. PA4157 shares high homology to the IclR family of transcriptional regulators which are known to regulate quorum sensing (QS), an elaborate cell-cell communication signaling system that uses quoromones. We postulated two hypotheses: 1) AmpR regulation of QS genes is mediated by PA4157, and 2) PA4157 may be involved in iron acquisition. To address the role of P A 415 7 we generated an in-frame chromosomal deletion of P A 415 7 in P. aeruginosa PA01 (PA0 PA4157). We compared PA0 PA4157 with its parent strain P A0 1 for its ability to produce quoromones using Chromobacterium violaceum as an indicator strain and LasA proteases using Staphylococcus aureus. We also tested its role in virulence using a Caenorhabditis elegans killing assay. Growth in iron-deficient media was also examined to determine if P A4157 has a potential role in iron uptake regulation. Our preliminary results suggest that P A 415 7 is not involved in quorum sensing regulation but does seem to exert a negative regulatory effect on iron uptake in P. aeruginosa P A0 1.

Pseudomonas aeruginosa AMPR transcriptional regulatory network

In Enterobacteriaceae, the transcriptional regulator AmpR, a member of the LysR family, regulates the expression of a chromosomal β-lactamase AmpC. The regulatory repertoire of AmpR is broader in Pseudomonas aeruginosa, an opportunistic pathogen responsible for numerous acute and chronic infections including cystic fibrosis. Previous studies showed that in addition to regulating ampC, P. aeruginosa AmpR regulates the sigma factor AlgT/U and production of some quorum sensing (QS)-regulated virulence factors. In order to better understand the ampR regulon, the transcriptional profiles generated using DNA microarrays and RNA-Seq of the prototypic P. aeruginosa PAO1 strain with its isogenic ampR deletion mutant, PAOΔampR were analyzed. Transcriptome analysis demonstrates that the AmpR regulon is much more extensive than previously thought influencing the differential expression of over 500 genes. In addition to regulating resistance to β-lactam antibiotics via AmpC, AmpR also regulates non-β-lactam antibiotic resistance by modulating the MexEF-OprN efflux pump. Virulence mechanisms including biofilm formation, QS-regulated acute virulence, and diverse physiological processes such as oxidative stress response, heat-shock response and iron uptake are AmpR-regulated. Real-time PCR and phenotypic assays confirmed the transcriptome data. Further, Caenorhabditis elegans model demonstrates that a functional AmpR is required for full pathogenicity of P. aeruginosa. AmpR, a member of the core genome, also regulates genes in the regions of genome plasticity that are acquired by horizontal gene transfer. The extensive AmpR regulon included other transcriptional regulators and sigma factors, accounting for the extensive AmpR regulon. Gene expression studies demonstrate AmpR-dependent expression of the QS master regulator LasR that controls expression of many virulence factors. Using a chromosomally tagged AmpR, ChIP-Seq studies show direct AmpR binding to the lasR promoter. The data demonstrates that AmpR functions as a global regulator in P. aeruginosa and is a positive regulator of acute virulence while negatively regulating chronic infection phenotypes. In summary, my dissertation sheds light on the complex regulatory circuit in P. aeruginosa to provide a better understanding of the bacterial response to antibiotics and how the organism coordinately regulates a myriad of virulence factors.

The Role of PP2A Regulatory Subunit B56 in Dictyostelium Discoideum Motility

Protein Phosphatase 2A, PP2A, is a heterotrimeric threonine/serine phosphatase system that is involved in a variety of cellular processes. This phosphatase is composed ofthree subunits: a catalytic subunit (C subunit), a scaffolding subunit (A subunit), and a regulatory subunit (B subunit). The regulatory subunit B is divided into four subclasses, B, B' (B56), B'' and B'' '. Studies showed that PP2A/B56 complexes regulate development of Dictyostelium and other metazoan cells. In addition to development, our experimental data suggest that PP2A/B56 complex also plays an important role in Dictyostelium cell motility. Cells lacking B56 was generated previously in our laboratory (Lee et al., 2008). Further studies showed that b56- cells are compromised in random cell motility compared to the wild type (AX3) cells. In contrast, b56 cells with re-introduced B56 displayed wild-type like motilities. Furthermore, one of the colleagues in our laboratory found that one of the Dictyostelium Ras species, RasG, associates with PP2A/B56 complex and RasG activation is compromised in b56- cells. Considering that Ras proteins are central in cellular motility regulation, PP2A/B56 complex may modulate cell motility through regulating Ras. We propose to determine if an introduction of constitutive active RasG proteins improves compromised b56- cell motility.

A systematic analysis of the role of GGDEF-EAL domain proteins in virulence and motility in Xanthomonas oryzae pv. oryzicola

The second messenger c-di-GMP is implicated in regulation of various aspects of the lifestyles and virulence of Gram-negative bacteria. Cyclic di-GMP is formed by diguanylate cyclases with a GGDEF domain and degraded by phosphodiesterases with either an EAL or HD-GYP domain. Proteins with tandem GGDEF-EAL domains occur in many bacteria, where they may be involved in c-di-GMP turnover or act as enzymatically-inactive c-di-GMP effectors. Here, we report a systematic study of the regulatory action of the eleven GGDEF-EAL proteins in Xanthomonas oryzae pv. oryzicola, an important rice pathogen causing bacterial leaf streak. Mutational analysis revealed that XOC_2335 and XOC_2393 positively regulate bacterial swimming motility, while XOC_2102, XOC_2393 and XOC_4190 negatively control sliding motility. The ΔXOC_2335/XOC_2393 mutant that had a higher intracellular c-di-GMP level than the wild type and the ΔXOC_4190 mutant exhibited reduced virulence to rice after pressure inoculation. In vitro purified XOC_4190 and XOC_2102 have little or no diguanylate cyclase or phosphodiesterase activity, which is consistent with unaltered c-di-GMP concentration in ΔXOC_4190. Nevertheless, both proteins can bind to c-di-GMP with high affinity, indicating a potential role as c-di-GMP effectors. Overall our findings advance understanding of c-di-GMP signaling and its links to virulence in an important rice pathogen.

Ocean acidification slows nitrogen fixation and growth in the dominant diazotroph Trichodesmium under low-iron conditions

Dissolution of anthropogenic CO(2) increases the partial pressure of CO(2) (pCO(2)) and decreases the pH of seawater. The rate of Fe uptake by the dominant N(2)-fixing cyanobacterium Trichodesmium declines as pH decreases in metal-buffered medium. The slower Fe-uptake rate at low pH results from changes in Fe chemistry and not from a physiological response of the organism. Contrary to previous observations in nutrient-replete media, increasing pCO(2)/decreasing pH causes a decrease in the rates of N(2) fixation and growth in Trichodesmium under low-Fe conditions. This result was obtained even though the bioavailability of Fe was maintained at a constant level by increasing the total Fe concentration at low pH. Short-term experiments in which pCO(2) and pH were varied independently showed that the decrease in N(2) fixation is caused by decreasing pH rather than by increasing pCO(2) and corresponds to a lower efficiency of the nitrogenase enzyme. To compensate partially for the loss of N(2) fixation efficiency at low pH, Trichodesmium synthesizes additional nitrogenase. This increase comes partly at the cost of down-regulation of Fe-containing photosynthetic proteins. Our results show that although increasing pCO(2) often is beneficial to photosynthetic marine organisms, the concurrent decreasing pH can affect primary producers negatively. Such negative effects can occur both through chemical mechanisms, such as the bioavailability of key nutrients like Fe, and through biological mechanisms, as shown by the decrease in N(2) fixation in Fe-limited Trichodesmium.

Experiment: Acidified seawater impacts sea urchin larvae pH regulatory systems relevant for calcification

Calcifying echinoid larvae respond to changes in seawater carbonate chemistry with reduced growth and developmental delay. To date, no information exists on how ocean acidification acts on pH homeostasis in echinoderm larvae. Understanding acid-base regulatory capacities is important because intracellular formation and maintenance of the calcium carbonate skeleton is dependent on pH homeostasis. Using H(+)-selective microelectrodes and the pH-sensitive fluorescent dye BCECF, we conducted in vivo measurements of extracellular and intracellular pH (pH(e) and pH(i)) in echinoderm larvae. We exposed pluteus larvae to a range of seawater CO(2) conditions and demonstrated that the extracellular compartment surrounding the calcifying primary mesenchyme cells (PMCs) conforms to the surrounding seawater with respect to pH during exposure to elevated seawater pCO(2). Using FITC dextran conjugates, we demonstrate that sea urchin larvae have a leaky integument. PMCs and spicules are therefore directly exposed to strong changes in pH(e) whenever seawater pH changes. However, measurements of pH(i) demonstrated that PMCs are able to fully compensate an induced intracellular acidosis. This was highly dependent on Na(+) and HCO(3)(-), suggesting a bicarbonate buffer mechanism involving secondary active Na(+)-dependent membrane transport proteins. We suggest that, under ocean acidification, maintained pH(i) enables calcification to proceed despite decreased pH(e). However, this probably causes enhanced costs. Increased costs for calcification or cellular homeostasis can be one of the main factors leading to modifications in energy partitioning, which then impacts growth and, ultimately, results in increased mortality of echinoid larvae during the pelagic life stage.

Characterization of three human sec14p-like proteins: a-Tocopherol

Three closely related human sec14p-like proteins (hTAP1, 2, and 3, or SEC14L2, 3, and 4, respectively) have been described. These proteins may participate in intracellular lipid transport (phospholipids, squalene, tocopherol analogues and derivatives) or influence regulatory lipid-dependent events. Here, we show that the three recombinant hTAP proteins associate with the Golgi apparatus and mitochondria, and enhance the in vitro transport of radioactively labeled α-tocopherol to mitochondria in the same order of magnitude as the human α-tocopherol transfer protein (α-TTP). hTAP1 and hTAP2 are expressed in several cell lines, whereas the expression level of hTAP3 is low. Expression of hTAP1 is induced in human umbilical cord blood-derived mast cells upon differentiation by interleukin 4. In tissues, the three hTAPs are detectable ubiquitously at low level; pronounced and localized expression is found for hTAP2 and hTAP3 in the perinuclear region in cerebellum, lung, liver and adrenal gland. hTAP3 is well expressed in the epithelial duct cells of several glands, in ovary in endothelial cells of small arteries as well as in granulosa and thecal cells, and in testis in Leydig cells. Thus, the three hTAPs may mediate lipid uptake, secretion, presentation, and sub-cellular localization in a tissue-specific manner, possibly using organelle- and enzyme-specific docking sites.

Nanotechnology in food industry II: risk assessment and legislation

Currently, there is increasing use of nanomaterials in the food industry thanks to the many advantages offered and make the products that contain them more competitive in the market. Their physicochemical properties often differ from those of bulk materials, which require specialized risk assessment. This should cover the risks to the health of workers and consumers as well as possible environmental risks. The risk assessment methods must go updating due to more widespread use of nanomaterials, especially now that are making their way down to consumer products. Today there is no specific legislation for nanomaterials, but there are several european dispositions and regulations that include them. This review gives an overview of the risk assessment and the existing current legislation regarding the use of nanotechnology in the food industry.