Site-specific N-Linked Glycosylation of Receptor Guanylyl Cyclase C Regulates Ligand Binding, Ligand-mediated Activation and Interaction with Vesicular Integral Membrane Protein 36, VIP36

Guanylyl cyclase C (GC-C) is a multidomain, membrane-associated receptor guanylyl cyclase. GC-C is primarily expressed in the gastrointestinal tract, where it mediates fluid-ion homeostasis, intestinal inflammation, and cell proliferation in a cGMP-dependent manner, following activation by its ligands guanylin, uroguanylin, or the heat-stable enterotoxin peptide (ST). GC-C is also expressed in neurons, where it plays a role in satiation and attention deficiency/hyperactive behavior. GC-C is glycosylated in the extracellular domain, and differentially glycosylated forms that are resident in the endoplasmic reticulum (130 kDa) and the plasma membrane (145 kDa) bind the ST peptide with equal affinity. When glycosylation of human GC-C was prevented, either by pharmacological intervention or by mutation of all of the 10 predicted glycosylation sites, ST binding and surface localization was abolished. Systematic mutagenesis of each of the 10 sites of glycosylation in GC-C, either singly or in combination, identified two sites that were critical for ligand binding and two that regulated ST-mediated activation. We also show that GC-C is the first identified receptor client of the lectin chaperone vesicular integral membrane protein, VIP36. Interaction with VIP36 is dependent on glycosylation at the same sites that allow GC-C to fold and bind ligand. Because glycosylation of proteins is altered in many diseases and in a tissue-dependent manner, the activity and/or glycan-mediated interactions of GC-C may have a crucial role to play in its functions in different cell types.

Development of Microfluidic Devices with the Use of Nanotechnology to Aid in the Analysis of Biological Systems Including Membrane Protein Separation, Single Cell Analysis, and Genetic Markers

Computation technology has dramatically changed the world around us; you can hardly find an area where cell phones have not saturated the market, yet there is a significant lack of breakthroughs in the development to integrate the computer with biological environments. This is largely the result of the incompatibility of the materials used in both environments; biological environments and experiments tend to need aqueous environments. To help aid in these development chemists, engineers, physicists and biologists have begun to develop microfluidics to help bridge this divide. Unfortunately, the microfluidic devices required large external support equipment to run the device. This thesis presents a series of several microfluidic methods that can help integrate engineering and biology by exploiting nanotechnology to help push the field of microfluidics back to its intended purpose, small integrated biological and electrical devices. I demonstrate this goal by developing different methods and devices to (1) separate membrane bound proteins with the use of microfluidics, (2) use optical technology to make fiber optic cables into protein sensors, (3) generate new fluidic devices using semiconductor material to manipulate single cells, and (4) develop a new genetic microfluidic based diagnostic assay that works with current PCR methodology to provide faster and cheaper results. All of these methods and systems can be used as components to build a self-contained biomedical device.

Outer membrane proteins induced by iron deficiency in Anabaena sp PCC 7120

Iron deficiency can induce cyanobacteria to synthesize siderophore receptor proteins on the outer membrane to enhance the uptake of iron. In this study, an outer membrane of high purity was prepared from Anabaena sp. PCC 7120 based on aqueous polymer two-phase partitioning and discontinuous sucrose density ultra-centrifugation, and the induction of outer membrane proteins by iron deficiency was investigated using 2-D gel electrophoresis. At least. five outer membrane proteins were newly synthesized or significantly up-regulated in cells transferred to iron-deficient conditions, which were all identified to be siderophore receptor proteins according to MALDI-TOF-MS analyses. Bacterial luciferase reporter genes luxAB were employed to monitor the transcription of the encoding genes. The genes were induced by iron deficiency at the transcriptional level in different responsive modes. Luciferase activity expressed from an iron-regulated promoter may be used as a bioreporter for utilizable iron in natural water samples. (C) 2009 National Natural Science Foundation of China and Chinese Academy of Sciences. Published by Elsevier Limited and Science in China Press. All rights reserved.

A TPR-family membrane protein gene is required for light-activated heterotrophic growth of the cyanobacterium Synechocystis sp PCC 6803

The unicellular cyanobacterium Synechocystis sp. PCC6803 can grow heterotrophically in complete darkness, given that a brief period of illumination is supplemented every day (light-activated heterotrophic growth, LAHG), or under very weak ( < 0.5 mumol m(-2) s(-1)) but continuous light. By random insertion of the genome with an antibiotic resistance cassette, mutants defective in LAHG were generated. In two identical mutants, sll0886, a tetratricopeptide repeat (TPR)-family membrane protein gene, was disrupted. Targeted insertion of sll0886 and three downstream genes showed that the phenotype was not due to a polar effect. The sll0886 mutant shows normal photoheterotrophic growth when the light intensity is at 2.5 mumol m(-2) s(-1) or above, but no growth at 0.5 mumol m(-2) s(-1). Homologs to sll0886 are also present in cyanobacteria that are not known of LAHG. sll0886 and homologs may be involved in controlling different physiological processes that respond to light of low fluence. (C) 2003 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.

Stress-induced outer membrane vesicle production by Pseudomonas aeruginosa.

As an opportunistic Gram-negative pathogen, Pseudomonas aeruginosa must be able to adapt and survive changes and stressors in its environment during the course of infection. To aid survival in the hostile host environment, P. aeruginosa has evolved defense mechanisms, including the production of an exopolysaccharide capsule and the secretion of a myriad of degradative proteases and lipases. The production of outer membrane-derived vesicles (OMVs) serves as a secretion mechanism for virulence factors as well as a general bacterial response to envelope-acting stressors. This study investigated the effect of sublethal physiological stressors on OMV production by P. aeruginosa and whether the Pseudomonas quinolone signal (PQS) and the MucD periplasmic protease are critical mechanistic factors in this response. Exposure to some environmental stressors was determined to increase the level of OMV production as well as the activity of AlgU, the sigma factor that controls MucD expression. Overexpression of AlgU was shown to be sufficient to induce OMV production; however, stress-induced OMV production was not dependent on activation of AlgU, since stress caused increased vesiculation in strains lacking algU. We further determined that MucD levels were not an indicator of OMV production under acute stress, and PQS was not required for OMV production under stress or unstressed conditions. Finally, an investigation of the response of P. aeruginosa to oxidative stress revealed that peroxide-induced OMV production requires the presence of B-band but not A-band lipopolysaccharide. Together, these results demonstrate that distinct mechanisms exist for stress-induced OMV production in P. aeruginosa.

Contribution of bacterial outer membrane vesicles to innate bacterial defense.

BACKGROUND: Outer membrane vesicles (OMVs) are constitutively produced by Gram-negative bacteria throughout growth and have proposed roles in virulence, inflammation, and the response to envelope stress. Here we investigate outer membrane vesiculation as a bacterial mechanism for immediate short-term protection against outer membrane acting stressors. Antimicrobial peptides as well as bacteriophage were used to examine the effectiveness of OMV protection. RESULTS: We found that a hyper-vesiculating mutant of Escherichia coli survived treatment by antimicrobial peptides (AMPs) polymyxin B and colistin better than the wild-type. Supplementation of E. coli cultures with purified outer membrane vesicles provided substantial protection against AMPs, and AMPs significantly induced vesiculation. Vesicle-mediated protection and induction of vesiculation were also observed for a human pathogen, enterotoxigenic E. coli (ETEC), challenged with polymyxin B. When ETEC with was incubated with low concentrations of vesicles concomitant with polymyxin B treatment, bacterial survival increased immediately, and the culture gained resistance to polymyxin B. By contrast, high levels of vesicles also provided immediate protection but prevented acquisition of resistance. Co-incubation of T4 bacteriophage and OMVs showed fast, irreversible binding. The efficiency of T4 infection was significantly reduced by the formation of complexes with the OMVs. CONCLUSIONS: These data reveal a role for OMVs in contributing to innate bacterial defense by adsorption of antimicrobial peptides and bacteriophage. Given the increase in vesiculation in response to the antimicrobial peptides, and loss in efficiency of infection with the T4-OMV complex, we conclude that OMV production may be an important factor in neutralizing environmental agents that target the outer membrane of Gram-negative bacteria.

Amino acid permeases require COPII components and the ER resident membrane protein Shr3p for packaging into transport vesicles in vitro.

In S. cerevisiae lacking SHR3, amino acid permeases specifically accumulate in membranes of the endoplasmic reticulum (ER) and fail to be transported to the plasma membrane. We examined the requirements of transport of the permeases from the ER to the Golgi in vitro. Addition of soluble COPII components (Sec23/24p, Sec13/31p, and Sar1p) to yeast membrane preparations generated vesicles containing the general amino acid permease. Gap1p, and the histidine permease, Hip1p. Shr3p was required for the packaging of Gap1p and Hip1p but was not itself incorporated into transport vesicles. In contrast, the packaging of the plasma membrane ATPase, Pma1p, and the soluble yeast pheromone precursor, glycosylated pro alpha factor, was independent of Shr3p. In addition, we show that integral membrane and soluble cargo colocalize in transport vesicles, indicating that different types of cargo are not segregated at an early step in secretion. Our data suggest that specific ancillary proteins in the ER membrane recruit subsets of integral membrane protein cargo into COPII transport vesicles.

Validation of membrane protein topology models by oxidative labeling and mass spectrometry

Computer-assisted topology predictions are widely used to build low-resolution structural models of integral membrane proteins (IMPs). Experimental validation of these models by traditional methods is labor intensive and requires modifications that might alter the IMP native conformation. This work employs oxidative labeling coupled with mass spectrometry (MS) as a validation tool for computer-generated topology models. ·OH exposure introduces oxidative modifications in solvent-accessible regions, whereas buried segments (e.g., transmembrane helices) are non-oxidizable. The Escherichia coli protein WaaL (O-antigen ligase) is predicted to have 12 transmembrane helices and a large extramembrane domain (Pérez et al., Mol. Microbiol. 2008, 70, 1424). Tryptic digestion and LC-MS/MS were used to map the oxidative labeling behavior of WaaL. Met and Cys exhibit high intrinsic reactivities with ·OH, making them sensitive probes for solvent accessibility assays. Overall, the oxidation pattern of these residues is consistent with the originally proposed WaaL topology. One residue (M151), however, undergoes partial oxidation despite being predicted to reside within a transmembrane helix. Using an improved computer algorithm, a slightly modified topology model was generated that places M151 closer to the membrane interface. On the basis of the labeling data, it is concluded that the refined model more accurately reflects the actual topology of WaaL. We propose that the combination of oxidative labeling and MS represents a useful strategy for assessing the accuracy of IMP topology predictions, supplementing data obtained in traditional biochemical assays. In the future, it might be possible to incorporate oxidative labeling data directly as constraints in topology prediction algorithms.

Nasal immunization with Burkholderia multivorans outer membrane proteins and the mucosal adjuvant adamantylamide dipeptide confers efficient protection against experimental lung infections with B. multivorans and B. cenocepacia

Chronic lung infection by opportunistic pathogens, such as Pseudomonas aeruginosa and members of the Burkholderia cepacia complex, is a major cause of morbidity and mortality in patients with cystic fibrosis. Outer membrane proteins (OMPs) of gram-negative bacteria are promising vaccine antigen candidates. In this study, we evaluated the immunogenicity, protection, and cross-protection conferred by intranasal vaccination of mice with OMPs from B. multivorans plus the mucosal adjuvant adamantylamide dipeptide (AdDP). Robust mucosal and systemic immune responses were stimulated by vaccination of naive animals with OMPs from B. multivorans and B. cenocepacia plus AdDP. Using a mouse model of chronic pulmonary infection, we observed enhanced clearance of B. multivorans from the lungs of vaccinated animals, which correlated with OMP-specific secretory immunoglobulin A responses. Furthermore, OMP-immunized mice showed rapid resolution of the pulmonary infection with virtually no lung pathology after bacterial challenge with B. multivorans. In addition, we demonstrated that administration of B. multivorans OMP vaccine conferred protection against B. cenocepacia challenge in this mouse infection model, suggesting that OMPs provide cross-protection against the B. cepacia complex. Therefore, we concluded that mucosal immunity to B. multivorans elicited by intranasal vaccination with OMPs plus AdDP could prevent early steps of colonization and infection with B. multivorans and also ameliorate lung tissue damage, while eliciting cross-protection against B. cenocepacia. These results support the notion that therapies leading to increased mucosal immunity in the airways may help patients with cystic fibrosis.

A comparison of the haemagglutinating and enzymic activities of Bacteroides fragilis whole cells and outer membrane vesicles

The haemagglutinating and enzymic activities of the obligately anaerobic pathogenic bacterium Bacteroides fragilis were examined. Outer membrane vesicles are released from the surface of B. fragilis. They can be detected by electron microscopy in ultrathin sections and bacterial suspensions after negative staining. Electron microscopy and immunogold labelling with a MAb specific for surface polysaccharide of B. fragilis confirmed that the vesicles carried outer membrane associated epitopes. The haemagglutinating activity of whole cells from populations of B. fragilis strains NCTC9343, BE3 and LS66 enriched by Percoll density gradient centrifugation for a large capsule (LC), electron dense layer (EDL); non-capsulate by light microscopy) and outer membrane vesicles (OMV) which had been purified by centrifugation from EDL-enriched populations were compared using human and horse erythrocytes. The enzymic activity of OMV, LC- and EDL-enriched populations, as detected by the API ZYM kit, was compared for strains NCTC 9343 and BE3. Purified OMV from the strains examined exhibited both haemagglutinating and enzymatic activity. Haemagglutination by the EDL-enriched population was sensitive to treatment with sodium periodate. The LC-enriched population haemagglutinated only after ultrasonic removal of the capsule. This indicates that the LC masks a haemagglutinin. The results suggest a potential role for OMV in the virulence of B. fragilis.

Pathogenic Yersinia enterocolitica strains increase the outer membrane permeability in response to environmental stimuli by modulating lipopolysaccharide fluidity and lipid A structure

Pathogenic biotypes of Yersinia enterocolitica (serotypes O:3, O:8, O:9, and O:13), but not environmental biotypes (serotypes O:5, O:6, O:7,8, and O:7,8,13,19), increased their permeability to hydrophobic probes when they were grown at pH 5.5 or in EGTA-supplemented (Ca(2+)-restricted) media at 37 degrees C. A similar observation was also made when representative strains of serotypes O:8 and O:5 were tested after brief contact with human monocytes. The increase in permeability was independent of the virulence plasmid. The role of lipopolysaccharide (LPS) in this phenomenon was examined by using Y. enterocolitica serotype O:8. LPS aggregates of bacteria grown in acidic or EGTA-supplemented broth took up more N-phenylnaphthylamine than LPS aggregates of bacteria grown in standard broth and also showed a marked increase in acyl chain fluidity which correlated with permeability, as determined by measurements obtained in the presence of hydrophobic dyes. No significant changes in O-antigen polymerization were observed, but lipid A acylation changed depending on the growth conditions. In standard medium at 37 degrees C, there were hexa-, penta-, and tetraacyl lipid A forms, and the pentaacyl form was dominant. The amount of tetraacyl lipid A increased in EGTA-supplemented and acidic media, and hexaacyl lipid A almost disappeared under the latter conditions. Our results suggest that pathogenic Y. enterocolitica strains modulate lipid A acylation coordinately with expression of virulence proteins, thus reducing LPS packing and increasing outer membrane permeability. The changes in permeability, LPS acyl chain fluidity, and lipid A acylation in pathogenic Y. enterocolitica strains approximate the characteristics in Yersinia pseudotuberculosis and Yersinia pestis and suggest that there is a common outer membrane pattern associated with pathogenicity.

Brucella abortus and its closest phylogenetic relative, Ochrobactrum spp., differ in outer membrane permeability and cationic peptide resistance

The outer membrane (OM) of the intracellular parasite Brucella abortus is permeable to hydrophobic probes and resistant to destabilization by polycationic peptides and EDTA. The significance of these unusual properties was investigated in a comparative study with the opportunistic pathogens of the genus Ochrobactrum, the closest known Brucella relative. Ochrobactrum spp. OMs were impermeable to hydrophobic probes and sensitive to polymyxin B but resistant to EDTA. These properties were traced to lipopolysaccharide (LPS) because (i) insertion of B. abortus LPS, but not of Escherichia coli LPS, into Ochrobactrum OM increased its permeability; (ii) permeability and polymyxin B binding measured with LPS aggregates paralleled the results with live bacteria; and (iii) the predicted intermediate results were obtained with B. abortus-Ochrobactrum anthropi and E. coli-O. anthropi LPS hybrid aggregates. Although Ochrobactrum was sensitive to polymyxin, self-promoted uptake and bacterial lysis occurred without OM morphological changes, suggesting an unusual OM structural rigidity. Ochrobactrum and B. abortus LPSs showed no differences in phosphate, qualitative fatty acid composition, or acyl chain fluidity. However, Ochrobactrum LPS, but not B. abortus LPS, contained galacturonic acid. B. abortus and Ochrobactrum smooth LPS aggregates had similar size and zeta potential (-12 to -15 mV). Upon saturation with polymyxin, zeta potential became positive (1 mV) for Ochrobactrum smooth LPS while remaining negative (-5 mV) for B. abortus smooth LPS, suggesting hindered access to inner targets. These results show that although Ochrobactrum and Brucella share a basic OM pattern, subtle modifications in LPS core cause markedly different OM properties, possibly reflecting the adaptive evolution of B. abortus to pathogenicity.

The outer membrane of Brucella ovis shows increased permeability to hydrophobic probes and is more susceptible to cationic peptides than are the outer membranes of mutant rough Brucella abortus strains

The permeability of the outer membrane (OM) to hydrophobic probes and its susceptibility to bactericidal cationic peptides were investigated for natural rough Brucella ovis and for mutant rough Brucella abortus strains. The OM of B. ovis displayed an abrupt and faster kinetic profile than rough B. abortus during the uptake of the hydrophobic probe N-phenyl-naphthylamine. B. ovis was more sensitive than rough B. abortus to the action of cationic peptides. Bactenecins 5 and 7 induced morphological alterations on the OMs of both rough Brucella strains. B. ovis lipopolysaccharide (LPS) captured considerably more polymyxin B than LPSs from both rough and smooth B. abortus strains. Polymyxin B, poly-L-lysine, and poly-L-ornithine produced a thick coating on the surfaces of both strains, which was more evident in B. ovis than in rough B. abortus. The distinct functional properties of the OMs of these two rough strains correlate with some structural differences of their OMs and with their different biological behaviors in animals and culture cells.