Membrane Porin Protein Research Articles

Neisseria Gonorrhoeae: Its Dominant Properties to Establish Contact and Attachment that Facilitate Epithelial Invasion and Colonization From a Biochemist Perspective

Aim: to discuss recent epidemiology of gonorrhoea and updates regarding its pathogenesis with a focus on biochemical aspects of contact and adhesion that preceded its epithelial invasion.
 Discussion: Neisseria gonorrhoeae, a Gram-negative obligate human pathogenic bacterium, infects human epithelial cells and causes sexually transmitted diseases in both males and females Gonorrhea rates are rising in many countries. It could lead to long-term health problems and even infertility. Vulnerable groups including men who have sex with men and sex workers appear to bear disproportionate burdens of gonorrhea. As N. gonorrhoeae advances through the steps of disease formation and pathogenesis (transmission, adherence, colonization and invasion, and also immune evasion), the bacterium expresses some virulence factors to facilitate its survival and replication; while at the same time keeping its existence barely invasive and almost undiscoverable by active immune cells. Adherence to epithelial cells becomes the first event that precedes invasion.
 Conclusion: Adhesion of gonococci to mucosal epithelial cells appears to be a critical step in the pathogenesis of gonococcal infection. Gonococci can adhere to a variety of human cells. Gonorrhea has multiple surface proteins that facilitate adhesion. N. gonorrhoeae utilize type IV pili and Opa, opacity-associated proteins, surface proteins involved in cellular attachment that preceded its invasion, Lipooligosaccharides (LOS) and also the outer membrane protein porin (POrB).

Porin 1 Modulates Autophagy in Yeast.

Autophagy is a cellular recycling program which efficiently reduces the cellular burden of ageing. Autophagy is characterised by nucleation of isolation membranes, which grow in size and further expand to form autophagosomes, engulfing cellular material to be degraded by fusion with lysosomes (vacuole in yeast). Autophagosomal membranes do not bud from a single cell organelle, but are generated de novo. Several lipid sources for autophagosomal membranes have been identified, but the whole process of their generation is complex and not entirely understood. In this study, we investigated how the mitochondrial outer membrane protein porin 1 (Por1), the yeast orthologue of mammalian voltage-dependent anion channel (VDAC), affects autophagy in yeast. We show that POR1 deficiency reduces the autophagic capacity and leads to changes in vacuole and lipid homeostasis. We further investigated whether limited phosphatidylethanolamine (PE) availability in por1∆ was causative for reduced autophagy by overexpression of the PE-generating phosphatidylserine decarboxylase 1 (Psd1). Altogether, our results show that POR1 deficiency is associated with reduced autophagy, which can be circumvented by additional PSD1 overexpression. This suggests a role for Por1 in Psd1-mediated autophagy regulation.

Fusobacterium nucleatum Extracellular Vesicles Modulate Gut Epithelial Cell Innate Immunity via FomA and TLR2.

Extracellular vesicles (EVs) derived from the gut microbiota are largely uncharacterized and their impacts on host intestinal physiology remain unresolved. Here, we isolated EVs from F. nucleatum for detailed characterization. Our analyses highlight the presence of the outer membrane protein porin FomA on EVs. Besides, we evaluated the impact of EVs on human intestinal epithelial cells (IECs) in a non-inflammatory context. Our results show no detrimental impact on the epithelial barrier. No internalization of EVs was observed. Moreover, we demonstrate that F. nucleatum EVs trigger innate immunity of IECs by promoting NF-κB activation via the dynamin-mediated endocytosis. The NF-κB activation was found to be TLR2-dependent yet, TLR4 was dispensable. Using competitive binding assays, we establish that FomA is involved in the NF-κB response. Taken together, our data indicate that EVs induce effects similar to those observed with whole F. nucleatum bacteria on IECs. In particular, our study highlights the role of TLR2 and FomA as major modulators of the gut epithelium immune responses to F. nucleatum.

Impact of methyl salicylate on storage quality, ethylene action, and protein profiling of ‘Zaosu’ pear (Pyrus bretschneideri)

‘Zaosu’ pear, an early-maturing cultivar harvested in summer in China, readily exhibits quality deterioration and senescence owing to the lack of refrigeration and effective postharvest technology. To explore possible molecular mechanisms of methyl salicylate (MeSA) in delaying senescence, ‘Zaosu’ pear was treated with 0.05 mmol L―1 MeSA and stored at 25 ± 2 °C. The effects on quality, ethylene synthesis and signaling elements, and protein profiling were evaluated. MeSA maintained fruit postharvest quality. In the treated fruit, the yellowing index, weight loss, and color L* value retained lower levels, and the yellowing index was 69 % of that of the control after 20 days, whereas the hue angle and texture properties (fracturability, firmness, and chewiness) had increased levels. The respiration rate and ethylene content, and ethylene biosynthesis and signaling components ACS4, ACO1, ACO4, ETR1, ETR2, ERS2, CTR1, EIN2, and EIL1, were inhibited and downregulated by MeSA, respectively, while ERS1 was upregulated. Thirty-eight different expression proteins were identified by 2-DE and MALDI-TOF/TOF MS analysis in the treated and untreated fruits. Among them, related ripening and senescence proteins 2-methylene-furan-3-one reductase, abscisic stress-ripening protein, annexin RJ4, and mitochondrial outer membrane protein porin were down-accumulated during storage, while electron transfer flavoprotein was up-accumulated later in the storage period. These results indicated that MeSA postponed the ripening and senescence of postharvest ‘Zaosu’ pear.

Chloroplast proteins involved in drought stress response in selected cultivars of common bean (Phaseolus vulgaris L.).

One of the major cell organelles, whose functions are affected during drought stress are chloroplasts. In this study, chloroplast proteome under drought was studied in two cultivars of common bean (Phaseolus vulgaris L), Tiber and more sensitive to drought, Starozagorski čern, which were subjected to drought for 6 and 13days. A comparative proteomic analysis with 2D-DIGE was performed on the isolated chloroplast proteins from leaves. Together, 44 proteins with changed abundance between control and stressed plants were identified with LC-MS/MS from both cultivars. The majority of the identified proteins were involved in photosynthetic processes. The results showed a decrease in abundance in different structure components of photosystem I and II, and ATP synthase, which may indicate a suppression of light-dependent reactions by drought stress. Similar proteomic response for both cultivars after 6 and 13days of drought was observed. Proteins with contrasting abundance patterns between the cultivars or proteins specific for only one cultivar, such as ferredoxin-NADP reductase, photosystem II stability/assembly factor HCF136, curvature thylakoid protein 1B, and plastidial membrane protein porin were pointed out as major identified proteins revealing differential abundance between the cultivars. Taken together, our results provide insight into the molecular response of chloroplasts in common bean under drought stress, whereas conclusions about the tolerance mechanisms require further studies.

Two outer membrane proteins are bovine lactoferrin-binding proteins in Mannheimia haemolytica A1.

Mannheimia haemolytica is a Gram negative bacterium that is part of the bovine respiratory disease, which causes important economic losses in the livestock industry. In the present work, the interaction between M. haemolytica A1 and bovine lactoferrin (BLf) was studied. This iron-chelating glycoprotein is part of the mammalian innate-immune system and is present in milk and mucosal secretions; Lf is also contained in neutrophils secondary granules, which release this glycoprotein at infection sites. It was evidenced that M. haemolytica was not able to use iron-charged BLf (BholoLf) as a sole iron source; nevertheless, iron-lacked BLf (BapoLf) showed a bactericidal effect against M. haemolytica with MIC of 4.88 ± 1.88 and 7.31 ± 1.62 μM for M. haemolytica strain F (field isolate) and M. haemolytica strain R (reference strain), respectively. Through overlay assays and 2-D electrophoresis, two OMP of 32.9 and 34.2 kDa with estimated IP of 8.18 and 9.35, respectively, were observed to bind both BapoLf and BholoLf; these OMP were identified by Maldi-Tof as OmpA (heat-modifiable OMP) and a membrane protein (porin). These M. haemolytica BLf binding proteins could be interacting in vivo with both forms of BLf depending on the iron state of the bovine.Electronic supplementary materialThe online version of this article (doi:10.1186/s13567-016-0378-1) contains supplementary material, which is available to authorized users.

Peculiarities and pitfalls of quantifying mitochondrial energy metabolism in the skin.

Peculiarities and pitfalls of quantifying mitochondrial energy metabolism in the skin.

Interaction of endothelial nitric oxide synthase with mitochondria regulates oxidative stress and function in fetal pulmonary artery endothelial cells.

An increase in oxygen tension at birth is one of the key signals that initiate pulmonary vasodilation in the fetal lung. We investigated the hypothesis that targeting endothelial nitric oxide synthase (eNOS) to the mitochondrial outer membrane regulates reactive oxygen species (ROS) formation in the fetal pulmonary artery endothelial cells (PAEC) during this transition. We isolated PAEC and pulmonary arteries from 137-day gestation fetal lambs (term = 144 days). We exposed PAEC to a simulated transition from fetal to (3% O2) to normoxic (21%) or hyperoxic (95% O2) postnatal Po2 or to the nitric oxide synthase (NOS) agonist ATP. We assessed the effect of O2 and ATP on eNOS interactions with the mitochondrial outer membrane protein porin and with the chaperone hsp90. We also investigated the effect of decoy peptides that blocked eNOS interactions with porin or hsp90 on PAEC angiogenesis and vasodilator function of pulmonary arteries. Transition of fetal PAEC from 3 to 21% O2 but not to 95% O2 or exposure to ATP increased eNOS association with hsp90 and porin. Decoy peptides that blocked eNOS interactions decreased NO release, increased O2 consumption and mitochondrial ROS levels, and impaired PAEC angiogenesis. Decoy peptides also inhibited the relaxation responses of pulmonary artery rings and dilation of resistance size pulmonary arteries to ATP. The mitochondrial-antioxidant mito-ubiquinone restored the response to ATP in decoy peptide-treated pulmonary arteries. These data indicate that targeting eNOS to mitochondria decreases endothelial oxidative stress and facilitates vasodilation in fetal pulmonary circulation at birth.

A ROLE OF PROTEINS OF THE OUTER MEMBRANE OF Shewanella oneidensis MR-1 BACTERIA IN FORMATION AND STABILIZATION OF SILVER SULFIDE NANOPARTICLES

Preparations of Ag 2 S nanoparticles obtained in equimolar AgNO3 and Na2S2O3 aqueous solutions in the presence of Shewanella oneidensis MR-1 bacteria cells have been shown to be stable in aqueous solutions. Proteins absorbed on the surface of these nanoparticles were identified for the first time by the MALDI TOF/TOF method. Among them, multi-heme cytochromes MtrC and OmcA, as well as a MtrB membrane protein porin that forms a complex with these cytochromes on the outer cell membrane were detected. It was shown that in the absence of the cells, an insoluble precipitate consisting of agglomerated Ag 2 S nanoparticles with broad size distribution was formed. The role of the identified proteins in the mechanism of the formation and stabilization of the Ag 2 S nanoparticles in this system is discussed.

In Vivo and In Vitro Characterization of the Immune Stimulating Activity of the Neisserial Porin PorB

Vaccines play a vital role in modern medicine. The development of novel vaccines for emerging and resistant pathogens has been aided in recent years by the use of novel adjuvants in subunit vaccines. A deeper understanding of the molecular pathways behind adjuvanticity is required to better select immunostimulatory molecules for use in individual vaccines. To this end, we have undertaken a study of the essential signaling pathways involved in the innate and adaptive immune responses to the Neisseria meningitidis outer membrane protein Porin B (PorB). We have previously demonstrated that PorB is an agonist of Toll-Like Receptor 2 (TLR2) and acts as an adjuvant in vaccines for protein, carbohydrate and lipopolysaccharide antigens using murine models. Here we demonstrate NFκB translocation following stimulation with PorB only occurs in the presence of TLR2. IL-6 and TNF-α secretion was shown to be MAPK dependent. Surface expression of activation markers on macrophages, including CD40, CD69, and CD86, was increased following PorB stimulation in vitro. Interestingly, some upregulation of CD54 and CD69 was still observed in macrophages obtained from TLR2 KO mice, indicating a possible non-TLR2 mediated activation pathway induced by PorB. In a murine vaccination model, using ovalbumin as the antigen and PorB as the adjuvant, a decreased antigen-specific IgG production was observed in TLR2 KO mice; adjuvant-dependent increased IgG production was entirely ablated in MyD88 KO mice. These observations demonstrate the importance of the above pathways to the adjuvant activity of PorB. The potential TLR2 independent effect is currently being explored.

Sap6, a secreted aspartyl proteinase, participates in maintenance the cell surface integrity of Candida albicans

BackgroundThe polymorphic species Candida albicans is the major cause of candidiasis in humans. The secreted aspartyl proteinases (Saps) of C. albicans, encoded by a family of 10 SAP genes, have been investigated as the virulent factors during candidiasis. However, the biological functions of most Sap proteins are still uncertain. In this study, we applied co-culture system of C. albicans and THP-1 human monocytes to explore the pathogenic roles and biological functions of Sap proteinases.ResultsAfter 1 hr of co-culture of C. albicans strains and THP-1 human monocytes at 37°C, more than 60% of the THP-1-engulfed wild type and Δsap5 Candida cells were developing long hyphae. However, about 50% of THP-1-engulfed Δsap6 Candida cells were generating short hyphae, and more dead Candida cells were found in Δsap6 strain that was ingested by THP-1 cells (about 15% in Δsap6 strain vs. 2 ~ 2.5% in SC5314 and Δsap5 strains). The immunofluorescence staining demonstrated that the Sap6 is the major hyphal tip located Sap protein under THP-1 phagocytosis. The sap6-deleted strains (Δsap6, Δsap4/6, and Δsap5/6) appeared slower growth on Congo red containing solid medium at 25°C, and the growth defect was exacerbated when cultured at 37°C in Congo red or SDS containing medium. In addition, more proteins were secreted from Δsap6 strain and the β-mercaptoethanol (β-ME) extractable surface proteins from Δsap6 mutant were more abundant than that of extracted from wild type strain, which included the plasma membrane protein (Pma1p), the ER-chaperone protein (Kar2p), the protein transport-related protein (Arf1p), the cytoskeleton protein (Act1), and the mitochondrial outer membrane protein (porin 1). Moreover, the cell surface accessibility was increased in sap6-deleted strains.ConclusionFrom these results, we speculated that the cell surface constitution of C. albicans Δsap6 strain was defect. This may cause the more accessible of β-ME to disulfide-bridged cell surface components and may weaken the resistance of Δsap6 strain encountering phagocytosis of THP-1 cells. Sap6 protein displays a significant function involving in maintenance the cell surface integrity.

APC Migration and antigen uptake is increased by the TLR2 ligand Neisserial Porin PorB (53.4)

Abstract TLR ligands play an essential role in identifying threats to the innate immune system, which can then activate the adaptive arm. Our lab has worked with the N. meningitidis outer membrane protein Porin B (PorB), a TLR2 ligand, and shown that it increases the adaptive immune response as measured by antibody production and T cell activity. In examining the adjuvant activity of PorB we now investigate the ability of PorB to increase APC antigen uptake and migration to effector draining lymph nodes. Using an in vitro model with a fluor-linked antigen we show that PorB increases the quantity of the model antigen ovalbumin taken up by the APC in a rate dependent manner. We are in the process of characterizing the intracellular compartmentalization of the processed antigen by fluorescent microscopy of BMDM. In vivo experiments were conducted using a hock injection model and tracking the movement of the antigen through its fluorescent tag. Draining lymph nodes were examined through flow cytometry and immunofluorescence of frozen node sections. Using the latter technique, we are studying changes to lymph node morphology with the inclusion of the TLR ligand. Confirming the in vitro results, the addition of PorB to a vaccine injection increases the degree of APC migration to draining lymph nodes and the carriage of the antigen by these cells. With these results we have identified characteristics of the TLR2 ligand PorB that may account for some of its efficacy as a vaccine adjuvant.

Loss of mitochondria in ganglioneuromas

A shift in cellular energy production from oxidative phosphorylation (OXPHOS) to glycolysis, even under aerobic conditions, is called the Warburg effect. To elucidate changes of the mitochondrial energy metabolism in ganglioneuroma (GN) individual OXPHOS enzymes were analyzed by activity assays and by immunohistochemical staining methods. GN (n=7) showed a significant reduction in the activity and content of OXPHOS enzymes. Citrate synthase activity was also severely diminished in GN compared to normal cortical kidney (p=0.0002) and adrenal (p=0.0024) tissues. Furthermore, the mitochondrial membrane protein porin was undetectable or significantly reduced. Accordingly, a reduction of the copy number of mitochondrial DNA was observed in GN compared to cortical kidney tissue. The striking decline of mitochondrial mass is specific for GN but not for neuroblastoma, in which a reduction of the OXPHOS complexes without reduction of mitochondrial mass was reported. Knowledge of the mechanism by which tumor cells achieve the Warburg effect will provide a starting point for functional studies aimed at restoring aerobic energy metabolism as a potential new therapeutic strategy to treat malignancies.

Impact of multidrug-resistant Pseudomonas aeruginosa infection on patient outcomes

Rates of antibiotic resistance in Pseudomonas aeruginosa are increasing worldwide. The multidrug-resistant (MDR) phenotype in P. aeruginosa could be mediated by several mechanisms including multidrug efflux systems, enzyme production, outer membrane protein (porin) loss and target mutations. Currently, no international consensus on the definition of multidrug resistance exists, making direct comparison of the literature difficult. Inappropriate empirical therapy has been associated with increased mortality in P. aeruginosa infections; delays in starting appropriate therapy may contribute to increased length of hospital stay and persistence of infection. In addition, worse clinical outcomes may be associated with MDR infections owing to limited effective antimicrobial options. This article aims to summarize the contemporary literature on patient outcomes following infections caused by drug-resistant P. aeruginosa. The impact of antimicrobial therapy on patient outcomes, mortality and morbidity; and the economic impact of MDR P. aeruginosa infections will be examined.

The morphology proteins Mdm12/Mmm1 function in the major β-barrel assembly pathway of mitochondria

The beta-barrel proteins of mitochondria are synthesized on cytosolic ribosomes. The proteins are imported by the translocase of the outer membrane (TOM) and the sorting and assembly machinery (SAM). It has been assumed that the SAM(core) complex with the subunits Sam35, Sam37 and Sam50 represents the last import stage common to all beta-barrel proteins, followed by splitting in a Tom40-specific route and a route for other beta-barrel proteins. We have identified new components of the beta-barrel assembly machinery and show that the major beta-barrel pathway extends beyond SAM(core). Mdm12/Mmm1 function after SAM(core) yet before splitting of the major pathway. Mdm12/Mmm1 have been known for their role in maintenance of mitochondrial morphology but we reveal assembly of beta-barrel proteins as their primary function. Moreover, Mdm10, which functions in the Tom40-specific route, can associate with SAM(core) as well as Mdm12/Mmm1 to form distinct assembly complexes, indicating a dynamic exchange between the machineries governing mitochondrial beta-barrel assembly. We conclude that assembly of mitochondrial beta-barrel proteins represents a major function of the morphology proteins Mdm12/Mmm1.

Tracking the Unfolding and Refolding Pathways of Outer Membrane Protein Porin fromParacoccus denitrificans

We have investigated outer membrane protein porin from Paracoccus denitrificans for its stability against heat and pH. Pathways of unfolding and refolding have been analyzed. Porin incubated at pH 12.5 and above undergoes a slow unfolding into an unordered structure. The unfolded protein could be refolded into a nativelike structure that is functionally active but with distinct deviation from the native protein. This nativelike structure exhibited an entirely different thermal stability. Although aggregation is normally considered a structural "dead-end", the possibility of opening an aggregated porin and forming a functionally active structure was analyzed here. Porin aggregates on heating above 86.2 degrees C. Incubating the heat-aggregated protein at high pH (> or = 12.5) leads to a slow opening of the protein into an unordered structure. It was possible to refold this unordered protein into a trimeric nativelike structure which was capable of forming active pores. However, the thermal stability of the refolded porin was unlike that of the native porin. To understand the basic mechanism behind the unfolding processes, the protein was subjected to heating at various pH values. It was observed that at pH > or = 12.5 the protein does not aggregate upon heating; instead, it opens into an unordered structure. We conclude that at high pH values, the electrostatic interactions of various amino acid residues are perturbed which leads to unfolding into an unordered structure. This study shows for the first time an entirely new unfolding and refolding pathway for porin.

Structure–function correlation of outer membrane protein porin from Paracoccus denitrificans

Porins from outer membrane of Gram-negative bacteria have a highly stable structure. Our previous studies on porin from Paracoccus denitrificans showed that the outer membrane protein porin is extremely stable toward heat, pH, and chemical denaturants. The major question we have addressed in this paper is whether the high stability of porin is a consequence of the beta-barrel structure and whether it is required for its function. To explain this we have analyzed two cases: first, we used porin wild-type and mutants and compared their structure and function; second, we compared the activity of porin preheated to different temperatures. Structural changes were monitored by infrared spectroscopy. We observed that the structural stability of porin is not equivalent to functional activity as minor alteration in the structure can result in drastic differences in the activity of porins.

Mass Tag-Assisted Identification of Naturally Processed HLA Class II-Presented Meningococcal Peptides Recognized by CD4+ T Lymphocytes

The meningococcal class I outer membrane protein porin A plays an important role in the development of T cell-dependent protective immunity against meningococcal serogroup B infection and is therefore a major component of candidate meningococcal vaccines. T cell epitopes from porin A are poorly characterized because of weak in vitro memory T cell responses against purified Ag and strain variation. We applied a novel strategy to identify relevant naturally processed and MHC class II-presented porin A epitopes, based on stable isotope labeling of Ag. Human immature HLA-DR1-positive dendritic cells were used for optimal uptake and MHC class II processing of (14)N- and (15)N-labeled isoforms of the neisserial porin A serosubtype P1.5-2,10 in bacterial outer membrane vesicles. HLA-DR1 bound peptides, obtained after 48 h of Ag processing, contained typical spectral doublets in mass spectrometry that could easily be assigned to four porin A regions, expressed at diverging densities ( approximately 30-4000 copies/per cell). Epitopes from two of these regions are recognized by HLA-DR1-restricted CD4(+) T cell lines and are conserved among different serosubtypes of meningococcal porin A. This mass tag-assisted approach provides a useful methodology for rapid identification of MHC class II presented bacterial CD4(+) T cell epitopes relevant for vaccine development.

Thermal stability of outer membrane protein porin from Paracoccus denitrificans: FT-IR as a spectroscopic tool to study lipid–protein interaction

Lipid protein interactions play a key role in the stability and function of various membrane proteins. Earlier we have reported the extreme thermal stability of porin from Paracoccus denitrificans reconstituted into liposomes. Here, we used Fourier transform infrared spectroscopy for a label free analysis of the global secondary structural changes and local changes in the tyrosine microenvironment. Our results show that a mixed lipid system (non-uniform bilayer) optimizes the thermal stability of porin as compared to the porin in pure lipids (uniform bilayer) or detergent micelles. This is in line with the fact that the bacterial outer membrane is a dynamic system made up of lipids of varying chain lengths, head groups and the barrel wall height contacting the membrane is uneven.

Investigation of the thermal stability of porin from Paracoccus denitrificans by site-directed mutagenesis and Fourier transform infrared spectroscopy.

The folding of membrane proteins was addressed using outer membrane protein porin from the soil bacterium Paracoccus denitrificans (P. den.). IR spectroscopy and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis were used to probe the effect of mutagenesis on the thermal stability of the protein. Secondary structure analysis by amide I ir spectroscopy showed that the wild-type protein was predominantly composed of beta-sheet, which supports the x-ray crystal structure information (A. Hirsch, J. Breed, K. Saxena, O.-M. H. Richter, B. Ludwig, K. Diederichs, and W. Welte, FEBS Letters, 1997, Vol. 404, pp. 208-210). The mutants E81Q, W74C, and E81Q/D148N were shown to have similar secondary structure composition as the wild type. Wild-type protein and the mutants in detergent micelles underwent irreversible denaturation as a result of heating. Transition temperature calculated from the amide I analysis revealed that mutant porins were slightly less stable compared to the wild type. The protein in micelles showed complete monomerization of the trimer above 85 degrees C. In native-like conditions (provided by liposomes), no change was observed in the secondary structure of the protein until 95 degrees C. This is supported by SDS-PAGE as no change in quaternary structure was observed, proving that the proteins are structurally thermostable in liposomes as compared to micelles. Our studies demonstrated that porins resistant to detergents and proteases are highly thermostable as well.