Neisseria meningitidis induces platelet inhibition and increases vascular endothelial permeability via nitric oxide regulated pathways

Summary

Despite antibiotic therapy, infections with Neisseria meningitidis still demonstrate a high rate of morbidity and mortality even in developed countries. The fulminant septicaemic course, named Waterhouse-Friderichsen syndrome, with massive haemorrhage into the adrenal glands and widespread petechial bleeding suggest pathophysiological inhibition of platelet function. Our data show that N. meningitidis produces the important physiological platelet inhibitor and cardiovascular signalling molecule nitric oxide (NO), also known as endothelium-derived relaxing factor (EDRF). N. meningitidis-derived NO inhibited ADPinduced platelet aggregation through the activation of soluble guanylyl cyclase (sGC) followed by an increase in platelet cyclic nucleotide levels and subsequent activation of platelet cGMP- and cAMP- depend- ent protein kinases (PKG and PKA). Furthermore, direct measurement of horseradish peroxidase (HRP) passage through a vascular endothelial cell monolayer revealed that N. meningitidis significantly increased endothelial monolayer permeability. Immunfluorescence analysis demonstrated NO dependent disturbances in the structure of endothelial adherens junctions after co-incubation with N. meningitidis. In contrast to platelet inhibition, the NO effects on HBMEC were not mediated by cyclic nucleotides. Our study provides evidence that NO plays an essential role in the pathophysiology of septicaemic meningococcal infection.

Keywords

Neisseria meningitidis - platelets - endothelial cells - nitric oxide

^* Equal contribution of these authors.

• References

• 1 Trotter CL, Chandra M, Cano R. et al. A surveillance network for meningococcal disease in Europe. FEMS Microbiol Rev 2007; 31: 27-36.
• 2 van Deuren M, Brandtzaeg P, van der Meer JW. Update on meningococcal disease with emphasis on pathogenesis and clinical management. Clin Microbiol Rev 2000; 13: 144-166.
• 3 Nassif X, Bourdoulous S, Eugene E. et al. How do extracellular pathogens cross the blood-brain barrier?. Trends Microbiol 2002; 10: 227-232.
• 4 Merz AJ, So M. Interactions of pathogenic neisseriae with epithelial cell membranes. Annu Rev Cell Dev Biol 2000; 16: 423-457.
• 5 Virji M. Pathogenic neisseriae: surface modulation, pathogenesis and infection control. Nat Rev Microbiol 2009; 7: 274-286.
• 6 Stevanin TM, Laver JR, Poole RK. et al. Metabolism of nitric oxide by Neisseria meningitidis modifies release of NO-regulated cytokines and chemokines by human macrophages. Microbes Infect 2007; 9: 981-987.
• 7 Stevanin TM, Moir JW, Read RC. Nitric oxide detoxification systems enhance survival of Neisseria meningitidis in human macrophages and in nasopharyngeal mucosa. Infect Immun 2005; 73: 3322-3329.
• 8 Tunbridge AJ, Stevanin TM, Lee M. et al. Inhibition of macrophage apoptosis by Neisseria meningitidis requires nitric oxide detoxification mechanisms. Infect Immun 2006; 74: 729-733.
• 9 Anjum MF, Stevanin TM, Read RC. et al. Nitric oxide metabolism in Neisseria meningitidis. J Bacteriol 2002; 184: 2987-2993.
• 10 Rock JD, Mahnane MR, Anjum MF. et al. The pathogen Neisseria meningitidis requires oxygen, but supplements growth by denitrification. Nitrite, nitric oxide and oxygen control respiratory flux at genetic and metabolic levels. Mol Microbiol 2005; 58: 800-809.
• 11 Vincent JL, Yagushi A, Pradier O. Platelet function in sepsis. Crit Care Med 2002; 30 (Suppl. 05) Suppl S313-317.
• 12 Baines PB, Hart CA. Severe meningococcal disease in childhood. Br J Anaesth 2003; 90: 72-83.
• 13 Fouassier M, Moreau D, Thiolliere F. et al. Evolution of thrombin formation and fibrinolysis markers, including thrombin-activatable fibrinolysis inhibitor, during severe meningococcemia. Pathophysiol Haemost Thromb 2005; 34: 284-287.
• 14 Aguilar-Garcia J, Olalla-Sierra J, Perea-Milla E. et al. Analysis of the epidemiological characteristics and prognostic factors in probable or confirmed invasive meningococcal disease in a cohort of adolescents and adults during an epidemic outbreak. Rev Clin Esp 2009; 209: 221-226.
• 15 Peters MJ, Heyderman RS, Faust S. et al. Severe meningococcal disease is characterized by early neutrophil but not platelet activation and increased formation and consumption of platelet-neutrophil complexes. J Leukocyte Biol 2003; 73: 722-730.
• 16 Mirlashari MR, Hagberg IA, Lyberg T. Platelet-platelet and platelet-leukocyte interactions induced by outer membrane vesicles from N. meningitidis. Platelets 2002; 13: 91-99.
• 17 Unkmeir A, Latsch K, Dietrich G. et al. Fibronectin mediates Opc-dependent internalization of Neisseria meningitidis in human brain microvascular endothelial cells. Mol Microbiol 2002; 46: 933-946.
• 18 Schubert-Unkmeir A, Sokolova O, Panzner U. et al. Gene expression pattern in human brain endothelial cells in response to Neisseria meningitidis. Infect Immun 2007; 75: 899-914.
• 19 Slanina H, Konig A, Hebling S. et al. Entry of Neisseria meningitidis into mammalian cells requires the Src family protein tyrosine kinases. Infect Immun 2010; 78: 1905-1914.
• 20 Sokolova O, Heppel N, Jagerhuber R. et al. Interaction of Neisseria meningitidis with human brain microvascular endothelial cells: role of MAP- and tyrosine kinases in invasion and inflammatory cytokine release. Cell Microbiol 2004; 6: 1153-1166.
• 21 Sa ECC, Griffiths NJ, Virji M. Neisseria meningitidis Opc invasin binds to the sulphated tyrosines of activated vitronectin to attach to and invade human brain endothelial cells. PLoS Pathog 2010; 6: e1000911
• 22 McGuinness BT, Clarke IN, Lambden PR. et al. Point mutation in meningococcal por A gene associated with increased endemic disease. Lancet 1991; 337: 514-517.
• 23 Parkhill J, Achtman M, James KD. et al. Complete DNA sequence of a serogroup A strain of Neisseria meningitidis Z2491. Nature 2000; 404: 502-506.
• 24 Claus H, Maiden MC, Maag R. et al. Many carried meningococci lack the genes required for capsule synthesis and transport. Microbiology 2002; 148: 1813-1819.
• 25 Kriz P, Musilek M, Skoczynska A. et al. Genetic and antigenic characteristics of Neisseria meningitidis strains isolated in the Czech Republic in 1997-1998. Eur J Clin Microbiol Infect Dis 2000; 19: 452-459.
• 26 Tettelin H, Saunders NJ, Heidelberg J. et al. Complete genome sequence of Neisseria meningitidis serogroup B strain MC58. Science 2000; 287: 1809-1815.
• 27 Unkmeir A, Kammerer U, Stade A. et al. Lipooligosaccharide and polysaccharide capsule: virulence factors of Neisseria meningitidis that determine meningococcal interaction with human dendritic cells. Infect Immun 2002; 70: 2454-2462.
• 28 Stins MF, Gilles F, Kim KS. Selective expression of adhesion molecules on human brain microvascular endothelial cells. J Neuroimmunol 1997; 76: 81-90.
• 29 Schwarz UR, Geiger J, Walter U. et al. Flow cytometry analysis of intracellular VASP phosphorylation for the assessment of activating and inhibitory signal transduction pathways in human platelets--definition and detection of ticlopidine/clopidogrel effects. Thromb Haemost 1999; 82: 1145-1152.
• 30 Markert T, Vaandrager AB, Gambaryan S. et al. Endogenous expression of type II cGMP-dependent protein kinase mRNA and protein in rat intestine. Implications for cystic fibrosis transmembrane conductance regulator. J Clin Invest 1995; 96: 822-830.
• 31 Stefanelli P, Colotti G, Neri A. et al. Molecular characterization of nitrite reductase gene (aniA) and gene product in Neisseria meningitidis isolates: is aniA essential for meningococcal survival?. IUBMB Life 2008; 60: 629-636.
• 32 Thomson MJ, Stevanin TM, Moir JW. Measuring nitric oxide metabolism in the pathogen Neisseria meningitidis. Methods Enzymol 2008; 437: 539-560.
• 33 Zumft WG. The biological role of nitric oxide in bacteria. Arch Microbiol 1993; 160: 253-264.
• 34 Horstrup K, Jablonka B, Honig-Liedl P. et al. Phosphorylation of focal adhesion vasodilator-stimulated phosphoprotein at Ser157 in intact human platelets correlates with fibrinogen receptor inhibition. Eur J Biochem 1994; 225: 21-27.
• 35 Schwarz UR, Walter U, Eigenthaler M. Taming platelets with cyclic nucleotides. Biochem Pharmacol 2001; 62: 1153-1161.
• 36 Butt E, Abel K, Krieger M. et al. cAMP- and cGMP-dependent protein kinase phosphorylation sites of the focal adhesion vasodilator-stimulated phosphoprotein (VASP) in vitro and in intact human platelets. J Biol Chem 1994; 269: 14509-14517.
• 37 Allen MJ, Coleman RA. Beta 2-adrenoceptors mediate a reduction in endothelial permeability in vitro. Eur J Pharmacol 1995; 274: 7-15.
• 38 Schubert-Unkmeir A, Konrad C, Slanina H. et al. Neisseria meningitidis induces brain microvascular endothelial cell detachment from the matrix and cleavage of occludin: a role for MMP-8. PLoS Pathog 2010; 6: e1000874
• 39 Schlegel N, Burger S, Golenhofen N. et al. The role of VASP in regulation of cAMP- and Rac 1-mediated endothelial barrier stabilization. Am J Physiol Cell Physiol 2008; 294: C178-188.
• 40 Bazzoni G, Dejana E. Pores in the sieve and channels in the wall: control of paracellular permeability by junctional proteins in endothelial cells. Microcirculation 2001; 8: 143-152.
• 41 Rabiet MJ, Plantier JL, Rival Y. et al. Thrombin-induced increase in endothelial permeability is associated with changes in cell-to-cell junction organization. Arterioscler Thromb Vasc Biol 1996; 16: 488-496.
• 42 Sukumaran SK, Prasadarao NV. Escherichia coli K1 invasion increases human brain microvascular endothelial cell monolayer permeability by disassembling vascular-endothelial cadherins at tight junctions. J Infect Dis 2003; 188: 1295-1309.
• 43 Alderton WK, Cooper CE, Knowles RG. Nitric oxide synthases: structure, function and inhibition. Biochem J 2001; 357: 593-615.
• 44 Draijer R, Vaandrager AB, Nolte C. et al. Expression of cGMP-dependent protein kinase I and phosphorylation of its substrate, vasodilator-stimulated phosphoprotein, in human endothelial cells of different origin. Circ Res 1995; 77: 897-905.
• 45 Nolte C, Eigenthaler M, Schanzenbacher P. et al. Endothelial cell-dependent phosphorylation of a platelet protein mediated by cAMP- and cGMP-elevating factors. J Biol Chem 1991; 266: 14808-14812.
• 46 Radomski MW, Rees DD, Dutra A. et al. S-nitroso-glutathione inhibits platelet activation in vitro and in vivo. Br J Pharmacol 1992; 107: 745-749.
• 47 Palmer RM, Ferrige AG, Moncada S. Nitric oxide release accounts for the biological activity of endothelium-derived relaxing factor. Nature 1987; 327: 524-526.
• 48 Wallis JP. Nitric oxide and blood: a review. Transfus Med 2005; 15: 1-11.
• 49 Feletou M, Bonnardel E, Canet E. Bradykinin and changes in microvascular permeability in the hamster cheek pouch: role of nitric oxide. Br J Pharmacol 1996; 118: 1371-1376.
• 50 Murohara T, Horowitz JR, Silver M. et al. Vascular endothelial growth factor/vascular permeability factor enhances vascular permeability via nitric oxide and prostacyclin. Circulation 1998; 97: 99-107.
• 51 Eigenthaler M, Nolte C, Halbrugge M. et al. Concentration and regulation of cyclic nucleotides, cyclic-nucleotide-dependent protein kinases and one of their major substrates in human platelets. Estimating the rate of cAMP-regulated and cGMP-regulated protein phosphorylation in intact cells. Eur J Biochem 1992; 205: 471-481.
• 52 Chen Y, Rosazza JP. Purification and characterization of nitric oxide synthase (NOSNoc) from a Nocardia species. J Bacteriol 1995; 177: 5122-5128.
• 53 Choi WS, Chang MS, Han JW. et al. Identification of nitric oxide synthase in Staphylococcus aureus. Biochem Biophys Res Commun 1997; 237: 554-558.
• 54 Sobko T, Reinders I C, Jansson E. et al. Gastrointestinal bacteria generate nitric oxide from nitrate and nitrite. Nitric Oxide 2005; 13: 272-278.
• 55 Barth KR, Isabella VM, Clark VL. Biochemical and genomic analysis of the denitrification pathway within the genus Neisseria. Microbiology 2009; 155: 4093-4103.
• 56 Kornelisse RF, Hazelzet JA, Hop WC. et al. Meningococcal septic shock in children: clinical and laboratory features, outcome, and development of a prognostic score. Clin Infect Dis 1997; 25: 640-646.
• 57 Fisch A, Michael-Hepp J, Meyer J. et al. Synergistic interaction of adenylate cyclase activators and nitric oxide donor SIN-1 on platelet cyclic AMP. Eur J Pharmacol 1995; 289: 455-461.
• 58 Nolte C, Eigenthaler M, Horstrup K. et al. Synergistic phosphorylation of the focal adhesion-associated vasodilator-stimulated phosphoprotein in intact human platelets in response to cGMP- and cAMP-elevating platelet inhibitors. Biochem Pharmacol 1994; 48: 1569-1575.
• 59 Anfossi G, Massucco P, Mularoni E. et al. Effects of forskolin and organic nitrate on aggregation and intracellular cyclic nucleotide content in human platelets. Gen Pharmacol 1994; 25: 1093-1100.
• 60 Shukla A, Dikshit M, Srimal RC. Nitric oxide-dependent blood-brain barrier permeability alteration in the rat brain. Experientia 1996; 52: 136-140.
• 61 Zhang Y, Zhao S, Gu Y. et al. Effects of peroxynitrite and superoxide radicals on endothelial monolayer permeability: potential role of peroxynitrite in preeclampsia. J Soc Gynecol Investig 2005; 12: 586-592.
• 62 Wojciak-Stothard B, Tsang LY, Haworth SG. Rac and Rho play opposing roles in the regulation of hypoxia/reoxygenation-induced permeability changes in pulmonary artery endothelial cells. Am J Physiol Lung Cell Mol Physiol 2005; 288: L749-760.
• 63 Dejana E, Bazzoni G, Lampugnani MG. Vascular endothelial (VE)-cadherin: only an intercellular glue?. Exp Cell Res 1999; 252: 13-19.
• 64 Wong RK, Baldwin AL, Heimark RL. Cadherin-5 redistribution at sites of TNF-alpha and IFN-gamma-induced permeability in mesenteric venules. Am J Physiol 1999; 276: H736-748.
• 65 Navarro P, Caveda L, Breviario F. et al. Catenin-dependent and -independent functions of vascular endothelial cadherin. J Biol Chem 1995; 270: 30965-30972.
• 66 Coureuil M, Mikaty G, Miller F. et al. Meningococcal type IV pili recruit the polarity complex to cross the brain endothelium. Science 2009; 325: 83-87.
• 67 Smolenski A, Poller W, Walter U. et al. Regulation of human endothelial cell focal adhesion sites and migration by cGMP-dependent protein kinase I. J Biol Chem 2000; 275: 25723-25732.