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Review Article - (2015) Volume 1, Issue 2

Enzymatic Pathways of Intracellular Survival, Replication and Phagosomal Escape of Francisella spp.: A Review

Payam Behzadi and Reza Ranjbar*
Molecular Biology Research Center, Baqiyatallah University of Medical Sciences, Tehran, Iran
Corresponding Author: Dr. Reza Ranjbar, Molecular Biology Research Center, Baqiyatallah University of Medical Sciences, Tehran, Iran, E-mail: ranjbarre@gmail.com
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Abstract

Francisella tularensis is an important microbial agent which causes the severe infection of tularemia. The intracellular life cycle of this bacterium is supported by a vast range of protective guards such as enzymes. Although several enzymes are recognized in different subspecies of the bacterium, acid phosphatase and superoxide dismutase are seen in all bacterial subspecies. On the other hand, the most important enzymatic system relating to host cells is NADPH oxidase. For this reason, the clear aim of this mini-review is to discuss about the intracellular life cycle of Francisella tularensis and important enzymatic machineries in association to bacterium and its host cells. As a result, all of the biological systems including bacterial (prokaryotic) and host (eukaryotic) cells are protected by their enzymatic machineries. There are a wide range of enzymes within organisms. But, NADPH oxidase (in host cells), acid phosphatase and superoxide dismutase (in all of Francisella tularensis subspecies) were the main enzymes in this discussion. In conclusion, detection and identification of the enzymatic machineries and their activities enables us to find out appropriate mechanisms for definite therapeutic methods.

Keywords
Francisella tularensis; Phagocyte; Enzyme

Introduction
The genus of Francisella involves Gram negative, non-motile, and facultative intracellular pathogenic bacteria. It may cause different types of zoonotic infection of tularemia including glandular, oculoglandular, ulceroglandular, oropharyngeal (gastrointestinal), respiratory (pneumonic), and typhoidal demonstrations [1-4].
Francisella genus includes two species of Francisella tularensis (F. tularensis) and F.philomiragia. Besides, F. tularensis involves four subspecies (spp.) of tularensis (type A), novicida, holarctica (type B) and mediasiatica. However, the classification of F. tularensis ssp. novicida and F.novicida is variable in different sources [2-6].
Transmission of tularemia is achieved via direct contact with insect vectors, infected animals, ingesting contaminated food or water, and inhalation of aerosolized bacteria [7-10].
The harshness of tularemia is directly associated with the bacterial cell number, the way of bacterial entrance, and the bacterial species and subspecies. The most severe form of tularemia is caused by F. tularensis subspecies (spp.) tularensis. With ignorance of bacterial entrance, the lethal dosage of F. tularensis ssp. tularensis is about 10 cells and the rate of mortality in untreated situation is up to 35%. Thus, this subspecies is recognized as an important case regarding to bioterrorism concern. The centers for infectious diseases control and prevention (CDC) have classified the life threatening bacterium of F. tularensis spp. tularensis in category A [2,3,6,9,11,12].
In accordance with previous studies, the intracellular life cycle of Francisella within a host cell begins by entrance into the phagocytes and continues via bioactivities and replication. The host cells consist of alveolar and dendritic cells, fibroblasts, hepatocytes, macrophages and polymorphonuclear leukocytes (neutrophils) [2,13-16].
The process of intracellular replication is known as an important factor regarding to F. tularensis pathogenicity. The formation of pseudopod loops in macrophages reveals the entrance of F. tularensis into the cell. The bacterial entrance is followed by phagosomal creation within the host cell. The maturation of phagosomes containing bacterial cells of F. tularensis may lead to bacterial degradation. This mechanism is observed for other intracellular pathogenic bacteria like Legionella pneumophila, Mycobacterium tuberculosis, Salmonella typhimurium, Coxiella, Shigella flexneri, Listeria monocytogenes, Bartonella spp., Chlamydia spp., Rickettsia spp., and Brucella spp. The proton ATPase pump enables maturated phagolysosome to have a successfully achieve bacterial degradation throughout the process of acidification. For preventing degradation and hydrolyzation of the trapped bacteria, each bacterial genus has its special defense system. Normally, the intracellular bacteria are equipped by type III secretion system or type IV to escape from bacterial degradation; but F. tularensis is excluded. Regarding to F. tularensis, several factors such as Francisella Pathogenicity Island (FPI) regulates phagosomal escape, ensures bacterial survival and supports bacterial proliferation [4,8,17-28].
In this review, the commonest enzymatic pathways relating to bacterial intracellular survival, replication and phagosomal escape in F. tularensis are discussed.

Francisella pathogenicity island and bacterial intracellular life cycle
The 30-33 Kb FPI consists of several important virulent genes (up to 19 genes) including iglA-D (responsible for intracellular growth of Francisella within macrophages), iglG (virulence factor), iglI and pdpA-E which are in association with bacterial intracellular activities and replication [8,18,19,27,29-34].
During the process of phagocytosis, Francisella bacterial cells are uptaken into the macrophages and kept within phagosomes. The outcome of phagosomal maturation is the appearance of a bactericidal structure of phagolysosome. While, several studies show that, prior to phagosomal maturation, Francisella bacterial cells break down phagosomal membrane via isoenzymes of acid phosphatase. The process of membrane disruption usually occurs up to 1 hour after phagocytosis and may lead to release of bacteria into the macrophage cytosol and is followed by bacterial replication. The processes of apoptosis or autophagy are seen as postinfectional phase in macrophages [5-42].
Moreover, FPI encodes type VI secretion system which contributes to bacterial escape from phagosomal space into the macrophage cytosol milieu [8,43,44].
Another survey shows that, the escaping stage within Francisella containing phagosome (FCP) occurs via the proton vacuolar ATPase pump (vATPase pump). This pump is actually in association with degradation of trapped bacteria within the space of phagosomes. But, the evolution of Francisella bacteria has provided them a suitable means for escaping from phagosome into the macrophage's cytosol. As mentioned above, the secretion system type VI is the evolved mechanism which disrupts the phagosomal membrane in parallel with vATPase proton pump activity. This process is followed by bacterial replication within the macrophage cytoplasm. Recent studies show that, the secretion system type VI inhibits the activity of vATPase proton pump. In another word, activation of secretion system type VI deactivates the role of vATPase proton pump and may lead to bacterial phagosomal escape [37,45-48].
The process of bacterial escaping relating to Francisella in neutrophils differs from macrophages. The bacteria prevent assembly of phagocytes' nicotinamide adenine dinucleotide phosphate (NADPH) oxidase and inactivation of NADPH oxidase leads to bacterial intracellular survival [3,14,49,50].
New studies indicate that the process of acidification of Francisella Containing Phagosomes (FCPs) within neutrophil milieu is in parallel with NADPH oxidase activity. The active membranal NADPH oxidase (in phagocytes) is able to convert molecular oxygen into reactive oxygen species (ROS) and other types of superoxide anions such as hypochlorous acid, hydrogen peroxide and peroxynitrite. The ROS protect phagocytes' intracellular milieu from outsiders. On the other hand, Francisella bacteria encompass a wide range of enzymes including acid phosphatase, superoxide dismutase, and catalase KatG (in some subspecies) are multifunctional tools that provide appropriate situations for bacterial intracellular survival, phagosomal escape and multiplication [4,42,51-57].

Acid phosphatase
Acid phosphatase enzymes are able to break down different substrates such as peptides and proteins which encompass phosphorylated amino acid of tyrosine, phosphorylated inositol, phospholipid, AMP, ATP, NADP and etc. [39,58].
Acid phosphatases are recognized as bacterial virulence factors in association with F. tularensis. F. tularensis possesses a number of genes including acpA-C and hap that are responsible for acid phosphatase enzymes. The acid phosphatase enzymes include AcpA-C and Hap is responsible for preventing NADPH oxidase assembly. Besides, the aforementioned enzymes are needed for bacterial intracellular survival and phagosomal escape. Acid phosphatases are able to prevent the respiratory burst within phagocytes like neutrophils [39,49,51,53].

Superoxide dismutase (SOD)
Superoxide dismutase enzymes catalyze produced superoxide anions into molecular oxygen and hydrogen peroxide throughout the process of dismutation in the presence of their metal cofactors including Mn-, Fe-, and CuZn. sodB and sodC genes are present in Francisella spp. which encode SODs. SODs are needed for pathogenesis and bacterial intracellular survival via detoxifying of ROS produced within phagocytes. The enzyme of catalase KatG, is responsible for H2O2 conversion into oxygen and water in mice [49,50,55].

NADPH oxidase
An assembled and activated NADPH oxidase is ready for destroying microbial agents. The enzyme is sensitive and it would be activated right after any induction. Recent investigations show that, NADPH oxidase is an electrogenic enzyme, which sends the produced electrons into the phagosomes. This enzyme is a component of a phagocytic cell that mediates the interaction of NADPH and molecular oxygen to produce ROS. This system is advanced defense machinery which is recognized within eosinophils, macrophages, monocytes, neutrophils. The superoxide anions may be converted into hydrogen peroxide by the catalytic activity of superoxide dismutase [49,59-63].
The NADPH oxidase enzyme is structurally composed of two membranous subunits of gp91PHOX (PHOX: Phagocyte Oxidase) and p22PHOX, three cytosolic soluble compartments of gp67PHOX, p40PHOX, p47PHOX and G protein [62,63].
The p22PHOX and gp91PHOX (flavocytochrome b558) are situated within the membrane. The gp91PHOX is the main core of NADPH oxidase and encompasses a cytosolic tail. The complex is able to attach to free cytosolic phosphorylated p47PHOX and other components like p40PHOX and G protein to provide an activated oxidase. The p47PHOX takes soluble cytosolic proteins into the membraneous proteins. The p47PHOX contributes to produce superoxide, but in the presence of high amount of gp67PHOX, there is no need for p47PHOX, except in neutrophils. gp67PHOX is an important part of NADPH oxidase that mediates superoxide formation throughout transmission of electrons from NADPH part to molecular oxygen [62-68].
Francisella bacteria are able to survive within phagocytes via blocking either the attachment of cytosolic soluble components of NADPH oxidase into the enzymatic proteins of phagosomal membranes or the process of phosphorylation p47PHOX and p40PHOX Indeed, the regulated activity of flavocytochrome is targeted by the Francisella [2,6,63].

Conclusion
Francisella tularensis is responsible for severe forms of tularemia. The Francisella enzymatic machinery is an essential defense system which guarantees the bacterial growth, survival, replication and phagosomal escaping process. Moreover, the host cells are protected with enzymatic machinery which supports them against foreigners including microbial infectious agents. Recognition of enzymatic systems belonging to host cells and bacterial pathogens may lead to provide more effective and appropriate antibiotics for a definite treatment.
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References

  1. Esmaeili S, Gooya MM, Shirzadi MR, Esfandiari B, Amiri FB, et al. (2014) Seroepidemiological survey of tularemia among different groups in western Iran. Int J Infect Dis 18: 27-31.
  2. Celli J, Zahrt TC (2013) Mechanisms of Francisellatularensis intracellular pathogenesis. Cold Spring Harbor perspectives in medicine 3:a010314.
  3. Oyston PC (2008) Francisellatularensis: unravelling the secrets of an intracellular pathogen. J Med Microbiol 57: 921-930.
  4. Barel M, Charbit A2 (2013) Francisellatularensis intracellular survival: To eat or to die. Microbes Infect .
  5. Sjöstedt III A. Family (2005)Francisellaceae fam. nov.Bergey's Manual of Systematic Bacteriology 2:199-200.
  6. Binesse J, Lindgren H, Lindgren L, Conlan W, Sjöstedt A3 (2015) Roles of reactive oxygen species-degrading enzymes of Francisellatularensis SCHU S4. Infect Immun 83: 2255-2263.
  7. Hightower J, Kracalik IT, Vydayko N, Goodin D, Glass G, et al. (2014) Historical distribution and host-vector diversity of Francisellatularensis, the causative agent of tularemia, in Ukraine. Parasit Vectors 7:1-6.
  8. Bröms JE, Sjöstedt A, Lavander M (2010) The Role of the FrancisellaTularensis Pathogenicity Island in Type VI Secretion, Intracellular Survival, and Modulation of Host Cell Signaling. Front Microbiol 1: 136.
  9. Su J, Yang J, Zhao D, Kawula TH, Banas JA, et al. (2007) Genome-wide identification of Francisellatularensis virulence determinants. Infect Immun 75: 3089-3101.
  10. Barel M, Ramond E, Gesbert G, Charbit A1 (2015) The complex amino acid diet of Francisella in infected macrophages. Front Cell Infect Microbiol 5: 9.
  11. Dennis DT, Inglesby TV, Henderson DA, Bartlett JG, Ascher MS, et al. (2001) Tularemia as a biological weapon: medical and public health management. JAMA 285: 2763-2773.
  12. Alm E, Advani A, Bråve A, Wahab T2 (2015) Draft Genome Sequence of Strain R13-38 from a Francisellatularensis Outbreak in Sweden. Genome Announc 3.
  13. Oyston PC, Sjostedt A, Titball RW (2004) Tularaemia: bioterrorism defence renews interest in Francisellatularensis. Nat Rev Microbiol 2: 967-978.
  14. McCaffrey RL, Allen L-AH (2006)Francisellatularensis LVS evades killing by human neutrophils via inhibition of the respiratory burst and phagosome escape. J Leukoc Biol 80:1224-1230.
  15. Hall JD, Craven RR, Fuller JR, Pickles RJ, Kawula TH (2007)Francisellatularensis replicates within alveolar type II epithelial cells in vitro and in vivo following inhalation. Infect Immun75:1034-1039.
  16. Hall JD, Woolard MD, Gunn BM, Craven RR, Taft-Benz S, et al. (2008) Infected-host-cell repertoire and cellular response in the lung following inhalation of FrancisellatularensisSchu S4, LVS, or U112. Infect Immun76:5843-5852.
  17. Tärnvik A (1989) Nature of protective immunity to Francisellatularensis. Rev Infect Dis 11: 440-451.
  18. Wehrly TD, Chong A, Virtaneva K, Sturdevant DE, Child R, et al. (2009) Intracellular biology and virulence determinants of Francisellatularensis revealed by transcriptional profiling inside macrophages. Cell Microbiol11:1128-1150.
  19. Meibom KL, Charbit A (2010) The unraveling panoply of Francisellatularensis virulence attributes. CurrOpinMicrobiol 13: 11-17.
  20. Meyer L (2015) The Francisella pathogenicity island: its role in type VI secretion and intracellular infection.
  21. Clemens DL, Lee BY, Horwitz MA (2005) Francisellatularensis enters macrophages via a novel process involving pseudopod loops. Infect Immun 73: 5892-5902.
  22. Santic M, Molmeret M, Klose KE, Abu Kwaik Y (2006) Francisellatularensis travels a novel, twisted road within macrophages. Trends Microbiol 14: 37-44.
  23. Brotcke A, Weiss DS, Kim CC, Chain P, Malfatti S, et al. (2006) Identification of MglA-regulated genes reveals novel virulence factors in Francisellatularensis. Infect Immun 74: 6642-6655.
  24. Kahn RA, Fu H, Roy CR (2002) Cellular hijacking: a common strategy for microbial infection. Trends Biochem Sci 27: 308-314.
  25. Duclos S, Desjardins M (2000) Subversion of a young phagosome: the survival strategies of intracellular pathogens. Cell Microbiol 2: 365-377.
  26. Hackstadt T (2000) Redirection of host vesicle trafficking pathways by intracellular parasites. Traffic 1:93-99.
  27. Nano FE, Zhang N, Cowley SC, Klose KE, Cheung KK, et al. (2004) A Francisellatularensis pathogenicity island required for intramacrophage growth. J Bacteriol 186: 6430-6436.
  28. Ray K, Marteyn B, Sansonetti PJ, Tang CM (2009) Life on the inside: the intracellular lifestyle of cytosolic bacteria. Nat Rev Microbiol 7: 333-340.
  29. Child R, Wehrly TD, Rockx-Brouwer D, Dorward DW, Celli J (2010) Acid phosphatases do not contribute to the pathogenesis of type A Francisellatularensis. Infect Immun 78: 59-67.
  30. Reilly TJ, Baron GS, Nano FE, Kuhlenschmidt MS (1996) Characterization and sequencing of a respiratory burst-inhibiting acid phosphatase from Francisellatularensis. J Biol Chem 271: 10973-10983.
  31. Gray CG, Cowley SC, Cheung KK, Nano FE (2002) The identification of five genetic loci of Francisellanovicida associated with intracellular growth. FEMS MicrobiolLett 215: 53-56.
  32. Nano FE, Schmerk C (2007) The Francisellapathogenicity island. Ann N Y AcadSci 1105: 122-137.
  33. Bröms JE, Meyer L, Sun K, Lavander M, Sjöstedt A (2012) Unique substrates secreted by the type VI secretion system of Francisellatularensis during intramacrophage infection. PLoS One 7: e50473.
  34. Filloux A, Hachani A, Bleves S (2008) The bacterial type VI secretion machine: yet another player for protein transport across membranes. Microbiology 154: 1570-1583.
  35. Clemens DL, Lee BY, Horwitz MA (2009) Francisellatularensisphagosomal escape does not require acidification of the phagosome. Infect Immun 77: 1757-1773.
  36. Chong A, Wehrly TD, Nair V, Fischer ER, Barker JR, et al. (2008) The early phagosomal stage of Francisellatularensis determines optimal phagosomal escape and Francisella pathogenicity island protein expression. Infect Immun76:5488-5499.
  37. Checroun C, Wehrly TD, Fischer ER, Hayes SF, Celli J (2006) Autophagy-mediated reentry of Francisellatularensis into the endocytic compartment after cytoplasmic replication. ProcNatlAcadSci U S A103:14578-1483.
  38. Clemens DL, Horwitz MA (2007) Uptake and intracellular fate of Francisellatularensis in human macrophages. Ann N Y AcadSci 1105: 160-186.
  39. Mohapatra NP, Soni S, Reilly TJ, Liu J, Klose KE, et al. (2008) Combined deletion of four Francisellanovicida acid phosphatases attenuates virulence and macrophage vacuolar escape. Infect Immun 76: 3690-3699.
  40. Baron GS, Nano FE (1998) MglA and MglB are required for the intramacrophage growth of Francisellanovicida. MolMicrobiol 29: 247-259.
  41. Navratil AR, Brummett AM, Bryan JD, Woolard MD (2014)Francisellatularensis LVS induction of prostaglandin biosynthesis by infected macrophages requires specific host phospholipases and lipid phosphatases. Infect Immun82:3299-3311.
  42. Mohapatra NP, Balagopal A, Soni S, Schlesinger LS, Gunn JS (2007) AcpA is a Francisella acid phosphatase that affects intramacrophage survival and virulence. Infect Immun 75: 390-396.
  43. Barker JR, Chong A, Wehrly TD, Yu JJ, Rodriguez SA, et al. (2009) The Francisellatularensispathogenicity island encodes a secretion system that is required for phagosome escape and virulence. MolMicrobiol74:1459-1470.
  44. de Bruin OM, Duplantis BN, Ludu JS, Hare RF, Nix EB, et al. (2011) The biochemical properties of the Francisella pathogenicity island (FPI)-encoded proteins IglA, IglB, IglC, PdpB and DotU suggest roles in type VI secretion. Microbiology 157: 3483-3491.
  45. Santic M, Asare R, Skrobonja I, Jones S, Abu Kwaik Y (2008) Acquisition of the vacuolar ATPase proton pump and phagosome acidification are essential for escape of Francisellatularensis into the macrophage cytosol. Infect Immun 76: 2671-2677.
  46. Asare R, Abu Kwaik Y (2007)Early trafficking and intracellular replication of Legionella longbeachaea within an ER-derived late endosome-like phagosome. Cell Microbiol. 9:1571-1587.
  47. Santic M, Molmeret M, Barker JR, Klose KE, Dekanic A, et al. (2007) A Francisellatularensis pathogenicity island protein essential for bacterial proliferation within the host cell cytosol. Cell Microbiol. 9:2391-2403.
  48. Straskova A, Stulik J (2012) Intracellular pathogenesis of Francisellatularensis. Milit Med SciLett81:27.
  49. Llewellyn AC (2012) Investigating Francisella intracellular survival mechanisms: Emory University.
  50. Pohanka M (2013) Role of oxidative stress in infectious diseases. A review. Folia Microbiol (Praha) 58: 503-513.
  51. Mohapatra NP, Soni S, Rajaram MV, Strandberg KL, Gunn JS (2013) Type A Francisellatularensis acid phosphatases contribute to pathogenesis. PLoS One 8: e56834.
  52. McRae S, Pagliai FA, Mohapatra NP, Gener A, Mahmou ASA, Gunn JS, et al. (2010) Inhibition of AcpA phosphatase activity with ascorbate attenuates Francisellatularensisintramacrophage survival. Journal of Biological Chemistry 285:5171-5177.
  53. Dai S, Mohapatra NP, Schlesinger LS, Gunn JS (2012) The acid phosphatase AcpA is secreted in vitro and in macrophages by Francisella spp. Infect Immun 80: 1088-1097.
  54. Bakshi CS, Malik M, Regan K, Melendez JA, Metzger DW, Pavlov VM, et al. (2006) Superoxide dismutase B gene (sodB)-deficient mutants of Francisellatularensis demonstrate hypersensitivity to oxidative stress and attenuated virulence. Journal of bacteriology 188:6443-6448.
  55. Melillo AA, Mahawar M, Sellati TJ, Malik M, Metzger DW, et al. (2009) Identification of Francisellatularensis live vaccine strain CuZn superoxide dismutase as critical for resistance to extracellularly generated reactive oxygen species. J Bacteriol 191: 6447-6456.
  56. Lindgren H, Honn M, Salomonsson E, Kuoppa K, Forsberg Å, Sjöstedt A (2011) Iron content differs between Francisellatularensis subspecies tularensis and subspecies holarctica strains and correlates to their susceptibility to H2O2-induced killing. Infection and immunity 79:1218-1224.
  57. Zahrt TC, Deretic V (2002) Reactive nitrogen and oxygen intermediates and bacterial defenses: unusual adaptations in Mycobacterium tuberculosis. Antioxid Redox Signal 4:141-159.
  58. Rossolini G, Schippa S, Riccio M, Berlutti F, Macaskie L, Thaller M (1998) Bacterial nonspecific acid phosphohydrolases: physiology, evolution and use as tools in microbial biotechnology. Cellular and Molecular Life Sciences CMLS 54:833-850.
  59. Ago T, Nunoi H, Ito T, Sumimoto H (1999) Mechanism for Phosphorylation-induced Activation of the Phagocyte NADPH Oxidase Protein p47 phoxtriple replacement of serines 30, 304, and 328 with aspartates disrupts the sh3 domain-mediated intramolecular interaction in p47 phox, thereby activating the oxidase. Journal of biological chemistry 274: 33644-33653.
  60. Batot G, Martel C, Capdeville N, Wientjes F, Morel F (1995) Characterization of Neutrophil NADPH Oxidase Activity Reconstituted in a Cell-Free Assay Using Specific Monoclonal Antibodies Raised Against Cytochrome b558. European Journal of Biochemistry 234: 208-215.
  61. Segal AW, Garcia R, Goldstone H, Cross AR, Jones OT (1981) Cytochrome b-245 of neutrophils is also present in human monocytes, macrophages and eosinophils. Biochem J 196: 363-367.
  62. Nunes P, Demaurex N, Dinauer MC (2013) Regulation of the NADPH oxidase and associated ion fluxes during phagocytosis. Traffic 14: 1118-1131.
  63. Han CH, Lee MH (2000) Expression and characterization of the flavoprotein domain of gp91phox. J Vet Sci 1: 19-26.
  64. Dang PM, Babior BM, Smith RM (1999) NADPH dehydrogenase activity of p67PHOX, a cytosolic subunit of the leukocyte NADPH oxidase. Biochemistry 38: 5746-5753.
  65. Dinauer MC, Pierce E, Bruns G, Curnutte J, Orkin S (1990) Human neutrophil cytochrome blight chain (p22-phox). Gene structure, chromosomal location, and mutations in cytochrome-negative autosomal recessive chronic granulomatous disease. Journal of Clinical Investigation 86: 1729.
  66. Lapouge K, Smith SJ, Groemping Y, Rittinger K (2002) Architecture of the p40-p47-p67phox complex in the resting state of the NADPH oxidase. A central role for p67phox. J Biol Chem 277: 10121-10128.
  67. Freeman JL, Lambeth JD (1996) NADPH oxidase activity is independent of p47phox in vitro. J Biol Chem 271: 22578-22582.