1887

Microbiology Society logo

Volume 168, Issue 8

Transcriptional regulation of the efflux pump operon: importance for antimicrobial resistance Free

This article is part of the Antimicrobial Efflux collection.

Abstract

This review focuses on the mechanisms of transcriptional control of an important multidrug efflux pump system (MtrCDE) possessed by , the aetiological agent of the sexually transmitted infection termed gonorrhoea. The operon that encodes this tripartite protein efflux pump is subject to both - and -acting transcriptional factors that negatively or positively influence expression. Critically, levels of MtrCDE can influence levels of gonococcal susceptibility to classical antibiotics, host-derived antimicrobials and various biocides. The regulatory systems that control can have profound influences on the capacity of gonococci to resist current and past antibiotic therapy regimens as well as virulence. The emergence, mechanisms of action and clinical significance of the transcriptional regulatory systems that impact expression in gonococci are reviewed here with the aim of linking bacterial antimicrobial resistance with multidrug efflux capability.

  • Received:
  • Accepted:
  • Published Online:
Keyword(s): antimicrobial resistance , efflux , Neisseria gonorrhoea , regulation and transcription
© Microbiology Society
Loading

Article metrics loading...

/content/journal/micro/10.1099/mic.0.001231
2022-08-02
2024-04-16
Loading full text...

Full text loading...

/deliver/fulltext/micro/168/8/mic001231.html?itemId=/content/journal/micro/10.1099/mic.0.001231&mimeType=html&fmt=ahah

References

  1. WHO World health organization.newsroom,fact sheets sexually transmitted infections (STIs); 2021 https://www.who.int/news-room/fact-sheets/detail/sexually-transmitted-infections-(stis)
  2. Unemo M, Del Rio C, Shafer WM. Antimicrobial resistance expressed by Neisseria gonorrhoeae: a major global public health problem in the 21st century. Microbiol Spectr 2016; 4: [View Article] [PubMed]
     [Google Scholar]
  3. Unemo M, Shafer WM. Antimicrobial resistance in Neisseria gonorrhoeae in the 21st century: past, evolution, and future. Clin Microbiol Rev 2014; 27:587–613 [View Article] [PubMed]
     [Google Scholar]
  4. Maness MJ, Sparling PF. Multiple antibiotic resistance due to a single mutation in Neisseria gonorrhoeae. J Infect Dis 1973; 128:321–330 [View Article] [PubMed]
     [Google Scholar]
  5. Guymon LF, Sparling PF. Altered crystal violet permeability and lytic behavior in antibiotic-resistant and -sensitive mutants of Neisseria gonorrhoeae. J Bacteriol 1975; 124:757–763 [View Article] [PubMed]
     [Google Scholar]
  6. Guymon LF, Walstad DL, Sparling PF. Cell envelope alterations in antibiotic-sensitive and-resistant strains of Neisseria gonorrhoeae. J Bacteriol 1978; 136:391–401 [View Article] [PubMed]
     [Google Scholar]
  7. Sarubbi FA, Sparling PF, Blackman E, Lewis E. Loss of low-level antibiotic resistance in Neisseria gonorrhoeae due to env mutations. J Bacteriol 1975; 124:750–756 [View Article] [PubMed]
     [Google Scholar]
  8. Eisenstein BI, Sparling PF. Mutations to increased antibiotic sensitivity in naturally-occurring gonococci. Nature 1978; 271:242–244 [View Article] [PubMed]
     [Google Scholar]
  9. Shafer WM, Guymon LF, Lind I, Sparling PF. Identification of an envelope mutation (env-10) resulting in increased antibiotic susceptibility and pyocin resistance in a clinical isolate of Neisseria gonorrhoeae. Antimicrob Agents Chemother 1984; 25:767–769 [View Article] [PubMed]
     [Google Scholar]
  10. George AM, Levy SB. Amplifiable resistance to tetracycline, chloramphenicol, and other antibiotics in Escherichia coli: involvement of a non-plasmid-determined efflux of tetracycline. J Bacteriol 1983; 155:531–540 [View Article] [PubMed]
     [Google Scholar]
  11. Pan W, Spratt BG. Regulation of the permeability of the gonococcal cell envelope by the mtr system. Mol Microbiol 1994; 11:769–775 [View Article] [PubMed]
     [Google Scholar]
  12. Hagman KE, Pan W, Spratt BG, Balthazar JT, Judd RC et al. Resistance of Neisseria gonorrhoeae to antimicrobial hydrophobic agents is modulated by the mtrRCDE efflux system. Microbiology 1995; 141 (Pt 3):611–622 [View Article]
     [Google Scholar]
  13. Robert Klein J, Henrich B, Plapp R. Plapp Molecular cloning of the EnvC gene of Escherichia coli. Curr Microbiol 1990; 21:341–347 [View Article]
     [Google Scholar]
  14. Ma D, Cook DN, Alberti M, Pon NG, Nikaido H et al. Molecular cloning and characterization of acrA and acrE genes of Escherichia coli. J Bacteriol 1993; 175:6299–6313 [View Article] [PubMed]
     [Google Scholar]
  15. Orth P, Schnappinger D, Hillen W, Saenger W, Hinrichs W. Structural basis of gene regulation by the tetracycline inducible Tet repressor-operator system. Nat Struct Biol 2000; 7:215–219 [View Article] [PubMed]
     [Google Scholar]
  16. Grkovic S, Brown MH, Roberts NJ, Paulsen IT, Skurray RA. QacR is a repressor protein that regulates expression of the Staphylococcus aureus multidrug efflux pump QacA. J Biol Chem 1998; 273:18665–18673 [View Article] [PubMed]
     [Google Scholar]
  17. Hagman KE, Lucas CE, Balthazar JT, Snyder L, Nilles M et al. The MtrD protein of Neisseria gonorrhoeae is a member of the resistance/nodulation/division protein family constituting part of an efflux system. Microbiology 1997; 143 (Pt 7):2117–2125 [View Article]
     [Google Scholar]
  18. Delahay RM, Robertson BD, Balthazar JT, Shafer WM, Ison CA. Involvement of the gonococcal MtrE protein in the resistance of Neisseria gonorrhoeae to toxic hydrophobic agents. Microbiology 1997; 143:2127–2133 [View Article]
     [Google Scholar]
  19. Koronakis V, Sharff A, Koronakis E, Luisi B, Hughes C. Crystal structure of the bacterial membrane protein TolC central to multidrug efflux and protein export. Nature 2000; 405:914–919 [View Article] [PubMed]
     [Google Scholar]
  20. Tsutsumi K, Yonehara R, Ishizaka-Ikeda E, Miyazaki N, Maeda S et al. Structures of the wild-type MexAB-OprM tripartite pump reveal its complex formation and drug efflux mechanism. Nat Commun 2019; 10:1520 [View Article] [PubMed]
     [Google Scholar]
  21. Lucas CE, Hagman KE, Levin JC, Stein DC, Shafer WM. Importance of lipooligosaccharide structure in determining gonococcal resistance to hydrophobic antimicrobial agents resulting from the mtr efflux system. Mol Microbiol 1995; 16:1001–1009 [View Article] [PubMed]
     [Google Scholar]
  22. Rouquette-Loughlin C, Stojiljkovic I, Hrobowski T, Balthazar JT, Shafer WM. Inducible, but not constitutive, resistance of gonococci to hydrophobic agents due to the MtrC-MtrD-MtrE efflux pump requires TonB-ExbB-ExbD proteins. Antimicrob Agents Chemother 2002; 46:561–565 [View Article] [PubMed]
     [Google Scholar]
  23. Shafer WM, Qu X, Waring AJ, Lehrer RI. Modulation of Neisseria gonorrhoeae susceptibility to vertebrate antibacterial peptides due to a member of the resistance/nodulation/division efflux pump family. Proc Natl Acad Sci U S A 1998; 95:1829–1833 [View Article] [PubMed]
     [Google Scholar]
  24. Veal WL, Yellen A, Balthazar JT, Pan W, Spratt BG et al. Loss-of-function mutations in the mtr efflux system of Neisseria gonorrhoeae. Microbiology 1998; 144:621–627 [View Article]
     [Google Scholar]
  25. Ma KC, Mortimer TD, Hicks AL, Wheeler NE, Sánchez-Busó L et al. Adaptation to the cervical environment is associated with increased antibiotic susceptibility in Neisseria gonorrhoeae. Nat Commun 2020; 11:4126 [View Article] [PubMed]
     [Google Scholar]
  26. Hagman KE, Shafer WM. Transcriptional control of the mtr efflux system of Neisseria gonorrhoeae. J Bacteriol 1995; 177:4162–4165 [View Article] [PubMed]
     [Google Scholar]
  27. Piddock LJV. Clinically relevant chromosomally encoded multidrug resistance efflux pumps in bacteria. Clin Microbiol Rev 2006; 19:382–402 [View Article] [PubMed]
     [Google Scholar]
  28. Folster JP, Johnson PJT, Jackson L, Dhulipali V, Dyer DW et al. MtrR modulates rpoH expression and levels of antimicrobial resistance in Neisseria gonorrhoeae. J Bacteriol 2009; 191:287–297 [View Article] [PubMed]
     [Google Scholar]
  29. Rouquette-Loughlin CE, Reimche JL, Balthazar JT, Dhulipala V, Gernert KM et al. Mechanistic basis for decreased antimicrobial susceptibility in a clinical isolate of Neisseria gonorrhoeae possessing a mosaic-Like mtr Efflux pump locus. mBio 2018; 9:e02281-18 [View Article] [PubMed]
     [Google Scholar]
  30. Veal WL, Nicholas RA, Shafer WM. Overexpression of the MtrC-MtrD-MtrE efflux pump due to an mtrR mutation is required for chromosomally mediated penicillin resistance in Neisseria gonorrhoeae. J Bacteriol 2002; 184:5619–5624 [View Article] [PubMed]
     [Google Scholar]
  31. Wadsworth CB, Arnold BJ, Sater MRA, Grad YH. Azithromycin resistance through interspecific acquisition of an epistasis-dependent efflux Pump component and transcriptional regulator in Neisseria gonorrhoeae. mBio 2018; 9:e01419-18 [View Article] [PubMed]
     [Google Scholar]
  32. Centers for Disease Control and Prevention (CDC) Update to CDC’s Sexually transmitted diseases treatment guidelines, 2010: oral cephalosporins no longer a recommended treatment for gonococcal infections. MMWR Morb Mortal Wkly Rep 2012; 61:590–594 [View Article] [PubMed]
     [Google Scholar]
  33. Trembizki E, Doyle C, Jennison A, Smith H, Bates J et al. A Neisseria gonorrhoeae strain with meningococcal mtrR sequence. J Med Microbiol 2014; 63:1113–1115 [View Article] [PubMed]
     [Google Scholar]
  34. Whiley DM, Kundu RL, Jennison AV, Buckley C, Limnios A et al. Azithromycin-resistant Neisseria gonorrhoeae spreading amongst men who have sex with men (MSM) and heterosexuals in New South Wales, Australia, 2017. J Antimicrob Chemother 2018; 73:1242–1246 [View Article] [PubMed]
     [Google Scholar]
  35. Demczuk W, Martin I, Peterson S, Bharat A, Van Domselaar G et al. Genomic epidemiology and molecular resistance mechanisms of azithromycin-resistant Neisseria gonorrhoeae in Canada from 1997 to 2014. J Clin Microbiol 2016; 54:1304–1313 [View Article] [PubMed]
     [Google Scholar]
  36. Johnson SR, Sandul AL, Parekh M, Wang SA, Knapp JS et al. Mutations causing in vitro resistance to azithromycin in Neisseria gonorrhoeae. Int J Antimicrob Agents 2003; 21:414–419 [View Article] [PubMed]
     [Google Scholar]
  37. Rouquette-Loughlin CE, Balthazar JT, Hill SA, Shafer WM. Modulation of the mtrCDE-encoded efflux pump gene complex of Neisseria meningitidis due to a correia element insertion sequence. Mol Microbiol 2004; 54:731–741 [View Article] [PubMed]
     [Google Scholar]
  38. Correia FF, Inouye S, Inouye M. A 26-base-pair repetitive sequence specific for Neisseria gonorrhoeae and Neisseria meningitidis genomic DNA. J Bacteriol 1986; 167:1009–1015 [View Article] [PubMed]
     [Google Scholar]
  39. Correia FF, Inouye S, Inouye M. A family of small repeated elements with some transposon-like properties in the genome of Neisseria gonorrhoeae. J Biol Chem 1988; 263:12194–12198 [PubMed]
     [Google Scholar]
  40. Buisine N, Tang CM, Chalmers R. Transposon-like correia elements: structure, distribution and genetic exchange between pathogenic Neisseria sp. FEBS Lett 2002; 522:52–58 [View Article] [PubMed]
     [Google Scholar]
  41. Warner DM, Shafer WM, Jerse AE. Clinically relevant mutations that cause derepression of the Neisseria gonorrhoeae MtrC-MtrD-MtrE Efflux pump system confer different levels of antimicrobial resistance and in vivo fitness. Mol Microbiol 2008; 70:462–478 [View Article] [PubMed]
     [Google Scholar]
  42. Ohneck EA, Zalucki YM, Johnson PJT, Dhulipala V, Golparian D et al. A novel mechanism of high-level, broad-spectrum antibiotic resistance caused by a single base pair change in Neisseria gonorrhoeae. mBio 2011; 2:e00187-11 [View Article] [PubMed]
     [Google Scholar]
  43. Ohneck EA, Goytia M, Rouquette-Loughlin CE, Joseph SJ, Read TD et al. Overproduction of the MtrCDE efflux pump in Neisseria gonorrhoeae produces unexpected changes in cellular transcription patterns. Antimicrob Agents Chemother 2015; 59:724–726 [View Article] [PubMed]
     [Google Scholar]
  44. Lucas CE, Balthazar JT, Hagman KE, Shafer WM. The MtrR repressor binds the DNA sequence between the mtrR and mtrC genes of Neisseria gonorrhoeae. J Bacteriol 1997; 179:4123–4128 [View Article] [PubMed]
     [Google Scholar]
  45. Beggs GA, Ayala JC, Kavanaugh LG, Read TD, Hooks GM et al. Structures of Neisseria gonorrhoeae MtrR-operator complexes reveal molecular mechanisms of DNA recognition and antibiotic resistance-conferring clinical mutations. Nucleic Acids Res 2021; 49:4155–4170 [View Article] [PubMed]
     [Google Scholar]
  46. Zalucki YM, Dhulipala V, Shafer WM. Dueling regulatory properties of a transcriptional activator (MtrA) and repressor (MtrR) that control efflux pump gene expression in Neisseria gonorrhoeae. mBio 2012; 3:e00446–12 [View Article] [PubMed]
     [Google Scholar]
  47. Beggs GA, Zalucki YM, Brown NG, Rastegari S, Phillips RK et al. Structural, biochemical, and In Vivo characterization of MtrR-mediated resistance to innate antimicrobials by the human athogen Neisseria gonorrhoeae. J Bacteriol 2019; 201:20 [View Article] [PubMed]
     [Google Scholar]
  48. Ayala JC, Shafer WM. Transcriptional regulation of a gonococcal gene encoding a virulence factor (L-lactate permease). PLoS Pathog 2019; 15:12 [View Article] [PubMed]
     [Google Scholar]
  49. Exley RM, Wu H, Shaw J, Schneider MC, Smith H et al. Lactate acquisition promotes successful colonization of the murine genital tract by Neisseria gonorrhoeae. Infect Immun 2007; 75:1318–1324 [View Article] [PubMed]
     [Google Scholar]
  50. Atack JM, Ibranovic I, Ong C-LY, Djoko KY, Chen NH et al. A role for lactate dehydrogenases in the survival of Neisseria gonorrhoeae in human polymorphonuclear leukocytes and cervical epithelial cells. J Infect Dis 2014; 210:1311–1318 [View Article] [PubMed]
     [Google Scholar]
  51. Ayala JC, Schmerer MW, Kersh EN, Unemo M, Shafer WM. Gonococcal clinical strains bearing a common gdhR Single nucleotide polymorphism that results in enhanced expression of the virulence Gene lctP frequently possess a mtrR promoter mutation that decreases antibiotic susceptibility. mBio 2022; 13:e0027622 [View Article] [PubMed]
     [Google Scholar]
  52. Laskos L, Ryan CS, Fyfe JAM, Davies JK. The RpoH-mediated stress response in Neisseria gonorrhoeae is regulated at the level of activity. J Bacteriol 2004; 186:8443–8452 [View Article] [PubMed]
     [Google Scholar]
  53. Gunesekere IC, Kahler CM, Powell DR, Snyder LAS, Saunders NJ et al. Comparison of the RpoH-dependent regulon and general stress response in Neisseria gonorrhoeae. J Bacteriol 2006; 188:4769–4776 [View Article] [PubMed]
     [Google Scholar]
  54. Cuthbertson L, Nodwell JR. The TetR family of regulators. Microbiol Mol Biol Rev 2013; 77:440–475 [View Article] [PubMed]
     [Google Scholar]
  55. Johnson PJT, Shafer WM. The transcriptional repressor, MtrR, of the mtrCDE efflux pump operon of Neisseria gonorrhoeae can also serve as an activator of “off target” gene (glnE) expression. Antibiotics 2015; 4:188–197 [View Article]
     [Google Scholar]
  56. Golparian D, Harris SR, Sánchez-Busó L, Hoffmann S, Shafer WM et al. Genomic evolution of Neisseria gonorrhoeae since the preantibiotic era (1928-2013): antimicrobial use/misuse selects for resistance and drives evolution. BMC Genomics 2020; 21:116 [View Article] [PubMed]
     [Google Scholar]
  57. Johnson PJT, Stringer VA, Shafer WM. Off-target gene regulation mediated by transcriptional repressors of antimicrobial efflux pump genes in Neisseria gonorrhoeae. Antimicrob Agents Chemother 2011; 55:2559–2565 [View Article] [PubMed]
     [Google Scholar]
  58. Mercante AD, Jackson L, Johnson PJT, Stringer VA, Dyer DW et al. MpeR regulates the mtr efflux locus in Neisseria gonorrhoeae and modulates antimicrobial resistance by an iron-responsive mechanism. Antimicrob Agents Chemother 2012; 56:1491–1501 [View Article] [PubMed]
     [Google Scholar]
  59. Yu C, McClure R, Nudel K, Daou N, Genco CA. Characterization of the Neisseria gonorrhoeae Iron and fur regulatory network. J Bacteriol 2016; 198:2180–2191 [View Article] [PubMed]
     [Google Scholar]
  60. Rouquette C, Harmon JB, Shafer WM. Induction of the mtrCDE-encoded efflux pump system of Neisseria gonorrhoeae requires MtrA, an AraC-like protein. Mol Microbiol 1999; 33:651–658 [View Article] [PubMed]
     [Google Scholar]
  61. Hobbs MM, Sparling PF, Cohen MS, Shafer WM, Deal CD et al. Experimental Gonococcal Infection in Male Volunteers: Cumulative Experience with Neisseria gonorrhoeae Strains FA1090 and MS11mkC. Front Microbiol 2011; 2:123 [View Article] [PubMed]
     [Google Scholar]
  62. Warner DM, Folster JP, Shafer WM, Jerse AE. Regulation of the MtrC-MtrD-MtrE efflux-pump system modulates the in vivo fitness of Neisseria gonorrhoeae. J Infect Dis 2007; 196:1804–1812 [View Article] [PubMed]
     [Google Scholar]
  63. Shafer WM, Balthazar JT, Hagman KE, Morse SA. Missense mutations that alter the DNA-binding domain of the MtrR protein occur frequently in rectal isolates of Neisseria gonorrhoeae that are resistant to faecal lipids. Microbiology 1995; 141 (Pt 4):907–911 [View Article]
     [Google Scholar]
  64. Olesky M, Zhao S, Rosenberg RL, Nicholas RA. Porin-mediated antibiotic resistance in Neisseria gonorrhoeae: ion, solute, and antibiotic permeation through PIB proteins with penB mutations. J Bacteriol 2006; 188:2300–2308 [View Article] [PubMed]
     [Google Scholar]
  65. Chen S, Connolly KL, Rouquette-Loughlin C, D’Andrea A, Jerse AE et al. Could dampening expression of the Neisseria gonorrhoeae mtrCDE-encoded efflux pump pe a strategy to preserve currently or resurrect formerly used antibiotics to reat Gonorrhea?. mBio 2019; 10:e01576-19 [View Article] [PubMed]
     [Google Scholar]
  66. Ohnishi M, Saika T, Hoshina S, Iwasaku K, Nakayama S et al. Ceftriaxone-resistant Neisseria gonorrhoeae, Japan. Emerg Infect Dis 2011; 17:148–149 [View Article] [PubMed]
     [Google Scholar]
  67. Gernert KM, Seby S, Schmerer MW, Thomas JC, Pham CD et al. Azithromycin susceptibility of Neisseria gonorrhoeae in the USA in 2017: a genomic analysis of surveillance data. Lancet Microbe 2020; 1:e154–e164 [View Article] [PubMed]
     [Google Scholar]
  68. Kersh EN, Allen V, Ransom E, Schmerer M, Cyr S et al. Rationale for a Neisseria gonorrhoeae usceptible-only interpretive breakpoint for Azithromycin. Clin Infect Dis 2020; 70:798–804 [View Article] [PubMed]
     [Google Scholar]
  69. Sánchez-Busó L, Cole MJ, Spiteri G, Day M, Jacobsson S et al. Europe-wide expansion and eradication of multidrug-resistant Neisseria gonorrhoeae lineages: a genomic surveillance study. Lancet Microbe 2022; 3:e452–e463 [View Article] [PubMed]
     [Google Scholar]
  70. St Cyr S, Barbee L, Workowski KA, Bachmann LH, Pham C et al. Update to CDC’s treatment guidelines for Gonococcal Infection, 2020. MMWR Morb Mortal Wkly Rep 2020; 69:1911–1916 [View Article] [PubMed]
     [Google Scholar]
  71. Evert BJ, Slesarenko VA, Punnasseril JM, Taha J, Zhan J et al. Self-inhibitory peptides Targeting the Neisseria gonorrhoeae MtrCDE Efflux pump increase antibiotic susceptibility. Antimicrob Agents Chemother 2022; 66:e0154221 [View Article]
     [Google Scholar]
  72. Chitsaz M, Booth L, Blyth MT, O’Mara ML, Brown MH. Multidrug resistance in Neisseria gonorrhoeae: identification of functionally important residues in the MtrD Efflux protein. mBio 2019; 10:e02277-19 [View Article]
     [Google Scholar]
  73. Ammerman L, Mertz SB, Park C, Wise JG. Transport dynamics of MtrD: An RND multidrug Efflux ump from Neisseria gonorrhoeae. Biochemistry 2021; 60:3098–3113 [View Article]
     [Google Scholar]
  74. Bolla JR, Su C-C, Do SV, Radhakrishnan A, Kumar N et al. Crystal structure of the Neisseria gonorrhoeae MtrD inner membrane multidrug efflux pump. PLoS One 2014; 9:e97903 [View Article]
     [Google Scholar]
  75. Lyu M, Moseng MA, Reimche JL, Holley CL, Dhulipala V et al. Cryo-EM structures of a Gonococcal multidrug Efflux pump illuminate a mechanism of drug recognition and esistance. mBio 2020; 11:e00996-20 [View Article]
     [Google Scholar]
  76. Berman HM, Westbrook J, Feng Z, Gilliland G, Bhat TN et al. The protein data bank. Nucleic Acids Res 2000; 28:235–242 [View Article] [PubMed]
     [Google Scholar]
  77. Lei H-T, Chou T-H, Su C-C, Bolla JR, Kumar N et al. Crystal structure of the open state of the Neisseria gonorrhoeae MtrE outer membrane channel. PLoS One 2014; 9:e97475 [View Article] [PubMed]
     [Google Scholar]
  78. Slynko V, Schubert M, Numao S, Kowarik M, Aebi M et al. NMR structure determination of a segmentally labeled glycoprotein using in vitro glycosylation. J Am Chem Soc 2009; 131:1274–1281 [View Article] [PubMed]
     [Google Scholar]
  79. Rouquette-Loughlin CE, Zalucki YM, Dhulipala VL, Balthazar JT, Doyle RG et al. Control of gdhR Expression in Neisseria gonorrhoeae via Autoregulation and a Master Repressor (MtrR) of a Drug Efflux Pump Operon. mBio 2017; 8:e00449-17 [View Article] [PubMed]
     [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/micro/10.1099/mic.0.001231
Loading
Transcriptional regulation of the mtrCDE efflux pump operon: importance for Neisseria gonorrhoeae antimicrobial resistance
Microbiology 168, 001231 (2022); https://doi.org/10.1099/mic.0.001231
/content/journal/micro/10.1099/mic.0.001231
/content/journal/micro/10.1099/mic.0.001231
Loading

Data & Media loading...

Most cited Most Cited RSS feed

This is a required field
Please enter a valid email address
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error