Identification of OmpA, a Protein Involved in Host Cell Invasion, by Multi-Phenotypic High-Content Screening

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Infectious diseases are among the major causes of mortality worldwide. Pathogens‚ invasion, colonization and persistence within their hosts depend on a tightly orchestrated cascade of events that are commonly referred to as host/pathogen interactions. These interactions are extremely diversified and every pathogen is characterized by its unique way of co-opting and manipulating host functions to its advantage. Understanding host/pathogen interactions is the key to face the threats imposed by infectious diseases and find alternative strategies to fight the emergence of multi-drug resistant pathogens. In this study, we have setup and validated a protocol for the rapid and unbiased identification of bacterial factors that regulate host/pathogen interactions. We have applied this method to the study of Coxiella burnetii, the etiological agent of the emerging zoonosis Q fever. We have isolated, sequenced and screened over 1000 bacterial mutations and identified genes important for Coxiella invasion and replication within host cells. Ultimately, we have characterized the first Coxiella invasin, which mediates bacterial internalization within non-phagocytic cells. Most importantly, our finding may lead to the development of a synthetic vaccine against Q fever.

Vyšlo v časopise: Identification of OmpA, a Protein Involved in Host Cell Invasion, by Multi-Phenotypic High-Content Screening. PLoS Pathog 10(3): e32767. doi:10.1371/journal.ppat.1004013
Kategorie: Research Article
prolekare.web.journal.doi_sk: https://doi.org/10.1371/journal.ppat.1004013

Souhrn

Zdroje

1. MaurinM, RaoultD (1999) Q fever. Clin Microbiol Rev 12 : 518–553.

2. Van SchaikEJ, ChenC, MertensK, WeberMM, SamuelJE (2013) Molecular pathogenesis of the obligate intracellular bacterium Coxiella burnetii. Nat Rev Microbiol 11 : 561–573 doi:10.1038/nrmicro3049

3. KazarJ (2005) Coxiella burnetii infection. Ann N Y Acad Sci 1063 : 105–114 doi:10.1196/annals.1355.018

4. McCaulTF, WilliamsJC (1981) Developmental cycle of Coxiella burnetii: structure and morphogenesis of vegetative and sporogenic differentiations. J Bacteriol 147 : 1063–1076.

5. VothDE, HeinzenRA (2007) Lounging in a lysosome: the intracellular lifestyle of Coxiella burnetii. Cell Microbiol 9 : 829–840 doi:10.1111/j.1462-5822.2007.00901.x

6. Arricau-BouveryN, RodolakisA (2005) Is Q Fever an emerging or re-emerging zoonosis? Vet Res 36 : 327–349 doi:10.1051/vetres

7. MadariagaMG, RezaiK, TrenholmeGM, WeinsteinRA (2003) Review Q fever: a biological weapon in your backyard. The Lancet infectious diseases 3 : 709–721.

8. HackstadtT, PeacockMG, HitchcockPJ, ColeRL (1985) Lipopolysaccharide Variation in Coxiella burnetii: Intrastrain Heterogeneity in Structure and Antigenicity. Infect Immun 48 : 359–365.

9. Moosa, HackstadtT (1987) Comparative virulence of intra -⁠ and interstrain lipopolysaccharide variants of Coxiella burnetii in the guinea pig model. Infect Immun 55 : 1144–1150.

10. AndohM, Russell-LodrigueKE, ZhangG, SamuelJE (2005) Comparative virulence of phase I and II Coxiella burnetii in immunodeficient mice. Ann N Y Acad Sci 1063 : 167–170 doi:10.1196/annals.1355.026

11. CapoC, LindbergFP, MeconiS, ZaffranY, TardeiG, et al. (1999) Subversion of monocyte functions by Coxiella burnetii: impairment of the cross-talk between alphavbeta3 integrin and CR3. J Immunol 163 : 6078–6085.

12. Russell-LodrigueKE, ZhangGQ, McMurrayDN, SamuelJE (2006) Clinical and pathologic changes in a guinea pig aerosol challenge model of acute Q fever. Infect Immun 74 : 6085–6091 doi:10.1128/IAI.00763-06

13. JensenTK, MontgomeryDL, JaegerPT, LindhardtT, AgerholmJS, et al. (2007) Application of fluorescent in situ hybridisation for demonstration of Coxiella burnetii in placentas from ruminant abortions. APMIS 115 : 347–353.

14. MeconiS, JacomoV, BoquetP (1998) Coxiella burnetii Induces Reorganization of the Actin Cytoskeleton in Human Monocytes. Infect Immun 66(11): 5527.

15. RosalesEM, AguileraMO, SalinasRP, CarminatiSa, ColomboMI, et al. (2012) Cortactin is involved in the entry of Coxiella burnetii into non-phagocytic cells. PLoS One 7: e39348 doi:10.1371/journal.pone.0039348

16. RomanoPS, GutierrezMG, BerónW, RabinovitchM, ColomboMI (2007) The autophagic pathway is actively modulated by phase II Coxiella burnetii to efficiently replicate in the host cell. Cell Microbiol 9 : 891–909 doi:10.1111/j.1462-5822.2006.00838.x

17. NewtonHJ, McDonoughJa, RoyCR (2013) Effector protein translocation by the Coxiella burnetii Dot/Icm type IV secretion system requires endocytic maturation of the pathogen-occupied vacuole. PLoS One 8: e54566 doi:10.1371/journal.pone.0054566

18. CareyKL, NewtonHJ, LührmannA, RoyCR (2011) The Coxiella burnetii Dot/Icm system delivers a unique repertoire of type IV effectors into host cells and is required for intracellular replication. PLoS Pathog 7: e1002056 doi:10.1371/journal.ppat.1002056

19. BeareP, GilkS, LarsonC, HillJ (2011) Dot/Icm Type IVB Secretion System Requirements for Coxiella burnetii Growth in Human Macrophages. MBio 2: e00175–11 doi:10.1128/mBio.00175-11

20. CampoyEM, MansillaME, ColomboMI (2013) Endocytic SNAREs are involved in optimal Coxiella burnetii vacuole development. Cell Microbiol 15 : 922–941 doi:10.1111/cmi.12087

21. VothDE, HoweD, HeinzenRA (2007) Coxiella burnetii inhibits apoptosis in human THP-1 cells and monkey primary alveolar macrophages. Infect Immun 75 : 4263–4271 doi:10.1128/IAI.00594-07

22. VothDE, HeinzenRA (2009) Sustained activation of Akt and Erk1/2 is required for Coxiella burnetii antiapoptotic activity. Infect Immun 77 : 205–213 doi:10.1128/IAI.01124-08

23. LührmannA, RoyCR (2007) Coxiella burnetii inhibits activation of host cell apoptosis through a mechanism that involves preventing cytochrome c release from mitochondria. Infect Immun 75 : 5282–5289 doi:10.1128/IAI.00863-07

24. LührmannA, NogueiraCV, CareyKL, RoyCR (2010) Inhibition of pathogen-induced apoptosis by a Coxiella burnetii type IV effector protein. Proc Natl Acad Sci U S A 107 : 18997–19001 doi:10.1073/pnas.1004380107

25. KlingenbeckL, EckartRA, BerensC, LührmannA (2012) The Coxiella burnetii type IV secretion system substrate CaeB inhibits intrinsic apoptosis at the mitochondrial level. Cell Microbiol [epub ahead of print]. doi:10.1111/cmi.12066

26. ChenC, BangaS, MertensK, WeberMM, GorbaslievaI, et al. (2010) Large-scale identification and translocation of type IV secretion substrates by Coxiella burnetii. Proc Natl Acad Sci U S A 107 : 21755–21760 doi:10.1073/pnas.1010485107

27. WeberMM, ChenC, RowinK, MertensK, GalvanG, et al. (2013) Identification of Coxiella burnetii type IV secretion substrates required for intracellular replication and Coxiella-containing vacuole formation. J Bacteriol 195 : 3914–3924 doi:10.1128/JB.00071-13

28. LifshitzZ, BursteinD, PeeriM, ZusmanT, SchwartzK, et al. (2013) Computational modeling and experimental validation of the Legionella and Coxiella virulence-related type-IVB secretion signal. Proc Natl Acad Sci U S A 110: E707–15 doi:10.1073/pnas.1215278110

29. PanX, LührmannA, SatohA, Laskowski-ArceMA, RoyCR (2008) Ankyrin repeat proteins comprise a diverse family of bacterial type IV effectors. Science 320 : 1651–1654 doi:10.1126/science.1158160

30. VothDE, HoweD, BearePA, VogelJP, UnsworthN, et al. (2009) The Coxiella burnetii Ankyrin Repeat Domain-Containing Protein Family Is Heterogeneous, with C-Terminal Truncations That Influence Dot/Icm-Mediated Secretion. J Bacteriol 191 : 4232–4242 doi:10.1128/JB.01656-08

31. VothDE, BearePa, HoweD, SharmaUM, SamoilisG, et al. (2011) The Coxiella burnetii cryptic plasmid is enriched in genes encoding type IV secretion system substrates. J Bacteriol 193 : 1493–1503 doi:10.1128/JB.01359-10

32. OmslandA, CockrellDC, HoweD, FischerER, VirtanevaK, et al. (2009) Host cell-free growth of the Q fever bacterium Coxiella burnetii. Proc Natl Acad Sci U S A 106 : 4430–4434 doi:10.1073/pnas.0812074106

33. BearePA, SandozKM, OmslandA, RockeyDD, HeinzenRA (2011) Advances in genetic manipulation of obligate intracellular bacterial pathogens. Front Microbiol 2 : 97 doi:10.3389/fmicb.2011.00097

34. BeareP, HoweD, CockrellD (2009) Characterization of a Coxiella burnetii ftsZ mutant generated by Himar1 transposon mutagenesis. J Bacteriol 191 : 1369–1381 doi:10.1128/JB.01580-08

35. BearePA, UnsworthN, AndohM, VothDE, OmslandA, et al. (2009) Comparative genomics reveal extensive transposon-mediated genomic plasticity and diversity among potential effector proteins within the genus Coxiella. Infect Immun 77 : 642–656 doi:10.1128/IAI.01141-08

36. BearePA, LarsonCL, GilkSD, HeinzenRA (2012) Two systems for targeted gene deletion in Coxiella burnetii. Appl Environ Microbiol 78 : 4580–4589 doi:10.1128/AEM.00881-12

37. VogelJP (2004) Turning a tiger into a house cat: using Legionella pneumophila to study Coxiella burnetii. Trends Microbiol 12 : 103–105.

38. ZamboniDS, McGrathS, RabinovitchM, RoyCR (2003) Coxiella burnetii express type IV secretion system proteins that function similarly to components of the Legionella pneumophila Dot/Icm system. Mol Microbiol 49 : 965–976 doi:10.1046/j.1365-2958.2003.03626.x

39. ZusmanT, YerushalmiG, SegalG (2003) Functional Similarities between the icm/dot Pathogenesis Systems of Coxiella burnetii and Legionella pneumophila. Infect Immun 71 : 3714–3723 doi:10.1128/IAI.71.7.3714

40. ZusmanT, AloniG, HalperinE, KotzerH, DegtyarE, et al. (2007) The response regulator PmrA is a major regulator of the icm/dot type IV secretion system in Legionella pneumophila and Coxiella burnetii. Mol Microbiol 63 : 1508–1523 doi:10.1111/j.1365-2958.2007.05604.x

41. SavojardoC, FariselliP, CasadioR (2013) BETAWARE: a machine-learning tool to detect and predict transmembrane beta-barrel proteins in prokaryotes. Bioinformatics 29 : 504–505 doi:10.1093/bioinformatics/bts728

42. NambaA, ManoN, TakanoH, BeppuT, UedaK, et al. (2008) OmpA is an adhesion factor of Aeromonas veronii, an optimistic pathogen that habituates in carp intestinal tract. J Appl Microbiol 105 : 1441–1451 doi:10.1111/j.1365-2672.2008.03883.x

43. SerinoL, NestaB, LeuzziR, FontanaMR, MonaciE, et al. (2007) Identification of a new OmpA-like protein in Neisseria gonorrhoeae involved in the binding to human epithelial cells and in vivo colonization. Mol Microbiol 64 : 1391–1403 doi:10.1111/j.1365-2958.2007.05745.x

44. FaganRP, LambertMa, SmithSGJ (2008) The hek outer membrane protein of Escherichia coli strain RS218 binds to proteoglycan and utilizes a single extracellular loop for adherence, invasion, and autoaggregation. Infect Immun 76 : 1135–1142 doi:10.1128/IAI.01327-07

45. BartraSS, GongX, LoricaCD, JainC, NairMKM, et al. (2012) The outer membrane protein A (OmpA) of Yersinia pestis promotes intracellular survival and virulence in mice. Microb Pathog 52 : 41–46 doi:10.1016/j.micpath.2011.09.009

46. MahawarM, AtianandMK, DotsonRJ, MoraV, RabadiSM, et al. (2012) Identification of a novel Francisella tularensis factor required for intramacrophage survival and subversion of innate immune response. J Biol Chem 287 : 25216–25229 doi:10.1074/jbc.M112.367672

47. DattaD, VaidehiN, FlorianoWB, KimKS, PrasadaraoNV, et al. (2003) Interaction of E. coli outer-membrane protein A with sugars on the receptors of the brain microvascular endothelial cells. Proteins 50 : 213–221 doi:10.1002/prot.10257

48. MarchC, MorantaD, RegueiroV, LlobetE, TomásA, et al. (2011) Klebsiella pneumoniae outer membrane protein A is required to prevent the activation of airway epithelial cells. J Biol Chem 286 : 9956–9967 doi:10.1074/jbc.M110.181008

49. SmithSGJ, MahonV, LambertMA, FaganRP (2007) A molecular Swiss army knife: OmpA structure, function and expression. FEMS Microbiol Lett 273 : 1–11 doi:10.1111/j.1574-6968.2007.00778.x

50. ConferAW, AyalewS (2013) The OmpA family of proteins: roles in bacterial pathogenesis and immunity. Vet Microbiol 163 : 207–222 doi:10.1016/j.vetmic.2012.08.019

51. SelvarajSK, PrasadaraoNV (2005) Escherichia coli K1 inhibits proinflammatory cytokine induction in monocytes by preventing NF-kB activation. Microbes and Infection 78 : 544–54 doi:10.1189/jlb.0904516.1

52. SoulasC, BaussantT (2000) Outer membrane protein A (OmpA) binds to and activates human macrophages. J Immunol 165 : 2335–2340.

53. KrüllM, BockstallerP, WuppermannFN, KluckenAC, MühlingJ, et al. (2006) Mechanisms of Chlamydophila pneumoniae-mediated GM-CSF release in human bronchial epithelial cells. Am J Respir Cell Mol Biol 34 : 375–382 doi:10.1165/rcmb.2004-0157OC

54. LavineMD, StrandMR (2002) Insect hemocytes and their role in immunity. Insect Biochem Mol Biol 32 : 1295–1309.

55. O'CallaghanD, VergunstA (2010) Non-mammalian animal models to study infectious disease: worms or fly fishing? Curr Opin Microbiol 79–85 doi:10.1016/j.mib.2009.12.005

56. FerrandonD, ImlerJ-L, HetruC, HoffmannJA (2007) The Drosophila systemic immune response: sensing and signalling during bacterial and fungal infections. Nat Rev Immunol 7 : 862–874 doi:10.1038/nri2194

57. VogelH, AltincicekB, GlöcknerG, VilcinskasA (2011) A comprehensive transcriptome and immune-gene repertoire of the lepidopteran model host Galleria mellonella. BMC Genomics 12 : 308 doi:10.1186/1471-2164-12-308

58. OlsenRJ, WatkinsME, CantuCC, BeresSB, MusserJM (2011) Virulence of serotype M3 Group A Streptococcus strains in wax worms (Galleria mellonella larvae). Virulence 2 : 111–119 doi:10.4161/viru.2.2.14338

59. ChampionOL, KarlyshevAV, SeniorNJ, WoodwardM, La RagioneR, et al. (2010) Insect infection model for Campylobacter jejuni reveals that O-methyl phosphoramidate has insecticidal activity. J Infect Dis 201 : 776–782 doi:10.1086/650494

60. BerginD, ReevesE (2005) Superoxide Production in Galleria mellonella Hemocytes: Identification of Proteins Homologous to the NADPH Oxidase Complex of Human Neutrophils. Infect Immun 73 : 4161–70 doi:10.1128/IAI.73.7.4161

61. MukherjeeK, AltincicekB, HainT, DomannE, VilcinskasA, et al. (2010) Galleria mellonella as a model system for studying Listeria pathogenesis. Appl Environ Microbiol 76 : 310–317 doi:10.1128/AEM.01301-09

62. HardingCR, SchroederGN, ReynoldsS, KostaA, CollinsJW, et al. (2012) Legionella pneumophila pathogenesis in the Galleria mellonella infection model. Infect Immun 80 : 2780–2790 doi:10.1128/IAI.00510-12

63. BrodinP, ChristopheT (2011) High-content screening in infectious diseases. Curr Opin Chem Biol 15 : 534–539 doi:10.1016/j.cbpa.2011.05.023

64. OmslandA, HeinzenRA (2011) Life on the outside: the rescue of Coxiella burnetii from its host cell. Annu Rev Microbiol 65 : 111–128 doi:10.1146/annurev-micro-090110-102927

65. PapenfortK, VogelJ (2010) Regulatory RNA in bacterial pathogens. Cell Host Microbe 8 : 116–127 doi:10.1016/j.chom.2010.06.008

66. Toledo-AranaA, RepoilaF, CossartP (2007) Small noncoding RNAs controlling pathogenesis. Curr Opin Microbiol 10 : 182–188 doi:10.1016/j.mib.2007.03.004

67. Pizarro-CerdáJ, CossartP (2006) Bacterial adhesion and entry into host cells. Cell 124 : 715–727 doi:10.1016/j.cell.2006.02.012

68. VeigaE, GuttmanJa, BonazziM, BoucrotE, Toledo-AranaA, et al. (2007) Invasive and adherent bacterial pathogens co-Opt host clathrin for infection. Cell Host Microbe 2 : 340–351 doi:10.1016/j.chom.2007.10.001

69. BonazziM, VasudevanL, MalletA, SachseM, SartoriA, et al. (2011) Clathrin phosphorylation is required for actin recruitment at sites of bacterial adhesion and internalization. 195 : 525–536 doi:10.1083/jcb.201105152

70. BonazziM, KühbacherA, Toledo-AranaA, MalletA, VasudevanL, et al. (2012) A common clathrin-mediated machinery co-ordinates cell-cell adhesion and bacterial internalization. Traffic 13 : 1653–1666 doi:10.1111/tra.12009

71. Pizarro-CerdáJ, BonazziM, CossartP (2010) Clathrin-mediated endocytosis: what works for small, also works for big. Bioessays 32 : 496–504 doi:10.1002/bies.200900172

72. PoreD, ChakrabartiMK (2013) Outer membrane protein A (OmpA) from Shigella flexneri 2a: a promising subunit vaccine candidate. Vaccine 31 : 3644–3650 doi:10.1016/j.vaccine.2013.05.100

73. HillmanRD, BaktashYM, MartinezJJ (2013) OmpA-mediated rickettsial adherence to and invasion of human endothelial cells is dependent upon interaction with α2β1 integrin. Cell Microbiol 15 : 727–741 doi:10.1111/cmi.12068

74. OjogunN, KahlonA, RaglandSA, TroeseMJ, MastronunzioJE, et al. (2012) Anaplasma phagocytophilum outer membrane protein A interacts with sialylated glycoproteins to promote infection of mammalian host cells. Infect Immun 80 : 3748–3760 doi:10.1128/IAI.00654-12

75. PopovVL, YuXJ, WalkerDH (2000) The 120 kDa outer membrane protein of Ehrlichia chaffeensis: preferential expression on dense-core cells and gene expression in Escherichia coli associated with attachment and entry. Microb Pathog 28 : 71–80 doi:10.1006/mpat.1999.0327

76. JeanninP, MagistrelliG, GoetschL, HaeuwJ-F, ThieblemontN, et al. (2002) Outer membrane protein A (OmpA): a new pathogen-associated molecular pattern that interacts with antigen presenting cells-impact on vaccine strategies. Vaccine 20 Suppl 4: A23–7.

77. OmslandA, BearePa, HillJ, CockrellDC, HoweD, et al. (2011) Isolation from animal tissue and genetic transformation of Coxiella burnetii are facilitated by an improved axenic growth medium. Appl Environ Microbiol 77 : 3720–3725 doi:10.1128/AEM.02826-10