title: Isolation of Streptococcus pneumoniae biofilm mutants and their characterization during nasopharyngeal colonization.
abstract: Asymptomatic colonization of the nasopharynx by Streptococcus pneumoniae precedes pneumococcal disease, yet pneumococcal colonization factors remain poorly understood. Many bacterial infections involve biofilms which protect bacteria from host defenses and antibiotics. To gain insight into the genetics of biofilm formation by S. pneumoniae, we conducted an in vitro screen for biofilm-altered mutants with the serotype 4 clinical isolate TIGR4. In a first screen of 6,000 mariner transposon mutants, we repeatedly isolated biofilm-overproducing acapsular mutants, suggesting that the capsule was antagonistic to biofilm formation. Therefore, we screened 6,500 additional transposon mutants in an S. pneumoniae acapsular background. Following this approach, we isolated 69 insertions in 49 different genes. The collection of mutants includes genes encoding bona fide and putative choline binding proteins, adhesins, synthases of membrane and cell wall components, extracellular and cell wall proteases, efflux pumps, ABC and PTS transporters, and transcriptional regulators, as well as several conserved and novel hypothetical proteins. Interestingly, while four insertions mapped to rrgA, encoding a subunit of a recently described surface pilus, rrgB and rrgC (encoding the other two pilus subunits) mutants had no biofilm defects, implicating the RrgA adhesin but not the pilus structure per se in biofilm formation. To correlate our findings to the process of colonization, we transferred a set of 29 mutations into the wild-type encapsulated strain and then tested the fitness of the mutants in vivo. Strikingly, we found that 23 of these mutants were impaired for nasopharyngeal colonization, thus establishing a link between biofilm formation and colonization.
authors.affiliation: Howard Hughes Medical Institute and Department of Molecular Biology and Microbiology, Tufts University, Boston, MA 02115, USA.
authors.forename: Ernesto J
authors.initials: EJ
authors.lastname: Muñoz-Elías
authors.forename: Joan
authors.initials: J
authors.lastname: Marcano
authors.forename: Andrew
authors.initials: A
authors.lastname: Camilli
mesh_terms: D018829:Adhesins, Bacterial / Q000235:genetics / Q000378:metabolism; D000818:Animals; D016667:Bacterial Capsules / Q000737:chemistry / Q000378:metabolism; D018441:Biofilms; D005260:Female; D005798:Genes, Bacterial; D051379:Mice; D008810:Mice, Inbred C57BL; D009154:Mutation; D009305:Nasopharynx / Q000382:microbiology*; D011008:Pneumococcal Infections / Q000235:genetics*; D016133:Polymerase Chain Reaction; D013296:Streptococcus pneumoniae / Q000302:isolation & purification* / Q000502:physiology*
chemical_list: D018829:Adhesins, Bacterial
references.citation: J Infect Dis. 1998 Feb;177(2):368-77
references.pmid: 9466523
references.citation: J Antimicrob Chemother. 1997 Oct;40(4):601-2
references.pmid: 9372435
references.citation: Nat Rev Microbiol. 2008 Apr;6(4):288-301
references.pmid: 18340341
references.citation: J Bacteriol. 2003 Jan;185(2):413-21
references.pmid: 12511486
references.citation: Microb Pathog. 1997 Sep;23(3):127-37
references.pmid: 9281471
references.citation: J Med Microbiol. 2006 Apr;55(Pt 4):355-363
references.pmid: 16533981
references.citation: Nucleic Acids Res. 1992 Nov 25;20(22):6103
references.pmid: 1461744
references.citation: J Bacteriol. 2006 Apr;188(7):2325-35
references.pmid: 16547018
references.citation: J Bacteriol. 2006 Aug;188(16):5945-57
references.pmid: 16885463
references.citation: Lancet Infect Dis. 2004 Mar;4(3):144-54
references.pmid: 14998500
references.citation: Mol Microbiol. 1998 May;28(3):449-61
references.pmid: 9632250
references.citation: J Bacteriol. 2008 Apr;190(8):2767-76
references.pmid: 18156255
references.citation: JAMA. 2006 Jul 12;296(2):202-11
references.pmid: 16835426
references.citation: Infect Immun. 2005 Aug;73(8):4653-67
references.pmid: 16040978
references.citation: Mol Microbiol. 2001 Dec;42(5):1223-32
references.pmid: 11886554
references.citation: Science. 2001 Jul 20;293(5529):498-506
references.pmid: 11463916
references.citation: Microbiology (Reading). 2007 Dec;153(Pt 12):4088-4097
references.pmid: 18048922
references.citation: J Bacteriol. 2000 May;182(10):2675-9
references.pmid: 10781532
references.citation: EMBO J. 1996 Oct 1;15(19):5470-9
references.pmid: 8895590
references.citation: J Bacteriol. 2008 Jan;190(1):221-30
references.pmid: 17981977
references.citation: Nat Rev Microbiol. 2003 Dec;1(3):219-30
references.pmid: 15035026
references.citation: Mol Microbiol. 1997 Sep;25(5):819-29
references.pmid: 9364908
references.citation: Infect Immun. 2006 Apr;74(4):2453-6
references.pmid: 16552078
references.citation: J Biol Chem. 2002 Jul 19;277(29):26652-61
references.pmid: 11953442
references.citation: J Bacteriol. 2004 Mar;186(5):1249-57
references.pmid: 14973035
references.citation: Trends Microbiol. 2005 Jan;13(1):27-33
references.pmid: 15639629
references.citation: J Antimicrob Chemother. 1986 Jul;18 Suppl A:35-45
references.pmid: 3745031
references.citation: Mol Microbiol. 2005 Sep;57(6):1545-56
references.pmid: 16135223
references.citation: Mol Microbiol. 2002 Sep;45(5):1389-406
references.pmid: 12207705
references.citation: Microbes Infect. 2006 Jan;8(1):145-53
references.pmid: 16182588
references.citation: Science. 1998 Apr 10;280(5361):295-8
references.pmid: 9535661
references.citation: J Exp Med. 1931 Mar 31;53(4):535-52
references.pmid: 19869863
references.citation: Mol Microbiol. 2007 Jun;64(6):1455-65
references.pmid: 17504273
references.citation: Mol Microbiol. 2003 Nov;50(4):1103-10
references.pmid: 14622402
references.citation: Infect Immun. 2003 May;71(5):2758-65
references.pmid: 12704150
references.citation: J Bacteriol. 2007 Nov;189(22):8270-6
references.pmid: 17586629
references.citation: J Dent Res. 1998 Mar;77(3):445-52
references.pmid: 9496917
references.citation: J Bacteriol. 2006 Sep;188(18):6680-7
references.pmid: 16952960
references.citation: Nucleic Acids Res. 2006 Jan 1;34(Database issue):D257-60
references.pmid: 16381859
references.citation: Infect Immun. 2001 Sep;69(9):5430-9
references.pmid: 11500414
references.citation: Trends Microbiol. 2005 Jan;13(1):20-6
references.pmid: 15639628
references.citation: Proc Natl Acad Sci U S A. 2006 Feb 21;103(8):2857-62
references.pmid: 16481624
references.citation: FEMS Microbiol Lett. 2008 Jan;278(2):231-5
references.pmid: 18053067
references.citation: J Bacteriol. 2007 May;189(9):3369-81
references.pmid: 17322316
references.citation: Proc Natl Acad Sci U S A. 1998 Jul 21;95(15):8927-32
references.pmid: 9671781
references.citation: Infect Immun. 2004 Jul;72(7):3902-6
references.pmid: 15213133
references.citation: J Bacteriol. 2006 Nov;188(22):7785-95
references.pmid: 16936041
references.citation: Infect Immun. 2001 Jun;69(6):3755-61
references.pmid: 11349040
references.citation: Infect Immun. 2007 Jan;75(1):83-90
references.pmid: 17088346
references.citation: Annu Rev Microbiol. 2000;54:49-79
references.pmid: 11018124
references.citation: Infect Immun. 2005 Feb;73(2):730-40
references.pmid: 15664911
references.citation: Infect Immun. 2001 Apr;69(4):2309-17
references.pmid: 11254588
references.citation: Infect Immun. 1994 Jun;62(6):2582-9
references.pmid: 8188381
references.citation: Infect Immun. 2007 Feb;75(2):958-65
references.pmid: 17130253
references.citation: J Bacteriol. 2005 Sep;187(17):6238-41
references.pmid: 16109966
references.citation: J Bacteriol. 2000 Mar;182(5):1374-82
references.pmid: 10671461
references.citation: Infect Immun. 2005 Oct;73(10):6945-51
references.pmid: 16177374
references.citation: Infect Immun. 1979 Feb;23(2):545-8
references.pmid: 33904
references.citation: J Clin Invest. 2003 Dec;112(11):1626-32
references.pmid: 14660736
references.citation: Infect Immun. 2004 Aug;72(8):4888-90
references.pmid: 15271955
references.citation: Infect Immun. 2000 Oct;68(10):5690-5
references.pmid: 10992472
references.citation: J Clin Invest. 2006 Aug;116(8):2297-2305
references.pmid: 16862214
references.citation: JAMA. 2002 Apr 3;287(13):1710-5
references.pmid: 11926896
references.citation: Infect Immun. 2003 Jan;71(1):218-25
references.pmid: 12496169
references.citation: Trends Microbiol. 2005 Jan;13(1):34-40
references.pmid: 15639630
references.citation: J Exp Med. 2002 Feb 4;195(3):359-65
references.pmid: 11828011
references.citation: Infect Immun. 2007 Jan;75(1):350-7
references.pmid: 17088353
references.citation: FEMS Microbiol Lett. 2005 Dec 1;253(1):151-4
references.pmid: 16246506
references.citation: Mol Microbiol. 2006 Sep;61(5):1196-210
references.pmid: 16925554
references.citation: Science. 2003 Jul 4;301(5629):105-7
references.pmid: 12843396
references.citation: J Bacteriol. 2008 Feb;190(4):1436-46
references.pmid: 18065546
references.citation: J Bacteriol. 2007 Mar;189(5):2030-8
references.pmid: 17189375
references.citation: Mol Microbiol. 2000 Jan;35(2):251-9
references.pmid: 10652087
references.citation: Mol Microbiol. 2007 Oct;66(2):329-40
references.pmid: 17850254
references.citation: Antimicrob Agents Chemother. 2001 Jun;45(6):1761-70
references.pmid: 11353623
references.citation: Infect Immun. 2006 Jul;74(7):4014-20
references.pmid: 16790774
references.citation: Cell Microbiol. 2007 Jul;9(7):1822-33
references.pmid: 17359232
references.citation: Mol Microbiol. 2007 May;64(4):968-83
references.pmid: 17501921
references.citation: Mol Microbiol. 2002 Mar;43(6):1367-78
references.pmid: 11952892
references.citation: Mol Microbiol. 1996 Jul;21(1):21-31
references.pmid: 8843431
references.citation: J Infect Dis. 1997 Apr;175(4):839-46
references.pmid: 9086139
references.citation: Vaccine. 2004 Jun 2;22(17-18):2209-20
references.pmid: 15149779
references.citation: J Clin Microbiol. 2007 Jan;45(1):97-101
references.pmid: 17093036
references.citation: Science. 1999 May 21;284(5418):1318-22
references.pmid: 10334980
references.citation: J Infect Dis. 1980 Dec;142(6):923-33
references.pmid: 7462701
references.citation: Nature. 2001 Oct 25;413(6858):860-4
references.pmid: 11677611
references.citation: J Bacteriol. 2005 Nov;187(21):7425-33
references.pmid: 16237026
references.citation: Infect Immun. 2005 Dec;73(12):8298-305
references.pmid: 16299327
references.citation: Res Microbiol. 2006 May;157(4):306-14
references.pmid: 16487683
references.citation: Appl Environ Microbiol. 2004 Aug;70(8):4980-8
references.pmid: 15294838
references.citation: J Bacteriol. 2005 Dec;187(24):8340-9
references.pmid: 16321938