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Infection and Immunity, November 2008, p. 5049-5061, Vol. 76, No. 11
0019-9567/08/$08.00+0     doi:10.1128/IAI.00425-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Isolation of Streptococcus pneumoniae Biofilm Mutants and Their Characterization during Nasopharyngeal Colonization{triangledown} ,{dagger}

Ernesto J. Muñoz-Elías,1 Joan Marcano,2 and Andrew Camilli1*

Howard Hughes Medical Institute and the Department of Molecular Biology and Microbiology, Tufts University, Boston, Massachusetts 02115,1 University of Puerto Rico, Mayagüez, Puerto Rico2

Received 4 April 2008/ Returned for modification 19 May 2008/ Accepted 3 September 2008

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.

* Corresponding author. Mailing address: Department of Molecular Biology and Microbiology, Tufts University, 136 Harrison Ave., Boston, MA 02111. Phone: (617) 636-2144. Fax: (617) 636-2175. E-mail: andrew.camilli{at}tufts.edu

{triangledown} Published ahead of print on 15 September 2008.

{dagger} Supplemental material for this article may be found at http://iai.asm.org/.

Editor: J. N. Weiser

Infection and Immunity, November 2008, p. 5049-5061, Vol. 76, No. 11
0019-9567/08/$08.00+0     doi:10.1128/IAI.00425-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.





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