The role of the LuxS quorum-sensing system in Streptococcus pneumoniae biofilms Pubblico

Abstract

The ability of Streptococcus pneumoniae to colonize the nasopharynx is an important risk
factor for pneumococcal disease and carriage. Biofilm production has been associated with the
persistence of other respiratory pathogens and S. pneumoniae is able to form biofilm structures
within the nasopharynx. Previous studies have demonstrated that the LuxS quorum-sensing
system plays a role in the ability of S. pneumoniae to persist within the nasopharynx of
experimentally infected mice. This quorum-sensing system has also been shown to play a role in
biofilm production among certain species of streptococci.

This thesis project investigated the role of the luxS gene in biofilm formation in S.
pneumoniae strain D39. Insertional inactivation was used to construct an isogenic mutant of
luxS. The luxS mutant genotype was confirmed using PCR and sequencing. Complemented
strain was prepared by cloning the luxS wild type gene in a Gram positive replicative plasmid.
Quantitative differences in biofilm production by wild type, mutant, and complemented strains
were assessed. The D39 luxS mutant produced ~80% less biofilm biomass than the wild type.
Complementation restored biofilm biomass to wild type levels. A series of experiments
demonstrated that, once produced, biofilm growth is in part due to expansion and reproduction of
the sessile cells, rather than recruitment of planktonic cells. These new insights into the genetics
of biofilm growth may allow researchers to better control pneumococcal infections and prevent
disease.

Table of Contents

Chapter 1: Streptococcus pneumoniae and biofilms

S. pneumoniae and disease
Carriage and persistence
Biofilms
Quorum-sensing
LuxS
Remaining scientific questions

Chapter 2: Construction and characterization of a lux S isogenic mutant
Introduction
Methods
Results
Discussion

Chapter 3: Role of biofilm cells on biofilm growth and development
Introduction
Methods
Results
Discussion

Chapter 4: Conclusions and Future Directions
Relationship between luxS and DNA competence
Biofilm formation and protein expression
Applications in Population Biology, Ecology, and Evolution

References


List of Tables and Figures

Table 1: Primers used in this study
Figure 1: Insertional inactivation of the luxS gene using a cassette containing an erythromycin resistance marker
Figure 2: Colony PCR of potential S. pneumoniae luxS mutants
Figure 3: PCR confirmation of the luxS mutant
Figure 4: Sequencing results
Figure 5: Biofilm formation by S. pneumoniae strain D39 is regulated by the luxS gene
Figure 6: Percentage of 24 hr biofilm biomass formed by S. pneumoniae D39 and SPJV05 luxS mutant
Figure 7: S. pneumoniae biofilm growth following removal of planktonic cells after 6 hr incubation
Table 2: Colony forming units/ml counts for 6 hr biofilms following removal of planktonic cells

About this Master's Thesis

Rights statement

• Permission granted by the author to include this thesis or dissertation in this repository. All rights reserved by the author. Please contact the author for information regarding the reproduction and use of this thesis or dissertation.

School	Laney Graduate School
Department	Biological and Biomedical Sciences
Subfield / Discipline	Population Biology, Ecology & Evolution
Degree	MS
Submission	Master's Thesis
Language	English
Research Field	Biology, Microbiology
Parola chiave	biofilms Streptococcus pneumoniae quorum-sensing biofilm regulation LuxS
Committee Chair / Thesis Advisor	Klugman, Keith P, Emory University Vidal Graniel, Jorge Eugenio, Emory University
Committee Members	Shafer, William M, Emory University Conneely, Karen N, Emory University

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