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Infection and Immunity, July 2003, p. 3757-3765, Vol. 71, No. 7
0019-9567/03/$08.00+0     DOI: 10.1128/IAI.71.7.3757-3765.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Effect of Heat Shock and Mutations in ClpL and ClpP on Virulence Gene Expression in Streptococcus pneumoniae

Hyog-Young Kwon,1 Seung-Whan Kim,1 Moo-Hyun Choi,1 A. David Ogunniyi,2 James C. Paton,2 Sin-Hee Park,1 Suhk-Neung Pyo,1 and Dong-Kwon Rhee1*

College of Pharmacy, Sungkyunkwan University, Suwon 440-746, Korea,1 School of Molecular and Biomedical Science, The University of Adelaide, Adelaide SA 5005, Australia2

Received 4 December 2002/ Returned for modification 27 March 2003/ Accepted 10 April 2003

Spread of Streptococcus pneumoniae from the nasopharynx to other host tissues would require the organism to adapt to a variety of environmental conditions. Since heat shock proteins are induced by environmental stresses, we investigated the effect of heat shock on ClpL and ClpP synthesis and the effect of clpL and clpP mutations on the expression of key pneumococcal virulence genes. Pulse labeling with [35S]methionine and chase experiments as well as immunoblot analysis demonstrated that ClpL, DnaK, and GroEL were stable. Purified recombinant ClpL refolded urea-denatured rhodanese in a dose-dependent manner, demonstrating ClpL's chaperone activity. Although growth of the clpL mutant was not affected at 30 or 37°C, growth of the clpP mutant was severely affected at these temperatures. However, both clpL and clpP mutants were sensitive to 43°C. Although it was further induced by heat shock, the level of expression of ClpL in the clpP mutant was high at 30°C, suggesting that ClpP represses expression of ClpL. Furthermore, the clpP mutation significantly attenuated the virulence of S. pneumoniae in a murine intraperitoneal infection model, whereas the clpL mutation did not. Interestingly, immunoblot and real-time reverse transcription-PCR analysis demonstrated that pneumolysin and pneumococcal surface antigen A were induced by heat shock in wild-type S. pneumoniae. Other virulence genes were also affected by heat shock and clpL and clpP mutations. Virulence gene expression seems to be modulated not only by heat shock but also by the ClpL and ClpP proteases.

* Corresponding author. Mailing address: College of Pharmacy, Sungkyunkwan University, Suwon, 440-746, South Korea. Phone: 82 31 2907707. Fax: 82 31 2928800. E-mail: dkrhee{at}skku.edu.

Editor: A. D. O'Brien

Infection and Immunity, July 2003, p. 3757-3765, Vol. 71, No. 7
0019-9567/03/$08.00+0     DOI: 10.1128/IAI.71.7.3757-3765.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.



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