Effect of heat shock and mutations in ClpL and ClpP on virulence gene expression in Streptococcus pneumoniae

Abstract

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 [(35)S]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 degrees C, growth of the clpP mutant was severely affected at these temperatures. However, both clpL and clpP mutants were sensitive to 43 degrees C. Although it was further induced by heat shock, the level of expression of ClpL in the clpP mutant was high at 30 degrees 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.

FIG. 1.

Physical map of S. pneumoniae clpL locus.

FIG. 2.

Transient induction and stability of S. pneumoniae ClpL after heat shock. (A) To determine induction kinetics, exponentially growing CP1200 cells (A₅₅₀ = 0.2) were pulse labeled for 10 min with [³⁵S]methionine, starting from the indicated time after the shift to 42°C. Two milliliters of cultures was harvested, and the cells were lysed by sonication in lysis buffer. The cell lysates were then analyzed by SDS-PAGE, and protein bands were visualized by autoradiography. (B) To determine the stability of heat shock proteins, exponentially growing CP1200 cells (A₅₅₀ = 0.2) were stressed at 42°C for 10 min and pulse labeled with [³⁵S]methionine at that time, and then the cell cultures were returned to 30°C, followed by chasing with excess nonradioactive methionine for the indicated times. Two milliliters of cultures was harvested, and the cells were lysed by sonication. The cell lysates were then analyzed by SDS-PAGE, and protein bands were visualized by autoradiography. (C) To determine induction of ClpP, exponentially growing CP1200 cells (A₅₅₀ = 0.2) were pulse labeled for 10 min with [³⁵S]methionine, starting from the indicated time after a shift to 42°C. The proteins from 2 ml of culture were separated by SDS-15% PAGE and visualized by autoradiography. A representative of duplicate experiments is shown. Lane C, not stressed. Numbers on top show time elapsed (minutes) after a return to the nonstress condition. The heavy arrows indicate major HSPs. Molecular sizes are indicated on the left.

FIG. 3.

Steady accumulation of ClpL after heat shock. (A) Immunoblot analysis of whole-cell lysates of exponentially growing S. pneumoniae CP1200 cells exposed to 42°C. S. pneumoniae cells grown at 30°C to an A₅₅₀ = 0.3 were heat shocked at 42°C for the indicated times. The culture was harvested and resuspended in lysis buffer. The cells were lysed by sonication. Then 10 μg of proteins was separated by SDS-PAGE and reacted with antisera to ClpL, DnaK, and GroEL. In the case of ClpP, 30 μg of proteins was used for SDS-PAGE, followed by immunoblot analysis. (B) Densitometric analysis of relative levels of ClpL, ClpP, DnaK, and GroEL after heat shock, as shown in panel A. The figure shows the standard deviation from three independent experiments.

FIG. 4.

Growth of D39 and its clpL and clpP mutants. Cultures of D39 and its isogenic clpL (HYK304) and clpP (HYK302) mutants were grown to an absorbance at 550 nm of 0.1. The temperature was then shifted from 37°C to 43°C, and the cultures were incubated for the indicated times.

FIG. 5.

Induction of ClpL in clpP mutant. Exponentially growing S. pneumoniae CP1200 (A₅₅₀ = 0.3) and its isogenic clpL and clpP derivatives were heat shocked at 42°C for 30 min. Proteins from 3 ml of culture were subjected to immunoblot analysis with either anti-ClpL or anti-ClpP polyclonal serum. The positions of ClpL and ClpP are indicated.

FIG. 6.

Induction of virulence-associated genes by heat shock. Exponentially growing encapsulated S. pneumoniae D39 (A₆₀₀ = 0.1) and its isogenic clpP (HYK302) and clpL (HYK304) derivatives were heat shocked at 42°C for 20 min. Then 0.6 ml of culture was centrifuged, and the cell pellets were resuspended in lysis buffer, followed by boiling for 3 min. Subsequently, cell lysates were subjected to immunoblot analysis with a mixture of polyclonal antisera raised against CbpA, pneumolysin, and PsaA. The relative positions of CbpA, pneumolysin (Ply), and PsaA are indicated.

FIG. 7.

Relative mRNA concentrations of cbpA, cps2A, ply, and psaA in D39 and the clpL and clpP mutants before and after heat shock as determined by real-time RT-PCR. Between RNA extracts, levels of individual mRNA species were corrected with reference to that obtained for the internal 16S rRNA control. Data points represent means ± standard deviation of quadruplicate samples from each RNA extract.

FIG. 8.

Survival times of mice after intraperitoneal challenge. Groups of 10 BALB/c mice were infected with approximately 10⁵ CFU of D39 or its clpP (HYK302) or clpL (HYK304) derivative. Each point represents one mouse. The horizontal lines denote the median survival time for each group.