Streptococcus pneumoniae PstS production is phosphate responsive and enhanced during growth in the murine peritoneal cavity

Abstract

Differential display-PCR (DDPCR) was used to identify a Streptococcus pneumoniae gene with enhanced transcription during growth in the murine peritoneal cavity. Northern dot blot analysis and comparative densitometry confirmed a 1.8-fold increase in expression of the encoded sequence following murine peritoneal culture (MPC) versus laboratory culture or control culture (CC). Sequencing and basic local alignment search tool analysis identified the DDPCR fragment as pstS, the phosphate-binding protein of a high-affinity phosphate uptake system. PCR amplification of the complete pstS gene followed by restriction analysis and sequencing suggests a high level of conservation between strains and serotypes. Quantitative immunodot blotting using antiserum to recombinant PstS (rPstS) demonstrated an approximately twofold increase in PstS production during MPC from that during CCs, a finding consistent with the low levels of phosphate observed in the peritoneum. Moreover, immunodot blot and Northern analysis demonstrated phosphate-dependent production of PstS in six of seven strains examined. These results identify pstS expression as responsive to the MPC environment and extracellular phosphate concentrations. Presently, it remains unclear if phosphate concentrations in vivo contribute to the regulation of pstS. Finally, polyclonal antiserum to rPstS did not inhibit growth of the pneumococcus in vitro, suggesting that antibodies do not block phosphate uptake; moreover, vaccination of mice with rPstS did not protect against intraperitoneal challenge as assessed by the 50% lethal dose.

FIG. 1

Sectional autoradiograph of S. pneumoniae DDPCR P6 indicating differentially amplified (3.2-fold) DDPCR product P6-1. Reactions using total RNA from both MPC and CC were done in duplicate with 100 and 200 ng of total RNA. As a negative control, reactions without RT are shown in the designated lanes (cont).

FIG. 2

RNA dot blot analysis of DDPCR product P6-1. Total RNA was isolated from S. pneumoniae cultured in MPCs and CCs and spotted in parallel onto duplicate nylon membranes in the indicated amounts. Membranes were hybridized with either radiolabeled pstS or a probe to pneumococcal DNA gyrase subunit A. Hybridization of DNA gyrase subunit A served as a control to ensure equal loading of RNA. These experiments were performed in triplicate. pstS in MPC and in CC demonstrated a 1.8-fold difference.

FIG. 3

(A) A Western blot analysis of pneumococcal WCL demonstrating specificity of the rPstS antiserum to native PstS (33 kDa) (1) and rPstS (49 kDa) (2). The 37-kDa protein (3) is believed to be a product of degradation. Kilodaltons are indicated on left. (B) PstS production during MPCs and CCs as determined by quantitative immunodot blot analyses. Note the 2.2-fold difference between PstS production during MPCs (n = 4) and that during CCs (n = 6). This difference was determined to be statistically significant using a Student's t test (two samples, assuming equal variances) (P = 3.0 × 10⁻³).

FIG. 4

PstS production after 100 min of growth in 1, 3, 10, and 30 mM P_i as determined by quantitative immunodot blot analysis. Columns designated with asterisk indicate a statistically significant difference between the samples and PstS production during growth in 1.0 mM P_i (D39, 3 mM [P = 0.019] and 30 mM [P = 0.014]; WU2, 30 mM [P = 0.049]; ATCC 6303, 3 mM [P = 0.003] and 10 mM [P = 0.002]; DW4.1, 3 mM [P = 0.005], 10 mM [P = 0.017] and 30 mM [P = 0.0002]; and DW 14.1, 30 mM [P = 0.004]). Statistical analysis was performed using Student's t test (paired two samples for means).

FIG. 5

Northern dot blot analysis examining pstS transcription of WU2 and R6x during growth in various phosphate concentrations. O.D., optical density.