Glutamate Dehydrogenase (GdhA) of Streptococcus pneumoniae Is Required for High Temperature Adaptation

Abstract

During its progression from the nasopharynx to other sterile and nonsterile niches of its human host, Streptococcus pneumoniae must cope with changes in temperature. We hypothesized that the temperature adaptation is an important facet of pneumococcal survival in the host. Here, we evaluated the effect of temperature on pneumococcus and studied the role of glutamate dehydrogenase (GdhA) in thermal adaptation associated with virulence and survival. Microarray analysis revealed a significant transcriptional response to changes in temperature, affecting the expression of 252 genes in total at 34°C and 40°C relative to at 37°C. One of the differentially regulated genes was gdhA, which is upregulated at 40°C and downregulated at 34°C relative to 37°C. Deletion of gdhA attenuated the growth, cell size, biofilm formation, pH survival, and biosynthesis of proteins associated with virulence in a temperature-dependent manner. Moreover, deletion of gdhA stimulated formate production irrespective of temperature fluctuation. Finally, ΔgdhA grown at 40°C was less virulent than other temperatures or the wild type at the same temperature in a Galleria mellonella infection model, suggesting that GdhA is required for pneumococcal virulence at elevated temperature.

Keywords: CcpA; Galleria mellonella; GdhA; Streptococcus pneumoniae; temperature; transcriptional expression.

FIG 1

Growth profiles of pneumococcal strains in CDM supplemented with 55 mM glucose at different temperatures. Error bars show the standard error of the mean for three individual measurements, each with three replicates (n = 9). Significant differences were seen comparing the growth rate of the mutant strain to the wild-type D39 or among themselves at tested temperatures using ANOVA followed by Tukey’s multiple-comparison test. ****, P < 0.0001.

FIG 2

Hemolytic activity of pneumococcal strains grown in CDM supplemented with 55 mM glucose at different temperatures. Hemolytic activity assay was performed as a measure of pneumolysin activity and done using 4% (vol/vol) defibrinated sheep blood. Significant differences were seen in pneumococcal strains and at different temperatures using ANOVA and Tukey's multiple-comparison tests. Error bars show the standard error of the mean for three individual measurements, each with three independent biological samples. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

FIG 3

To access capsule, we measured glucuronic acid concentration of pneumococcal strains grown in CDM supplemented with 55 mM glucose at different temperatures. Significant differences were seen by comparing the amount of capsular polysaccharide produced among pneumococcal strains at different temperatures using ANOVA and Tukey's multiple-comparison tests. Error bars show the standard error of the mean for three individual measurements, each with three independent experiments. *, P < 0.05; **, P < 0.01; ****, P < 0.0001.

FIG 4

Biofilm formation of pneumococcal strains at different temperatures. Biofilms were measured using crystal violet assay on overnight cultures grown in static conditions. All values are expressed as optical density of stained adherent cells at 595 nm. Each column represents means of three individual measurements each with triplicates with their standard error of means. Mean differences in biofilm formation of the mutant strain were compared to the wild-type strain and among themselves at tested temperatures using ANOVA and Tukey's multiple-comparison tests. *, P < 0.05, ***, P < 0.01; ****, P < 0.001.

FIG 5

Acid tolerance of pneumococcal strains at 34°C, 37°C, or 40°C. Data represent the mean of three independent experiments, each with three replicates (n = 9). Mean differences in growth profile of the mutant strain were compared to the wild-type strain at different pH and temperature conditions using two-way ANOVA and Tukey's multiple-comparison tests. *, P < 0.05; ****, P < 0.0001.

FIG 6

Survival of G. mellonella infected with 5 × 10⁵ CFU/larvae. Each dot represents the number of dead larvae for individual group (n = 10) at 34°C (blue), 37°C (black), or 40°C (red). Significant differences in mortality numbers are seen comparing with the D39 wild-type strain with the mutant strain and among themselves at tested temperatures using ANOVA and Tukey’s multiple-comparison tests. Error bars show the standard error of the mean. *, P < 0.05; ***, P < 0.001; ****, P < 0.0001.

FIG 7

Measurement of metabolic end products in pneumococcal strains grown to mid-exponential phase in CDM supplemented with 55 mM glucose at different temperatures. The number of products normalized against 10⁸ CFU. Values (mM) are mean of three independent experiments, each with three replicates and expressed for 10⁸ CFU. ±, SEM; LDH, lactate dehydrogenase; PFL, pyruvate-formate lyase; ACK, acetate kinase.

FIG 8

β-Galactosidase activity of pneumococcal strains grown in CDM supplemented with 55 mM glucose. The activity is expressed in nmol p-nitrophenol/min/ml using mid-exponential-phase cultures. In the ΔccpA, the expression of PgdhA was significantly lower at 37°C than in the WT. Values are average of at least three independent experiments, each with three replicates. Error bars indicate the SEM. *, P < 0.05; **, P < 0.01; ****, P < 0.0001.