Arginine Decarboxylase Is Essential for Pneumococcal Stress Responses

Abstract

Polyamines such as putrescine, cadaverine, and spermidine are small cationic molecules that play significant roles in cellular processes, including bacterial stress responses and host-pathogen interactions. Streptococcus pneumoniae is an opportunistic human pathogen, which causes several diseases that account for significant morbidity and mortality worldwide. As it transits through different host niches, S. pneumoniae is exposed to and must adapt to different types of stress in the host microenvironment. We earlier reported that S. pneumoniae TIGR4, which harbors an isogenic deletion of an arginine decarboxylase (ΔspeA), an enzyme that catalyzes the synthesis of agmatine in the polyamine synthesis pathway, has a reduced capsule. Here, we report the impact of arginine decarboxylase deletion on pneumococcal stress responses. Our results show that ΔspeA is more susceptible to oxidative, nitrosative, and acid stress compared to the wild-type strain. Gene expression analysis by qRT-PCR indicates that thiol peroxidase, a scavenger of reactive oxygen species and aguA from the arginine deiminase system, could be important for peroxide stress responses in a polyamine-dependent manner. Our results also show that speA is essential for endogenous hydrogen peroxide and glutathione production in S. pneumoniae. Taken together, our findings demonstrate the critical role of arginine decarboxylase in pneumococcal stress responses that could impact adaptation and survival in the host.

Keywords: Streptococcus pneumoniae; acid stress; arginine decarboxylase; nitrosative stress; oxidative stress; polyamines.

Figure 1

Hydrogen peroxide susceptibility of Streptococcus pneumoniae TIGR4, ΔspeA, and pABG5-speA. Graph (A) shows bacterial sensitivity to 2.5 mM and 5.0 mM H₂O₂ at 15 min post-exposure. Graph (B) shows bacterial sensitivity to 2.5 mM and 5.0 mM H₂O₂ at 30 min post-exposure. The results represent an average of three independent experiments. The percentage survival relative to untreated controls is shown as a bar with the standard error of the mean, with * indicating p ≤ 0.05 and ** representing p ≤ 0.001, determined by Student’s t-test.

Figure 2

Potassium tellurite susceptibility of S. pneumoniae TIGR4, ΔspeA, and pABG5-speA. Graph (A) shows bacterial sensitivity to 0.1 mM and 0.2 mM potassium tellurite at 15 min post-exposure. Graph (B) shows bacterial sensitivity to 0.1 mM and 0.2 mM potassium tellurite at 30 min post-exposure. The results represent an average of three independent experiments. The percentage survival relative to untreated controls is shown as a bar with the standard error of the mean, with * indicating p ≤ 0.05 and ** representing p ≤ 0.001, determined by Student’s t-test.

Figure 3

S-nitrosoglutathione susceptibility assay of S. pneumoniae TIGR4, ΔspeA, and pABG5-speA. The graph shows bacterial sensitivity to 2.5 mM GSNO at 15–60 min post-exposure. The results represent an average of three independent experiments. The percentage survival relative to untreated controls is shown as a bar with the standard error of the mean, with * indicating p ≤ 0.05 and ** representing p ≤ 0.001, determined by Student’s t-test.

Figure 4

Intracellular pH of S. pneumoniae TIGR4 and ΔspeA. Three replicates of TIGR4 and ΔspeA were loaded with 5 mM of pH-sensitive fluorescence dye 2′-7′-bis(carboxyethyl)-5(6)-carboxyfluorescein - acetoxymethyl (BCECF-AM), washed with phosphate-buffered saline (PBS), and re-energized with 10% glucose, and baseline fluorescence readings were established in the first 5 min. Controls were supplemented with 10 µM carbonyl cyanide 3-chlorophenylhydrazone (CCCP) as a protonophore (triangles), and fluorescence of samples, including untreated ones (squares), was measured for an additional 5 min. The pH_i of untreated TIGR4 (blue squares) and ΔspeA (red squares) was 7.5 and 7.3, respectively. Then, 20 µM of nigericin was added to both treated and untreated samples to dissipate transmembrane gradients over the last 5 min. Graphs represent the mean of three independent experiments. Statistical significance was determined by Student’s t-test at a significance level of p ≤ 0.01.

Figure 5

Growth of S. pneumoniae TIGR4, ΔspeA, and pABG5-speA cultured in Todd–Hewitt broth with 0.5% yeast extract (THY) at varying pH was monitored by measuring absorbance at 600 nm and is shown in blue, red, and green, respectively, while the blank is shown in black circles in graph B. Graph (A) shows growth of the strains at pH 7.4 (squares) and 5.7 (triangles), and (B) shows growth of the strains at pH 7.4 (squares) and 6.0 (triangles). The results represent an average of three independent experiments. Statistical significance was determined by Student’s t-test at a significance level of p ≤ 0.05 and p ≤ 0.01.