SP_0916 Is an Arginine Decarboxylase That Catalyzes the Synthesis of Agmatine, Which Is Critical for Capsule Biosynthesis in Streptococcus pneumoniae

Abstract

The global burden of invasive pneumococcal diseases, including pneumonia and sepsis, caused by Streptococcus pneumoniae, a Gram-positive bacterial pathogen, remains a major global health risk. The success of pneumococcus as a pathogen can be attributed to its ability to regulate the synthesis of capsular polysaccharide (CPS) during invasive disease. We previously reported that deletion of a putative lysine decarboxylase (LDC; ΔSP_0916) in pneumococcal serotype 4 (TIGR4) results in reduced CPS. SP_0916 locus is annotated as either an arginine or a LDC in pneumococcal genomes. In this study, by biochemical characterization of the recombinant SP_0916, we determined the substrate specificity of SP_0916 and show that it is an arginine decarboxylase (speA/ADC). We also show that deletion of the polyamine transporter (potABCD) predicted to import putrescine and spermidine results in reduced CPS, while deletion of spermidine synthase (speE) for the conversion of putrescine to spermidine had no impact on the capsule. Targeted metabolomics identified a correlation between reduced levels of agmatine and loss of capsule in ΔspeA and ΔpotABCD, while agmatine levels were comparable between the encapsulated TIGR4 and ΔspeE. Exogenous supplementation of agmatine restored CPS in both ΔpotABCD and ΔspeA. These results demonstrate that agmatine is critical for regulating the CPS, a predominant virulence factor in pneumococci.

Keywords: Streptococcus pneumoniae; agmatine; capsular polysaccharide; metabolomics; polyamines.

Figure 1

Gel electrophoresis and enzyme kinetics of SP_0916. (A) Overexpressed and purified recombinant SP_0916 was resolved by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and stained with Coomassie Brilliant Blue R-250 for visualization of the 54 KDa protein. The ladder lane on the left is the protein marker showing the molecular mass standards. (B) Enzyme kinetics for the conversion of arginine to agmatine, lysine to cadaverine, and ornithine to putrescine were performed in triplicate using liquid chromatography-mass spectrometry (LC-MS). The change in velocity with increase in substrate concentrations was fitted with Sigma Plot v.12 to estimate the kinetic parameters using the Michaelis-Menten equation by non-linear regression method. The K _m of SP_0916 with the substrates is ornithine (3.55 ± 0.28 mM) > lysine (1.61 ± 0.28 mM) > arginine (0.11 ± 0.02 mM), indicating that arginine is the preferred substrate of this decarboxylase. High catalytic efficiency (k_cat/K_m) for the SP_0916-catalyzed conversion of arginine to agmatine (4.0 × 10⁵ min⁻¹mM⁻¹) compared to low k_cat/K_m for conversion of lysine to cadaverine (1.7 × 10⁴ min⁻¹mM⁻¹) and ornithine to putrescine (4.8 × 10³ min⁻¹mM⁻¹) show that arginine is the preferred substrate for SP_0916. k_cat/K_m values are obtained using the means for each kinetic parameter.

Figure 2

Immunoblot analysis of total capsular polysaccharide (CPS) in WT, polyamine synthesis/transport deletion and complemented strains. All strains were cultured to mid-log phase and total CPS isolated from equal number of cells for each strain. Representative immunoblots using serotype 4 specific antibody from three independent experiments are shown. (A) CPS from unencapsulated T4R, TIGR4, ΔspeA (Nakamya et al., 2018), polyamine transport deficient ΔpotABCD and ΔspeE. (B) CPS from T4R, TIGR4, ΔpotABCD, and ΔpotABCD complement.

Figure 3

Measurement of surface-exposed phosphocholine (PC) in TIGR4 and deletion strains. Pneumococci were grown to exponential phase and PC was measured by flow cytometry. PC was stained with an unconjugated Kappa murine myeloma IgA anti-phosphocholine antibody, followed by detection with a phycoerythrin (PE)-conjugated rat anti-mouse secondary antibody. Samples were fixed in 2% paraformaldehyde and histogram heights read in blue laser 2 channel (BL2-H) on an Attune Acoustic Focusing Cytometer. The gate was set based on a negative control that was treated with secondary antibody only. Representative histogram overlay plots, from three independent experiments, of the fluorescence intensity of murine myeloma IgA antibody binding to exposed PC on TIGR4 (red), ΔspeE (yellow), and ΔpotABCD (blue) are shown.

Figure 4

Polyamine transport and synthesis pathways. Cadaverine is synthesized by decarboxylation of lysine by putative lysine decarboxylase (LDC), while CadB serves as an antiporter for cadaverine and lysine exchange. Arginine decarboxylation by arginine decarboxylase (ADC) generates agmatine, a precursor for putrescine. Enzymatic activities of agmatine deiminase (AguA) and carbon-nitrogen hydrolase family protein (AguB) convert agmatine to putrescine in two steps or agmatine directly to putrescine by agmatinase (SpeB). The conversion of ornithine directly to putrescine by ornithine decarboxylase (ODC) is considered to be the main evolutionary pathway of polyamine biosynthesis in most organisms, but this gene is not currently annotated in pneumococcal genomes (shown in red). Ornithine/arginine antiporter (ArcD) regulates intracellular concentrations of arginine and ornithine. Putrescine can be converted to spermidine and spermine in sequential steps using decarboxylated S-adenosylmethionine [dcSAM, catalyzed by adenosylmethionine decarboxylase (AdoMetDC)] from methionine as a methyl donor by the enzymatic activity of spermidine synthase (SpeE). Alternatively, putrescine can be converted to carboxyspermidine and spermidine by carboxyspermidine dehydrogenase (CASDH) and carboxyspermidine decarboxylase (NspC), respectively. Pneumococcal genomes encode a single polyamine transporter (PotABCD) that is predicted to import spermidine and putrescine. Polyamine catabolism includes acetylation and thereby sequestration by a polyamine acetyltransferase (AT). Polyamine oxidase (APAO) that catalyzes the reverse reactions, i.e., generates free polyamines from acetylated forms (and other enzymes shown in red), are not annotated in the genome, at present.

Figure 5

Agmatine is critical for capsule biosynthesis in pneumococcus. Total CPS isolated from ΔspeA and ΔpotABCD cultured in THY or THY supplemented with agmatine [20 mM of agmatine, a quarter of minimum inhibitory concentration (MIC)] was spotted on a nitrocellulose membrane. Probing with anti-serotype 4 specific antibody and a horseradish peroxidase (HRP)-conjugated secondary antibody, the membrane was developed with enhanced chemiluminescence (ECL) detection and scanned using a ChemiDoc XRS+ with Image Lab software (Bio-Rad, Hercules, CA, United States). Agmatine successfully restored capsule in the two unencapsulated ΔspeA and ΔpotABCD strains in three independent experiments.