Arginine catabolic mobile element encoded speG abrogates the unique hypersensitivity of Staphylococcus aureus to exogenous polyamines

Abstract

Polyamines, including spermine (Spm) and spermidine (Spd), are aliphatic cations that are reportedly synthesized by all living organisms. They exert pleiotropic effects on cells and are required for efficient nucleic acid and protein synthesis. Here, we report that the human pathogen Staphylococcus aureus lacks identifiable polyamine biosynthetic genes, and consequently produces no Spm/Spd or their precursor compounds putrescine and agmatine. Moreover, while supplementing defined medium with polyamines generally enhances bacterial growth, Spm and Spd exert bactericidal effects on S. aureus at physiological concentrations. Small colony variants specifically lacking menaquinone biosynthesis arose after prolonged Spm exposure and exhibited reduced polyamine sensitivity. However, other respiratory-defective mutants were no less susceptible to Spm implying menaquinone itself rather than general respiration is required for full Spm toxicity. Polyamine hypersensitivity distinguishes S. aureus from other bacteria and is exhibited by all tested strains save those belonging to the USA-300 group of community-associated methicillin-resistant S. aureus (CA-MRSA). We identified one gene within the USA-300-specific arginine catabolic mobile element (ACME) encoding a Spm/Spd N-acetyltransferase that is necessary and sufficient for polyamine resistance. S. aureus encounters significant polyamine levels during infection; however, the acquisition of ACME encoded speG allows USA-300 clones to circumvent polyamine hypersensitivity, a peculiar trait of S. aureus.

Figure 1. Arginine-dependent polyamine biosynthetic pathways

Four different biosynthetic pathways exist across all three kingdoms of life for the conversion of l-arginine to putrescine. Putrescine is then converted to spermidine through the addition of an aminopropyl group from decarboxylated S-adenosylmethionine (DeC-AdoMet). Glyphs in the lower right depict pathways present in E. coli as well as humans, green lines indicate enzymes in the above scheme that are present, red = absent. Abbreviations: ARG (Arginase), ODC (Ornithine decarboxylase), SPS (Spermidine synthase), S-MeAd (S-methyladenosine), SDC (S-adenosylmethionine decarboxylase), S-AdoMet (S-adenosylmethionine), ADC (Arginine decarboxylase), AUH (Agmatine ureohydrolase, Agmatinase), CPA (N-Carbamoylputrescine amidohydrolase) and PTC (Putrescine transcarbamoylase).

Figure 2. Bactericidal effects of spermine on S. aureus

A. Zones of inhibition around discs supplemented with 20 µl of 250 mM spermine across various species and S. aureus strains. (*) denotes mean zone radii significantly (p ≤ 0.01, n ≥ 3) smaller than that of S. aureus COL (Students t-test). None of these strains exhibited zones around discs supplemented with 20 µl of 250 mM NaOH. B. Viable cfu of S. aureus strain COL over a 24-hour exposure to 5 mM Spermine.

Figure 3. Spontaneous spermine-resistance results primarily in menadione-requiring small colony variants (SCVs)

A. Proportions of 47 characterized spontaneous spermine-resistant clones that were SCVs (89%) versus mutants exhibiting normal growth (11%). Normal growth could be restored to 83% of these SCVs by supplementing 8 µg·ml⁻¹ of menadione. The remaining strains were not rescued by supplementing menadione, heme, thiamine or thymidine. Spermine-resistant mutants were isolated by plating 10⁹ cfu of S. aureus strain Newman on BHI plates supplemented with 20 mM Spermine. B. Zones of inhibition around 250 mM spermine discs using S. aureus strain Newman and isogenic SCV mutants; menD::Er^R and ΔhemB. (*) indicates that the only spermine zone of the menD::Er^R mutant was significantly smaller than that observed in WT (p ≤ 0.05, n ≥ 3, Student’s t-test) C. Supplementing BHI medium with 8 µg·ml⁻¹ menadione restored WT growth, normal pigmentation and full spermine susceptibility to a menD::Er^R mutant of S. aureus strain Newman.

Figure 4. CA-MRSA isolates belonging to the USA-300 PFGE-type exhibit full spermine resistance due to an ACME encoded spermine/spermidine acetyltransferase (speG)

TOP: Schematic of the 33 gene ACME island found in nearly every USA-300 clone harboring the SCCMEC type IVa. BOTTOM: speG is necessary and sufficient for USA300 spermine-resistance. Spermine inhibition zones of USA-300 strain SF8300 and isogenic ΔACME and ΔspeG strains as well as MRSA isolate COL and ΔACME harboring a plasmid-borne speG allele.

Figure 5. SpeG spermine-acetyltransferase activity is required for USA300 spermine-resistance

Growth of USA300 in the presence of 5 mM Spermine requires speG and can be inhibited by the addition of 10 µM Berenil, a spermine/spermidine acetyltransferase inhibitor. This concentration of Berenil had negligible effects on the growth of USA300 in the absence of spermine.