Characterization of a novel two-component regulatory system, HptRS, the regulator for the hexose phosphate transport system in Staphylococcus aureus

Abstract

Hexose phosphate is an important carbon source within the cytoplasm of host cells. Bacterial pathogens that invade, survive, and multiply within various host epithelial cells exploit hexose phosphates from the host cytoplasm through the hexose phosphate transport (HPT) system to gain energy and synthesize cellular components. In Escherichia coli, the HPT system consists of a two-component regulatory system (UhpAB) and a phosphate sensor protein (UhpC) that tightly regulate expression of a hexose phosphate transporter (UhpT). Although growing evidence suggests that Staphylococcus aureus also can invade, survive, and multiply within various host epithelial cells, the genetic elements involved in the HPT system in S. aureus have not been characterized yet. In this study, we identified and characterized the HPT system in S. aureus that includes the hptRS (a novel two-component regulatory system), the hptA (a putative phosphate sensor), and the uhpT (a hexose phosphate transporter) genes. The hptA, hptRS, and uhpT markerless deletion mutants were generated by an allelic replacement method using a modified pMAD-CM-GFPuv vector system. We demonstrated that both hptA and hptRS are required to positively regulate transcription of uhpT in response to extracellular phosphates, such as glycerol-3-phosphate (G3P), glucose-6-phosphate (G6P), and fosfomycin. Mutational studies revealed that disruption of the hptA, hptRS, or uhpT gene impaired the growth of bacteria when the available carbon source was limited to G6P, impaired survival/multiplication within various types of host cells, and increased resistance to fosfomycin. The results of this study suggest that the HPT system plays an important role in adaptation of S. aureus within the host cells and could be an important target for developing novel antistaphylococcal therapies.

FIG 1

Identification of a putative hexose phosphate transport system in S. aureus. (A) A schematic map of the genomic organization of the hexose phosphate transport system in S. aureus LAC and its mutant derivatives; (B) a PCR confirmation of a deletion mutant of HptA (ΔHptA mutant), HptRS (ΔHptRS mutant), and UhpT (ΔUhpT mutant) and the corresponding complemented strains, pMK4-HptA, pMK4-HptRS, and pMK4-UhpT, respectively.

FIG 2

Role of the HPT system in growth under nutrition specifically limited to glucose-6-phosphate. Results shown are the growth kinetics of the wild-type, ΔHptA, ΔHptRS, and ΔUhpT strains in MH2 (A) or in chemically defined medium (CDM) supplemented with G6P (B), G3P (C), or glucose (Glu) (D) as the sole carbon source. Results shown are combined from triplicate measurements from three independent experiments. The asterisk indicates statistical difference from the wild type at P values of <0.001.

FIG 3

Role of HptA and HptRS in transcriptional regulation of the UhpT in response to extracellular G3P and G6P. Transcription of UhpT in the wild-type, ΔHptA, and ΔHptRS strains, cultured in MH2 broth (A) or in CDM supplemented with G6P (B), G3P (C), or glucose (Glu) (D) as the sole carbon source, was measured using qRT-PCR. Results shown are combined from triplicate measurements from two independent experiments.

FIG 4

Role of HptRS in intracellular survival/multiplication of S. aureus. A cell monolayer of murine bone marrow-derived macrophages (BMDM), human monocytic cells (THP-1), and human alveolar epithelial cells (A549) was infected with the wild-type or ΔHptRS strains at an MOI of 5. After 30 min of infection, extracellular bacteria were removed by gentamicin treatment, and survival/multiplication of S. aureus within the host cells were measured at the indicated time points. Results shown are combined from triplicate measurements from three independent experiments. The asterisk indicates statistical difference from the wild type at P values of <0.05.

FIG 5

Role of HptRS and UhpT in fosfomycin sensitivity. The fosfomycin MIC of the wild-type, ΔHptRS, and ΔUhpT strains was determined using the Etest by following the manufacturer's instructions.

FIG 6

Effect of an exposure to fosfomycin on transcription of genes related to fosfomycin resistance. The wild-type and ΔHptRS strains were grown in MH2 broth (A) or in MH2 broth supplemented with a high level of fosfomycin (128 μg/ml) for 6 h (B). Transcription of genes related to fosfomycin resistance was measured using qRT-PCR. Results shown are combined from triplicate measurements from two independent experiments.