Influence of the phosphoenolpyruvate:carbohydrate phosphotransferase system on toxin gene expression and virulence in Bacillus anthracis

Abstract

AtxA, the master virulence gene regulator of Bacillus anthracis, is a PRD-Containing Virulence Regulator (PCVR) as indicated by the crystal structure, post-translational modifications and activity of the protein. PCVRs are transcriptional regulators, named for PTS Regulatory Domains (PRDs) subject to phosphorylation by the phosphoenolpyruvate phosphotransferase system (PEP-PTS) and for their impact on virulence gene expression. Here we present data from experiments employing physiological, genetic and biochemical approaches that support a model in which the PTS proteins HPr and Enzyme I (EI) are required for transcription of the atxA gene, rather than phosphorylation of AtxA. We show that atxA transcription is reduced 2.5-fold in a mutant lacking HPr and EI, and that this change is sufficient to affect anthrax toxin production. Mutants harboring HPr proteins altered for phosphotransfer activity were unable to restore atxA transcription to parent levels, suggesting that phosphotransfer activity of HPr and EI is important for regulation of atxA. In a mouse model for anthrax, a HPr^- EI^- mutant was attenuated for virulence. Virulence was restored by expressing atxA from an alternative, PTS-independent, promoter. Our data support a model in which HPr transfers a phosphate to an unidentified downstream transcriptional regulator to influence atxA gene transcription.

Fig. 1.

Toxin production by parent and mutant strains. Culture lysates and supernates were obtained at late exponential phase (4 h) of growth from cultures grown in CACO₃. Samples of culture supernatants were concentrated 10X and subjected to SDSPAGE followed by western blot with α-LF serum, and α⁻THE™His mAb to detect HPr and EI. ΔptsHI (UT439) was complemented with EV (empty vector pAW285) or genes ptsH (pUTE1143), ptsI (pUTE1144) or ptsHI (pUTE1145) as indicated.

Fig. 2.

AtxA activity at the lef promoter in the presence of PTS sugars. Cultures of the B. anthracis reporter strain UT376(pUTE991) were grown in CACO₃ containing 0.1% of glycerol and 0.1% of added sugar as indicated. Cells were collected from cultures at late exponential phase (4 h growth) for AtxA activity assays and western blotting with α-THE™His antibody. AtxA activity was assessed as β-galactosidase activity from a Plef-lacZ transcriptional fusion. Data represent the average of three independent experiments. Error bars represent standard deviation.

Fig. 3.

In vitro phosphorylation assay with the PTS proteins HPr and EI. Proteins present in each lane are indicated by ‘+’. Hexa-His EI, HPr, GlcT, and AtxA were induced in B. anthracis and purified via nickel-affinity purification. ³²P-PEP was mixed with the proteins and incubated at 37°C for 30 min. A. Samples were separated on 10% of poly-acrylamide-SDS gels and stained with Coumassie blue. B. The proteins were subjected to SDS-PAGE, dried and exposed to a phosphor-imaging screen. Proteins present in each lane are indicated by the tables. ¹Ten-fold more AtxA was added to the reaction. ²Purified AtxA was treated with calf-intestine alkaline phosphatase followed by removal of the phosphatase prior to incubation.

Fig. 4.

AtxA activity at the lef promoter in the absence of the PTS. B. anthracis lef-lacZ reporter strains UT376(pUTE991)(PTS+), UT417(pUTE991)(PTS−) and UT408 (PTS−) were grown in CACO₃. Strains harboring pUTE991 express atxA from an IPTG-inducible promoter. Strain UT408 expresses atxA from its native promoter. Cells were collected at late exponential phase for β-galactosidase assays (AtxA activity) and for western blotting of cell lysates with α-THE™His antibody (AtxA level). β-galactosidase activity represents the average of three experiments. Error bars represent standard deviation. A representative western is shown.

Fig. 5.

Transcription of PatxA-lacZ. Cultures of parent, PTS⁻ mutant, and PTS complementation strains carrying the PatxA-lacZ reporter pUTE843 were grown in CACO₃ to late exponential phase and collected for β-galactosidase assays and western blotting. A. β-gal activity from the PatxA-lacZ transcriptional fusion. *p value < 0.05, **p value < 0.01. B. Western blot of concentrated culture supernatants with anti-LF antibody.

Fig. 6.

Transcription of PatxA-lacZ in the presence of PTS sugars. Cultures of the parent strain carrying the PatxA-lacZ reporter pUTE843 were grown in CACO₃ containing 0.1% of glycerol and 0.1% of added sugar as indicated. Cells were collected from cultures at late exponential phase (4 h growth) for β-galactosidase assays. Data represent the average of three independent experiments. Error bars indicate one standard deviation.

Fig. 7.

Relative expression of lacZ driven by atxA promoter regions P1+P2 (−770 bp to translational start) and P1 alone (−72 bp to translational start). A. Schematic representation of atxA promoter region. B. Cultures of B. anthracis strains harboring PatxA-lacZ constructs were grown in casamino acids medium containing 0.2% of glucose and 0.9% of bicarbonate in 5% of atmospheric CO₂. Cells were collected from cultures at late exponential phase (4 h growth) for analysis of β-galactosidase activity representative of activity from the atxA promoter regions. Data represent the average of three independent experiments. Error bars indicate standard deviation.

Fig. 8.

Virulence of parent and ptsHI mutants. A. Survival curves of mice infected intravenously with vegetative B. anthracis are shown. A/J mice were injected i.v. with 2.75 × 10³ CFU of the parent (black solid line; n = 6), 4.5 × 10³ CFU of parent constitutively expressing atxA (ANR-1 atxA*) (black dashed line; n = 6), 3 × 10³ CFU of ptsHI-null (gray solid line; n = 6), or 3 × 10³ CFU of ptsHI atxA* (gray dashed line, n = 6) vegetative cells. B. CFU g⁻¹ of tissue collected. Two sample permutation analysis was performed to compare each strain to the other. **p < 0.05.

Fig. 9.

Alignment of PTS regulation domains. Modified from Stulke et al. (1998). Amino acid sequences denoted ‘I’ correspond to PRDs proximal to the amino-terminus. Sequences denoted ‘II’ correspond to PRDs, proximal to the carboxy-terminus. Bracketed groups 1, 2 and 3 indicate: PCVRs in Gram-positive organisms; PRD-containing transcription activators; and PRD-containing transcription antiterminators respectively. Highly conserved amino acids are highlighted in black. Residues with similarity are highlighted in gray. Red boxes indicate published data suggesting phosphorylation of the indicated histidine.