Regulons and protein-protein interactions of PRD-containing Bacillus anthracis virulence regulators reveal overlapping but distinct functions

Abstract

Bacillus anthracis produces three regulators, AtxA, AcpA and AcpB, which control virulence gene transcription and belong to an emerging class of regulators termed 'PCVRs' (Phosphoenolpyruvate-dependent phosphotransferase regulation Domain-Containing Virulence Regulators). AtxA, named for its control of toxin gene expression, is the master virulence regulator and archetype PCVR. AcpA and AcpB are less well studied. Reports of PCVR activity suggest overlapping function. AcpA and AcpB independently positively control transcription of the capsule biosynthetic operon capBCADE, and culture conditions that enhance AtxA level or activity result in capBCADE transcription in strains lacking acpA and acpB. We used RNA-Seq to assess the regulons of the paralogous regulators in strains constructed to express individual PCVRs at native levels. Plasmid and chromosome-borne genes were PCVR controlled, with AtxA, AcpA and AcpB having a ≥ 4-fold effect on transcript levels of 145, 130 and 49 genes respectively. Several genes were coregulated by two or three PCVRs. We determined that AcpA and AcpB form homomultimers, as shown previously for AtxA, and we detected AtxA-AcpA heteromultimers. In co-expression experiments, AcpA activity was reduced by increased levels of AtxA. Our data show that the PCVRs have specific and overlapping activity and that PCVR stoichiometry and potential heteromultimerization can influence target gene expression.

Fig. 1. Amino acid sequence alignment of B. anthracis PCVRs

Domains identified in the AtxA crystal structure are shown as solid bars directly above the respective amino acid sequence. Histidines at positions 199 and 379 within AtxA, which have been shown to be subject to phosphorylation, are shown with black background. Potential sites for histidine phosphorylation within the putative PRDs of AcpA and AcpB are shown with a gray background. Amino acid identity and similarity are denoted by asterisk (full conservation), colon (strongly similar properties), or period (weakly similar properties). Alignments were completed using the Clustal X2 program.

Fig. 2. Venn diagrams of PCVR regulons

(A) Gene expression changes of ≥2-fold, ≥4-fold, and ≥16-fold in the atxAacpAacpB-null strain (UTA40) complemented with AtxA, AcpA or AcpB. (B) Gene expression changes of ≥4-fold organized by genetic element.

Fig. 3. Sequencing reads mapping to PCVR-regulated loci

Bedgraphs show normalized sequencing reads mapped to pXO1 (A), pXO2 (B), and the chromosome (C). Regions enriched for PCVR-regulated genes indicated above graph in kilobases.

Fig. 4. Edema Factor Production by individual PCVRs

Expression of recombinant atxA, acpA, and acpB was induced with IPTG to yield either native or overexpressed steady state protein levels (Native - 5 μM, 5 μM, 100 μM respectively; Overexpressed - 50 μM, 50 μM, 500 μM respectively) during growth in CA medium supplemented with dissolved bicarbonate in 5% CO₂ atmosphere. Samples of culture supernates were subjected to slot blot Western analysis using rabbit anti-EF serum raised against B. anthracis edema factor.

Fig. 5. Capsule production by individual PCVRs

Expression of recombinant atxA, acpA, and acpB was induced with IPTG to yield native steady state protein levels (5 μM, 5 μM, 100 μM respectively) during growth in CA medium supplemented with dissolved bicarbonate in 5% CO₂ atmosphere (CACO₃)(A). (B) Expression of recombinant atxA induced with 30 μM IPTG in UTA40 during growth in CACO₃ in 20% CO₂. (C) Overexpression of atxA with 50 μM IPTG during culture in CACO₃ in 5% CO₂. The UTA40 derivatives were induced at early exponential phase (2h; OD₆₀₀ 0.25–0.35) in during growth in CACO₃. Samples were collected at the transition to stationary phase (4h; OD₆₀₀ 1.2–1.8), stained with India Ink, and visualized using DIC microscopy.

Fig. 6. Homomultimerization AcpA and AcpB

Lysates from B. anthracis atxA-null pXO1+ pXO2- strains (UT423) containing plasmids that encode IPTG-inducible (A) AcpA-His (pUTE1090), AcpA-FLAG (pUTE1079), or GFP-FLAG (pUTE1013); (B) AcpB-His (pUTE1091), AcpB-FLAG (pUTE1093), or GFP-FLAG (pUTE1013) were co-incubated as indicated, then co-affinity purified with Ni²⁺-NTA resin. Proteins present in the mixed lysates prior to (Load, lanes 1–3) and after purification (Eluate, lanes 4–6) were subjected to SDS-PAGE and Western blot with α-His and α-FLAG antibodies as indicated. Arrows indicate the predicted sizes of AcpA-His, AcpA-FLAG, AcpB-His, AcpB-FLAG, and GFP-FLAG; (C) FLAG-tagged AcpA (pUTE1079) and AcpB (pUTE1093) were induced by IPTG in a B. anthracis atxA-null pXO1+ pXO2- strain. Lysates were incubated with or without the crosslinking agent BMH and subjected to SDS-PAGE and Western blot. Proteins were detected with α-FLAG antibody. (D) Affinity purified AcpA-His from B. anthracis ANR-1 (pUTE1090) incubated with or without BMH and subjected to SDS-PAGE and Western blot. Proteins were detected with α-His antibody.

Fig. 7. Activity and multimerization of AcpA and AcpB EIIB-like domain truncation mutants

UT423 strains expressing AcpA-ΔEIIB-His (pUTE1125), AcpA-FLAG (pUTE1079), AcpB-ΔEIIB-His (pUTE1126), or AcpB-FLAG (pUTE1093) were cultured in CA medium supplemented with dissolved bicarbonate in 5% CO₂ atmosphere and induced with 30–50 μM IPTG. A. Cell lysates containing IPTG-induced proteins were treated with crosslinking agent BMH or vehicle alone (DMSO). Molecular weights of protein standards are listed. B. The β-galactosidase activity of B. anthracis strains harboring the PcapB-lacZ reporter and IPTG-induced AcpA and AcpB variants was determined as previously described (Miller, 1972).. Errors represent ±1 SD.

Fig. 8. Heteromultimerization by PCVRs

Lysates from B. anthracis atxA-null pXO1+ pXO2- strains (UT423) containing plasmids that encode IPTG-inducible AtxA-His (pUTE991), AcpA-FLAG (pUTE1079), or GFP-FLAG (pUTE1013); were co-incubated as indicated, then co-affinity purified with Ni²⁺-NTA resin. Proteins present in the mixed lysates prior to (Load, lanes 1–3) and after purification (Eluate, lanes 4–6) were subjected to SDS-PAGE and Western blot with α-His and α-FLAG antibodies as indicated. Arrows indicate the predicted sizes of AtxA, AcpA, and GFP.

Fig. 9. AtxA effect on AcpA and AcpB activity

UT423 strains co-expressing AtxA-His (pUTE991) from an IPTG inducible promoter and AcpA-FLAG (pUTE1099) or AcpB-FLAG (pUTE1100) from a xylose inducible promoter were cultured in CA medium supplemented with dissolved bicarbonate in 5% CO₂ atmosphere. Across six cultures IPTG was added in the indicated concentrations to incrementally increase AtxA-His expression, while AcpA-FLAG or AcpB-FLAG expression levels were kept constant with 1% xylose in all cultures. Asterisks represent a significant decrease in activity at the indicated [IPTG] compared to 10 μM IPTG (P value <0.05). β-galactosidase activity of these strains harbouring the PcapB-lacZ reporter was determined as described previously (Miller, 1972).. Error bars represent ±1 SD.