Enterococcal Rgg-like regulator ElrR activates expression of the elrA operon

Abstract

The Enterococcus faecalis leucine-rich protein ElrA promotes virulence by stimulating bacterial persistence in macrophages and production of the interleukin-6 (IL-6) cytokine. The ElrA protein is encoded within an operon that is poorly expressed under laboratory conditions but induced in vivo. In this study, we identify ef2687 (renamed elrR), which encodes a member of the Rgg (regulator gene for glucosyltransferase) family of putative regulatory proteins. Using quantitative reverse transcription-PCR, translational lacZ fusions, and electrophoretic mobility shift assays, we demonstrate that ElrR positively regulates expression of elrA. These results correlate with the attenuated virulence of the ΔelrR strain in a mouse peritonitis model. Virulence of simple and double elrR and elrA deletion mutants also suggests a remaining ElrR-independent expression of elrA in vivo and additional virulence-related genes controlled by ElrR.

Fig 1

Gene organization and natural deletion of the elrA operon in E. faecalis isolates. (A) Schematic representation of the chromosomal region of the elrA operon. Putative rho-independent transcriptional terminators are indicated as lollipops. The promoter region of the elrA operon is represented by a black-headed arrow. (B) Schematic representation of the predicted ElrR product. HTH indicates the helix-turn-helix domain. G, R, and Y indicate glycine, arginine, and tyrosine residues, respectively. Numbers refer to the amino acid positions in ElrR.

Fig 2

Western immunoblot analysis of ElrR-6×His under laboratory growth conditions from OG1RF derivative strains. Cytoplasmic and cell wall proteins from strains expressing chromosomal ElrR (lane 1), chromosomal ElrR-6×His (lane 2), and ElrR-6×His under the control of P_aphA3 on a plasmid (lane 3) were extracted. Equivalent amounts of proteins were analyzed using an antipolyhistidine polyclonal rabbit antibody. The band corresponding to ElrR-6×His is indicated by an arrow. Other bands are nonspecific and correspond to cross-reacting signal.

Fig 3

Schematic representation of lacZ translational fusions and their relative β-galactosidase activities. At top is shown the genomic organization of the elrR-elrA region with the P_elrA promoter, and the potential rho-independent transcription terminator is indicated by a lollipop. DNA fragments encompassing the elrA upstream region were fused to the lacZ coding sequence. Transcription from tetM of the vector pVE14174 was arrested by rho-independent transcription terminators, Term (black lollipop). Ratios of β-galactosidase activities were normalized to the activity measured for P_elrA::lacZ (1.29 ± 0.24 Miller units). Data provided are the averages of five independent experiments.

Fig 4

ElrR specifically binds the elrA promoter region. (Top) Map of the intergenic region upstream of the E. faecalis elrA operon, with the ORFs elrR and elrA. The transcription orientation of elrA and elrR is indicated by thick arrows, and the thin arrow indicates the promoter of the elrA operon (P_elrA). The lollipop indicates a 50-bp inverted repeat at the DNA level and the predicted transcription terminator. Lines below represent DNA probes used for EMSAs; numbers correspond to the location of the extremities relative to the start codon of the elrA gene. (Bottom) EMSA of elrA promoter region with ElrR. In the left panel, 3 ng of a 0.5-kb fluorescently labeled DNA fragment encompassing the upstream region of P_elrA (S for “specific”) and 5 ng of a fluorescently labeled DNA control fragment (NS for “nonspecific”), corresponding to a 0.3-kb internal fragment of the elrR gene, were incubated with 0, 1, 1.5, and 2 μM purified ElrR (lanes 1 to 4, respectively). An excess of cold S (lanes 5 and 6) and NS (lanes 7 and 8) DNA probes was added to the binding reaction mixtures in the absence (lanes 5 and 7) or presence (lanes 6 and 8) of 1.5 μM ElrR. In the right panel, EMSA of DNA probes encompassing nested fragments of the P_elrA region that were incubated without (−) or with (+) 1.5 μM ElrR. S and 1 to 4 correspond to the DNA probes shown on the map above.

Fig 5

Schematic representation of the lacZ translational fusions and their corresponding β-galactosidase activities with elrR provided in trans. At the top, the elrA promoter region is schematized with the two transcription start sites t1 and t2 and the 50-bp inverted repeat indicated by the lollipop as defined previously (6). DNA fragments encompassing elrA promoter regions were fused to the lacZ coding sequence in two vectors. Transcription from tetM of vector pVE14189 was stopped by rho-independent transcription terminators, Term (black lollipop). tetM of vector pVE14195 was reversed and Term terminators were removed compared to pVE14189. β-Galactosidase activities of strains expressing elrR (ElrR⁺⁺) and plasmid control (Vector) are the averages of at least three independent experiments. nd, not done.

Fig 6

Nucleotide sequence of elrA promoter region. Lowercase letters indicate elrR and elrA coding sequences; the stop and start codons are in bold. The 50-bp inverted repeat is indicated by convergent arrows. Numbering refers to the translation start site of elrA (+1). The two transcriptional start sites t1 and t2 of elrA previously mapped are shown (6); t2 was not found under the conditions used in this work. The putative −35 and −10 boxes are underlined. The ribosome binding site for elrA is shown by italics. Shaded letters correspond to extremities of DNA probes used for EMSA (Fig. 4).

Fig 7

Effect of elrR inactivation on E. faecalis virulence. Kaplan-Meier survival analysis in a mouse peritonitis model with the OG1RF wild-type strain (open circles), the ΔelrA mutant strain (squares), the ΔelrR mutant strain (inverted triangles), the ΔelrR ΔelrA double mutant strain (diamonds), and the elrR-complemented strain (closed circles). Data were pooled from two independent experiments, except for the ΔelrR-complemented strain. A total of 20 mice were infected intraperitoneally with 1.4 × 10⁹ or 0.85 × 10⁹, 1.3 × 10⁹ or 0.9 × 10⁹, 1.5 × 10⁹ or 1.1 × 10⁹, and 1.3 × 10⁹ or 0.98 × 10⁹ CFU of OG1RF, ΔelrA, ΔelrR, and ΔelrR ΔelrA strains, respectively. A total of 10 mice were infected with 1.6 × 10⁹ CFU of the ΔelrR-complemented strain. For each pairwise comparison, OG1RF/ΔelrR, ΔelrR/ΔelrR ΔelrA, and ΔelrA/ΔelrR ΔelrA, P values were <0.0001, <0.005, and <0.15, respectively.