clpB, a class III heat-shock gene regulated by CtsR, is involved in thermotolerance and virulence of Enterococcus faecalis

Abstract

Here, we transcriptionally and phenotypically characterized the clpB gene from Enterococcus faecalis. Northern blot analysis identified a monocistronic mRNA strongly induced at 48 and 50 °C. In silico analysis identified that the clpB gene encodes a protein of 868 aa with a predicted molecular mass of approximately 98 kDa, presenting two conserved ATP-binding domains. Sequence analysis also identified a CtsR-binding box upstream of the putative -10 sequence, and inactivation of the ctsR gene resulted in an approximately 2-log increase in clpB mRNA expression, confirming ClpB as a member of the CtsR regulon. While expression of clpB was induced by heat stress, a ΔclpB strain grew relatively well under many different stressful conditions, including elevated temperatures. However, expression of ClpB appears to play a major role in induced thermotolerance and in pathogenesis, as assessed by using the Galleria mellonella virulence model.

Fig. 1.

Structural features of the ClpB proteins of E. faecalis. (a) Amino acid sequence deduced from the E. faecalis clpB gene indicating the consensus sequences. (b) Consensus motifs of the ClpB proteins. ATP-1 and ATP-2 represent the Walker-type nucleotide-binding sites: Walker-A is a glycine-containing segment and Walker-B represents hydrophobic segment motifs. I, N-terminal; II and III, middle; and IV and V, C-terminal consensus sequences.

Fig. 2.

Nucleotide sequence of the promoter region and the 5′ coding region of the E. faecalis clpB gene. Regulatory sequences, in bold type and underlined, are as follows: one putative CtsR element (consensus sequence GGTCAAANANGGTCAAA) indicated with an arrow below, the putative −35 and −10 regions, the initiation codon ATG and its ribosome-binding site (RBS), and a second putative initiation codon GTG and its related RBS (boxed).

Fig. 3.

Induction of clpB mRNA and ClpB in response to heat shock. (a) Northern blot analysis of the E. faecalis clpB gene. In order to stimulate the production of the heat-shock mRNA, the cells were either cultivated at 37 °C (control cells) or stressed by upshift to 42, 45, 48 or 50 °C for 10 min. Total RNA was separated under denaturing conditions, transferred to a nitrocellulose membrane, and hybridized to a clpB-specific probe (left). A parallel gel was stained with ethidium bromide to show equal loading of the slots (right). (b) Influence of supra-optimum temperatures on protein synthesis. Wild-type cells were either incubated at 37 °C (control) or treated at 42, 45, 48, 50, 52 or 55 °C in the presence of [³⁵S]methionine. Labelled proteins were separated by SDS-PAGE and analysed by autoradiography. The ClpB protein is indicated. DnaK and GroEL, which were previously identified, are also indicated (Laport et al., 2001). (c) Identification of the ClpB protein. Wild-type (wt) cells were subjected to heat shock at 45 °C in the presence of [³⁵S]methionine. Labelled proteins were separated by SDS-PAGE and analysed by either autoradiogram (³⁵S-Met) or Western blotting using polyclonal antibodies raised against Synechococcus spp. ClpB diluted 1 : 1000. (d) Western blotting of wild-type (wt) or ΔclpB cells subjected to heat shock at 45 °C showing the cross-reaction of the ClpB polyclonal antibody.

Fig. 4.

ClpB is important for development of thermotolerance. Cultures of wild-type OG1RF, ΔclpB, the complemented cΔclpB and nisin-induced cΔclpB strains were grown exponentially at 37 °C to OD₆₀₀∼0.4. At this point, cultures were pre-incubated at 50 °C for 30 min prior to exposure to 60 °C (a lethal temperature) for 30 and 60 min. Cell survival was evaluated by plating diluted samples on BHI plates. The result presented is a representative of five independent experiments; error bars, sd.

Fig. 5.

Killing of G. mellonella larvae infected with E. faecalis OG1RF and ΔclpB at 37 °C. Survival (Kaplan–Meyer) plots of larvae injected with OG1RF (•) and ΔclpB (▪) strains. Larvae injected with saline solution (▾) or heat-killed OG1RF (⧫) were used as controls with minimum killing. The experiments were repeated three times, and the results are representative of a typical experiment. Compared with the wild-type OG1RF strain, ΔclpB showed attenuated virulence (P≤0.001).