Role of Streptococcus intermedius DnaK chaperone system in stress tolerance and pathogenicity

Abstract

Streptococcus intermedius is a facultatively anaerobic, opportunistic pathogen that causes purulent infections and abscess formation. The DnaK chaperone system has been characterized in several pathogenic bacteria and seems to have important functions in stress resistance and pathogenicity. However, the role of DnaK in S. intermedius remains unclear. Therefore, we constructed a dnaK knockout mutant that exhibited slow growth, thermosensitivity, accumulation of GroEL in the cell, and reduced cytotoxicity to HepG2 cells. The level of secretion of a major pathogenic factor, intermedilysin, was not affected by dnaK mutation. We further examined the function and property of the S. intermedius DnaK chaperone system by using Escherichia coli ΔdnaK and ΔrpoH mutant strains. S. intermedius DnaK could not complement the thermosensitivity of E. coli ΔdnaK mutant. However, the intact S. intermedius DnaK chaperone system could complement the thermosensitivity and acid sensitivity of E. coli ΔdnaK mutant. The S. intermedius DnaK chaperone system could regulate the activity and stability of the heat shock transcription factor σ(32) in E. coli, although S. intermedius does not utilize σ(32) for heat shock transcription. The S. intermedius DnaK chaperone system was also able to efficiently eliminate the aggregated proteins from ΔrpoH mutant cells. Overall, our data showed that the S. intermedius DnaK chaperone system has important functions in quality control of cellular proteins but has less participation in the modulation of expression of pathogenic factors.

Fig. 1

Multiple sequence alignment of DnaK, DnaJ, and GrpE. a Part of the ATPase domains of DnaK from S. intermedius (SI DnaK), B. subtilis (Bs DnaK), and E. coli (Ec DnaK) were aligned with the CLUSTAL W program. b The N-terminal regions of DnaJ including the J domain (underlined) and G/F region (overlined) from S. intermedius (SI DnaJ), B. subtilis (Bs DnaJ), and E. coli (Ec DnaJ) were aligned. c The C-terminal regions of GrpE from S. intermedius (SI GrpE), B. subtilis (Bs GrpE), and E. coli (Ec GrpE) were aligned. The GrpE protein signature motif is underlined. Identical amino acid residues to S. intermedius DnaK, DnaJ, or GrpE are shown in bold

Fig. 2

Immunoblotting analysis and stress-sensitivity of S. intermedius ΔdnaK mutant. a Immunoblotting analysis of ΔdnaK R37 and its complemented strain. Whole-cell extracts (10 μg) were separated by 12% SDS–PAGE. Immunodetection was carried out with anti-T. halophilus DnaK antiserum or anti-GroEL antiserum. b Spot test for the examination of thermosensitivity. Spotted plates were incubated for 48 h at the indicated temperature. c Spot test for the examination of acid tolerance. Spotted plates were incubated for 48 h at 30°C. M molecular weight marker; WT UNS38; ΔdnaK, ΔdnaK R37; Comp. ΔdnaK the complemented strain of ΔdnaK

Fig. 3

Effect of dnaK null mutation on cell growth and secretion of ILY. a Growth curves of UNS38, ΔdnaK R37, and its complemented strain. Strains were cultured in BHI medium and the OD₆₀₀ measured at the indicated time points. The graphical data are the mean values ± standard deviation of at least four replicated independent experiments. b Hemolytic activity in the culture supernatant. Strains were cultured in BHI medium 24 h at 37°C, and the culture supernatants collected. Culture supernatant standardized at OD₆₀₀ was diluted from 200- to 3,200-fold by 2-fold serial dilutions, and the cytolytic activity of ILY in the diluted culture supernatant was estimated by hemolysis assay. Solid circle UNS38; open triangle ΔdnaK R37; open square the complemented strain

Fig. 4

Cytotoxic effect on HepG2 cells of ΔdnaK R37 and its complemented strain. Cytotoxic effects were observed over 4 days post-bacterial infection. Solid circle UNS38; open triangle ΔdnaK R37; open square the complemented strain

Fig. 5

Complementation of E. coli ΔdnaK by S. intermedius DnaK chaperone system. a Spot test for determining thermosensitivity. Cells were cultured for 24 h at 30°C and then spotted on LB agar plates containing the indicated amounts of IPTG. Spotted plates were incubated 24 h at the indicated temperatures. b Spot test for the detection of acid tolerance. Spotted plates were incubated for 48 h at 30°C. MC4100 + Vec MC4100 transformed with plasmid pZE13; Ec ΔdnaK + Vec E. coli ΔdnaK mutant transformed with plasmid pZE13; Ec ΔdnaK + pZE13 EcK E. coli ΔdnaK mutant transformed with plasmid pZE13 EcK; Ec ΔdnaK + pZE13 SiK E. coli ΔdnaK mutant transformed with plasmid pZE13 SiK; Ec ΔdnaK + pZE13 SiEK E. coli ΔdnaK mutant transformed with plasmid pZE13 SiEK; Ec ΔdnaK + pZE13 SiKJ E. coli ΔdnaK mutant transformed with plasmid pZE13 SiKJ; Ec ΔdnaK + pZE13 SiEKJ E. coli ΔdnaK mutant transformed with plasmid pZE13 SiEKJ

Fig. 6

Function of S. intermedius DnaK chaperone system in E. coli. a Cellular level of GroEL and σ³² in E. coli ΔdnaK mutants in the presence or absence of the S. intermedius DnaK chaperone system. Cells were grown in LB medium containing the indicated concentrations of IPTG for 4 h at 30°C, the OD₆₀₀ of the cultures measured and standardized amounts of the cell lysates analyzed by 12% SDS–PAGE. Immunodetection was carried out with anti-E. coli GroEL or anti-σ³² antiserum. M molecular weight marker; MC4100 + Vec MC4100 transformed with a plasmid pZE13; Ec ΔdnaK + Vec E. coli ΔdnaK mutant transformed with a plasmid pZE13; Ec ΔdnaK + pZE13 SiEKJ E. coli ΔdnaK mutant transformed with a plasmid pZE13 SiEKJ. b Amounts of aggregated protein in E. coli ΔrpoH mutants in the presence or absence of the S. intermedius DnaK chaperone system. Cells were grown in LB medium containing the indicated concentrations of IPTG for 3 h at 30°C and further cultured at 42°C for 1 h. Aggregated proteins were isolated as described in “Materials and methods.” The amount of aggregated protein was quantified by the Bradford assay reagent and calculated in relation to total protein content (set at 100%). Each value is the average from at least three different experiments. Ec ΔrpoH + Vec E. coli ΔrpoH mutant transformed with a plasmid pZE13; Ec ΔrpoH + pZE13 SiEKJ E. coli ΔrpoH mutant transformed with a plasmid pZE13 SiEKJ