ClpB enhances thermotolerance in Campylobacter jejuni through protein disaggregation independent of DnaK

Abstract

Campylobacter jejuni is a leading cause of foodborne infections worldwide and primarily transmitted to humans through the consumption of contaminated poultry meat. To enhance Campylobacter-associated food safety, it is critical to understand how C. jejuni survives during the thermal processing of poultry products. In this study, we monitored the survival of 86 C. jejuni strains during heat treatment and observed that some strains exhibited elevated heat tolerance. Notably, multilocus sequence typing clonal complex (CC)-443 and CC-607 were dominant among heat-tolerant strains, while the CC-21 strains were mostly heat-sensitive, indicating phylogenetic association with thermotolerance. We also investigated the function of heat shock chaperones in the thermotolerance of C. jejuni. Among several knockout mutants of heat shock chaperones, a mutant lacking clpB exhibited significantly lower survival than the wild type under heat treatment. Moreover, we observed a significantly higher accumulation of protein aggregates in the absence of ClpB, demonstrating that ClpB functions as a disaggregase during heat exposure. Additionally, ClpB from the heat-tolerant CC-443 group possessed distinct amino acid substitutions in the functional nucleotide-binding domain compared to ClpB in other CC groups. Interestingly, despite the well-known interaction of these proteins in many other bacteria, a two-hybrid assay demonstrated that ClpB of C. jejuni does not bind to DnaK, suggesting that C. jejuni may have a distinct mechanism for protein disaggregation and stress tolerance. Our findings demonstrate that ClpB plays a crucial role in the thermotolerance of C. jejuni through unique protein disaggregation mechanisms during poultry processing.IMPORTANCEThis study unveils a distinctive mechanism of thermotolerance involving protein disaggregation in Campylobacter jejuni, a major foodborne pathogen. Understanding C. jejuni's ability to withstand heat stress is crucial for comprehending the occurrence of Campylobacter infections resulting from the consumption of contaminated poultry meat. Our research elucidates the roles of heat shock proteins, particularly ClpB, in the thermotolerance of C. jejuni. These findings significantly contribute to our fundamental understanding of bacterial physiology related to stress tolerance, which has important implications for public health and food safety.

Keywords: Campylobacter; ClpB; multilocus sequence typing (MLST); protein disaggregation; thermotolerance.

Fig 1

Differential survival of 87 C. jejuni strains under heat stress and variations in the distribution of multilocus sequence typing (MLST) clonal complexes (CCs) between heat-sensitive and heat-tolerant strains. (A) Viable counts of 87 C. jejuni strains were measured at 0, 20, 40, and 60 min of exposure to 50°C in Mueller-Hinton broth. The experiment was repeated three times, and each dot represents the average of three replicates. The dotted line indicates the detection limit (200 CFU/mL). (B) Log reduction values of heat-sensitive and heat-tolerant strains after 60 min of heat treatment. In panels A and B, colored circles represent C. jejuni strains as follows: orange, C. jejuni isolates (n = 86); pink, heat-sensitive strains (n = 43); blue, heat-tolerant strains (n = 43); and black, reference strain (C. jejuni NCTC 11168). The experiment was performed in triplicate with consistent results. Black lines indicate mean values. (C) Comparison of MLST CC proportions between heat-sensitive (n = 43) and heat-tolerant strains (n = 43). Student’s t-test was used to compare viabilities between heat-sensitive and heat-tolerant strains. A χ test was conducted to compare the proportions of CCs. Significance levels are denoted as follows: *, P < 0.05; **, P < 0.01; ****, P < 0.0001. CC, clonal complex; UA, unassigned to any defined CC.

Fig 2

Contribution of clpB to thermotolerance in C. jejuni. (A) Survival of C. jejuni strains was measured during 60 min of exposure to 50°C. The dotted line indicates the detection limit (200 CFU/mL). (B) Viability of C. jejuni strains measured after exposure to scalding water at 50°C for 5 min. The dotted line indicates the detection limit (100 CFU/mL). Data shown are representative of three independent experiments with similar results. Error bars represent standard errors of the means. Statistical analysis was performed using Student’s t-test to compare viabilities between WT and indicated strains at each sampling time point. Significance levels are denoted as follows: *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; ns, non-significant; N.D., not detected; WT, C. jejuni NCTC 11168 wild type; ΔclpB, ΔclpB mutant; clpB comp, clpB-complemented strain; ΔdnaK, ΔdnaK mutant; dnaK comp, dnaK-complemented strain; ΔgroESL, ΔgroESL mutant; groESL comp, groESL-complemented strain.

Fig 3

C. jejuni ClpB does not interact with DnaK. (A) Bacterial adenylate cyclase two-hybrid (BACTH) assays demonstrating heterotypic interactions between ClpB and DnaK of C. jejuni. Positive (T18-ClpB of E. coli versus T28-DnaK of E. coli) and negative controls (T18-empty vector versus T28-empty vector) are shown for comparison. (B) Quantification of β-galactosidase activity from the BACTH interactions shown in panel A, expressed in Miller units. The experiment was performed in triplicate. Error bars represent standard errors of the means. Statistical analysis was conducted using Student’s t-test. Significance levels are denoted as follows: ***, P < 0.001; ns, non-significant.

Fig 4

Protein disaggregation by ClpB of aggregates formed under heat stress in C. jejuni. (A) Comparison of aggregated protein levels during heat treatment at 50°C for 20 min. The fluorescence intensity of the WT strain before heat treatment (0 min) was set as 1. (B) Confocal microscopy images of protein aggregates using SYTO 9 dye (green) and PROTEOSTAT dye (red) in samples heat-treated for 20 min. Merged images of SYTO 9 and PROTEOSTAT dye staining are shown on the right. (C) Cross-sectional transmission electron microscopy (TEM) images of C. jejuni strains before (Initial) and after heat treatment for 20 min at 50°C (heat stress). Protein aggregates are indicated by orange arrows. (D) Aggregates per field of view obtained from cross-sectional TEM images of C. jejuni strains before (0 min) and after heat treatment at 50°C for 20 min. The data shown are representative of three independent experiments with similar results. Error bars represent standard errors of the means. Statistical analysis was performed using Student’s t-test. Significance levels are denoted as follows: *, P < 0.05; **, P < 0.01; ns, non-significant. WT, C. jejuni NCTC 11168 wild type; ΔclpB, ΔclpB mutant; clpB comp, clpB-complemented strain.

Fig 5

Analysis of ClpB amino acid sequences from C. jejuni isolates. (A) Phylogenetic tree based on ClpB amino acid sequences from 86 C. jejuni isolates. CC, clonal complex; UA, unassigned to any defined CC.