Levels Of DnaK Research Articles

Lacticaseibacillus paracasei AD22 Stress Response in Brined White Cheese Matrix: In Vitro Probiotic Profiles and Molecular Characterization.

Functionalizing foods involve discovering and integrating new candidate health-promoting bacteria into the food matrix. This study aimed (i) to reveal the probiotic potential of autochthonous Lacticaseibacillus paracasei AD22 by a series of in vitro tests and molecular characterization and (ii) to evaluate its application to the matrix of brined white cheese, which is the most common cheese in Türkiye, in terms of survival and stress response. To evaluate in vitro probiotic characteristics, L. paracasei AD22 was exposed to functional, technological, and safety tests. Pilot scale production was conducted to integrate L. paracasei AD22 into the brined white cheese matrix. The expression levels of stress-related genes (dnaK, groES, ftsH, argH, and hsp20) were detected by reverse-transcriptase polymerase chain reaction to determine the transcriptional stress response during ripening. The presence of genes encoding stress-related proteins was determined by whole-genome sequence analysis using a subsystem approach; the presence of antibiotic resistance and virulence genes was determined by ResFinder4.1 and VirulenceFinder 2.0 databases. The BAGEL4 database determined the presence of bacteriocin clusters. L. paracasei AD22 was found to survive in pH 2 and medium with 12% NaCl and did not cause hemolysis. Adhesion of the strain to Caco2 cells was 76.26 ± 4.81% and it had coaggregation/autoaggregation properties. It was determined that L. paracasei AD22 exceeded 7 log cfu/g in the cheese matrix at the end of the ripening period. Total mesophilic aerobes decreased in the cheese inoculated with L. paracasei AD22 after the 45th day of ripening. While hsp20 and groES genes were downregulated during ripening, argH was upregulated. Both downregulation and upregulation were observed in dnaK and ftsH. Fold changes indicating the expression levels of dnaK, groES, ftsH, argH, and hsp20 genes were not statistically significant during ripening (p > 0.05). Whole-genome sequence profiles revealed that the strain did not contain antibiotic and virulence genes but bacteriocin clusters encoding Enterolysin A (Class III bacteriocin), Carnosine CP52 (class II bacteriocin), Enterocin X beta chain (Class IIc bacteriocin), and the LanT region. Subsystems approach manifested that the most functional part of the genomic distribution belonged to metabolism, protein processing, and stress response functions. The study findings highlight that L. paracasei AD22 will provide biotechnological innovation as a probiotic adjunct because it contains tolerance factors and probiotic characteristics to produce new functional foods.

Expression of DnaK and HtrA genes under high temperatures and their impact on thermotolerance of a Salmonella serotype isolated from tahini product

BackgroundSalmonella is considered to be the second largest source of infection in food-borne diseases. It is also considered one of the most important dangers particularly in the meat and dairy industry. Therefore, the main objective of our study was to determine the relationship between thermotolerance of a Salmonella serotype and the expression of DnaK and HtrA genes. ResultsIn this study, expression of the two genes DnaK and HtrA was compared under four different temperatures 37 °C, 42 °C, 50 °C, and 55 °C in two serotypes of Salmonella enterica subsp. enterica. One of them was isolated from tahini product and identified as Salmonella enterica subsp. enterica serovar choleraesuis. This identified serotype was found to be more thermotolerant than the second serotype (Salmonella enterica subsp. enterica serovar typhimurium (ATCC 13311)), which was used as reference. This conclusion was based on D and Z values, which were used to compare thermoresistance ability of the two serotypes under four different temperatures 60 °C, 65 °C, 70 °C, and 75 °C. In addition, the results of qRT-PCR showed that after 43 °C (induction temperature), the relative expression (fold change) of DnaK and HtrA genes increased up to 5 and 47, respectively, comparing to their expression at 37 °C. ConclusionsThermotolerance of the identified S. choleraesuis serotype showed significantly high expression levels of DnaK and HtrA genes.

How Do Chaperones Protect a Cell's Proteins from Oxidative Damage?

The accumulation of protein damage in aging organisms is thought to contribute to many aging-related diseases. Yet the properties determining which proteins are most susceptible remain poorly understood. Are certain conformations more vulnerable? Which chaperones are the main guardians? We address these questions with a system-wide model of E.coli proteostasis. By predicting how proteins with different folding properties respond to each chaperone's concentration, the model computes "damage fingerprints" that identify unfolded conformations as the major damage target. What matters most is not a protein's stability or difficulty of folding, but its dwell time in an unfolded state. The main guardian chaperone is DnaK because its client proteins spend more time unfolded than clients of GroEL, providing a mechanism for why the cell's capacity to handle stress is more sensitive to DnaK levels. Also, we find that chaperones are protectors, not recyclers. This model describes how cells resist stress and indicates that designing chaperone-targeting drugs may require whole-cell, system-wide modeling.

Genome Shuffling of Stenotrophomonas maltophilia OK-5 for Improving the Degradation of Explosive RDX (Hexahydro-1,3,5-trinitro-1,3,5-triazine)

A genome-shuffled Stenotrophomonas maltophilia strain showing the enhanced ability of RDX degradation was constructed, and its characteristics were compared with those of the wild-type one. The shuffled strain was able to completely degrade 25, 50, and 75µM RDX (hexahydro-1,3,5-trinitro-1,3,5-triazine) within 10, 30, and 50days, respectively. However, it took 30 and 70days for the wild-type strain to degrade 25 and 50µM RDX, respectively, and at day 70, the strain degraded only 67% of 75µM RDX. The shuffled strain reached its maximum growth at 50-60days and exhibited approximately 1.5-fold increased cell numbers. SEM revealed more severe damage on the surface of the wild-type cells compared to the genome-shuffled cells. The mRNA levels of dnaK and groEL encoding the heat shock proteins were increased by 2.5-fold and fourfold, and DnaK and GroEL proteins were more highly produced in the shuffled cells. In addition, the mRNA levels of pnrB encoding a TNT nitroreductase, and algA involved in exopolymer biosynthesis, were slightly higher in the shuffled strain, but not as high as those of dnaK and groEL. These results indicate that the genome shuffling rendered the shuffled cells more resistant to RDX stress. A proteomic comparison revealed changes in the production levels of certain proteins including nitrate and cell protection, particularly those involved in metabolism. These proteomic analyses provide clues for understanding the improved RDX degradation by the genome-shuffled S. maltophilia strain.

Association of DnaK and GroEL with Antimicrobial Resistance in Salmonella Abortusovis

Multidrug-resistant (MDR) strains of Salmonella are now encountered frequently and the rates of multidrug-resistance have increased considerably in recent years. The association of DnaK and GroEL molecular chaperones with the stress response induced by antibiotics has been mentioned for the most frequently isolated serovar, S. enterica subsp. enterica Typhimurium, but similar studies on S. Abortusovis were not published, so far. In the present work, the induction of DnaK and GroEL in S. Abortusovis cells under stress conditions caused by heat shock and by various antibiotics respectively was studied by Western blot and real time RT-PCR methods. The transcription and translation analyses showed increased levels of DnaK and GroEL after a temperature shift from 37 °C to 42 °C, and after the exposure of a multiresistant S. Abortusovis strain to subinhibitory concentrations of various antimicrobials (nalidixic acid, streptomycin, kanamycin, tetracycline and gentamicin). The data presented here may provide a starting point for future research regarding the role of S. Abortusovis molecular chaperones in the stress response induced by antibiotics.

HSPA5/Dna K may be a useful target for human disease therapies.

The chaperone protein HSPA5/Dna K is conserved throughout evolution from higher eukaryotes down to prokaryotes. The celecoxib derivative OSU-03012 (also called AR-12) interacts with Viagra or Cialis in eukaryotic cells to rapidly reduce HSPA5 levels as well as blunt the functions of many other chaperone proteins. Because multiple chaperones are modulated in eukaryotes, the expression of cell surface virus receptors is reduced and because HSPA5 in blocked viruses cannot efficiently replicate. Because DnaK levels are reduced in prokaryotes by OSU-03012, the levels of DnaK chaperone proteins such as Rec A decline, which is associated with bacterial cell death and a resensitization of so-called drug-resistant superbugs to standard of care antibiotics. In Alzheimer's disease, HSPA5 has been shown to play a supportive role for the progression of tau phosphorylation and neurodegeneration. Thus, in eukaryotes, HSPA5 represents a target for anticancer, antiviral, and anti-Alzheimer's therapeutics and in prokaryotes, DnaK and bacterial phosphodiesterases represent novel antibiotic targets that should be exploited in the future by pharmaceutical companies.

Induction of DnaK and GroEL in Brucella Ovis under Various Stress Conditions

Brucella ovis is a facultative intracellular pathogen, able to survive and replicate within macrophages. The role of molecular chaperones in Brucella intracellular survival has been previously mentioned, but nothing was published on B. ovis in this respect, as far as we know. In the present work, in order to understand any possible contribution of the molecular chaperones DnaK and GroEL for Brucella ovis stress survival and intracellular persistance, the expression on different culture media under different stress conditions were analysed at the transcriptional and translational levels and compared with that of bacteria grown inside murine J774A.1 cells. The transcription analyses showed different levels of dnaK and groEL induction under various stress conditions (e.g., macrophages survival, acidic pH shock, high temperature, oxidative and osmotic shock) and a significant influence of the media composition. These results were consistent with the level of DnaK and GroEL expression analysed by the traditional proteomic methods. Here we demonstrate the molecular chaperones involvement in macrophage-B. ovis interaction, their expression under stress conditions suggesting the chaperones role in the bacterial resistance to such unfavourable environmental conditions.

Induction of DnaK upon <i>γ</i>-Irradiation at High and Low Rates in <i>Escherichia coli</i>

DnaK is implicated in protein folding, repair and degradation. Its protective role during heat shock is well documented and many other stresses can also induce its production. Using a competitive ELISA, intracellular DnaK concentrations were determined in Escherichia coli ATCC 25922 exposed to a γ-irradiation dose of 0.3 KGy applied either at high (8 × 10-2 KGy/min) or low rates (3 × 10-3 KGy/min) and with or without a recuperation period of 22 h at 37℃ post-treatment. All four irradiation treatments reduced cell counts similarly and significantly compared to the control (P < 0.0001). However, the highest DnaK concentration was observed in cells irradiated at low rate without recuperation (105,416 molecules/cell; P = 0.0001). Furthermore, DnaK levels remained higher than the control (38,500 molecules/ cell) after the recuperation period (P < 0.05). Variation in the intracellular DnaK concentration indicates that the bacterial stress response was modulated differently according to the irradiation treatment (P = 0.0001).

Proteomic analysis of responses of a new probiotic bacterium Lactobacillus casei Zhang to low acid stress

Tolerance to acid is an important feature for probiotic bacteria during transition through the gastrointestinal tract. Proteomics analysis of a new probiotic bacterium, Lactobacillus casei Zhang, was performed upon 30-min exposure to low acid stress (pH 2.5 vs. pH 6.4) using two-dimensional electrophoresis. Out of 33 protein spots that showed changes of expression between the two pHs, 22 showed 1.5-fold higher expression at pH 2.5 than at pH 6.4, whereas five spots had expression decreased by 1.5-fold at pH 2.5. There were also six protein spots that were exclusively present on different pH maps. Further analysis showed that eight of the enhanced proteins, NagA, NagB, PGM, GlmM, LacC, TDP, GALM and PtsI, were involved in carbohydrate catabolism. Moreover, quantitative RT-PCR showed that the mRNA expression levels of dnaK, nagB, galm, estC, tuf and luxS were consistent with changes in protein expression. We postulate that there might be some relationship between differentially expressed proteins and acid tolerance in L. casei Zhang.

Inhibitory Effects of Low-Energy Pulsed Ultrasonic Stimulation on Cell Surface Protein Antigen C through Heat Shock Proteins GroEL and DnaK inStreptococcus mutans

This study concerns the use of low-energy pulsed ultrasound as nondestructive photodynamic antimicrobial therapy for controlling dental plaque. We examined the antibacterial and bactericidal effects of low-energy pulsed ultrasound on mutans streptococci and its inhibitory effects on bacterial cell adhesion of Streptococcus mutans. The results indicated weak antibacterial and bactericidal effects. However, ultrasonic stimulation for less than 20 min markedly decreased bacterial cell adhesion. To analyze the mechanism underlying the inhibitory effect, we examined cell surface protein antigen C (PAc) and glucosyltransferase I (GTF-I) expression in S. mutans. The levels of PAc gene and protein expression were markedly decreased by ultrasonic stimulation for 20 min. However, no change in GTF-I expression was observed. The expression of stress response heat shock proteins GroEL and DnaK was also examined. GroEL and DnaK levels were significantly decreased by ultrasonic stimulation, and the expression of the PAc protein was also diminished upon the addition of GroEL or DnaK inhibitors without ultrasonic stimulation. These observations suggest that the expression of the PAc protein in S. mutans may be dependent on heat shock proteins. Thus, low-energy pulsed ultrasound decreases bacterial adhesion by the inhibitory effect on the PAc protein and heat shock protein expression and may be useful as photodynamic antimicrobial chemotherapy in controlling dental plaque.

Dual regulation of dnaK and groE operons by HrcA and Ca++ in Streptococcus pneumoniae

The dnaK and groE operons in Streptococcus pneumoniae are repressed by HrcA in the presence of Ca(++). However, it is unclear how HrcA and Ca(++) regulate the dnaK and groE operons. This study examined the dual regulation of the dnaK and groE operons in S. pneumoniae by HrcA and Ca(++). At 30 degrees C, the hrcA mutant showed a constitutively higher level of dnaK expression at both the protein and mRNA levels than that of the wild type whereas the level of groEL expression was relatively unchanged. On the other hand, the levels of both dnaK and groEL transcripts were increased after heat shock but the hrcA mutant was not sensitive to heat shock. Immunoblot analysis of the cells pretreated with the Ca(++) chelator, BAPTA-AM, revealed the induction of HrcA and GroEL at both 30 degrees C and 42 degrees C. However, at longer preincubation time with BAPTA-AM, GroEL was further induced but the level of HrcA decreased suggesting that Ca(++) regulates the dnaK and groE operons differently. Overall, Ca(++) and HrcA differentially regulate the transcription of the dnaK and groE operons in S. pneumoniae. These results are expected to contribute to resolving the relationship between DnaK/GroEL expression and the pathogenesis in S. pneumoniae.

Requirements for Surface Expression and Function of Adhesin P1 from Streptococcus mutans

In this report, we define requirements for the successful translocation and functional maturation of the adhesin P1 of Streptococcus mutans. Conformational epitopes recognized by anti-P1 monoclonal antibodies (MAbs) were further characterized, thus facilitating the use of particular MAbs as tools to monitor the locations of various forms of the protein. We show that correct localization of P1 is dependent on structural features of the molecule itself, including a requisite A region-P region intramolecular interaction that occurs within the cell prior to secretion. P1 also was shown to be affected by several members of the protein-folding-secretion-turnover apparatus. It does not achieve a fully functional form in the absence of the trigger factor PPIase homolog RopA, and its translocation is delayed when DnaK levels are limited. In addition, dnaK message levels are differentially altered in the presence of P1 lacking the alanine-rich compared to the proline-rich repeat domains. Lastly, nonsecreted P1 lacking the P region accumulates within the cell in the absence of htrA, implying an intracellular HtrA protease function in the degradation and turnover of this particular internal-deletion polypeptide. However, the opposite effect is seen for full-length P1, suggesting a sensing mechanism and substrate-dependent alteration in HtrA's function and effect that is consistent with its known ability to switch between chaperone and protease, depending on environmental perturbations.

Role of the chaperone DnaK in protein solubility and conformational quality in inclusion body-formingEscherichia colicells

Misfolding-prone proteins produced in bacteria usually fail to adopt their native conformation and aggregate. In cells producing folding-reluctant protein species, folding modulators are supposed to be limiting, a fact that enhances protein deposition. Therefore, coproducing DnaK or other main chaperones along with the target protein has been a common approach to gain solubility, although with very inconsistent and often discouraging results. In an attempt to understand the reason for this inconsistency, the impact of exogenous DnaK (encoded in an accompanying plasmid) on two protein features observed as indicators of protein quality, namely solubility and functionality, has been analysed here through the specific fluorescence emission of a reporter Green Fluorescent Protein (GFP). Intriguingly, GFP solubility is strongly dependent on its own yield but poorly affected by DnaK levels. On the contrary, the specific fluorescence of both soluble and insoluble GFP populations is simultaneously modulated by the availability of DnaK, with a profile that is clearly dissimilar to that shown by protein solubility. Therefore, solubility, not being coincident with the biological activity of the target protein, might not be a robust indicator of protein quality.

Response to alkaline stress by root canal bacteria in biofilms

To determine whether bacteria isolated from infected root canals survive alkaline shifts better in biofilms than in planktonic cultures. Clinical isolates of Enterococcus faecalis, Lactobacillus paracasei, Olsenella uli, Streptococcus anginosus, S. gordonii, S. oralis and Fusobacterium nucleatum in biofilm and planktonic cultures were stressed at pH 10.5 for 4 h, and cell viability determined using the fluorescent staining LIVE/DEAD BacLight bacterial viability kit. In addition, proteins released into extracellular culture fluids were identified by Western blotting. Enterococcus faecalis, L. paracasei, O. uli and S. gordonii survived in high numbers in both planktonic cultures and in biofilms after alkaline challenge. S. anginosus, S. oralis and F. nucleatum showed increased viability in biofilms compared with planktonic cultures. Alkaline exposure caused all planktonic cultures to aggregate into clusters and resulted in a greater extrusion of cellular proteins compared with cells in biofilms. Increased levels of DnaK, HPr and fructose-1,6-bisphosphate aldolase were observed in culture fluids, especially amongst streptococci. In general, bacteria isolated from infected roots canals resisted alkaline stress better in biofilms than in planktonic cultures, however, planktonic cells appeared to use aggregation and the extracellular transport of specific proteins as survival mechanisms.

Physiologic Effects of Forced Down-Regulation of dnaK and groEL Expression in Streptococcus mutans

Strains of Streptococcus mutans lacking DnaK or GroEL appear not to be isolable. To better distinguish the roles played by these chaperones/chaperonins in the physiology of S. mutans, we created a knockdown strategy to lower the levels of DnaK by over 95% in strain SM12 and the level of GroEL about 80% in strain SM13. Interestingly, GroEL levels were approximately twofold higher in SM12 than in the parent strain, but the levels of DnaK were not altered in the GroEL knockdown strain. Both SM12 and SM13 grew slower than the parent strain, had a strong tendency to aggregate in broth culture, and showed major changes in their proteomes. Compared with the wild-type strain, SM12 and SM13 had impaired biofilm-forming capacities when grown in the presence of glucose. The SM12 strain was impaired in its capacity to grow at 44 degrees C or at pH 5.0 and was more susceptible to H(2)O(2), whereas SM13 behaved like the wild-type strain under these conditions. Phenotypical reversions were noted for both mutants when cells were grown in continuous culture at a low pH, suggesting the occurrence of compensatory mutations. These results demonstrate that DnaK and GroEL differentially affect the expression of key virulence traits, including biofilm formation and acid tolerance, and support that these chaperones have evolved to accommodate unique roles in the context of this organism and its niche.

Independent regulation of H-NS-mediated silencing of the bgl operon at two levels: upstream by BglJ and LeuO and downstream by DnaKJ

Silencing of the Escherichia coli bgl operon by the histone-like nucleoid-structuring protein H-NS occurs at two levels. Binding of H-NS upstream of the promoter represses transcription initiation, whilst binding within the coding region is also proposed to repress transcription elongation. The latter, downstream level of repression is counteracted by the protease Lon and, thus, silencing of the bgl operon is more effective in lon mutants. Transposon-mutagenesis screens for suppression of this lon phenotype on bgl were performed and insertion mutations disrupting rpoS and crl were obtained, as well as mutations mapping upstream of the open reading frames of bglJ, leuO and dnaK. In rpoS and crl mutants, bgl promoter activity is known to be higher. Likewise, as shown here, bgl promoter activity is increased in the bglJ and leuO mutants, which express BglJ and LeuO constitutively. However, BglJ and LeuO have no impact on downstream repression. A dnaKJ mutant was isolated for the first time in the context of the bgl operon. The mutant expresses lower levels of DnaK than the wild-type. Interestingly, in this dnaKJ : : miniTn10 mutant, downstream repression of bgl by H-NS is less effective, whilst upstream repression by H-NS remains unaffected. Together, the data show that the two levels of bgl silencing by H-NS are regulated independently.

Acid resistance variability among isolates of Salmonella enterica serovar Typhimurium DT104

Acid resistance could be an indicator of virulence since acid resistant strains are able to better survive the human stomach passage and in macrophages. We studied the acid resistance of several Salmonella Typhimurium DT104 strains isolated from food and humans and identified cellular parameters contributing to the enhanced acid resistance of these isolates. Acid resistance was tested in 37 Salmonella enterica Typhimurium serovar DT104 (S. Typhimurium DT104) strains. Acid adaptation at pH 5 followed by exposure for 2 h at pH 2.5 in the 27 human, nine nonhuman, and in two reference strains, revealed strong variation of acid survival. After 2 h at pH 2.5 six strains of S. Typhimurium DT104 were considered high acid resistant as they displayed a level of survival >10%, 14 strains were considered intermediate acid resistant (level of survival was <10% and >0.01%) and 19 strains were considered low acid resistant (level of survival <0.01%). Six strains were selected for further studies and proteomics revealed a relatively high amount of phase 2 flagellin in an acid-sensitive strain and a relatively high amount of the beta component of the H(+)/ATPase in an acid-resistant strain. Two strains were slightly more heat resistant possibly as the result of increased levels of DnaK or GroEL. A significant difference could be detected between human and food isolates regarding their acid resistance; all high acid-resistant strains were human isolates. S. Typhimurium DT104 is known for two decades and has a great impact on human health causing serious food-borne diseases. Our results suggest the existence of a positive correlation between acid resistance and pathogenicity in S. Typhimurium DT104 as all high acid-resistant strains were isolated from humans.

Proteolysis in prokaryotes: protein quality control and regulatory principles.

Proteolysis in prokaryotes: protein quality control and regulatory principles.

Small heat shock proteins, ClpB and the DnaK system form a functional triade in reversing protein aggregation.

Small heat shock proteins (sHsps) can efficiently prevent the aggregation of unfolded proteins in vitro. However, how this in vitro activity translates to function in vivo is poorly understood. We demonstrate that sHsps of Escherichia coli, IbpA and IbpB, co-operate with ClpB and the DnaK system in vitro and in vivo, forming a functional triade of chaperones. IbpA/IbpB and ClpB support independently and co-operatively the DnaK system in reversing protein aggregation. A delta ibpAB delta clpB double mutant exhibits strongly increased protein aggregation at 42 degrees C compared with the single mutants. sHsp and ClpB function become essential for cell viability at 37 degrees C if DnaK levels are reduced. The DnaK requirement for growth is increasingly higher for delta ibpAB, delta clpB, and the double delta ibpAB delta clpB mutant cells, establishing the positions of sHsps and ClpB in this chaperone triade.

Downregulation of the heat shock response is independent of DnaK and sigma32 levels in Caulobacter crescentus.

Expression of heat shock genes in Gram-negative proteobacteria is positively modulated by the transcriptional regulator RpoH, the sigma(32) subunit of RNA polymerase (RNAP). In this study we investigated the chaperones DnaK/DnaJ and GroES/GroEL as possible modulators of the heat response in Caulobacter crescentus. We have shown that cells overexpressing DnaK show poor induction of heat shock protein (HSP) synthesis, even though sigma(32) levels present a normal transient increase upon heat stress. On the other hand, depletion of DnaK led to higher levels of sigma(32) and increased transcription of HSP genes, at normal growth temperature. In contrast, changes in the amount of GroES/EL had little effect on sigma(32) levels and HSP gene transcription. Despite the strong effect of DnaK levels on the induction phase of the heat shock response, downregulation of HSP synthesis was not affected by changes in the amount this chaperone. Thus, we propose that competition between sigma(32) and sigma(73), the major sigma factor, for the core RNAP could be the most important factor controlling the shut-off of HSP synthesis during recovery phase. In agreement with this hypothesis, we have shown that expression of sigma(73) gene is heat shock inducible.