Evaluating the ability of different chaperones in improving soluble expression of a triple-mutated human interferon gamma in Escherichia coli.

Human interferon gamma (hIFN-γ) plays a pivotal role as a soluble cytokine with diverse functions in both innate and adaptive immunity. In a previous investigation, we pinpointed three critical amino acid residues, i.e., threonine (T) 27, phenylalanine (F) 29, and leucine (L) 30, on the IFN-γ structure, which are integral to the epitope recognized by anti-IFN-γ autoantibodies. It is crucial to impede the interaction between this epitope and autoantibodies for effective therapy in adult-onset immunodeficiency (AOID). However, the challenge arises from the diminished solubility of the T27AF29L30A mutant in Escherichia coli BL21(DE3). This study delves into a targeted strategy aimed at improving the soluble expression of IFN-γ T27AF29AL30A. This is achieved through the utilization of five chaperone plasmids: pG-KJE8, pKJE7, pGro7, pG-Tf2, and pTf16. These plasmids, encoding cytoplasmic chaperones, are co-expressed with the IFN-γ mutant in E. coli BL21(DE3), and we meticulously analyze the proteins in cell lysate and inclusion bodies using SDS-PAGE and Western blotting. Our findings reveal the remarkable efficacy of pG-KJE8, which houses cytoplasmic chaperones DnaK-DnaJ-GrpE and GroEL-GroES, in significantly enhancing the solubility of IFN-γ T27AF29AL30A. Importantly, this co-expression not only addresses solubility concerns but also preserves the functional dimerized structure, as confirmed by sandwich ELISA. This promising outcome signifies a significant step forward in developing biologic strategies for AOID.

Gut microbiome diversity within Clostridia is negatively associated with human obesity.

Clostridia are abundant in the human gut and comprise families associated with host health such as Oscillospiraceae, which has been correlated with leanness. However, culturing bacteria within this family is challenging, leading to their detection primarily through 16S rRNA amplicon sequencing, which has a limited ability to unravel diversity at low taxonomic levels, or by shotgun metagenomics, which is hindered by its high costs and complexity. In this cross-sectional study involving 114 Colombian adults, we used an amplicon-based sequencing strategy with alternative markers-gyrase subunit B (gyrB) and DNA K chaperone heat protein 70 (dnaK)-that evolve faster than the 16S rRNA gene. Comparing the diversity and abundance observed with the three markers in our cohort, we found a reduction in the diversity of Clostridia, particularly within Lachnospiraceae and Oscillospiraceae among obese individuals [as measured by the body mass index (BMI)]. Within Lachnospiraceae, the diversity of Ruminococcus_A negatively correlated with BMI. Within Oscillospiraceae, the genera CAG-170 and Vescimonas also exhibited this negative correlation. In addition, the abundance of Vescimonas was negatively correlated with BMI. Leveraging shotgun metagenomic data, we conducted a phylogenetic and genomic characterization of 120 metagenome-assembled genomes from Vescimonas obtained from a larger sample of the same cohort. We identified 17 of the 72 reported species. The functional annotation of these genomes showed the presence of multiple carbohydrate-active enzymes, particularly glycosyl transferases and glycoside hydrolases, suggesting potential beneficial roles in fiber degradation, carbohydrate metabolism, and butyrate production. IMPORTANCE: The gut microbiota is diverse across various taxonomic levels. At the intra-species level, it comprises multiple strains, some of which may be host-specific. However, our understanding of fine-grained diversity has been hindered by the use of the conserved 16S rRNA gene. While shotgun metagenomics offers higher resolution, it remains costly, may fail to identify specific microbes in complex samples, and requires extensive computational resources and expertise. To address this, we employed a simple and cost-effective analysis of alternative genetic markers to explore diversity within Clostridia, a crucial group within the human gut microbiota whose diversity may be underestimated. We found high intra-species diversity for certain groups and associations with obesity. Notably, we identified Vescimonas, an understudied group. Making use of metagenomic data, we inferred functionality, uncovering potential beneficial roles in dietary fiber and carbohydrate degradation, as well as in short-chain fatty acid production.

Application of a new designed high resolution melting analysis for mycobacterial species identification.

The Non-tuberculous mycobacterial (NTM) isolates should be distinguished from tuberculosis and identified at the species level for choosing an appropriate treatment plan. In this study, two molecular methods were used to differentiate NTM species, including a new designed High Resolution Melting (HRM) and Multilocus Sequence Analysis (MLSA). Seventy-five mycobacterial isolates were evaluated by sequencing four genes ( MLSA) and a HRM assay specifically targeting atpE was designed to rapidly and accurately identify and differentiate mycobacterium species. Out of 70 NTM isolates, 66 (94.3%), 65 (92.9%), 65 (92.9%) and 64 (91.4%) isolates were identified to the species level by PCR of atpE, tuf, rpoB and dnaK genes. We could identify 100% of the isolates to the species level (14 different species) by MLSA. By using HRM assay, all NTM isolates were identified and classified into eight groups, in addition, Mycobacterium tuberculosis and Nocardia were also detected simultaneously. The MLSA technique was able to differentiate all 14 species of NTM isolates. According to the results, the HRM assay is a rapid and beneficial method for identifying NTM, M. tuberculosis (MTB), and Nocardia isolates without sequencing.

Mechanism of chaperone coordination during cotranslational protein folding in bacteria.

Protein folding is assisted by molecular chaperones that bind nascent polypeptides during mRNA translation. Several structurally distinct classes of chaperones promote de novo folding, suggesting that their activities are coordinated at the ribosome. We used biochemical reconstitution and structural proteomics to explore the molecular basis for cotranslational chaperone action in bacteria. We found that chaperone binding is disfavored close to the ribosome, allowing folding to precede chaperone recruitment. Trigger factor recognizes compact folding intermediates that expose an extensive unfolded surface, and dictates DnaJ access to nascent chains. DnaJ uses a large surface to bind structurally diverse intermediates and recruits DnaK to sequence-diverse solvent-accessible sites. Neither Trigger factor, DnaJ, nor DnaK destabilize cotranslational folding intermediates. Instead, the chaperones collaborate to protect incipient structure in the nascent polypeptide well beyond the ribosome exit tunnel. Our findings show how the chaperone network selects and modulates cotranslational folding intermediates.

Biochemical characterization of ClpB and DnaK from Anaplasma phagocytophilum.

Anaplasma phagocytophilum is an intracellular tick-transmitted bacterial pathogen that infects neutrophils in mammals and causes granulocytic anaplasmosis. In this study, we investigated the molecular chaperones ClpB and DnaK from A. phagocytophilum. In Escherichia coli, ClpB cooperates with DnaK and its co-chaperones DnaJ and GrpE in ATP-dependent reactivation of aggregated proteins. Since ClpB is not produced in metazoans, it is a promising target for developing antimicrobial therapies, which generates interest in studies on that chaperone's role in pathogenic bacteria. We found that ClpB and DnaK are transcriptionally upregulated in A. phagocytophilum 3-5 days after infection of human HL-60 and tick ISE6 cells, which suggests an essential role of the chaperones in supporting the pathogen's intracellular life cycle. Multiple sequence alignments show that A. phagocytophilum ClpB and DnaK contain all structural domains that were identified in their previously studied orthologs from other bacteria. Both A. phagocytophilum ClpB and DnaK display ATPase activity, which is consistent with their participation in the ATP-dependent protein disaggregation system. However, despite a significant sequence similarity between the chaperones from A. phagocytophilum and those from E. coli, the former were not as effective as their E. coli orthologs during reactivation of aggregated proteins in vitro and in supporting the survival of E. coli cells under heat stress. We conclude that the A. phagocytophilum chaperones might have evolved with distinct biochemical properties to maintain the integrity of pathogenic proteins under unique stress conditions of an intracellular environment of host cells.

Bacterial DnaK reduces the activity of anti-cancer drugs cisplatin and 5FU.

BACKGROUND: Chemotherapy is a primary treatment for cancer, but its efficacy is often limited by cancer-associated bacteria (CAB) that impair tumor suppressor functions. Our previous research found that Mycoplasma fermentans DnaK, a chaperone protein, impairs p53 activities, which are essential for most anti-cancer chemotherapeutic responses. METHODS: To investigate the role of DnaK in chemotherapy, we treated cancer cell lines with M. fermentans DnaK and then with commonly used p53-dependent anti-cancer drugs (cisplatin and 5FU). We evaluated the cells' survival in the presence or absence of a DnaK-binding peptide (ARV-1502). We also validated our findings using primary tumor cells from a novel DnaK knock-in mouse model. To provide a broader context for the clinical significance of these findings, we investigated human primary cancer sequencing datasets from The Cancer Genome Atlas (TCGA). We identified F. nucleatum as a CAB carrying DnaK with an amino acid composition highly similar to M. fermentans DnaK. Therefore, we investigated the effect of F. nucleatum DnaK on the anti-cancer activity of cisplatin and 5FU. RESULTS: Our results show that both M. fermentans and F. nucleatum DnaKs reduce the effectiveness of cisplatin and 5FU. However, the use of ARV-1502 effectively restored the drugs' anti-cancer efficacy. CONCLUSIONS: Our findings offer a practical framework for designing and implementing novel personalized anti-cancer strategies by targeting specific bacterial DnaKs in patients with poor response to chemotherapy, underscoring the potential for microbiome-based personalized cancer therapies.

Hsp90, a team player in protein quality control and the stress response in bacteria.

SUMMARYHeat shock protein 90 (Hsp90) participates in proteostasis by facilitating protein folding, activation, disaggregation, prevention of aggregation, degradation, and protection against degradation of various cellular proteins. It is highly conserved from bacteria to humans. In bacteria, protein remodeling by Hsp90 involves collaboration with the Hsp70 molecular chaperone and Hsp70 cochaperones. In eukaryotes, protein folding by Hsp90 is more complex and involves collaboration with many Hsp90 cochaperones as well as Hsp70 and Hsp70 cochaperones. This review focuses primarily on bacterial Hsp90 and highlights similarities and differences between bacterial and eukaryotic Hsp90. Seminal research findings that elucidate the structure and the mechanisms of protein folding, disaggregation, and reactivation promoted by Hsp90 are discussed. Understanding the mechanisms of bacterial Hsp90 will provide fundamental insight into the more complex eukaryotic chaperone systems.

DnaK duplication and specialization in bacteria correlates with increased proteome complexity.

The chaperone 70 kDa heat shock protein (Hsp70) is important for cells from bacteria to humans to maintain proteostasis, and all eukaryotes and several prokaryotes encode Hsp70 paralogs. Although the mechanisms of Hsp70 function have been clearly illuminated, the function and evolution of Hsp70 paralogs is not well studied. DnaK is a highly conserved bacterial Hsp70 family. Here, we show that dnaK is present in 98.9% of bacterial genomes, and 6.4% of them possess two or more DnaK paralogs. We found that the duplication of dnaK is positively correlated with an increase in proteomic complexity (proteome size, number of domains). We identified the interactomes of the two DnaK paralogs of Myxococcus xanthus DK1622 (MxDnaKs), which revealed that they are mostly nonoverlapping, although both prefer α and ß domain proteins. Consistent with the entire M. xanthus proteome, MxDnaK substrates have both significantly more multi-domain proteins and a higher isoelectric point than that of Escherichia coli, which encodes a single DnaK homolog. MxDnaK1 is transcriptionally upregulated in response to heat shock and prefers to bind cytosolic proteins, while MxDnaK2 is downregulated by heat shock and is more associated with membrane proteins. Using domain swapping, we show that the nucleotide-binding domain and the substrate-binding ß domain are responsible for the significant differences in DnaK interactomes, and the nucleotide binding domain also determines the dimerization of MxDnaK2, but not MxDnaK1. Our work suggests that bacterial DnaK has been duplicated in order to deal with a more complex proteome, and that this allows evolution of distinct domains to deal with different subsets of target proteins.IMPORTANCEAll eukaryotic and ~40% of prokaryotic species encode multiple 70 kDa heat shock protein (Hsp70) homologs with similar but diversified functions. Here, we show that duplication of canonical Hsp70 (DnaK in prokaryotes) correlates with increasing proteomic complexity and evolution of particular regions of the protein. Using the Myxococcus xanthus DnaK duplicates as a case, we found that their substrate spectrums are mostly nonoverlapping, and are both consistent to that of Escherichia coli DnaK in structural and molecular characteristics, but show differential enrichment of membrane proteins. Domain/region swapping demonstrated that the nucleotide-binding domain and the ß substrate-binding domain (SBDß), but not the SBDα or disordered C-terminal tail region, are responsible for this functional divergence. This work provides the first direct evidence for regional evolution of DnaK paralogs.

Mycoplasma DnaK expression increases cancer development in vivo upon DNA damage.

Well-controlled repair mechanisms are involved in the maintenance of genomic stability, and their failure can precipitate DNA abnormalities and elevate tumor risk. In addition, the tumor microenvironment, enriched with factors inducing oxidative stress and affecting cell cycle checkpoints, intensifies DNA damage when repair pathways falter. Recent research has unveiled associations between certain bacteria, including Mycoplasmas, and various cancers, and the causative mechanism(s) are under active investigation. We previously showed that Mycoplasma fermentans DnaK, an HSP70 family chaperone protein, hampers the activity of proteins like PARP1 and p53, crucial for genomic integrity. Moreover, our analysis of its interactome in human cancer cell lines revealed DnaK's engagement with several components of DNA-repair machinery. Finally, in vivo experiments performed in our laboratory using a DnaK knock-in mouse model generated by our group demonstrated that DnaK exposure led to increased DNA copy number variants, indicative of genomic instability. We present here evidence that expression of DnaK is linked to increased i) incidence of tumors in vivo upon exposure to urethane, a DNA damaging agent; ii) spontaneous DNA damage ex vivo; and iii) expression of proinflammatory cytokines ex vivo, variations in reactive oxygen species levels, and increased ß-galactosidase activity across tissues. Moreover, DnaK was associated with increased centromeric instability. Overall, these findings highlight the significance of Mycoplasma DnaK in the etiology of cancer and other genetic disorders providing a promising target for prevention, diagnostics, and therapeutics.

Bacterial heat shock protein genes during induction chemotherapy in pediatric patients with acute lymphoblastic leukemia.

Background: Heat shock proteins (HSP) protect cancer cells. Gastrointestinal bacteria contain HSP genes and can release extracellular vesicles which act as biological shuttles. Stress from treatment may result in a microbial community with more HSP genes, which could contribute to circulating HSP levels. Methods: The authors examined the abundance of five bacterial HSP genes pre-treatment and during induction in stool sequences from 30 pediatric acute lymphoblastic leukemia patients. Results: Decreased mean HTPG counts (p = 0.0024) pre-treatment versus induction were observed. During induction, HTPG, Shannon diversity and Bacteroidetes decreased (p = 7.5e-4; 1.1e-3; 8.6e-4), while DNAK and Firmicutes increased (p = 6.9e-3; 9.2e-4). Conclusion: Understanding microbial HSP gene community changes with treatment is the first step in determining if bacterial HSPs are important to the tumor microenvironment and leukemia treatment.

SMR peptide antagonizes Staphylococcus aureus biofilm formation.

The emergence and international dissemination of multi-drug resistant Staphylococcus aureus (S. aureus) strains challenge current antibiotic-based therapies, representing an urgent threat to public health worldwide. In the U.S. alone, S. aureus infections are responsible for 11,000 deaths and 500,000 hospitalizations annually. Biofilm formation is a major contributor to antibiotic tolerance and resistance-induced delays in empirical therapy with increased infection severity, frequency, treatment failure, and mortality. Developing novel treatment strategies to prevent and disrupt biofilm formation is imperative. In this article, we test the Secretion Modification Region (SMR) peptides for inhibitory effects on resistant S. aureus biofilm-forming capacity by targeting the molecular chaperone DnaK. The dose effect of SMR peptides on biofilm formation was assessed using microtiter plate methods and confocal microscopy. Interaction between the antagonist and DnaK was determined by immune precipitation with anti-Flag M2 Affinity and Western blot analysis. Increasing SMR peptide concentrations exhibited increasing blockade of S. aureus biofilm formation with significant inhibition found at 18 µM, 36 µM, and 72 µM. This work supports the potential therapeutic benefit of SMR peptides in reducing biofilm viability and could improve the susceptibility to antimicrobial agents.IMPORTANCEThe development of anti-biofilm agents is critical to restoring bacterial sensitivity, directly combating the evolution of resistance, and overall reducing the clinical burden related to pervasive biofilm-mediated infections. Thus, in this study, the SMR peptide, a novel small molecule derived from the HIV Nef protein, was preliminarily explored for anti-biofilm properties. The SMR peptide was shown to effectively target the molecular chaperone DnaK and inhibit biofilm formation in a dose-dependent manner. These results support further investigation into the mechanism of SMR peptide-mediated biofilm formation and inhibition to benefit rational drug design and the identification of therapeutic targets.

Tandem repeats of highly bioluminescent NanoLuc are refolded noncanonically by the Hsp70 machinery.

Chaperones are a large family of proteins crucial for maintaining cellular protein homeostasis. One such chaperone is the 70 kDa heat shock protein (Hsp70), which plays a crucial role in protein (re)folding, stability, functionality, and translocation. While the key events in the Hsp70 chaperone cycle are well established, a relatively small number of distinct substrates were repetitively investigated. This is despite Hsp70 engaging with a plethora of cellular proteins of various structural properties and folding pathways. Here we analyzed novel Hsp70 substrates, based on tandem repeats of NanoLuc (Nluc), a small and highly bioluminescent protein with unique structural characteristics. In previous mechanical unfolding and refolding studies, we have identified interesting misfolding propensities of these Nluc-based tandem repeats. In this study, we further investigate these properties through in vitro bulk experiments. Similar to monomeric Nluc, engineered Nluc dyads and triads proved to be highly bioluminescent. Using the bioluminescence signal as the proxy for their structural integrity, we determined that heat-denatured Nluc dyads and triads can be efficiently refolded by the E. coli Hsp70 chaperone system, which comprises DnaK, DnaJ, and GrpE. In contrast to previous studies with other substrates, we observed that Nluc repeats can be efficiently refolded by DnaK and DnaJ, even in the absence of GrpE co-chaperone. Taken together, our study offers a new powerful substrate for chaperone research and raises intriguing questions about the Hsp70 mechanisms, particularly in the context of structurally diverse proteins.

DnaK2 Mediates a Negative Feedback Regulation of the Heat Shock Responsive Hik2-Rre1 Two-Component System in the Cyanobacterium Synechococcus Elongatus PCC 7942.

The two-component system (TCS) is a conserved signal transduction module in bacteria. The Hik2-Rre1 system is responsible for transcriptional activation upon high-temperature shift as well as plastoquinone-related redox stress in the cyanobacterium Synechococcus elongatus PCC 7942. As heat-induced de novo protein synthesis was previously shown to be required to quench the heat-activated response, we investigated the underlying mechanism in this study. We found that the heat-inducible transcription activation was alleviated by the overexpression of dnaK2, which is an essential homolog of the highly conserved HSP70 chaperone and whose expression is induced under the control of the Hik2-Rre1 TCS. Phosphorylation of Rre1 correlated with transcription of the regulatory target hspA. The redox stress response was found to be similarly repressed by dnaK2 overexpression. Considered together with the previous information, we propose a negative feedback mechanism of the Hik2-Rre1-dependent stress response that maintains the cellular homeostasis mediated by DnaK2.

Insertion of GGMP repeat residues of Plasmodium falciparum Hsp70-1 in the lid of DnaK adversely impacts client recognition.

Although Hsp70 is a conserved molecular chaperone, it exhibits some degree of functional specialisation across species. Features of Hsp70 regulating its functional specialisation remain to be fully established. We previously demonstrated that E. coli Hsp70 (DnaK) exhibits functional features that distinguishes it from PfHsp70-1, a canonical cytosolic Hsp70 of Plasmodium falciparum. One of the defining features of PfHsp70-1 is that it possesses GGMP repeat residues located in its C-terminal lid segment, while DnaK lacks this motif. Previously, we demonstrated that the insertion of GGMP repeat residues of PfHsp70-1 into E. coli DnaK abrogates the chaperone activity of DnaK. However, the role of the GGMP motif in regulating Hsp70 function remains to be fully understood. To explore the function of this motif, we expressed recombinant forms of wild type DnaK and its GGMP insertion motif, DnaK-G and systematically characterised the structure-function features of the two proteins using in silico analysis, biophysical approaches and an in cellulo complementation assay. Our findings demonstrated that the GGMP inserted in DnaK compromised various functional features such as nucleotide binding, allostery, substrate binding affinity and cellular proteome client selectivity. These findings thus, highlight the GGMP motif of Hsp70 as an important functional module.

New insights into the structure and function of the complex between the Escherichia coli Hsp70, DnaK, and its nucleotide-exchange factor, GrpE.

The 70 kDa heat shock proteins (Hsp70s) play a pivotal role in many cellular functions using allosteric communication between their nucleotide-binding domain (NBD) and substrate-binding domain, mediated by an interdomain linker, to modulate their affinity for protein clients. Critical to modulation of the Hsp70 allosteric cycle, nucleotide-exchange factors (NEFs) act by a conserved mechanism involving binding to the ADP-bound NBD and opening of the nucleotide-binding cleft to accelerate the release of ADP and binding of ATP. The crystal structure of the complex between the NBD of the Escherichia coli Hsp70, DnaK, and its NEF, GrpE, was reported previously, but the GrpE in the complex carried a point mutation (G122D). Both the functional impact of this mutation and its location on the NEF led us to revisit the DnaK NBD/GrpE complex structurally using AlphaFold modeling and validation by solution methods that report on protein conformation and mutagenesis. This work resulted in a new model for the DnaK NBD in complex with GrpE in which subdomain IIB of the NBD rotates more than in the crystal structure, resulting in an open conformation of the nucleotide-binding cleft, which now resembles more closely what is seen in other Hsp/NEF complexes. Moreover, the new model is consistent with the increased ADP off-rate accompanying GrpE binding. Excitingly, our findings point to an interdomain allosteric signal in DnaK triggered by GrpE binding.

DnaK promotes autophosphorylation of DYRK1A and its family kinases in Escherichia coli-based cell-free protein expression.

Dual-specificity tyrosine phosphorylation-regulated kinase 1A (DYRK1A) is one of the drug target kinases involved in neurological disorders. DYRK1A phosphorylates substrate proteins related to disease progression in an intermolecular manner. Meanwhile, DYRK1A intramolecularly phosphorylates its own residues on key segments during folding process, which is required for its activation and stabilization. To reproduce the autophosphorylation in vitro, DYRK1A was expressed in Escherichia coli-based cell-free protein synthesis system. Although this system was useful for investigating autophosphorylation of serine residue at position 97 (Ser97) in DYRK1A, only a small fraction of the synthesized protein was successfully autophosphorylated. In this study, we found that the addition of DnaK, a bacterial HSP70 chaperone, to cell-free expression of DYRK1A promoted its Ser97 autophosphorylation. Structure prediction with AlphaFold2 indicates that Ser97 forms a hydrogen bond within an α-helix structure, indicating a possibility that DnaK unfolds the α-helix and maintains the structure around Ser97 in a conformation susceptible to phosphorylation. In addition, DnaK promoted phosphorylation of DYRK1B and HIPK2, but not DYRK2 and DYRK4, suggesting a sequence selectivity in the action of DnaK. This study provides a facile method for promoting autophosphorylation of DYRK family kinases in cell-free protein expression.

WGS-based screening of the co-chaperone protein DjlA-induced curved DNA binding protein A (CbpA) from a new multidrug-resistant zoonotic mastitis-causing Klebsiella pneumoniae strain: a novel molecular target of selective flavonoids.

The research aimed to establish a multidrug-resistant Klebsiella pneumoniae-induced genetic model for mastitis considering the alternative mechanisms of the DjlA-mediated CbpA protein regulation. The Whole Genome Sequencing of the newly isolated K. pneumoniae strain was conducted to annotate the frequently occurring antibiotic resistance and virulence factors following PCR and MALDI-TOF mass-spectrophotometry. Co-chaperon DjlA was identified and extracted via restriction digestion on PAGE. Based on the molecular string property analysis of different DnaJ and DnaK type genes, CbpA was identified to be regulated most by the DjlA protein during mastitis. Based on the quantum tunnel-cluster profiles, CbpA was modeled as a novel target for diversified biosynthetic, and chemosynthetic compounds. Pharmacokinetic and pharmacodynamic analyses were conducted to determine the maximal point-specificity of selective flavonoids in complexing with the CbpA macromolecule at molecular docking. The molecular dynamic simulation (100 ns) of each of the flavonoid-protein complexes was studied regarding the parameters RMSD, RMSF, Rg, SASA, MMGBSA, and intramolecular hydrogen bonds; where all of them resulted significantly. To ratify all the molecular dynamic simulation outputs, the potential stability of the flavonoids in complexing with CbpA can be remarked as Quercetin > Biochanin A > Kaempherol > Myricetin, which were all significant in comparison to the control Galangin. Finally, a comprehensive drug-gene interaction pathway for each of the flavonoids was developed to determine the simultaneous and quantitative-synergistic effects of different operons belonging to the DnaJ-type proteins on the metabolism of the tested pharmacophores in CbpA. Considering all the in vitro and in silico parameters, DjlA-mediated CbpA can be a novel target for the tested flavonoids as the potential therapeutics of mastitis as futuristic drugs.

Immunogenicity and Protection by DnaK and SpaA Recombinant Proteins Against Erysipelothrix Rhusiopathiae in a Murine Model.

BACKGROUND/AIMS: Swine erysipelas is a disease caused by Erysipelothrix rhusiopathiae, a Gram-positive bacillus, which has great economic importance because it leads to the loss of the swine herd. To control this disease, animals are immunized with a cellular vaccine of killed or attenuated E. rhusiopathiae, but even with herd vaccination, cases of swine erysipelas outbreaks have been reported in the United States, China and Japan, leading to the search for other antigenic components of the bacteria that may promote greater protection against E. rhusiopathiae. The surface protein SpaA from E. rhusiopathiae has been shown to be a candidate to constitute a subunit vaccine, since it has already been reported to induce a host immune response against the bacterium. DnaK, a hsp70 molecular chaperone, also seems to be a good candidate in the composition of a vaccine, as it has been demonstrated to be an antigenic protein of the bacteria. METHODS: This work evaluated the immunogenicity and protection induced by the E. rhusiopathiaee SpaA and DnaK recombinant proteins in a murine model, by intramuscular administration to mice with two doses of 100 µg at 21-day interval between them. The candidate proteins were tested either separately and together, compared with the commercial vaccine and the non-vaccination condition, and mice were challenged with a virulent strain of E. rhusiopathiae. Serum was collected to assess the produced antibodies and peripheral blood cells, whereas spleen and kidney tissues were assayed for E. rhusiopathiae presence by colony counting. RESULTS: A survival curve of the animals was performed, which confirmed the protection induced by the proteins. IgG antibodies increased in the animal serum inoculated with the proteins when compared to the control, and a significant delay in disease symptoms was observed. CONCLUSION: These results suggest that E. rhusiopathiae DnaK and SpaA are immunogenic in mice and interfere with the disease development.

Impact of DnaK on protein expression and deamidation modification in Cronobacter sakazakii: insights from proteomic analysis.

This study explores the influence of DnaK on intracellular proteins in Cronobacter sakazakii, a Gram-negative bacterium known for causing infections, particularly in neonates. Proteomic and post-translational modification analyses were performed, revealing a potential link between DnaK and protein deamidation.

Down-Regulation of HSP by Pd-Cu Nanozymes for NIR Light Triggered Mild-Temperature Photothermal Therapy Against Wound Bacterial Infection: In vitro and in vivo Assessments.

Purpose: We aimed to develop an oxidative-stress-activated palladium-copper nanozyme to reduce bacterial's heat sensitivity by down-regulating heat shock proteins to overcome the shortcomings of conventional photothermal antimicrobial therapy and achieve mild photothermal bactericidal efficacy. Methods: We first synthesized palladium-copper nanozymes (PC-NPs) by hydration and used transmission electron microscopy, X-ray diffraction, and Fourier transform infrared spectroscopy to demonstrate their successful preparation. Their photothermal therapy (PTT) and chemo-dynamic therapy (CDT) activities were then determined by a series of photothermal performance tests and peroxidase-like performance tests, and the destruction of heat shock proteins by reactive oxygen species (ROS) was verified at the protein level by Western Blotting tests, providing a basis for the effective bacteria-killing by the mild-temperature photothermal treatment subsequently applied. We also validated this promising programmed and controlled antimicrobial treatment with palladium-copper nanozymes by in vivo/in vitro antimicrobial assays. A hemolysis assay, MTT cytotoxicity test and histopathological analysis were also performed to assess the in vivo safety of PC-NPs. Results: In the micro-acidic environment of bacterial infection, PC-NPs showed peroxidase-like activity that broke down the H2O2 at the wound into hydroxyl radicals and down-regulated bacterial heat shock proteins. The application of PC-NPs increased bacteria's sensitivity to subsequent photothermal treatment, enabling the elimination of bacteria via mild photothermal treatment. Conclusion: The programmed synergistic catalytic enhancement of CDT and mild photothermal therapy achieves the most efficient killing of bacteria and their biofilms, which brings future thinking in the relationship between heat shock proteins and oxidative stress damage in bacteria.