Search

GroEL, a chaperone protein responsible for peptide and denatured protein folding, undergoes substantial conformational changes driven by ATP binding and hydrolysis during folding. Utilizing these conformational changes, we demonstrated the GroEL-mediated regioselective photocyclodimerization of 2-anthracenecarboxylic acid (AC) using ATP hydrolysis as an external stimulus. We designed and prepared an optimal GroEL mutant to employ in a docking simulation that has been actively used in recent years. Based on the large difference in the motif of hydrogen bonds between AC and GroEL mutant compared with the wild-type, we predicted that GroELMEL, in which the 307‒309th amino acid residues were mutated to Ala, could alter the orientation of bound AC in GroEL. The GroELMEL-mediated photocyclodimerization of AC can be used for regioselective inversion upon ATP addition to a moderate extent. [Display omitted] [ABSTRACT FROM AUTHOR]

The Escherichia coli GroEL/ES chaperonin system facilitates protein folding in an ATP‐driven manner. There are <100 obligate clients of this system in E. coli although GroEL can interact and assist the folding of a multitude of proteins in vitro. It has remained unclear, however, which features distinguish obligate clients from all the other proteins in an E. coli cell. To address this question, we established a system for selecting mutations in mouse dihydrofolate reductase (mDHFR), a GroEL interactor, that diminish its dependence on GroEL for folding. Strikingly, both synonymous and non‐synonymous codon substitutions were found to reduce mDHFR's dependence on GroEL. The non‐synonymous substitutions increase the rate of spontaneous folding whereas computational analysis indicates that the synonymous substitutions appear to affect translation rates at specific sites. [ABSTRACT FROM AUTHOR]

Chaperonins from psychrophilic bacteria have been shown to exist as single-ring complexes. This deviation from the standard double-ring structure has been thought to be a beneficial adaptation to the cold environment. Here we show that Cpn60 from the psychrophile Pseudoalteromonas haloplanktis (Ph) maintains its double-ring structure also in the cold. A strongly reduced ATPase activity keeps the chaperonin in an energy-saving dormant state, until binding of client protein activates it. Ph Cpn60 in complex with co-chaperonin Ph Cpn10 efficiently assists in protein folding up to 55 °C. Moreover, we show that recombinant expression of Ph Cpn60 can provide its host Escherichia coli with improved viability under low temperature growth conditions. These properties of the Ph chaperonin may make it a valuable tool in the folding and stabilization of psychrophilic proteins. [ABSTRACT FROM AUTHOR]

Whole genome sequencing (WGS) of Mycobacterium avium complex (MAC) isolates in the clinical laboratory setting allows for rapid and reliable subspecies identification of a closely related complex of human pathogens. We developed a bioinformatics pipeline for accurate subspecies identification and tested 74 clinical MAC isolates from various anatomical sites. We demonstrate that reliable subspecies level identification of these common and clinically significant MAC isolates, including M. avium subsp. hominissuis (most dominant in causing lower respiratory tract infections in our cohort), M. avium subsp. avium, M. intracellulare subsp. intracellulare, and M. intracellulare subsp. chimaera, can be achieved by analysis of only two marker genes (rpoB and groEL/hsp65). We then explored the relationship between these subspecies and anatomical site of infection. Further, we conducted an in silico analysis and showed our algorithm also performed well for M. avium subsp. paratuberculosis but failed to consistently identify M. avium subsp. silvaticum and M. intracellulare subsp. yongonense, likely due to a lack of available reference genome sequences; all the 3 subspecies were not found in our clinical isolates and rarely reported to cause human infections. Accurate MAC subspecies identification may provide the tool and opportunity for better understanding of the disease-subspecies dynamics in MAC infections.

In this study, the lactic acid bacteria from the feces of Uygur mother-infant pairs in Hotan, Xinjiang, was isolated and identified. The bacteriostatic performance of the isolates for diarrhea pathogenic bacteria was assessed, and the probiotic properties were investigated in vitro. According to the groEL gene sequencing, 113 isolates of lactic acid bacteria representing 15 species were isolated from 19 mother-infant pairs, with Ligilactobacillus salivarius and Companilactobacillus farciminis being the dominant species, and 29 strains had inhibition effect on all the 6 pathogenic bacteria were screened from all strains. Among the strains with antibacterial activity, Companilactobacillus farciminis HTb36X-2 had the highest survival rate of 69.50% after being treated with simulated gastrointestinal juices for 4 h. Limosilactobacillus reuteri HTb34X5-6 and HTb34X5-1 had the highest auto-aggregation and hydrophobicity, with the hydrophobicity of 31.67% and 29.00%, and auto-aggregation of 81.32% and 70.91%, respectively. Combined with antibiotic resistance and carbohydrate metabolism of all 29 strains, a total of 7 strains of Companilactobacillus farciminis HTb36X-2, Lacticaseibacillus paracasei HTb6X-6, Lacticaseibacillus casei 17X-4, 17X-5, Limosilactobacillus reuteri HTb34X-5-1, HTb34X-5-6, Limosilactobacillus fermentum 10D12-2 were screened as potential probiotics, which might take a foundation for the development of probiotic and products with anti-diarrhea function.

We found that GroEL in Porphyromonas gingivalis accelerated tumor growth and increased mortality in tumor‐bearing mice; GroEL promoted proangiogenic function, which may be the reason for promoting tumor growth. To understand the regulatory mechanisms by which GroEL increases the proangiogenic function of endothelial progenitor cells (EPCs), we explored in this study. In EPCs, MTT assay, wound‐healing assay, and tube formation assay were performed to analyze its activity. Western blot and immunoprecipitation were used to study the protein expression along with next‐generation sequencing for miRNA expression. Finally, a murine tumorigenesis animal model was used to confirm the results of in vitro. The results indicated that thrombomodulin (TM) direct interacts with PI3K/Akt to inhibit the activation of signaling pathways. When the expression of TM is decreased by GroEL stimulation, molecules in the PI3K/Akt signaling axis are released and activated, resulting in increased migration and tube formation of EPCs. In addition, GroEL inhibits TM mRNA expression by activating miR‐1248, miR‐1291, and miR‐5701. Losing the functions of miR‐1248, miR‐1291, and miR‐5701 can effectively alleviate the GroEL‐induced decrease in TM protein levels and inhibit the proangiogenic abilities of EPCs. These results were also confirmed in animal experiments. In conclusion, the intracellular domain of the TM of EPCs plays a negative regulatory role in the proangiogenic capabilities of EPCs, mainly through direct interaction between TM and PI3K/Akt to inhibit the activation of signaling pathways. The effects of GroEL on tumor growth can be reduced by inhibiting the proangiogenic properties of EPCs through the inhibition of the expression of specific miRNAs. [ABSTRACT FROM AUTHOR]

Protein complexes are essential for proteins' folding and biological function. Currently, native analysis of large multimeric protein complexes remains challenging. Structural biology techniques are time‐consuming and often cannot monitor the proteins' dynamics in solution. Here, a capillary electrophoresis‐mass spectrometry (CE–MS) method is reported to characterize, under near‐physiological conditions, the conformational rearrangements of ∽1 MDa GroEL upon complexation with binding partners involved in a protein folding cycle. The developed CE–MS method is fast (30 min per run), highly sensitive (low‐amol level), and requires ∽10 000‐fold fewer samples compared to biochemical/biophysical techniques. The method successfully separates GroEL14 (∽800 kDa), GroEL7 (∽400 kDa), GroES7 (∽73 kDa), and NanA4 (∽130 kDa) oligomers. The non‐covalent binding of natural substrate proteins with GroEL14 can be detected and quantified. The technique allows monitoring of GroEL14 conformational changes upon complexation with (ATPγS)4–14 and GroES7 (∽876 kDa). Native CE‐pseudo‐MS3 analyses of wild‐type (WT) GroEL and two GroEL mutants result in up to 60% sequence coverage and highlight subtle structural differences between WT and mutated GroEL. The presented results demonstrate the superior CE–MS performance for multimeric complexes' characterization versus direct infusion ESI–MS. This study shows the CE–MS potential to provide information on binding stoichiometry and kinetics for various protein complexes. [ABSTRACT FROM AUTHOR]

Simple Summary: Camel anaplasmosis is a recent emerging disease with potential zoonotic concerns. There is poor understanding of the epidemiology of anaplasmosis in camels and, in particular of Candidatus Anaplasma camelii, which is detected in several countries including Saudi Arabia, Iran, Kenya, and Morocco. Most studies of anaplasmosis in camels relied on microscopy and serology for diagnosis, and few used molecular approaches. The present work characterizes Anaplasmataceae strains circulating in the Camelus dromedarius reservoir in the United Arab Emirates (UAE) using PCR, sequencing, and phylogenetic analysis for the first time to provide information about the largely neglected disease they cause. Between 2019 to 2023, thirty-five whole-blood samples (35/287 = 12.2%) tested positive for Anaplasmataceae spp. by PCR assay targeting the groEL gene. Of these, only nine positive samples (9/35 = 25.7%) were sequenced using groEL gene primers. A GenBank BLAST analysis revealed that all strains were 100% identical to the Candidatus Anaplasma camelii reference sequence available in the GenBank nucleotide database. The recent emergence of anaplasmosis in camels has raised global interest in the pathogenicity and zoonotic potential of the pathogen causing it and the role of camels as reservoir hosts. In the United Arab Emirates (UAE), molecular studies and genetic characterization of camel-associated Anaplasma species are limited. This study aimed to characterize molecularly Anaplasmataceae strains circulating in dromedary camels in the UAE. Two hundred eighty-seven whole-blood samples collected from dromedary camels across regions of the Abu Dhabi Emirate were received between 2019 and 2023 at the Abu Dhabi Agriculture and Food Safety Authority (ADAFSA) veterinary laboratories for routine diagnosis of anaplasmosis. The animals were sampled based on field clinical observation by veterinarians and their tentative suspicion of blood parasite infection on the basis of similar clinical symptoms as those caused by blood parasites in ruminants. The samples were screened for Anaplasmataceae by PCR assay targeting the groEL gene. Anaplasmataceae strains were further characterized by sequencing and phylogenetic analysis of the groEL gene. Thirty-five samples (35/287 = 12.2%) tested positive for Anaplasmataceae spp. by PCR assay. Nine positive samples (9/35 = 25.7%) were sequenced using groEL gene primers. GenBank BLAST analysis revealed that all strains were 100% identical to the Candidatus A. camelii reference sequence available in the GenBank nucleotide database. Phylogenetic analysis further indicated that the sequences were close to each other and were located in one cluster with Candidatus A. camelii sequences detected in Saudi Arabia, Morocco, and the UAE. Pairwise alignment showed that the UAE sequences detected in this study were completely identical and shared 100% identity with Candidatus A. camelii from Morocco and Saudi Arabia and 99.5% identity with Candidatus A. camelii from the UAE. This study demonstrates the presence of Candidatus A. camelii in UAE dromedary camels. Further critical investigation of the clinical and economical significance of this pathogen in camels needs to be carried out. [ABSTRACT FROM AUTHOR]

This study was conducted to isolate and identify Bifidobacterium from the feces of Kazakh school-age children in Yining, Xinjiang and evaluate the in vitro probiotic characteristics of B. longum subsp. longum isolates. By groEL gene sequencing and repetitive element sequence-based polymerase chain reaction (rep-PCR) fingerprinting, 416 Bifidobacterium strains were identified to belong to B. longum, B. bifidum, B. pseudocatenulatum, B. catenulatum and B. breve. According to the fingerprints of B. longum subsp. longum, 27 genotypes showed genetic differences between individual strains and the coexistence of multiple strains in the gut was found. The in vitro experimental results showed that out of 27 representative strains, strains 2B3-21, 1B23-11, 2B33-3, and 1B68-16 were optimal in acid and bile salt tolerance, strains 1B68-16, 2B13-5, 2B33-3, and 1B39-2 had broad-spectrum antibacterial properties, and strains 1B38-1, 2B33-3, 1B68-16, and 2B13-28 showed a strong antioxidant capacity. Considering the antibiotic resistance of all strains and their ability to utilize plant-derived glycans, strains 1B38-1 and 2B13-28 were selected to assess their in vivo probiotic potentials. This study may lay the foundation for the development of excellent probiotics and related products for populations from specific areas.

Simple Summary: Bacteria continuously circulate in the natural environment and frequently cross into the human environment. While some of these bacteria can have a positive effect on human health, they are often accompanied by pathogenic forms. The aim of the present study was to more closely examine the zoonotic, tick-borne bacterium Anaplasma phagocytophilum, the causative agent of human and animal anaplasmosis. The study examined tissue samples from roe deer and red deer. Anaplasma phagocytophilum samples were genotyped based on DNA amplification, sequencing and phylogenetic analysis. Our results showed that detected bacteria from red deer belong to ecotype I and cluster I, which is why those bacteria are potentially pathogenic to humans. Detected bacteria from roe deer were mainly in ecotype II and clusters II and III, which means those bacteria are not pathogenic for humans. Background: The Gram-negative bacterium Anaplasma phagocytophilum is an intracellular pathogen and an etiological agent of human and animal anaplasmosis. Its natural reservoir comprises free-ranging ungulates, including roe deer (Capreolus capreolus) and red deer (Cervus elaphus). These two species of deer also constitute the largest group of game animals in Poland. The aim of the study was to genotype and perform a phylogenetic analysis of A. phagocytophilum strains from roe deer and red deer. Methods: Samples were subjected to PCR amplification, sequencing, and phylogenetic analysis of strain-specific genetic markers (groEL, ankA). Results: Five haplotypes of the groEL gene from A. phagocytophilum and seven haplotypes of ankA were obtained. The phylogenetic analysis classified the groEL into ecotypes I and II. Sequences of the ankA gene were classified into clusters I, II, and III. Conclusions: Strains of A. phagocytophilum from red deer were in the same ecotype and cluster as strains isolated from humans. Strains of A. phagocytophilum from roe deer represented ecotypes (I, II) and clusters (II, III) that were different from those isolated from red deer, and these strains did not show similarity to bacteria from humans. However, roe deer can harbor nonspecific strains of A. phagocytophilum more characteristic to red deer. It appears that the genetic variants from red deer can be pathogenic to humans, but the significance of the variants from roe deer requires more study. [ABSTRACT FROM AUTHOR]

Copyright of Shipin Kexue/ Food Science is the property of Food Science Editorial Department and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

Abstract Protein complexes are essential for proteins' folding and biological function. Currently, native analysis of large multimeric protein complexes remains challenging. Structural biology techniques are time‐consuming and often cannot monitor the proteins' dynamics in solution. Here, a capillary electrophoresis‐mass spectrometry (CE–MS) method is reported to characterize, under near‐physiological conditions, the conformational rearrangements of ∽1 MDa GroEL upon complexation with binding partners involved in a protein folding cycle. The developed CE–MS method is fast (30 min per run), highly sensitive (low‐amol level), and requires ∽10 000‐fold fewer samples compared to biochemical/biophysical techniques. The method successfully separates GroEL14 (∽800 kDa), GroEL7 (∽400 kDa), GroES7 (∽73 kDa), and NanA4 (∽130 kDa) oligomers. The non‐covalent binding of natural substrate proteins with GroEL14 can be detected and quantified. The technique allows monitoring of GroEL14 conformational changes upon complexation with (ATPγS)4–14 and GroES7 (∽876 kDa). Native CE‐pseudo‐MS3 analyses of wild‐type (WT) GroEL and two GroEL mutants result in up to 60% sequence coverage and highlight subtle structural differences between WT and mutated GroEL. The presented results demonstrate the superior CE–MS performance for multimeric complexes' characterization versus direct infusion ESI–MS. This study shows the CE–MS potential to provide information on binding stoichiometry and kinetics for various protein complexes.

Background: Anaplasma phagocytophilum is a Gram-negative obligate intracellular bacterium that replicates in neutrophil granulocytes. It is transmitted by ticks of the Ixodes ricinus complex and causes febrile illness called granulocytic anaplasmosis primarily in humans, horses, dogs, sheep, cattle and goats. In comparison, clinically apparent disease has been described rarely in cats especially compared to dogs and horses. It is currently unknown whether cats are less susceptible to A. phagocytophilum or whether granulocytic anaplasmosis might be underdiagnosed in cats. Methods: To address this question, we examined clinical signs and laboratory findings in seven A. phagocytophilum infected cats from Germany and Switzerland. We then genetically characterized feline A. phagocytophilum strains and compared them to those from other hosts showing clinically apparent disease. For this purpose, ankA-based, groEL-based and multilocus sequence typing (MLST) were applied. Furthermore, the concordance between these typing methods was assessed. Results: Fever, lethargy and anorexia were the most common clinical signs in cats suffering from granulocytic anaplasmosis. The most frequent laboratory finding was thrombocytopenia. All three typing methods consistently indicated that the A. phagocytophilum strains found infecting cats are the same as those that cause disease in humans, dogs and horses. In general, the three typing methods applied exhibited high concordance. Conclusions: The genetic characterization of the feline A. phagocytophilum strains indicates that strain divergence is not the explanation for the fact that granulocytic anaplasmosis is much less frequently diagnosed in cats than in dogs and horses. Otherwise, it may be possible that cats are less susceptible to the same strains than dogs and horse are. However, due to the unspecific clinical signs, it should be considered that granulocytic anaplasmosis may be under-diagnosed in cats. [ABSTRACT FROM AUTHOR]

GroEL is a chaperonin that helps other proteins fold correctly. However, alternative activities, such as acting as an insect toxin, have also been discovered. This work evaluates the chaperonin and insecticidal activity of different GroEL proteins from entomopathogenic nematodes on G. mellonella. The ability to synergize with the ExoA toxin of Pseudomonas aeruginosa was also investigated. The GroELXn protein showed the highest insecticidal activity among the different GroELs. In addition, it was able to significantly activate the phenoloxidase system of the target insects. This could tell us about the mechanism by which it exerts its toxicity on insects. GroEL proteins can enhance the toxic activity of the ExoA toxin, which could be related to its chaperonin activity. However, there is a significant difference in the synergistic effect that is more related to its alternative activity as an insecticidal toxin. [ABSTRACT FROM AUTHOR]

Profiling bacterial populations in mixed communities is a common task in microbiology. Sequencing of 16S small subunit ribosomal-RNA (16S rRNA) gene amplicons is a widely accepted and functional approach but relies on amplification primers and cannot quantify isotope incorporation. Tandem mass spectrometry proteotyping is an effective alternative for taxonomically profiling microorganisms. We suggest that targeted proteotyping approaches can complement traditional population analyses. Therefore, we describe an approach to assess bacterial community compositions at the family level using the taxonomic marker protein GroEL, which is ubiquitously found in bacteria, except a few obligate intracellular species. We refer to our method as GroEL-proteotyping. GroEL-proteotyping is based on high-resolution tandem mass spectrometry of GroEL peptides and identification of GroEL-derived taxa via a Galaxy workflow and a subsequent Python-based analysis script. Its advantage is that it can be performed with a curated and extendable sample-independent database and that GroEL can be pre-separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) to reduce sample complexity, improving GroEL identification while simultaneously decreasing the instrument time. GroEL-proteotyping was validated by employing it on a comprehensive raw dataset obtained through a metaproteome approach from synthetic microbial communities as well as real human gut samples. Our data show that GroEL-proteotyping enables fast and straightforward profiling of highly abundant taxa in bacterial communities at reasonable taxonomic resolution. [ABSTRACT FROM AUTHOR]

The molecular chaperone GroEL of C. sakazakii, a highly conserved protein encoded by the gene grol, has the basic function of responding to heat shock, thus enhancing the bacterium's adaptation to dry and high-temperature environments, which poses a threat to food safety and human health. Our previous study demonstrated that GroEL was found in the bacterial membrane fraction and caused a strong immune response in C. sakazakii. In this study, we tried to elucidate the subcellular location and virulent effects of GroEL. In live C. sakazakii cells, GroEL existed in both the soluble and insoluble fractions. To study the secretory mechanism of GroEL protein, a non-reduced Western immunoblot was used to analyze the form of the protein, and the result showed that the exported GroEL protein was mainly in monomeric form. The exported GroEL could also be located on bacterial surface. To further research the virulent effect of C. sakazakii GroEL, an indirect immunofluorescence assay was used to detect the adhesion of recombinant GroEL protein to HCT-8 cells. The results indicated that the recombinant GroEL protein could adhere to HCT-8 cells in a short period of time. The recombinant GroEL protein could activate the NF-κB signaling pathway to release more pro-inflammatory cytokines (TNF-α, IL-6 and IL-8), downregulating the expression of tight-junction proteins (claudin-1, occluding, ZO-1 and ZO-2), which collectively resulted in dose-dependent virulent effects on host cells. Inhibition of the grol gene expression resulted in a significant decrease in bacterial adhesion to and invasion of HCT-8 cells. Moreover, the deficient GroEL also caused slow growth, decreased biofilm formation, defective motility and abnormal filamentation of the bacteria. In brief, C. sakazakii GroEL was an important virulence factor. This protein was not only crucial for the physiological activity of C. sakazakii but could also be secreted to enhance the bacterium's adhesion and invasion capabilities. [ABSTRACT FROM AUTHOR]

Dietary shifts can have a direct impact on the gut microbiome by preferentially selecting for microbes capable of utilizing the various dietary nutrients. The intake of dietary fiber has decreased precipitously in the last century, while consumption of processed foods has increased. Fiber, or microbiota-accessible carbohydrates (MACs), persist in the digestive tract and can be metabolized by specific bacteria encoding fiber-degrading enzymes. The digestion of MACs results in the accumulation of short-chain fatty acids (SCFAs) and other metabolic by-products that are critical to human health. Here, we implemented a 2-week dietary fiber intervention aiming for 40 to 50 g of fiber per day within the context of a course-based undergraduate research experience (CURE) (n = 20). By coupling shotgun metagenomic sequencing and targeted gas chromatography-mass spectrometry (GC-MS), we found that the dietary intervention significantly altered the composition of individual gut microbiomes, accounting for 8.3% of the longitudinal variability within subjects. Notably, microbial taxa that increased in relative abundance as a result of the diet change included known MAC degraders (i.e., Bifidobacterium and Lactobacillus). We further assessed the genetic diversity within Bifidobacterium, assayed by amplification of the groEL gene. Concomitant with microbial composition changes, we show an increase in the abundance of genes involved in inositol degradation. Despite these changes in gut microbiome composition, we did not detect a consistent shift in SCFA abundance. Collectively, our results demonstrate that on a short-term timescale of 2 weeks, increased fiber intake can induce compositional changes of the gut microbiome, including an increase in MAC-degrading bacteria.IMPORTANCE A profound decrease in the consumption of dietary fiber in many parts of the world in the last century may be associated with the increasing prevalence of type II diabetes, colon cancer, and other health problems. A typical U.S. diet includes about ∼15 g of fiber per day, far less fiber than the daily recommended allowance. Changes in dietary fiber intake affect human health not only through the uptake of nutrients directly but also indirectly through changes in the microbial community and their associated metabolism. Here, we conducted a 2-week diet intervention in healthy young adults to investigate the impact of fiber consumption on the gut microbiome. Participants increased their average fiber consumption by 25 g/day on average for 2 weeks. The high-fiber diet intervention altered the gut microbiome of the study participants, including increases in known fiber-degrading microbes, such as Bifidobacterium and Lactobacillus.

The recent emergence of anaplasmosis in camels has raised global interest in the pathogenicity and zoonotic potential of the pathogen causing it and the role of camels as reservoir hosts. In the United Arab Emirates (UAE), molecular studies and genetic characterization of camel-associated Anaplasma species are limited. This study aimed to characterize molecularly Anaplasmataceae strains circulating in dromedary camels in the UAE. Two hundred eighty-seven whole-blood samples collected from dromedary camels across regions of the Abu Dhabi Emirate were received between 2019 and 2023 at the Abu Dhabi Agriculture and Food Safety Authority (ADAFSA) veterinary laboratories for routine diagnosis of anaplasmosis. The animals were sampled based on field clinical observation by veterinarians and their tentative suspicion of blood parasite infection on the basis of similar clinical symptoms as those caused by blood parasites in ruminants. The samples were screened for Anaplasmataceae by PCR assay targeting the groEL gene. Anaplasmataceae strains were further characterized by sequencing and phylogenetic analysis of the groEL gene. Thirty-five samples (35/287 = 12.2%) tested positive for Anaplasmataceae spp. by PCR assay. Nine positive samples (9/35 = 25.7%) were sequenced using groEL gene primers. GenBank BLAST analysis revealed that all strains were 100% identical to the Candidatus A. camelii reference sequence available in the GenBank nucleotide database. Phylogenetic analysis further indicated that the sequences were close to each other and were located in one cluster with Candidatus A. camelii sequences detected in Saudi Arabia, Morocco, and the UAE. Pairwise alignment showed that the UAE sequences detected in this study were completely identical and shared 100% identity with Candidatus A. camelii from Morocco and Saudi Arabia and 99.5% identity with Candidatus A. camelii from the UAE. This study demonstrates the presence of Candidatus A. camelii in UAE dromedary camels. Further critical investigation of the clinical and economical significance of this pathogen in camels needs to be carried out.

Tick-borne Coxiella spp. are emerging in novel regions infecting different hosts, but information regarding their occurrence is limited. The purpose of this study was the molecular screening of Coxiella spp. in various ticks infesting goats, sheep, camels, cattle, wild mice, and domestic fowls (Gallus gallus domesticus) in various districts of Khyber Pakhtunkhwa, Pakistan. Morphologically identified tick species were confirmed by obtaining their cox1 sequences and were molecularly screened for Coxiella spp. by sequencing GroEL fragments. Almost 345 out of 678 (50.9%) hosts were infested by nine tick species. Regarding the age groups, the hosts having an age >3 years were highly infested (192/345, 55.6%), while gender-wise infestation was higher in female hosts (237/345, 68.7%). In collected ticks, the nymphs were outnumbered (613/1,119, 54.8%), followed by adult females (293/1,119, 26.2%) and males (213/1,119, 19.7%). A total of 227 ticks were processed for molecular identification and detection of Coxiella spp. The obtained cox1 sequences of nine tick species such as Hyalomma dromedarii, Hyalomma anatolicum, Haemaphysalis cornupunctata, Haemaphysalis bispinosa, Haemaphysalis danieli, Haemaphysalis montgomeryi, Rhipicephalus haemaphysaloides, Rhipicephalus microplus, and Argas persicus showed maximum identities between 99.6% and 100% with the same species and in the phylogenetic tree, clustered to the corresponding species. All the tick species except Ha. danieli and R. microplus were found positive for Coxiella spp. (40/227, 17.6%), including Coxiella burnetii (15/40, 6.7%), Coxiella endosymbionts (14/40, 6.3%), and different Coxiella spp. (11/40, 4.9%). By the BLAST results, the GroEL fragments of Coxiella spp. showed maximum identity to C. burnetii, Coxiella endosymbionts, and Coxiella sp., and phylogenetically clustered to the corresponding species. This is the first comprehensive report regarding the genetic characterization of Coxiella spp. in Pakistan’s ticks infesting domestic and wild hosts. Proper surveillance and management measures should be undertaken to avoid health risks. [ABSTRACT FROM AUTHOR]

Herein, we report an ATP‐responsive nanoparticle (GroELNP) whose surface is fully covered with the biomolecular machine "chaperonin protein GroEL". GroELNP was synthesized by DNA hybridization between a gold NP with DNA strands on its surface and GroEL carrying complementary DNA strands at its apical domains. The unique structure of GroELNP was visualized by transmission electron microscopy including under cryogenic conditions. The immobilized GroEL units retain their machine‐like function and enable GroELNP to capture denatured green fluorescent protein and release it in response to ATP. Interestingly, the ATPase activity of GroELNP per GroEL was 4.8 and 4.0 times greater than those of precursor cysGroEL and its DNA‐functionalized analogue, respectively. Finally, we confirmed that GroELNP could be iteratively extended to double‐layered (GroEL)2 ${{^{({\rm GroEL}){_{2}}}}}$ NP. [ABSTRACT FROM AUTHOR]

Mycobacterium tuberculosis encodes two chaperonin proteins, MtbCpn60.1 and MtbCpn60.2, that share substantial sequence similarity with the Escherichia coli chaperonin, GroEL. However, unlike GroEL, MtbCpn60.1 and MtbCpn60.2 purify as lower‐order oligomers. Previous studies have shown that MtbCpn60.2 can functionally replace GroEL in E. coli, while the function of MtbCpn60.1 remained an enigma. Here, we demonstrate the molecular chaperone function of MtbCpn60.1 and MtbCpn60.2, by probing their ability to assist the folding of obligate chaperonin clients, DapA, FtsE and MetK, in an E. coli strain depleted of endogenous GroEL. We show that both MtbCpn60.1 and MtbCpn60.2 support cell survival and cell division by assisting the folding of DapA and FtsE, but only MtbCpn60.2 completely rescues GroEL‐depleted E. coli cells. We also show that, unlike MtbCpn60.2, MtbCpn60.1 has limited ability to support cell growth and proliferation and assist the folding of MetK. Our findings suggest that the client pools of GroEL and MtbCpn60.2 overlap substantially, while MtbCpn60.1 folds only a small subset of GroEL clients. We conclude that the differences between MtbCpn60.1 and MtbCpn60.2 may be a consequence of their intrinsic sequence features, which affect their thermostability, efficiency, clientomes and modes of action. [ABSTRACT FROM AUTHOR]

Analysis of proteins and complexes under native mass spectrometric (MS) and solution conditions was typically performed using time-of-flight (ToF) analyzers, due to their routine high m/z transmission and detection capabilities. However, over recent years, the ability of Orbitrap-based mass spectrometers to transmit and detect a range of high molecular weight species is well documented. Herein, we describe how a 15 Tesla Fourier transform ion cyclotron resonance mass spectrometer (15 T FT-ICR MS) is more than capable of analyzing a wide range of ions in the high m/z scale (>5000), in both positive and negative instrument polarities, ranging from the inorganic cesium iodide salt clusters; a humanized IgG1k monoclonal antibody (mAb; 148.2 kDa); an IgG1-mertansine drug conjugate (148.5 kDa, drug-to-antibody ratio; DAR 2.26); an IgG1-siRNA conjugate (159.1 kDa; ribonucleic acid to antibody ratio; RAR 1); the membrane protein aquaporin-Z (97.2 kDa) liberated from a C8E4 detergent micelle; the empty MSP1D1-nanodisc (142.5 kDa) and the tetradecameric chaperone protein complex GroEL (806.2 kDa; GroEL dimer at 1.6 MDa). We also investigate different regions of the FT-ICR MS that impact ion transmission and desolvation. Finally, we demonstrate how the transmission of these species and resultant spectra are highly consistent with those previously generated on both quadrupole-ToF (Q-ToF) and Orbitrap instrumentation. This report serves as an impactful example of how FT-ICR mass analyzers are competitive to Q-ToFs and Orbitraps for high mass detection at high m/z.

Background and Aim: Bacillus cereus is accountable for several outbreaks of diseases spread by food. Therefore, the study aimed to use routine culture methods and direct PCR to detect the foodborne bacterial pathogen and its enterotoxins. Materials and Methods: In the present study, a total of 75 Kibda sandwiches, Sausage sandwiches, Chicken Luncheons, Beef Meat luncheons, and Chicken shawarma (Fifteen samples of each) were collected from different places in Kafr El-Sheikh governorate, Egypt, from July to September 2022. isolation, identification, and rapid analysis by PCR were done to find Foodborne bacterial pathogens in samples. Results: Bacterial isolation revealed 17 positive samples from different food types. From 17 infected samples, 40% were Kibda, 26.6% were Sausage, 20% were Chicken luncheon, and 13.3% were positive for Meat luncheon and Chicken shawarma sandwiches. Using PCR to identify B. cereus from positive isolates (group A), 8 isolates were detected having groEL, nhe & cytK genes amplified at 533, 766, and 421 bp, respectively. Also, the PCR, which was used to detection of Bacillus cereus directly in positive samples (group B) and revealed that 8 B. cereus in samples with its enterotoxins genes nhe & cytK, while group C which representssome random food samples of negative isolation resultsrevealed that 3 samples were infected by B. cereus. Conclusion: PCR assay was a sensitive & specific diagnostic tool in detecting Bacillus cereus with its enterotoxins genes directly from food samples, even in the presence of low numbers of B. cereus bacteria that traditional isolation and identification methods cannot detect. [ABSTRACT FROM AUTHOR]

Although diverse immunomodulatory reactions of probiotic bacteria have been reported, this effect via Bacillus subtilis natto remains unclear, despite its long consumption history in Japan and usage in Natto production. Hence, we performed a comparative analysis of the immunomodulatory activities of 23 types of B. subtilis natto isolated from Natto products to elucidate the key active components. Among the isolated 23 strains, the supernatant from B. subtilis strain 1 fermented medium showed the highest induction of anti-inflammatory IL-10 and pro-inflammatory IL-12 in THP-1 dendritic cells (THP-1 DC) after co-incubation. We isolated the active component from strain 1 cultured medium and employed DEAE-Sepharose chromatography with 0.5 M NaCl elution for fractionation. IL-10-inducing activity was specific to an approximately 60 kDa protein, GroEL, which was identified as a chaperone protein and was significantly reduced with anti-GroEL antibody. Differential expression analysis of strains 1 and 15, which had the lowest cytokine-producing activity, showed a higher expression of various genes involved in chaperones and sporulation in strain 1. Furthermore, GroEL production was induced in spore-forming medium. The present study is the first to show that the chaperone protein GroEL, secreted by B. subtilis natto during sporulation, plays a crucial role in IL-10 and IL-12 production in THP-1 DC. [ABSTRACT FROM AUTHOR]

Tick-borne Coxiella spp. are emerging in novel regions infecting different hosts, but information regarding their occurrence is limited. The purpose of this study was the molecular screening of Coxiella spp. in various ticks infesting goats, sheep, camels, cattle, wild mice, and domestic fowls (Gallus gallus domesticus) in various districts of Khyber Pakhtunkhwa, Pakistan. Morphologically identified tick species were confirmed by obtaining their cox1 sequences and were molecularly screened for Coxiella spp. by sequencing GroEL fragments. Almost 345 out of 678 (50.9%) hosts were infested by nine tick species. Regarding the age groups, the hosts having an age >3 years were highly infested (192/345, 55.6%), while gender-wise infestation was higher in female hosts (237/345, 68.7%). In collected ticks, the nymphs were outnumbered (613/1,119, 54.8%), followed by adult females (293/1,119, 26.2%) and males (213/1,119, 19.7%). A total of 227 ticks were processed for molecular identification and detection of Coxiella spp. The obtained cox1 sequences of nine tick species such as Hyalomma dromedarii, Hyalomma anatolicum, Haemaphysalis cornupunctata, Haemaphysalis bispinosa, Haemaphysalis danieli, Haemaphysalis montgomeryi, Rhipicephalus haemaphysaloides, Rhipicephalus microplus, and Argas persicus showed maximum identities between 99.6% and 100% with the same species and in the phylogenetic tree, clustered to the corresponding species. All the tick species except Ha. danieli and R. microplus were found positive for Coxiella spp. (40/227, 17.6%), including Coxiella burnetii (15/40, 6.7%), Coxiella endosymbionts (14/40, 6.3%), and different Coxiella spp. (11/40, 4.9%). By the BLAST results, the GroEL fragments of Coxiella spp. showed maximum identity to C. burnetii, Coxiella endosymbionts, and Coxiella sp., and phylogenetically clustered to the corresponding species. This is the first comprehensive report regarding the genetic characterization of Coxiella spp. in Pakistan's ticks infesting domestic and wild hosts. Proper surveillance and management measures should be undertaken to avoid health risks.

Background: The Gram-negative bacterium Anaplasma phagocytophilum is an intracellular pathogen and an etiological agent of human and animal anaplasmosis. Its natural reservoir comprises free-ranging ungulates, including roe deer (Capreolus capreolus) and red deer (Cervus elaphus). These two species of deer also constitute the largest group of game animals in Poland. The aim of the study was to genotype and perform a phylogenetic analysis of A. phagocytophilum strains from roe deer and red deer. Methods: Samples were subjected to PCR amplification, sequencing, and phylogenetic analysis of strain-specific genetic markers (groEL, ankA). Results: Five haplotypes of the groEL gene from A. phagocytophilum and seven haplotypes of ankA were obtained. The phylogenetic analysis classified the groEL into ecotypes I and II. Sequences of the ankA gene were classified into clusters I, II, and III. Conclusions: Strains of A. phagocytophilum from red deer were in the same ecotype and cluster as strains isolated from humans. Strains of A. phagocytophilum from roe deer represented ecotypes (I, II) and clusters (II, III) that were different from those isolated from red deer, and these strains did not show similarity to bacteria from humans. However, roe deer can harbor nonspecific strains of A. phagocytophilum more characteristic to red deer. It appears that the genetic variants from red deer can be pathogenic to humans, but the significance of the variants from roe deer requires more study.

Chaperonin (GroEL, Hsp60) is a molecular chaperone involved in maintaining cellular protein homeostasis. It interacts with unfolded and misfolded proteins to assist in their folding. In this article, structures, functions, regulation, and significance of chaperonin in cyanobacteria will be reviewed. There are multiple kinds of groEL genes in cyanobacteria in contrast to the Escherichia coli chaperonin paradigm. The cyanobacterial groEL1 gene forms an operon with the single groES gene, similar to the E. coli groESL operon. In contrast, the cyanobacterial groEL2 gene is monocistronic. The regulation of expression and function of the groEL1 and groEL2 genes are mutually distinct. Transcription of the groESL1 operon and groEL2 gene is induced not only by heat but also by light. Unlike the E. coli groESL operon, the expression of the cyanobacterial groESL1 operon and groEL2 is not controlled by the transcription factor sigma32. Cis-regulatory elements such as K-box and CIRCE regulate it positively and/or negatively. Combinations of the elements in groESL1 and groEL2 are evolutionarily diversified. Functional studies suggested that GroEL1 is equivalent to E. coli GroEL, which is essential, whereas GroEL2 is nonessential but plays an important role under stress. The absence of GroEL2 affects proteome and phosphoproteome under stress conditions. Moreover, GroEL1 and GroEL2 are structurally different. We propose that the groEL2 gene is an outcome of neofunctionalization after groESL operon duplication. The groEL1 gene retains the original function essential for cellular activities under both normal and stress conditions, whereas groEL2 acquires a novel, beneficial function required under stress. [ABSTRACT FROM AUTHOR]

The pioneering studies carried out on heat shock-induced synthesis of specific proteins in the early 1970s did not identify any Hsp60 family protein in Drosophila. By the early 1980s, although the members of Hsp60 family of heat shock proteins (Hsp) were identified in a wide range of eukaryotes as homologs of the bacterial GroEL, none was known in Drosophila. The existence of the Hsp60 family protein was serendipitously revealed in Drosophila in my laboratory in 1989. Contrary to the earlier reports that all tissues in flies display the canonical heat shock response, the larval Malpighian tubules (MT) did not show induction of any of the major Hsps but synthesis of a putative Hsp60 family protein was found to be the most abundant in this tissue. A few years later, we identified this MT-specific heat shock-induced protein to indeed be a member of the Hsp60/chaperonin family. The Drosophila genome sequence projects subsequently revealed four putative Hsp60 gene sequences in the D. melanogaster genome. The present historical perspective chronicles contributions from my and other laboratories that unraveled several aspects of intriguing biology of the multiple Hsp60 genes in D. melanogaster, and highlights challenging questions awaiting future studies. [ABSTRACT FROM AUTHOR]

In this study, the presence, prevalence, and genotypes of Anaplasma phagocytophilum, A. ovis, and A. capra in sheep were investigated based on 16 S SSU rRNA, groEL, and gtlA gene-specific polymerase chain reaction (PCR), respectively. The sequences of the genes were used for detection of the phylogenetic position of the species. Additionally, a restriction fragment length polymorphism (RFLP) were carried out for discrimination of A. phagocytophilum and related variants (A. phagocytophilum-like 1 and 2). The prevalence of Anaplasma spp. was found as 25.8% (101/391), while it was found that A. ovis, A. phagocytophilum-like 1, and A. capra are circulating in the sheep herds in Kyrgyzstan, according to the PCRs, RFLP and the partial DNA sequencing results. The positivity rates of A. phagocytophilum-like 1, A. ovis, and A. capra genotype-1 were 6.9, 22.5, and 5.3%, respectively. A total of 32 (8.2%) sheep were found to be mix infected. Moreover, phylogenetic analyses and sequence comparison with those available in the GenBank showed that A. capra formed two distinct genetic groups (A. capra genotype-1 and A. capra genotype-2). Considering the zoonotic potential of these species, it may be necessary to make changes in the interpretation of anaplasmosis cases in animals and there is a need for further studies to determine the pathogenicity of the species/genotypes circulating in animals. [ABSTRACT FROM AUTHOR]

Abstract Background The quasi-enveloped picornavirus, Hepatitis A Virus (HAV), causes acute hepatitis in humans and infects approximately 1.5 million individuals a year, which does not include the asymptomatically infected population. Several severe outbreaks in developing nations in recent years have highlighted the reduction in HAV endemicity, which increases the risk of infections in the vulnerable population. The current HAV vaccines are based on growing wildtype or attenuated virus in cell culture, which raises the cost of production. For generation of cheaper, subunit vaccines or strategies for antibody-based diagnostics, production of viral structural proteins in recombinant form in easily accessible expression systems is a priority. Results We attempted several strategies for recombinant production of one of the major capsid proteins VP1, from HAV, in the E. coli expression system. Several efforts resulted in the formation of soluble aggregates or tight association of VP1 with the bacterial chaperone GroEL. Correctly folded VP1 was eventually generated in a discrete oligomeric form upon purification of the protein from inclusion bodies and refolding. The oligomers resemble oligomers of capsid proteins from other picornaviruses and appear to have the correct secondary and antigenic surface structure. Conclusions VP1 oligomers generated in the bacterial expression system can be utilized for understanding the molecular pathway of HAV capsid assembly and may also have potential biomedical usages in prevention and diagnostics of HAV infections.

Chaperonins are evolutionarily conserved proteins that facilitate polypeptide assemblies. The most extensively studied chaperonin is GroEL, which plays a crucial role in Escherichia coli. In addition to its chaperone activity, the RNA cleavage activity of GroEL has also been proposed. However, direct evidence of GroEL as a ribonuclease (RNase) and its physiological significance has not been fully elucidated. Here, we characterized the role of GroEL in E. coli as an RNase distinct from RNase E/G activity using in vivo reporter assays, in vitro cleavage assays with varying reaction times, divalent ions, and 5′ phosphorylation status. GroEL bound to single-stranded RNA at nanomolar concentrations. Functional analysis of GroEL chaperonin-defective mutants and segments identified specific regions, and the chaperone active status of GroEL is not a necessary factor for RNase activity. Additionally, RNase activity of GroEL was attenuated by co-overexpression with GroES. Finally, we characterized potential transcripts regulated by GroEL and the conserved RNase activity of GroEL in Shigella flexneri. Our findings indicate that GroEL is a novel post-transcriptional regulator in bacteria. • GroEL functions as an innate RNA-binding RNase. • Chaperone active status of GroEL is not a determinant for RNase activity. • Heat shock-induced production of GroEL exhibits RNase activity in vivo. • RNase activity of GroEL is regulated by co-expression of GroES. • GroEL regulates gene expression at the post-transcriptional level and its activity is conserved in Shigella. [ABSTRACT FROM AUTHOR]

The molecular chaperone GroEL, commonly found in various bacterial species, exhibits heightened expression levels in response to high temperatures and increased levels of oxygen free radicals. Limited literature currently exists on the probiotic role of GroEL in invertebrates. This study sought to explore how the surface protein GroEL from Lactobacillus plantarum Ep-M17 impacts the intestinal barrier function of Penaeus vannamei. Through pull-down and immunofluorescence assays, the interaction between GroEL and Act1 in the gastrointestinal tract of P. vannamei was confirmed. Results from bacterial binding assays demonstrated that rGroEL can bind to pathogens like Vibrio parahaemolyticus E1 (V. p -E1). In vitro experiments revealed that the administration of rGroEL significantly decreased the levels of inflammatory cytokines induced by pathogens while preserving the integrity of tight junctions between intestinal epithelial cells and reducing bacteria-induced apoptosis. Additionally, rGroEL notably lessened the intestinal loading of V. p -E1 in P. vannamei , downregulated immune-related gene expression, and upregulated BCL/BAX expression in the intestines following V. p -E1 challenge. Mechanistic investigations further showed that rGroEL treatment effectively suppressed the expression and phosphorylation of proteins involved in the NF-κB and PI3K-AKT-mTOR signalling pathways in the intestines of bacteria-infected P. vannamei. Furthermore, GroEL reinforces protection against bacterial infections by enhancing the phagocytic and anti-apoptotic capabilities of P. vannamei hemocytes. These results suggest that GroEL may impede the interaction between pathogens and the intestinal mucosa through its competitive binding characteristics, ultimately reducing bacterial infections. [ABSTRACT FROM AUTHOR]

GroEL is a chaperonin that helps other proteins fold correctly. However, alternative activities, such as acting as an insect toxin, have also been discovered. This work evaluates the chaperonin and insecticidal activity of different GroEL proteins from entomopathogenic nematodes on G. mellonella. The ability to synergize with the ExoA toxin of Pseudomonas aeruginosa was also investigated. The GroELXn protein showed the highest insecticidal activity among the different GroELs. In addition, it was able to significantly activate the phenoloxidase system of the target insects. This could tell us about the mechanism by which it exerts its toxicity on insects. GroEL proteins can enhance the toxic activity of the ExoA toxin, which could be related to its chaperonin activity. However, there is a significant difference in the synergistic effect that is more related to its alternative activity as an insecticidal toxin.

The molecular chaperone GroEL of C. sakazakii, a highly conserved protein encoded by the gene grol, has the basic function of responding to heat shock, thus enhancing the bacterium’s adaptation to dry and high-temperature environments, which poses a threat to food safety and human health. Our previous study demonstrated that GroEL was found in the bacterial membrane fraction and caused a strong immune response in C. sakazakii. In this study, we tried to elucidate the subcellular location and virulent effects of GroEL. In live C. sakazakii cells, GroEL existed in both the soluble and insoluble fractions. To study the secretory mechanism of GroEL protein, a non-reduced Western immunoblot was used to analyze the form of the protein, and the result showed that the exported GroEL protein was mainly in monomeric form. The exported GroEL could also be located on bacterial surface. To further research the virulent effect of C. sakazakii GroEL, an indirect immunofluorescence assay was used to detect the adhesion of recombinant GroEL protein to HCT-8 cells. The results indicated that the recombinant GroEL protein could adhere to HCT-8 cells in a short period of time. The recombinant GroEL protein could activate the NF-κB signaling pathway to release more pro-inflammatory cytokines (TNF-α, IL-6 and IL-8), downregulating the expression of tight-junction proteins (claudin-1, occluding, ZO-1 and ZO-2), which collectively resulted in dose-dependent virulent effects on host cells. Inhibition of the grol gene expression resulted in a significant decrease in bacterial adhesion to and invasion of HCT-8 cells. Moreover, the deficient GroEL also caused slow growth, decreased biofilm formation, defective motility and abnormal filamentation of the bacteria. In brief, C. sakazakii GroEL was an important virulence factor. This protein was not only crucial for the physiological activity of C. sakazakii but could also be secreted to enhance the bacterium’s adhesion and invasion capabilities.

Rickettsioses, caused by intracellular bacteria of the rickettsia genus, pose a global health threat, particularly affecting underserved communities. Delays in diagnosis and treatment due to limited healthcare access result in severe cases and fatalities. To address this, a multi-epitope vaccine targeting OmpB and GroEL proteins in epidemic and endemic typhus rickettsia was developed using immunoinformatics techniques. T-cell and B-cell epitopes were predicted from OmpB and GroEL proteins, resulting in 5 CTL, 4 HTL, and 4 B-cell epitopes. These epitopes were linked appropriately and combined with suitable adjuvants to construct the vaccine. The vaccine's physicochemical properties and tertiary structure were validated within acceptable ranges. Molecular docking analysis demonstrated favorable binding with TLR4 receptors and MHC molecules, further confirmed by molecular dynamics simulation. Immune simulation analysis predicted T-cell, IFN, and IL-2 responses upon vaccine administration. The vaccine sequence was then optimized and cloned into a plasmid vector pET-23a(+) at the Hind and AlwNI restriction sites. In this in silico experiment, a multi-epitope vaccine derived from OmpB and GroEL proteins is presented, the first validation steps of which have been completed. Subject to successful in vivo studies, this vaccine could prove to be a promising therapeutic strategy against rickettsiosis and address a critical public health challenge.

Research objective: in a prospective controlled study to investigate the role of HSP60, GroEl and other infertility factors as predictors of successful IVF outcome. Materials and methods. 106 female patients were divided into two groups: 54 individuals who received conventional treatment for infertility (using ICSI techniques for IVF) and 52 individuals who received conventional therapy with intravenous IgG, enoxaparin and aspirin. All collected blood samples were tested for HSP60 and GroEl antibodies using immunofluorescence and ELISA techniques at the time of admission, after treatment (and before embryo transfer), and after embryo transfer. We analyzed the factors that can be helpful as prognostic parameters to estimate the risk of implantation failure. Results. The risk of implantation failure is predicted when HSP60 level decreases from the first to the second measurement by less than 0.02 optical density units, with a sensitivity of 62% (95% confidence interval (CI) 47.2–75.3), and a specificity of 87.5% (95% CI 75.9–94.8), the positive predictive value was 81.6% (95% CI 68.2–90.2), the negative predictive value was 72.1% (95% CI 64.1–78.8). The GroEl value for the second dimension was more than 0.411 optical density units, which suggests a risk of treatment failure with a sensitivity of 64% (95% CI 49.2–77.1) and a specificity of 85.6% (95% CI 73.8–93.6), the positive predictive value was 80.0% (95% CI 67.1–88.7), the negative predictive value was 72.7% (95% CI 64.5–79.7). The highest (p < 0.05) value was observed at the beginning of treatment, and the lowest (p < 0.05) – during the third measurement. Treatment of the underlying cause of infertility led to a decrease in HSP60 and GroEl levels, which ensured a positive in vitro fertilization result. It was found that HSP60 and GroEl have a strong association with embryo implantation. The risk of implantation failure was strongly associated with twelve factors, the area under the curve (AUC) was 0.85 (95% CI 0.76–0.91). Conclusions. HSP60 and GroEl are good prognostic factors for predicting a successful IVF outcome in patients undergoing infertility treatment. The measurement of these parameters during the initial infertility examination may help in the immediate diagnosis of autoimmune infertility. Embryo implantation is a multifactorial process. The risk of implantation failure should be evaluated with multiple factors (twelve factors).

A recent elegant cryo-electron tomography study of the populations of different GroEL-GroES chaperonins complexes in whole bacterial cells (Wagner, Carvajal et al. 2024) contributes to the resolution of a long-standing debate about their mechanism, and reconciles three-decade-old results from in vitro biochemical studies, with new, refined in situ observations. Biochemists working with purified proteins often wonder if their findings faithfully reflect the situation in the crowded environment of cells, when their proteins mingle with concentrated metabolites and bump into membranes and thousands of different unrelated proteins. Here, cryo-electron tomography confirmed that careful in vitro protein biochemistry research still has a bright future., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Introduction Burkholderia cepacia complex(BCC) is one of the most common polymyxin-resistant Gram-negative bacilli isolated in the clinical microbiological laboratory. They are often underreported when conventional biochemicals are used for identification, due to their similarity to other non-fermenting bacilli. It is essential to identify BCC using simple biochemical tests with good reliability to ease the identification of BCC in resource-limited settings and initiate treatment. Objectives We aim to identify isolates belonging to BCC from other non-fermenters using simple conventional biochemical tests, automated methods, and polymerase chain reaction (PCR) and analyze antibiotic profiles and treatment outcomes in patients with BCC infection. Materials and methods All samples received at the clinical microbiology laboratory for bacterial culture from March 2023 to March 2024 were included in this study. Samples that grew non-lactose fermenting colonies on MacConkey agar, which were resistant to polymyxin B 300 (PB300) units, were further identified using conventional biochemicals, and lysine decarboxylase test, ornithine decarboxylase test, arginine dihydrolase test, ortho-nitrophenyl-β-galactoside (ONPG) test, urea hydrolysis, triple disc screening test (polymyxin B 300 units, amoxicillin-clavulanic acid 20/10 mcg (AMC20/10), and gentamicin 10 mcg (GEN10)), VITEK MS (bioMérieux, Marcy-l'Etoile, France), VITEK 2 Gram-Negative (GN) Identification (ID) Card (bioMérieux), and PCR targeting groEL sequence was performed for Burkholderia isolates. The antibiotic susceptibility pattern was analyzed using VITEK AST (bioMérieux). Basic patient details were collected from the medical records. Results Using conventional biochemicals and automated methods, 60 isolates were identified belonging to the genus Burkholderia , among which 46 (76.6%) belonged to Burkholderia cepacia complex. The concordance for genus-level identification of Burkholderia with conventional biochemicals, VITEK MS, VITEK 2, and groEL PCR was found to be 100%. Species-level identification using the listed conventional biochemicals was 28.2% as compared to VITEK MS.All the isolates were susceptible to ceftazidime (n=46, 100%). Risk factors included diabetes mellitus (n=19, 41.3%) and indwelling devices such as central venous catheters (CVCs), urinary catheters, and mechanical ventilation. Recovery was seen in 40 (86.9%) patients on treatment with recommended antibiotics. Conclusion Our study shows that the identification of the genus Burkholderia by conventional biochemicals was as efficient as automated and molecular methods. Species identification of BCC was better with automated systems. Infections with BCC commonly occurred in patients with diabetes mellitus and indwelling devices. Treatment with recommended antibiotics showed high recovery rates., Competing Interests: Human subjects: Consent was obtained or waived by all participants in this study. The Institutional Research Ethics Committee of Sri Ramachandra Institute of Higher Education and Research issued approval IEC No: CSP-MED/23/MAR/85/61. Animal subjects: All authors have confirmed that this study did not involve animal subjects or tissue. Conflicts of interest: In compliance with the ICMJE uniform disclosure form, all authors declare the following: Payment/services info: All authors have declared that no financial support was received from any organization for the submitted work. Financial relationships: All authors have declared that they have no financial relationships at present or within the previous three years with any organizations that might have an interest in the submitted work. Other relationships: All authors have declared that there are no other relationships or activities that could appear to have influenced the submitted work., (Copyright © 2024, Periaiah et al.)

Chaperonins are biological nanomachines that help newly translated proteins to fold by rescuing them from kinetically trapped misfolded states. Protein folding assistance by the chaperonin machinery is obligatory in vivo for a subset of proteins in the bacterial proteome. Chaperonins are large oligomeric complexes, with unusual seven fold symmetry (group I) or eight/nine fold symmetry (group II), that form double-ring constructs, enclosing a central cavity that serves as the folding chamber. Dramatic large-scale conformational changes, that take place during ATP-driven cycles, allow chaperonins to bind misfolded proteins, encapsulate them into the expanded cavity and release them back into the cellular environment, regardless of whether they are folded or not. The theory associated with the iterative annealing mechanism, which incorporated the conformational free energy landscape description of protein folding, quantitatively explains most, if not all, the available data. Misfolded conformations are associated with low energy minima in a rugged energy landscape. Random disruptions of these low energy conformations result in higher free energy, less folded, conformations that can stochastically partition into the native state. Two distinct mechanisms of annealing action have been described. Group I chaperonins (GroEL homologues in eubacteria and endosymbiotic organelles), recognize a large number of misfolded proteins non-specifically and operate through highly coordinated cooperative motions. By contrast, the less well understood group II chaperonins (CCT in Eukarya and thermosome/TF55 in Archaea), assist a selected set of substrate proteins. Sequential conformational changes within a CCT ring are observed, perhaps promoting domain-by-domain substrate folding. Chaperonins are implicated in bacterial infection, autoimmune disease, as well as protein aggregation and degradation diseases. Understanding the chaperonin mechanism and the specific proteins they rescue during the cell cycle is important not only for the fundamental aspect of protein folding in the cellular environment, but also for effective therapeutic strategies.

The Escherichia coli chaperonin GroEL/ES (GroE) is one of the most extensively studied molecular chaperones. So far, ~80 proteins in E. coli are identified as GroE substrates that obligately require GroE for folding in vivo. In GroE-depleted cells, these substrates, when overexpressed, tend to form aggregates, whereas the GroE substrates expressed at low or endogenous levels are degraded, probably due to misfolded states. However, the protease(s) involved in the degradation process has not been identified. We conducted a mass-spectrometry-based proteomics approach to investigate the effects of three ATP-dependent proteases, Lon, ClpXP, and HslUV, on the E. coli proteomes under GroE-depleted conditions. A label-free quantitative proteomic method revealed that Lon protease is the dominant protease that degrades the obligate GroE substrates in the GroE-depleted cells. The deletion of DnaK/DnaJ, the other major E. coli chaperones, in the ∆lon strain did not cause major alterations in the expression or folding of the obligate GroE substrates, supporting the idea that the folding of these substrates is predominantly dependent on GroE. [ABSTRACT FROM AUTHOR]

The closely related members of the Bacillus cereus -group can mainly only be differentiated by whole genome sequencing. Among them, there are potentially toxin-producing bacteria. When consumed with food, these can cause vomiting or diarrhea and abdominal cramps. To date, although no EU-wide threshold exists, a bacterial count of 105 CFU/g can be regarded as critical. Specific and rapid detection of the bacteria is difficult due to their close relationship, and no loop-mediated isothermal amplification (LAMP) assay has been developed so far to detect potentially toxin-producing members of the B. cereus -group. Aim of this study was to develop a LAMP method to detect critical cell counts specifically and rapidly of potentially non-haemolytic enterotoxin (NHE)-producing cells of this group. A two-step LAMP assay was developed. First, the target sequence groEL was used to determine the representatives of the B. cereus -group. Second, since bacteria in which nheB is present are basically capable of producing enterotoxins, this gene was chosen for detection. The specificity of the developed assay was 100% for B. cereus -group isolates and 93.7% for the detection of nheB. The analytical sensitivity was 0.1 pg DNA/μl. Using simplified DNA extraction by boiling, cell-based sensitivity was determined. Targeting groEL and nheB , 11.35–27.05 CFU/reaction and 11.35–270.5 CFU/reaction were detectable, respectively. Artificially contaminated samples were investigated to prove the application in foods. Direct detection of the critical value of B. cereus -group cells was possible in 83.3% of samples and detecting the toxin-gene 50% thereof. After a 6-h incubation period, the detection rate increased to 100 and 91.7%, respectively. Additionally, 100 natively contaminated food samples were tested, also quantitatively and culturally. Samples with relevant contamination levels were reliably detected using groEL -LAMP. After a 6-h incubation period, isolates bearing the toxin gene nheB could also be reliably detected. In addition, colony material was boiled and used as a LAMP template for simple detection. Specificity for the B. cereus -group was 100 and 93.22% detecting nheB. The study demonstrated that screening of food samples with the groEL / nheB -LAMP assay can be performed within 1 day, making it possible to detect critical levels of potentially NHE-toxin-producing cells of the B. cereus -group. [ABSTRACT FROM AUTHOR]

The closely related members of the Bacillus cereus-group can mainly only be differentiated by whole genome sequencing. Among them, there are potentially toxin-producing bacteria. When consumed with food, these can cause vomiting or diarrhea and abdominal cramps. To date, although no EU-wide threshold exists, a bacterial count of 105 CFU/g can be regarded as critical. Specific and rapid detection of the bacteria is difficult due to their close relationship, and no loop-mediated isothermal amplification (LAMP) assay has been developed so far to detect potentially toxin-producing members of the B. cereus-group. Aim of this study was to develop a LAMP method to detect critical cell counts specifically and rapidly of potentially non-haemolytic enterotoxin (NHE)-producing cells of this group. A two-step LAMP assay was developed. First, the target sequence groEL was used to determine the representatives of the B. cereus-group. Second, since bacteria in which nheB is present are basically capable of producing enterotoxins, this gene was chosen for detection. The specificity of the developed assay was 100% for B. cereus-group isolates and 93.7% for the detection of nheB. The analytical sensitivity was 0.1 pg DNA/μl. Using simplified DNA extraction by boiling, cell-based sensitivity was determined. Targeting groEL and nheB, 11.35–27.05 CFU/reaction and 11.35–270.5 CFU/reaction were detectable, respectively. Artificially contaminated samples were investigated to prove the application in foods. Direct detection of the critical value of B. cereus-group cells was possible in 83.3% of samples and detecting the toxin-gene 50% thereof. After a 6-h incubation period, the detection rate increased to 100 and 91.7%, respectively. Additionally, 100 natively contaminated food samples were tested, also quantitatively and culturally. Samples with relevant contamination levels were reliably detected using groEL-LAMP. After a 6-h incubation period, isolates bearing the toxin gene nheB could also be reliably detected. In addition, colony material was boiled and used as a LAMP template for simple detection. Specificity for the B. cereus-group was 100 and 93.22% detecting nheB. The study demonstrated that screening of food samples with the groEL/nheB-LAMP assay can be performed within 1 day, making it possible to detect critical levels of potentially NHE-toxin-producing cells of the B. cereus-group.

Abstract Background Leptospirosis is a zoonotic disease caused by infection with spirochetes from Leptospira genus. It has been classified into at least 17 pathogenic species, with more than 250 serologic variants. This wide distribution may be a result of leptospiral ability to colonize the renal tubules of mammalian hosts, including humans, wildlife, and many domesticated animals. Previous studies showed that the expression of proteins belonging to the microbial heat shock protein (HSP) family is upregulated during infection and also during various stress stimuli. Several proteins of this family are known to have important roles in the infectious processes in other bacteria, but the role of HSPs in Leptospira spp. is poorly understood. In this study, we have evaluated the capacity of the protein GroEL, a member of HSP family, of interacting with host proteins and of stimulating the production of cytokines by macrophages. Results The binding experiments demonstrated that the recombinant GroEL protein showed interaction with several host components in a dose-dependent manner. It was also observed that GroEL is a surface protein, and it is secreted extracellularly. Moreover, two cytokines (tumor necrosis factor-α and interleukin-6) were produced when macrophages cells were stimulated with this protein. Conclusions Our findings showed that GroEL protein may contribute to the adhesion of leptospires to host tissues and stimulate the production of proinflammatory cytokines during infection. These features might indicate an important role of GroEL in the pathogen-host interaction in the leptospirosis.

Although diverse immunomodulatory reactions of probiotic bacteria have been reported, this effect via Bacillus subtilis natto remains unclear, despite its long consumption history in Japan and usage in Natto production. Hence, we performed a comparative analysis of the immunomodulatory activities of 23 types of B. subtilis natto isolated from Natto products to elucidate the key active components. Among the isolated 23 strains, the supernatant from B. subtilis strain 1 fermented medium showed the highest induction of anti-inflammatory IL-10 and pro-inflammatory IL-12 in THP-1 dendritic cells (THP-1 DC) after co-incubation. We isolated the active component from strain 1 cultured medium and employed DEAE-Sepharose chromatography with 0.5 M NaCl elution for fractionation. IL-10-inducing activity was specific to an approximately 60 kDa protein, GroEL, which was identified as a chaperone protein and was significantly reduced with anti-GroEL antibody. Differential expression analysis of strains 1 and 15, which had the lowest cytokine-producing activity, showed a higher expression of various genes involved in chaperones and sporulation in strain 1. Furthermore, GroEL production was induced in spore-forming medium. The present study is the first to show that the chaperone protein GroEL, secreted by B. subtilis natto during sporulation, plays a crucial role in IL-10 and IL-12 production in THP-1 DC.

Simple Summary: The populations of bison and moose, the largest wild ruminants in Poland, are growing every year. These animals return to Polish forests after many years of risk of extinction or, as in the case of European bison, are reintroduced step by step. Unfortunately, they are still rare and require close health surveillance and monitoring. One serious threat to their health and life is the bacterial parasite Anaplasma phagocytophilum. Moose and bison can also be sources of pathogenic bacteria for humans, which can be transferred through tick bites. In line with the World Health Organization (WHO) "OneHealth" program, indicating that animal health affects human health and vice versa, the following study examines the occurrence of Anaplasma phagocytophilum bacteria in European bison and Eurasian moose populations using molecular biology tools. Our results provide useful data regarding Anaplasma phagocytophilum that could be used in future strategies for the diagnosis and treatment of people and animals. Wild large ungulates, like European bison (Bison bonasus) and Eurasian moose (Alces alces), form an important part of the circulation of Anaplasma phagocytophilum, a Gram-negative, intracellular, tick-transmitted bacterium, in the natural environment. Bison and moose tissue samples were subjected to 16S rDNA, groEL and ankA partial gene marker amplification with specific primers using various variants of PCR. Out of 42 examined individuals, Anaplasma sp. were detected in 4/13 Eurasian moose (31%) and 7/29 European bison (24%). In addition, 12 groEL and 5 ankA partial gene positive samples were obtained from the examined animals. The phylogenetic analysis of the groEL partial gene classified samples from European bison to ecotype I, and samples from Eurasian moose to ecotype I and II; the analysis of the ankA partial gene assigned the samples to clusters I and IV. This study extends knowledge about A. phagocytophilum in wild large ungulates in Poland. This is the first report about the occurrence of Anaplasma sp. in one of the largest populations of free living European bison in the world. Our findings confirm that strains of A. phagocytophilum from Bison bonasus and Alces alces may constitute a natural reservoir of pathogenic HGA Anaplasma strains. [ABSTRACT FROM AUTHOR]

Chaperones play a vital role in the life of cells by facilitating the correct folding of other proteins and maintaining them in a functional state, being themselves, as a rule, more stable than the rest of cell proteins. Their functional properties naturally tempt investigators to actively adapt them for biotechnology needs. This review will mostly focus on the applications found for the bacterial chaperonin GroE and its counterparts from other organisms, in biotechnology or for research purposes, both in their engineered or intact versions. [ABSTRACT FROM AUTHOR]