Extracellular Membrane Vesicles of Escherichia coli Induce Apoptosis of CT26 Colon Carcinoma Cells.

Escherichia coli (E. coli) is commonly utilized as a vehicle for anti-tumor therapy due to its unique tumor-targeting capabilities and ease of engineering modification. To further explore the role of E. coli in tumor treatment, we consider that E. coli outer membrane vesicles (E. coli-OMVs) play a crucial role in the therapeutic process. Firstly, E. coli-OMVs were isolated and partially purified by filtration and ultracentrifugation, and were characterized using techniques such as nanoparticle tracking analysis (NTA), transmission electron microscopy (TEM) and Western Blot (WB). The obtained extracellular nanoparticles, containing OMVs, were found to inhibited the growth of CT26 tumor in mice, while the expression of Bax protein was increased and the expression of Bcl-2 protein decreased. In vitro experiments showed that E. coli-OMVs entered CT26 cells and inhibited cell proliferation, invasion and migration. In addition, in the presence of E. coli-OMVs, we observed an increase in apoptosis rate and a decrease in the ratio of Bcl-2/Bax. These data indicate that E. coli-OMVs inhibits the growth of CT26 colon cancer by inducing apoptosis of CT26 cells. These findings propose E. coli-OMVs as a promising therapeutic drug for colorectal cancer (CRC), providing robust support for further research in related fields.

Antibacterial and therapeutic effects of low energy shock waves on uropathogenic E. coli investigated by in vitro and in vivo cystitis rat model.

AIMS: Low-energy shock waves (LESWs) are known to alter cell-membrane permeability. This study aimed to investigate the effect of LESWs on Escherichia coli and E. coli-induced cystitis in rats. MAIN METHODS: Standardized suspensions of E. coli ATCC25922 were treated with or without LESWs (100 or 300 pulses; 0.12 mJ/mm2; 2 pulses/s) followed by bacterial counting, an antibiotic sensitivity test, and gene ontology analysis and gene-set enrichment analysis. Intravesical administration of saline or E. coli (0.5 mL with 108 CFU/mL) for 30 min was performed in female Sprague-Dawley rats. The rats were treated with or without LESWs (300 pulses; 0.12 mJ/mm2; 2 pulses/s) on days 4 and 5. The changes in inflammatory reactions, uroplakin IIIa staining, and correlation with urodynamic findings were assessed on day 8. KEY FINDINGS: LESW treatment induced a decrease in CFU and the autoaggregation rate and increased the inhibition zone sizes in a cefazolin-sensitivity study. These changes were associated with gene expression in regulation of cellular membrane components, biofilm formation, and the ATP-binding cassette transporter pathway. E. coli induced bladder hyperactivity and an inflammatory reaction as well as decreased uroplakin IIIa staining; these effects were partially reversed by LESW treatment. SIGNIFICANCE: The LESW antibacterial effect occurs by altering bacterial cell-membrane gene expression, enhancing antibiotic sensitivity, and inhibiting bladder inflammatory reaction and overactivity. These findings support the potential benefits of LESWs for treatment of recurrent or refractory bacterial cystitis.

Rapid detection of pathogenic E. coli based on CRISPR Cas system.

Access to safe and nutritious food is critical for maintaining life and supporting good health. Eating food that is contaminated with pathogens leads to serious diseases ranging from diarrhea to cancer. Many foodborne infections can cause long-term impairment or even death. Hence, early detection of foodborne pathogens such as pathogenic Escherichia coli strains is essential for public safety. Conventional methods for detecting these bacteria are based on culturing on selective media and following standard biochemical identification. Despite their accuracy, these methods are time-consuming. PCR-based detection of pathogens relies on sophisticated equipment and specialized technicians which are difficult to find in areas with limited resources. Whereas CRISPR technology is more specific and sensitive for identifying pathogenic bacteria because it employs programmable CRISPR-Cas systems that target particular DNA sequences, minimizing non-specific binding and cross-reactivity. In this project, a robust detection method based on CRISPR-Cas12a sensing was developed, which is rapid, sensitive and specific for detection of pathogenic E. coli isolates that were collected from the fecal samples from adult goats from 17 farms in Tennessee. Detection reaction contained amplified PCR products for the pathogenic regions, reporter probe, Cas12a enzyme, and crRNA specific to three pathogenic genes-stx1, stx2, and hlyA. The CRISPR reaction with the pathogenic bacteria emitted fluorescence when excited under UV light. To evaluate the detection sensitivity and specificity of this assay, its results were compared with PCR based detection assay. Both methods resulted in similar results for the same samples. This technique is very precise, highly sensitive, quick, cost effective, and easy to use, and can easily overcome the limitations of the present detection methods. This project can result in a versatile detection method that is easily adaptable for rapid response in the detection and surveillance of diseases that pose large-scale biosecurity threats to human health, and plant and animal production.

Anti-Microbial, Thermal, Mechanical, and Gas Barrier Properties of Linear Low-Density Polyethylene Extrusion Blow-Molded Bottles.

Microbial contamination can occur on the surfaces of blow-molded bottles, necessitating the development and application of effective anti-microbial treatments to mitigate the hazards associated with microbial growth. In this study, new methods of incorporating anti-microbial particles into linear low-density polyethylene (LLDPE) extrusion blow-molded bottles were developed. The anti-microbial particles were thermally embossed on the external surface of the bottle through two particle deposition approaches (spray and powder) over the mold cavity. The produced bottles were studied for their thermal, mechanical, gas barrier, and anti-microbial properties. Both deposition approaches indicated a significant enhancement in anti-microbial activity, as well as barrier properties, while maintaining thermal and mechanical performance. Considering both the effect of anti-microbial agents and variations in tensile bar weight and thickness, the statistical analysis of the mechanical properties showed that applying the anti-microbial agents had no significant influence on the tensile properties of the blow-molded bottles. The external fixation of the particles over the surface of the bottles would result in optimum anti-microbial activity, making it a cost-effective solution compared to conventional compounding processing.

The application of organoids in cancers associated with pathogenic infections.

Cancers associated with pathogen infections are gradually becoming important threats to human health globally, and it is of great significance to study the mechanisms of pathogen carcinogenesis. Current mechanistic studies rely on animal and two-dimensional (2D) cell culture models, but traditional methods have been proven insufficient for the rapid modeling of diseases caused by new pathogens. Therefore, research focus has shifted to organoid models, which can replicate the structural and genetic characteristics of the target tissues or organs in vitro, providing new platforms for the study of pathogen-induced oncogenic mechanisms. This review summarizes the application of organoid technology in the studies of four pathogen-associated cancers: gastric cancer linked to Helicobacter pylori, liver cancer associated with hepatitis B virus or hepatitis C virus, colorectal cancer caused by Escherichia coli, and cervical cancer related to human papillomavirus. This review also proposes several limitations of organoid technology to optimize organoid models and advance the treatment of cancer associated with pathogen infections in the future.

Study on Cloning and Expression of TNF-α Variants in E. coli: Production, Purification, and Interaction with Anti-TNF-α Inhibitors.

BACKGROUND: TNF-α is a proinflammatory cytokine and plays a role in cell proliferation, differentiation, survival, and death pathways. When administered at high doses, it may cause damage to the tumor vasculature, thereby increasing the permeability of the blood vessels. Therefore, monitoring the dose and the response of the TNF-α molecule is essential for patients' health. OBJECTIVES: This study aimed to clone, express, and purify the active form of the TNF-α protein, which can interact with various anti-TNF-α inhibitors with high efficiency. METHODS: Recombinant DNA technology was used to clone three different versions of codon-optimized human TNF-α sequences to E. coli. Colony PCR protocol was used for verification and produced proteins were analyzed through SDS-PAGE and western blot. Size exclusion chromatography was used to purify sTNF-α. ELISA techniques were used to analyze and compare binding efficiency of sTNF-α against three different standards. RESULTS: Under native condition (25°C), interaction between sTNF-α and anti-TNF-α antibody was 3,970, compared to positive control. The interaction was 0,587, whereas it was 0,535 for TNF- α and anti-TNF-α antibodies under denaturing conditions (37°C). F7 of sTNF-α (920 µg/mL) had the same/higher binding efficiency to adalimumab, etanercept, and infliximab, compared to commercial TNF-α. CONCLUSION: This study was the first to analyze binding efficiency of homemade sTNF-α protein against three major TNF-α inhibitors (adalimumab, etanercept, and infliximab) in a single study. The high binding efficiency of sTNF-α with adalimumab, etanercept, and infliximab, evidenced in this study supports the feasibility of its use in therapeutic applications, contributing to more sustainable, cost-effective, and independent healthcare system.

In silico mining and identification of a novel lipase from Paenibacillus larvae: Rational protein design for improving catalytic performance.

Lipases play a vital role in various biological processes, from lipid metabolism to industrial applications. However, the ever-evolving challenges and diverse substrates necessitate the continual exploration of novel high-performance lipases. In this study, we employed an in silico mining approach to search for lipases with potential high sn-1,3 selectivity and catalytic activity. The identified novel lipase, PLL, from Paenibacillus larvae subsp. larvae B-3650 exhibited a specific activity of 111.2 ± 5.5 U/mg towards the substrate p-nitrophenyl palmitate (pNPP) and 6.9 ± 0.8 U/mg towards the substrate olive oil when expressed in Escherichia coli (E. coli). Computational design of cysteine mutations was employed to enhance the catalytic performance of PLL. Superior stability was achieved with the mutant K7C/A386C/H159C/K108C (2M3/2M4), showing an increase in melting temperature (Tm) by 1.9°C, a 2.05-fold prolonged half-life at 45°C, and no decrease in enzyme activity. Another mutant, K7C/A386C/A174C/A243C (2M1/2M3), showed a 4.9-fold enhancement in specific activity without compromising stability. Molecular dynamics simulations were conducted to explore the mechanisms of these two mutants. Mutant 2M3/2M4 forms putative disulfide bonds in the loop region, connecting the N- and C-termini of PLL, thus enhancing overall structural rigidity without impacting catalytic activity. The cysteines introduced in mutant 2M1/2M3 not only form new intramolecular hydrogen bonds but also alter the polarity and volume of the substrate-binding pocket, facilitating the entry of large substrate pNPP. These results highlight an efficient in silico exploration approach for novel lipases, offering a rapid and efficient method for enhancing catalytic performance through rational protein design.

A very complicated UTI: malakoplakia following E. coli urinary tract infection.

Malakoplakia is a rare inflammatory disorder believed to result from a defect in macrophage phagocytic function triggering a granulomatous reaction. It can present with genitourinary, gastrointestinal, or cutaneous manifestations in immunocompromised or, less commonly, immunocompetent hosts. We describe a case of renal malakoplakia in a young, otherwise healthy patient presenting with nephromegaly and sepsis following an E. coli urinary tract infection. We discuss diagnosis and management, including antibiotic selection and the decision to pursue nephrectomy. This case highlights the potential for kidney recovery with prolonged antibiotic therapy in conjunction with adjunct immunomodulatory therapies and source control.

Presence of intracellular bacterial communities in uroepithelial cells, a potential reservoir in symptomatic and non-symptomatic people.

BACKGROUND: Urinary tract infection is one of the most common infections in humans, affecting women in more proportion. The bladder was considered sterile, but it has a urinary microbiome. Moreover, intracellular bacteria (IB) were observed in uroepithelial cells from children and women with urinary tract infections (UTIs). Here, we evaluated the presence of IB in urine from healthy people and patients with UTI symptoms. METHODS: Midstream urine was self-collected from 141 donors, 77 females and 64 males; 72 belonged to the asymptomatic group and 69 were symptomatic. IB was characterized by a culture-dependent technique and visualized by confocal microscopy. Urine was also subjected to the classical uroculture and isolated bacteria were identified by MALDI-TOF. RESULTS: One-hundred and fifteen uroculture were positive. A significant association was observed between the presence of symptoms and IB (P = 0.007). Moreover, a significant association between the presence of IB, symptoms and being female was observed (P = 0.03). From the cases with IB, Escherichia coli was the most frequent microorganism identified (34.7%), followed by Stenotrophomonas maltophilia (14.2%), Staphylococcus spp (14.2%), and Enterococcus faecalis (10.7%). Intracellular E. coli was associated with the symptomatic group (P = 0.02). Most of the intracellular Staphylococcus spp. were recovered from the asymptomatic group (P = 0.006). CONCLUSIONS: Intracellular bacteria are present in patients with UTI but also in asymptomatic people. Here, we report for the first time, the presence of S. maltophilia, Staphylococcus spp., and Enterobacter cloacae as intracellular bacteria in uroepithelial cells. These findings open new insights into the comprehension of urinary tract infections, urinary microbiome and future therapies. Uroculture as the gold standard could not be enough for an accurate diagnosis in recurrent or complicated cases.

Nonclinical Evaluation of Single-Mutant E. coli Asparaginases Obtained by Double-Mutant Deconvolution: Improving Toxicological, Immune and Inflammatory Responses.

Acute lymphoblastic leukaemia is currently treated with bacterial L-asparaginase; however, its side effects raise the need for the development of improved and efficient novel enzymes. Previously, we obtained low anti-asparaginase antibody production and high serum enzyme half-life in mice treated with the P40S/S206C mutant; however, its specific activity was significantly reduced. Thus, our aim was to test single mutants, S206C and P40S, through in vitro and in vivo assays. Our results showed that the drop in specific activity was caused by P40S substitution. In addition, our single mutants were highly stable in biological environment simulation, unlike the double-mutant P40S/S206C. The in vitro cell viability assay demonstrated that mutant enzymes have a higher cytotoxic effect than WT on T-cell-derived ALL and on some solid cancer cell lines. The in vivo assays were performed in mice to identify toxicological effects, to evoke immunological responses and to study the enzymes' pharmacokinetics. From these tests, none of the enzymes was toxic; however, S206C elicited lower physiological changes and immune/allergenic responses. In relation to the pharmacokinetic profile, S206C exhibited twofold higher activity than WT and P40S two hours after injection. In conclusion, we present bioengineered E. coli asparaginases with high specific enzyme activity and fewer side effects.

RAPSYN-mediated neddylation of BCR-ABL alternatively determines the fate of Philadelphia chromosome-positive leukemia.

Philadelphia chromosome-positive (Ph+) leukemia is a fatal hematological malignancy. Although standard treatments with tyrosine kinase inhibitors (TKIs) have achieved remarkable success in prolonging patient survival, intolerance, relapse, and TKI resistance remain serious issues for patients with Ph+ leukemia. Here, we report a new leukemogenic process in which RAPSYN and BCR-ABL co-occur in Ph+ leukemia, and RAPSYN mediates the neddylation of BCR-ABL. Consequently, neddylated BCR-ABL enhances the stability by competing its c-CBL-mediated degradation. Furthermore, SRC phosphorylates RAPSYN to activate its NEDD8 E3 ligase activity, promoting BCR-ABL stabilization and disease progression. Moreover, in contrast to in vivo ineffectiveness of PROTAC-based degraders, depletion of RAPSYN expression, or its ligase activity decreased BCR-ABL stability and, in turn, inhibited tumor formation and growth. Collectively, these findings represent an alternative to tyrosine kinase activity for the oncoprotein and leukemogenic cells and generate a rationale of targeting RAPSYN-mediated BCR-ABL neddylation for the treatment of Ph+ leukemia.

Chronic myeloid leukemia (CML for short) accounts for about 15% of all blood cancers diagnosed in adults in the United States. The condition is characterized by the overproduction of immature immune cells that interfere with proper blood function. It is linked to a gene recombination (a type of mutation) that leads to white blood cells producing an abnormal 'BCR-ABL' enzyme which is always switched on. In turn, this overactive protein causes the cells to live longer and divide uncontrollably. Some of the most effective drugs available to control the disease today work by blocking the activity of BCR-ABL. Yet certain patients can become resistant to these treatments over time, causing them to relapse. Other approaches are therefore needed to manage this disease; in particular, a promising avenue of research consists in exploring whether it is possible to reduce the amount of the enzyme present in diseased cells. As part of this effort, Zhao, Dai, Li, Zhang et al. focused on RAPSYN, a scaffolding protein previously unknown in CML cells. In other tissues, it has recently been shown to participate in neddylation ­ a process by which proteins receive certain chemical 'tags' that change the way they behave. The experiments revealed that, compared to healthy volunteers, RAPSYN was present at much higher levels in the white blood cells of CML patients. Experimentally lowering the amount of RAPSYN in CML cells led these to divide less quickly ­ both in a dish and when injected in mice, while also being linked to decreased levels of BCR-ABL. Additional biochemical experiments indicated that RAPSYN sticks with BCR-ABL to add chemical 'tags' that protect the abnormal protein against degradation, therefore increasing its overall levels. Finally, the team showed that SRC, an enzyme often dysregulated in emerging cancers, can activate RAPSYN's ability to conduct neddylation; such mechanism could promote BCR-ABL stabilization and, in turn, disease progression. Taken together, these experiments indicate a new way by which BCR-ABL levels are controlled. Future studies should investigate whether RAPSYN also stabilizes BCR-ABL in patients whose leukemias have become resistant to existing drugs. Eventually, RAPSYN may offer a new target for overcoming drug-resistance in CML patients.

The αC-ß4 loop controls the allosteric cooperativity between nucleotide and substrate in the catalytic subunit of protein kinase A.

Allosteric cooperativity between ATP and substrates is a prominent characteristic of the cAMP-dependent catalytic subunit of protein kinase A (PKA-C). This long-range synergistic action is involved in substrate recognition and fidelity, and it may also regulate PKA's association with regulatory subunits and other binding partners. To date, a complete understanding of this intramolecular mechanism is still lacking. Here, we integrated NMR(Nuclear Magnetic Resonance)-restrained molecular dynamics simulations and a Markov State Model to characterize the free energy landscape and conformational transitions of PKA-C. We found that the apoenzyme populates a broad free energy basin featuring a conformational ensemble of the active state of PKA-C (ground state) and other basins with lower populations (excited states). The first excited state corresponds to a previously characterized inactive state of PKA-C with the αC helix swinging outward. The second excited state displays a disrupted hydrophobic packing around the regulatory (R) spine, with a flipped configuration of the F100 and F102 residues at the αC-ß4 loop. We validated the second excited state by analyzing the F100A mutant of PKA-C, assessing its structural response to ATP and substrate binding. While PKA-CF100A preserves its catalytic efficiency with Kemptide, this mutation rearranges the αC-ß4 loop conformation, interrupting the coupling of the two lobes and abolishing the allosteric binding cooperativity. The highly conserved αC-ß4 loop emerges as a pivotal element to control the synergistic binding of nucleotide and substrate, explaining how mutations or insertions near or within this motif affect the function and drug sensitivity in homologous kinases.

Directed Evolution of a Bacterial Leucyl tRNA in Mammalian Cells for Enhanced Noncanonical Amino Acid Mutagenesis.

The Escherichia coli leucyl-tRNA synthetase (EcLeuRS)/tRNAEcLeu pair has been engineered to genetically encode a structurally diverse group of enabling noncanonical amino acids (ncAAs) in eukaryotes, including those with bioconjugation handles, environment-sensitive fluorophores, photocaged amino acids, and native post-translational modifications. However, the scope of this toolbox in mammalian cells is limited by the poor activity of tRNAEcLeu. Here, we overcome this limitation by evolving tRNAEcLeu directly in mammalian cells by using a virus-assisted selection scheme. This directed evolution platform was optimized for higher throughput such that the entire acceptor stem of tRNAEcLeu could be simultaneously engineered, which resulted in the identification of several variants with remarkably improved efficiency for incorporating a wide range of ncAAs. The advantage of the evolved leucyl tRNAs was demonstrated by expressing ncAA mutants in mammalian cells that were challenging to express before using the wild-type tRNAEcLeu, by creating viral vectors that facilitated ncAA mutagenesis at a significantly lower dose and by creating more efficient mammalian cell lines stably expressing the ncAA-incorporation machinery.

Contribution of pks+ Escherichia coli (E. coli) to Colon Carcinogenesis.

Colorectal cancer (CRC) stands as a significant global health concern, ranking second in mortality and third in frequency among cancers worldwide. While only a small fraction of CRC cases can be attributed to inherited genetic mutations, the majority arise sporadically due to somatic mutations. Emerging evidence reveals gut microbiota dysbiosis to be a contributing factor, wherein polyketide synthase-positive Escherichia coli (pks+ E. coli) plays a pivotal role in CRC pathogenesis. pks+ bacteria produce colibactin, a genotoxic protein that causes deleterious effects on DNA within host colonocytes. In this review, we examine the role of the gut microbiota in colon carcinogenesis, elucidating how colibactin-producer bacteria induce DNA damage, promote genomic instability, disrupt the gut epithelial barrier, induce mucosal inflammation, modulate host immune responses, and influence cell cycle dynamics. Collectively, these actions foster a microenvironment conducive to tumor initiation and progression. Understanding the mechanisms underlying pks+ bacteria-mediated CRC development may pave the way for mass screening, early detection of tumors, and therapeutic strategies such as microbiota modulation, bacteria-targeted therapy, checkpoint inhibition of colibactin production and immunomodulatory pathways.

Scyllatoxin-based peptide design for E. coli expression and HIV gp120 binding.

Targeting the hydrophobic Phe43 pocket of HIV's envelope glycoprotein gp120 is a critical strategy for antiviral interventions due to its role in interacting with the host cell's CD4. Previous inhibitors, including small molecules and CD4 mimetic peptides based on scyllatoxin, have demonstrated significant binding and neutralization capabilities but were often chemically synthesized or contained non-canonical amino acids. Microbial expression using natural amino acids offers advantages such as cost-effectiveness, scalability, and efficient production of fusion proteins. In this study, we enhanced the previous scyllatoxin-based synthetic peptide by substituting natural amino acids and successfully expressed it in E. coli. The peptide was optimized by mutating the C-terminal amidated valine to valine and glutamine, and by reducing the disulfide bonds from three to two. Circular dichroism confirmed proper secondary structure formation, and fluorescence polarization analysis revealed specific, concentration-dependent binding to HIV gp120, supported by molecular dynamics simulations. These findings indicate the potential for scalable microbial production of effective antiviral peptides, with significant applications in pharmaceutical development for HIV treatment.

Identification of a copper-responsive small molecule inhibitor of uropathogenic Escherichia coli.

Urinary tract infections (UTIs) are a major global health problem and are caused predominantly by uropathogenic Escherichia coli (UPEC). UTIs are a leading cause of prescription antimicrobial use. Incessant increase in antimicrobial resistance in UPEC and other uropathogens poses a serious threat to the current treatment practices. Copper is an effector of nutritional immunity that impedes the growth of pathogens during infection. We hypothesized that copper would augment the toxicity of select small molecules against bacterial pathogens. We conducted a small molecule screening campaign with a library of 51,098 molecules to detect hits that inhibit a UPEC ΔtolC mutant in a copper-dependent manner. A molecule, denoted as E. coli inhibitor or ECIN, was identified as a copper-responsive inhibitor of wild-type UPEC strains. Our gene expression and metal content analysis results demonstrate that ECIN works in concert with copper to exacerbate Cu toxicity in UPEC. ECIN has a broad spectrum of activity against pathogens of medical and veterinary significance including Acinetobacter baumannii, Pseudomonas aeruginosa, and methicillin-resistant Staphylococcus aureus. Subinhibitory levels of ECIN eliminate UPEC biofilm formation. Transcriptome analysis of UPEC treated with ECIN reveals induction of multiple stress response systems. Furthermore, we demonstrate that L-cysteine rescues the growth of UPEC exposed to ECIN. In summary, we report the identification and characterization of a novel copper-responsive small molecule inhibitor of UPEC.IMPORTANCEUrinary tract infection (UTI) is a ubiquitous infectious condition affecting millions of people annually. Uropathogenic Escherichia coli (UPEC) is the predominant etiological agent of UTI. However, UTIs are becoming increasingly difficult to resolve with antimicrobials due to increased antimicrobial resistance in UPEC and other uropathogens. Here, we report the identification and characterization of a novel copper-responsive small molecule inhibitor of UPEC. In addition to E. coli, this small molecule also inhibits pathogens of medical and veterinary significance including Acinetobacter baumannii, Pseudomonas aeruginosa, and methicillin-resistant Staphylococcus aureus.

Escherichia coli monothiol glutaredoxin GrxD replenishes Fe-S clusters to the essential ErpA A-type carrier under low iron stress.

Iron-sulfur (Fe-S) clusters are required for essential biological pathways, including respiration and isoprenoid biosynthesis. Complex Fe-S cluster biogenesis systems have evolved to maintain an adequate supply of this critical protein cofactor. In Escherichia coli, two Fe-S biosynthetic systems, the "housekeeping" Isc and "stress responsive" Suf pathways, interface with a network of cluster trafficking proteins, such as ErpA, IscA, SufA, and NfuA. GrxD, a Fe-S cluster-binding monothiol glutaredoxin, also participates in Fe-S protein biogenesis in both prokaryotes and eukaryotes. Previous studies in E. coli showed that the ΔgrxD mutation causes sensitivity to iron depletion, spotlighting a critical role for GrxD under conditions that disrupt Fe-S homeostasis. Here, we utilized a global chemoproteomic mass spectrometry approach to analyze the contribution of GrxD to the Fe-S proteome. Our results demonstrate that (1) GrxD is required for biogenesis of a specific subset of Fe-S proteins under iron-depleted conditions, (2) GrxD is required for cluster delivery to ErpA under iron limitation, (3) GrxD is functionally distinct from other Fe-S trafficking proteins, and (4) GrxD Fe-S cluster binding is responsive to iron limitation. All these results lead to the proposal that GrxD is required to maintain Fe-S cluster delivery to the essential trafficking protein ErpA during iron limitation conditions.

Avaliação das boas práticas de fabricação e da contaminação de preparações cárneas de restaurantes institucionais e comerciais na cidade de Americana, SP / Evaluation of good manufacturing practices and contamination in meat preparations from institutional and commercial restaurants in the city of Americana/SP

Diante do ritmo acelerado da vida contemporânea, observa-se um aumento na tendência dos indivíduos em optar por realizar suas refeições fora de casa. A carne, reconhecida como um componente essencial na alimentação dos brasileiros, está suscetível à contaminação pois apresenta ambiente favorável à proliferação de microrganismos patogênicos. Fazendo-se necessária uma análise de contaminação pós-produção afim de evitar Doenças Transmitidas por Alimentos. No presente estudo objetivouse avaliar as boas práticas de fabricação e contaminação de preparações de carne bovina assada, de restaurantes particulares e institucionalizados no município de Americana-SP. Amostras de carne prontas para o consumo foram obtidas de seis estabelecimentos comerciais e seis institucionais. Durante a coleta, foram verificadas as temperaturas e realizadas análises de conformidades com a RDC n° 275, de 2002. As amostras foram examinadas para detectar a presença ou ausência de E. coli e coliformes termotolerantes a 45° C. Para a análise foi realizada a técnica de tubos múltiplos para quantificar a totalidade dos coliformes. Observou-se que, conforme estipulado pela Resolução n°43 de 2015, nenhuma das amostras oriundas de restaurantes comerciais, e a maioria das provenientes de restaurantes institucionais, atingiram as temperaturas requeridas. No que concerne à identificação de E. coli através de testes microbiológicos, foi constatado que seis amostras de restaurantes comerciais e quatro de restaurantes institucionais testaram positivo para a presença deste microrganismo. Conclui-se que as amostras de restaurantes comerciais apresentaram níveis de contaminação superiores em comparação com as amostras de restaurantes institucionais.

Given the fast-paced rhythm of contemporary life, there is an increase in individuals choosing to have their meals outside the home. Meat, recognized as an essential component in the Brazilian diet, is susceptible to contamination as it provides a favorable environment for the proliferation of pathogenic microorganisms. It is necessary to conduct post-production contamination analysis to prevent Foodborne Diseases. This study aimed to evaluate the good manufacturing practices and contamination of roasted beef preparations from private and institutional restaurants in the city of Americana-SP. Samples of ready-to-eat meat were obtained from six commercial establishments and six institutional ones. During collection, temperatures were checked, and conformity analyses were conducted according to RDC No. 275, 2002. The samples were examined for the presence or absence of E. coli and thermotolerant coliforms at 45°C using the multiple tube technique to quantify the total coliforms. It was observed that, as stipulated by Resolution No. 43, 2015, none of the samples from commercial restaurants and the majority from institutional restaurants reached the required temperatures. Regarding the identification of E. coli through microbiological tests, it was found that six samples from commercial restaurants and four from institutional ones tested positive for the presence of this microorganism. It is concluded that samples from commercial restaurants showed higher contamination levels compared to institutional restaurant samples.

Effects of tobacco smoke on the expression of virulence genes in Escherichia coli.

It is widely acknowledged that smoking exacerbates the severity of infectious diseases. A presumed mechanism involves the damage inflicted by tobacco smoke on the organs of host organisms. In this study, an alternative hypothesis was explored: smoking enhances the virulence of bacteria. This possibility was investigated using Escherichia coli as the model bacteria and Drosophila as the host organism. Our inquiry focused on the potential gene expression changes in E. coli subsequent to exposure to tobacco smoke extracts. Analysis of the transcription promoter activity of genes encoding proteins within the E. coli two-component system, a regulatory machinery governing gene expression, revealed the suppression of thirteen out of 23 promoters in response to tobacco smoke extracts. Subsequently, Drosophila was infected with E. coli exposed to tobacco smoke extracts or left untreated. Interestingly, there were no significant differences observed in the survival periods of Drosophila following infection with E. coli, whether treated or untreated with tobacco smoke extracts. Contrary to the initial hypothesis, the findings suggest that while tobacco smoke extracts alter gene expression in E. coli, these changes do not appear to impact bacterial virulence. Although this study has illuminated the influence of tobacco smoke extracts on the gene expression of E. coli, further analyses are necessary to elucidate the implications of these changes. Nevertheless, the results imply that smoking affects not only host organisms but may also exert influence on invading bacteria.

The Next-Generation Probiotic E. coli 1917-pSK18a-MT Ameliorates Cadmium-Induced Liver Injury by Surface Display of Metallothionein and Modulation of Gut Microbiota.

Cadmium (Cd) is recognized as being linked to several liver diseases. Currently, due to the limited spectrum of drugs available for the treatment of Cd intoxication, developing and designing antidotes with superior detoxification capacity and revealing their underlying mechanisms remains a major challenge. Therefore, we developed the first next-generation probiotic E. coli 1917-pSK18a-MT that delivers metallothionein (MT) to overcome Cd-induced liver injury in C57BL/6 mice by utilizing bacterial surface display technology. The results demonstrate that E. coli 1917-pSK18a-MT could efficiently express MT without altering the growth and probiotic properties of the strain. Moreover, we found that E. coli 1917-pSK18a-MT ameliorated Cd contamination-induced hepatic steatosis, inflammatory cell infiltration, and liver fibrosis by decreasing the expression of aminotransferases along with inflammatory factors. Activation of the Nrf2-Keap1 signaling pathway also further illustrated the hepatoprotective effects of the engineered bacteria. Finally, we showed that E. coli 1917-pSK18a-MT improved the colonic barrier function impaired by Cd induction and ameliorated intestinal flora dysbiosis in Cd-poisoned mice by increasing the relative abundance of the Verrucomicrobiota. These data revealed that the combination of E. coli 1917 and MT both alleviated Cd-induced liver injury to a greater extent and restored the integrity of colonic epithelial tissues and bacterial dysbiosis.