Search Results (163)

Staphylococcus aureus is an important pathogen that can cause widespread infections as well as severe outbreaks of food poisoning. Recent studies have drawn attention to foodborne pathogens such as S. aureus endowed with the ability to form biofilms and increase resistance to antimicrobial agents as well as environmental stress, posing challenges to food safety. The Clp (caseinolytic protease) protein complex plays a crucial role in energy-dependent protein hydrolysis processes. This mechanism is a common way to maintain intracellular homeostasis and regulation in both prokaryotic and eukaryotic cells, especially under stress conditions. In S. aureus, multiple genes encoding Clp ATPase homologues have been identified: clpC, clpB, clpY, clpX, and clpL. This study investigated the roles of clpC in stress tolerance and biofilm formation of foodborne S. aureus RMSA24 isolated from raw milk. Our results showed that the deletion of the clpC gene significantly reduced the bacterium’s tolerance to heat, desiccation, hydrogen peroxide, and high osmotic pressure compared to wild type (WT). Furthermore, the clpC knockout mutant also exhibited a marked decrease in biofilm formation using Crystal Violet Staining (CVS) and Scanning Electron Microscopy (SEM). Finally, compared to WT, there was a total of 102 DEGs (differentially expressed genes), with a significant downregulation of genes related to biofilm formation (isaA and spa) and heat-shock response (clpP and danJ). These findings suggest that clpC regulates environmental tolerance in S. aureus by modulating the expression of stress- and biofilm-related genes, positioning it as a potential biomarker and a novel target for controlling contamination in the food industry. Full article

Komagataeibacter species are the best producers of bacterial nanocellulose membranes (BNC)—amazing biomaterial with unique properties and applications in the medical and food industries. The molecular mechanisms of BNC synthesis control remain poorly understood and the need for BNC production and structure improvement is growing. Looking for the genes significant for biosynthesis, we studied the unexplored effect of ClpP proteolytic subunit inactivation on Komagataeibacter xylinus E₂₅ cell morphology and production of BNC. A mutant with a disrupted clpP gene and a complemented strain were obtained. The colonies of the mutant cells, in contrast to the wild-type and complemented ones, were smaller, irregular, and were surrounded by a polymeric noncellulosic envelope. Additionally, the mutant cells were longer and organized in chains, showing different growth and production dynamics of BNC when grown under standard conditions. We also observed worse growth and production of BNC at 5 °C above optimal temperature and in the presence of increased levels of ethanol. E₂₅ mutant cells were characterized by lower viability under stress conditions. The 3D microstructure of BNC displayed thicker fibers and denser packing and contained more hard-to-extract exopolysaccharides (HE-EPSs). Based on the outcomes, we conclude that the effect of ClpP on K. xylinus decreased resistance to stress and lowered the BNC production level. Full article

Background: Sepsis-induced lung injury is a major clinical challenge, frequently complicated by pulmonary microthrombosis, which exacerbates disease severity and worsens prognosis. Recent studies suggest that proprotein convertase subtilisin/kexin type 9 (PCSK9) is involved in inflammatory and thrombotic processes. However, the contribution of PCSK9 to pulmonary microthrombosis in sepsis remains unclear. Methods: Adult male C57BL/6J mice were subjected to cecal ligation and puncture (CLP) to induce sepsis. Twelve hours post-CLP, mice received either recombinant PCSK9 (1 μg, i.p.) or the PCSK9 inhibitor evolocumab (10 mg/kg, i.p.). Platelet activation and pulmonary microthrombosis were assessed using hematoxylin–eosin staining, immunofluorescence, and thromboelastography. Results: Administration of PCSK9 significantly enhanced platelet activation and increased neutrophil extracellular trap (NET) formation, promoting pulmonary microthrombosis in septic mice. In contrast, administration of evolocumab effectively attenuated platelet activation, reduced NET formation, and alleviated pulmonary microthrombosis. Conclusions: PCSK9 exacerbates sepsis-induced pulmonary microthrombosis by promoting platelet activation and NET formation. Targeting PCSK9 may represent a novel therapeutic strategy for preventing thrombotic complications in sepsis-induced lung injury. Full article

Listeria monocytogenes is a foodborne pathogen of significant public health concern due to its high environmental resilience and ability to cause severe infections in vulnerable populations. The objective of the present study is to characterize foodborne strains of Listeria monocytogenes isolated between 2020 and 2024 in northwestern Italy. Lm was detected through isolation, biochemical confirmation and molecular serogrouping. Next generation sequencing (NGS) analysis was used to characterize the strains in terms of virulence and antibiotic resistance. A total of 39 positive samples were identified from various food matrices, including meat products, fish, cheeses and ready-to-eat foods. The most frequently detected serogroups were IIc and IIa, with a notable presence of the highly virulent IVb group. Next-generation sequencing (NGS) was applied to all isolates, revealing the presence of virulence genes associated with the LIPI-1 island and internalins. In addition to pathogenicity islands, genes related to stress resistance (clpCEP, Gad A, GadB, GadC), biofilm production (agrA, flaA, degU, hfq) and sortase-mediated anchoring of surface protein (strA, strB) have been identified. The presence of antibiotic resistance genes was confirmed, with all isolates harboring the fosX gene. Moreover, four isolates exhibited resistance determinants against antibiotics belonging to two different classes: tetracyclines (tetM) and lincosamides (lsa(A)). Multilocus sequence typing (MLST) showed that clonal complex CC9 was the most prevalent among the isolates. Further, cgMLST and SNP analyses identified a principal cluster of closely related strains, which were isolated from meat products. These findings highlight the need for continuous surveillance of L. monocytogenes. Full article

Drug-resistant Mycobacterium tuberculosis (Mtb) remains a major global health challenge, prompting the need for new therapeutics targeting essential bacterial proteins. The caseinolytic protein C1 (ClpC1) is a promising drug target, and accurate measurement of its ATPase activity is critical for understanding drug mechanisms. We optimized a sensitive luminescence-based ATPase assay and evaluated ClpC1 constructs with various tag positions and truncations. N-terminal tagging significantly impaired enzymatic activity, whereas C-terminal tagging had no effect; truncated domains showed reduced activity compared to native full-length (FL) ClpC1. Using the native FL-ClpC1, we assessed ecumicin (ECU) and five analogs via ATPase activity and surface plasmon resonance (SPR), using rufomycin (RUF) and cyclomarin A (CYMA) as controls. RUF and CYMA bound tightly (K_D = 0.006–0.023 µM) and inhibited Mtb growth (MIC₉₀ = 0.02–0.094 µM) but modestly stimulated ATPase activity (≤2-fold). In contrast, ECU and its analogs strongly enhanced ATPase activity (4–9-fold) despite slightly weaker binding (K_D = 0.042–0.80 µM) and growth inhibition (MIC₉₀ = 0.19 µM). The partial correlation among AC₅₀, K_D, and MIC values highlights the complementary value of enzymatic, biophysical, and cellular assays. Our assay platform enables mechanistic characterization of ClpC1-targeting compounds and supports rational antitubercular drug development. Full article

Septic shock has an unacceptably high mortality rate and unmet need for new therapeutics. Murine models are crucial for research, yet methodologies often differ. This study characterised standard- and high-grade caecal ligation and puncture (CLP) murine models of septic shock by integrating ultraminiature arterial telemetry with comprehensive plasma biomarker analysis. Standard-grade and high-grade CLP was performed in 8–10 week old, male C57BL/6 mice (n = 98), with a subset implanted with arterial telemetry to monitor real-time circulatory function. Plasma markers of inflammation and organ damage were measured at multiple intervals up to 168 h post-CLP. Standard-grade and high-grade CLP showed distinct progressions; episodes of hypotension began 5–6 h after CLP in 30% of standard-grade and all high-grade CLP mice, with respective 168 h mortality of 40% and 71%. Recurrent episodes of hypotension 5–39 h after CLP were universally lethal. The coincidence of hypotension and elevated plasma lactate defined the onset of septic shock after high-grade CLP, which was always lethal. Inflammatory cytokines and markers of liver, renal, and cardiac damage were markedly elevated to 168 h after high-grade CLP, in contrast to standard-grade CLP, which returned to baseline by 48 h. Elevated plasma IL-6, TNFα, and corticosterone, along with reduced albumin, were significantly correlated with mortality. In conclusion, this research refines murine CLP models by providing a precise, dynamic map of the progression to septic shock. The high-grade CLP model consistently models early and late-stage physiological deterioration and serves as a robust model for evaluating the efficacy of novel therapies aimed at human septic shock. Full article

Diketopiperazines (DKPs) are biologically important cyclic dipeptides widespread in nature, associated primarily with microorganisms. This is the case for the 2,5-DKP derivative cyclo(L-Leu-L-Pro) (cLP), also known as gancidin W or PPDHMP, identified from a variety of bacteria and fungi, and occasionally found in food products. The present review retraces the discovery of cLP, its identification in living species, its chemical syntheses, and its biochemical properties. In bacteria, cLP is often associated with other DKPs to serve as a defense element against other microorganisms and/or as a regulator of bacterial growth. cLP plays a role in quorum-sensing and functions as an anticariogenic and antifungal agent. The antimicrobial mechanism of action and molecular targets of cLP are evoked. The interest in cLP for combatting certain parasitic diseases, such as malaria, and cancers is discussed. The capacity of cLP to interact with CD151 and to down-regulate the expression of this tetraspanin can be exploited to reduce tumor dissemination and metastases. The review sheds light on the pharmacology and specific properties of cyclo(L-Leu-L-Pro), which can be useful for the development of a novel therapeutic approach for different human pathologies. It is also of interest to help define the bioactivity and mechanisms of action of closely related DKP-based natural products. Full article

Sepsis is a life-threatening condition driven by a dysregulated host response to infection, with high mortality and few treatment options. Decades of failed drug development underscore the urgent need for therapies with novel mechanisms of action. Vimentin, an intermediate filament protein, acts as a network hub that senses and integrates cellular signals. Its involvement in key sepsis pathologies, including infection, hyperinflammation, immunosuppression, coagulopathy and metabolic dysregulation, positions it as a potential therapeutic target. This study evaluated the efficacy of ALD-R491, a novel small-molecule vimentin modulator, in a murine model of polymicrobial sepsis induced by cecal ligation and puncture (CLP). Mice received ALD-R491 prophylactically or therapeutically, alone or with ceftriaxone. The treatment significantly reduced serum levels of key biomarkers of sepsis, including C-reactive protein (CRP), lactate (Lac), tumor necrosis factor-alpha (TNF-α) and interleukin-6 (IL-6), and dose-dependently improved the survival of septic mice. Organ-specific analysis confirmed the effects of ALD-R491 in mitigating hyperinflammation and multi-organ injury. The treatment reduced pulmonary edema and inflammation; preserved liver tissue architecture and improved hepatic function with lowered alanine aminotransferase/aspartate aminotransferase (ALT/AST); decreased kidney tubular damage; and improved renal function with lowered creatinine/blood urea nitrogen (BUN). These preclinical findings indicate that the vimentin-targeting agent ALD-R491 represents a promising therapeutic candidate for sepsis and merits further clinical investigation. Full article

Dental caries, a prevalent biofilm-mediated chronic disease, causes enamel demineralization, pulp infection, and systemic complications. Dental plaque biofilm is the initiating factor for the occurrence and development of caries. Streptococcus mutans is an opportunistic pathogen linked to the structure and ecology of dental plaque biofilms. The molecular mechanism of S. mutans during biofilm ontogeny in driving cariogenesis has been extensively elucidated. Here, we observed that asiatic acid is a potent biofilm disruptor that selectively dismantles S. mutans biofilm architectures, prompting us to investigate its mechanism. The minimum biofilm inhibition concentration (MBIC) of asiatic acid on S. mutans was 62.5 μM, but the MBIC level did not substantially impede planktonic growth. Using the static active-attachment model, it was demonstrated that asiatic acid significantly reduced biofilm biomass (p < 0.001) and extracellular polysaccharides (EPS) content (p < 0.001), while concurrently diminishing acid production (p = 0.017) and metabolic activity (p = 0.014). Confocal and scanning electron microscopy further confirmed structural disintegration, including bacterial detachment and reduced biofilm thickness. Transcriptome analysis of S. mutans biofilm treated with asiatic acid revealed 454 differentially expressed genes (adjusted p < 0.05, |log₂FC| ≥ 1). Notably, genes related to the CiaRH two-component system (ciaR, ciaH), a central regulatory hub for biofilm maturation and acid tolerance. This disruption initiates a downstream cascade, causing a coordinated downregulation of critical gene clusters essential for virulence and pathogenesis, including stress response (htrA, clpP, groEL, dnaK), and the glucan-binding protein gene (gbpC) essential for biofilm structural integrity. These findings provide the first mechanistic evidence linking asiatic acid to transcriptional reprogramming in S. mutans biofilm, offering a novel ecological strategy for caries prevention by targeting key regulatory pathways. Full article

Cold stress limits the growth and productivity of Hevea brasiliensis, the primary source of natural rubber. This study investigated early transcriptomic responses in Reyan ‘7-33-97’ seedlings exposed to 4 °C, 10 °C, and 15 °C for 1, 2, and 4 h with room temperature (25 °C) as the control. RNA sequencing identified 9894 differentially expressed genes (DEGs), with the most significant transcriptional changes observed at 10 °C, indicating that genes to resist cold stress could not be mobilized at 4 °C, resulting in poor cold resistance of the rubber tree. KEGG enrichment analysis of DEGs between 10 °C (2 h) and 4 °C (2 h) revealed that genes involved in tryptophan metabolism (HbKynL.x1, HbKynL, HbCLP1, HbCLP2) and carbon metabolism (TCH4, XTH23), which contribute to cell wall modification, exhibited higher expression at 10 °C. Gene Ontology enrichment analysis highlighted significant involvement of “thylakoid,” “photosystems,” and “photosynthetic membrane,” alongside molecular functions such as “xyloglucan transferase activity” and “transcriptional regulator activity.” The interacting network of key pathways, including carbon metabolism (ko01200) and carbon fixation (ko00710) pathways, was sorted out, highlighting their integration with plant hormone signal transduction. Complex signaling networks, including MAPK, and kynurenine pathways coordinate the expression of cold-responsive genes and protective proteins, and it was confirmed and speculated that there is crosstalk response in cold defense mechanisms. Furthermore, 61 DEGs were associated with antioxidant processes, including major catalase and peroxidase enzymes. Our study shows that rubber trees physiological activities that respond to low-temperature signals cannot be carried out normally at 4 °C. The newly discovered metabolic pathway and the reason for abnormal cold signal transduction at low temperatures are the focus of future research on cold resistance. Full article

Tuberculosis (TB), an infection caused by Mycobacterium tuberculosis, affects nearly one-third of the world’s population. It is estimated that TB infects around ten million people worldwide, with no less than two million fatalities annually. It is one of the treatable infections due to improved diagnostic tools and therapeutic agents. However, the disease remains a threat to humankind due to the emergence of multidrug- and extensively drug-resistant strains of M. tuberculosis. This has driven many researchers to look for new antitubercular medications with better efficacy, safety, and affordability. As has always been the case, natural products have provided huge potential as a source of remedies for various infectious and non-infectious diseases. This review aims to report discoveries and updates of antitubercular natural products with minimum inhibitory concentration (MIC) values of less than or 10 µg/mL or 50 µM and selectivity indices of greater than 10. The review discusses 36 naturally occurring compounds from various classes, isolated from both terrestrial and aquatic organisms, including higher plants and microorganisms. Perusal of the literature reveals that most of these promising compounds are alkaloids, terpenoids, steroids, and peptides. Rufomycin I, a cyclic heptapeptide from Streptomyces sp., showed potent activity against drug-sensitive and isoniazid-resistant M. tuberculosis H37Rv (MIC < 0.004 µM), surpassing isoniazid (MIC = 0.23 µM), likely by inhibiting ClpC1 transcription. Hapalindole A also displayed strong activity (MIC < 0.6 µM). Current TB drugs have become less effective; therefore, natural products such as hapalindole A and rufomycin I, owing to their potent activity, selectivity, and novelty, are increasingly recognized as potential lead compounds against TB. Full article

Sepsis-induced myocardial injury is age-related and leads to increased mortality. Considering the importance of mitochondrial dysfunction in cardiac impairment, we aimed to investigate whether aging exacerbates the cardiac mitochondrial metabolic response to inflammation, thus leading to increased cardiac dysfunction in the elderly. Cecal ligation and puncture (CLP) was conducted in young adult (12–18 weeks) and aged (19–21 months) male C57BL/6 mice. Cardiac function was detected 20 h post-CLP. Additionally, cardiomyocytes isolated from young adult and aged male mice were used for assessments of mitochondrial respiratory function +/– TNFα or LPS. Protein levels of oxidative phosphorylation (OXPHOS), NADPH oxidase (NOX)2, NOX4, phosphor-STAT3 and STAT3 were determined in mouse hearts 24 h post-CLP and in cardiomyocytes following inflammatory stimuli. CLP significantly reduced cardiac contractility in both young and aged mice, with a higher incidence and greater severity of cardiac functional depression in the older group. Mitochondrial respiratory capacity was decreased in cardiomyocytes derived from aged mice, with increased susceptible to inflammatory toxic effects compared to those from young adult mice. The age-dependent changes were observed in myocardial OXPHOS complexes and NOX4. Importantly, CLP led to a significant increase in OXPHOS protein levels in the hearts of older mice, suggesting a possible compensatory response to decreased mitochondrial metabolic function and a greater potential for reactive oxygen species (ROS) generation. Our findings highlight that the response of aging-impaired mitochondria to inflammation may underlie the worsened cardiac functional depression in the aged group during sepsis. Full article

Nitric oxide (NO) has a dual effect on mitochondria. Incubating liver mitochondria with NO improves oxidative phosphorylation (OXPHOS) efficiency by decreasing state 4 respiration more than ATP synthesis and preventing mitochondrial permeability transition pore (mPTP) opening. We evaluated the effect of L-arginine (L-arg), an NO donor, on isolated liver mitochondrial respiration and mPTP in sepsis. Male mice were subjected to cecal ligation and perforation (CLP) with saline resuscitation or sham. After 8, 24, and 48 h, with and without L-arg, we measured isolated liver mitochondrial respiration and cytochrome c oxidase (COX) activity using polarographic methods and calcium retention capacity (CRC) to assess the mPTP and NO metabolites via the Griess reaction. Mitochondrial NO synthase (mtNOS) was identified by Western blot. CLP decreased state 3 respiration at 24 and 48 h, decreased COX activity at 8, 24, and 48 h, and increased state 4 respiration and decreased the respiratory control ratio (RCR) and CRC at 48 h. L-arg increased NO levels at 8 h, decreased state 4 respiration more than state 3 respiration (−39% versus −12%) at 48 h, decreased the CRC in the CLP groups at 24 and 48 h, but did not improve RCR. Our data suggests that L-arg does not restore liver mitochondrial OXPHOS efficiency or prevent mPTP opening in the late or recovery phases of sepsis. Full article

Clostridioides difficile is a Gram-positive bacterium recognized for its ability to produce toxins and form spores. It is mainly accountable for the majority of instances of antibiotic-related diarrhea. Background. Bacterial persister represent a minor fraction of the population that shows temporary tolerance to bactericidal agents, and they pose considerable medical issues because of their link to the rise of antibiotic resistance and challenging chronic or recurrent infections. Our previous research has shown a persister-like phenotype associated with treatments that include pefloxacin. Nonetheless, the mechanism is still mostly unclear, mainly because of the difficulty in isolating this small group of cells. Objectives. To enhance the understanding of C. difficile persister cells, we made an enrichment and characterization of these cells from bacterial cultures during the exponential phase under pefloxacin treatment and lysis treatment. Results. We demonstrate the appearance of cells with lower metabolism and DNA damage. Furthermore, we noted the participation of toxin–antitoxin systems and Clp proteases in the generation of persister cells. Conclusions. This work demonstrates the formation of C. difficile persister cells triggered by a lethal concentration of pefloxacin. Full article

Pyrazinamide (PZA) is a key component of tuberculosis treatment, with drug resistance (PZA^R) primarily related to pncA mutations. However, discordance between phenotypic resistance and conventional pncA-based molecular diagnostics challenges diagnostic accuracy. This study investigates discrepancies between phenotypic and genotypic resistance profiles among Mycobacterium tuberculosis (Mtb) clinical isolates. Fifty-three Mtb isolates from Guangzhou Chest Hospital were tested for PZA resistance using the BACTEC MGIT 960 system and PZase activity assay. Thirty-one phenotypically PZA^R strains were genetically assessed by Sanger sequencing of PZA^R-associated customary genes. Five pncA-wild-type PZA^R strains were investigated through whole-genome sequencing. ClpC1P1P2 activity was evaluated by proteolytic degradation assay. Notably, 26/31 of the PZA^R strains harbored mutations in pncA and/or its upstream region, aligning PZase activity and phenotypic profiles. However, five PZA^R strains lacked pncA mutations. The WGS of five discordant strains revealed four novel mutations (Gly58Ser, Val63Ala, Ala567Val, and Pro796Leu) across ClpC1 domains. Incorporating clpC1 mutations improved molecular diagnostic sensitivity and accuracy from 48.3% and 69.8% (pncA alone) to 100%. This is the first report from southern China that identifies novel clpC1 mutations in wild-type pncA PZA^R Mtb isolates. Our findings underscore the limitations of pncA-targeted diagnostics and support the integration of WGS and clpC1 analysis in molecular diagnostics to prevent false-negative diagnoses and improve clinical outcomes. Full article