Search Results (346)

Bacterial pathogens exploit host-derived nutrients to coordinate metabolism and virulence determinants to optimize fitness in vivo. In Pseudomonas aeruginosa, GlnK is a central regulator of nitrogen metabolism. It senses the intracellular nitrogen status by integrating 2-oxoglutarate (2-OG) and glutamine signals, which in turn triggers its uridylylation and conformational changes. This reversible post-translational modification modulates its interaction with target proteins, thereby precisely regulating carbon-nitrogen metabolic homeostasis and enabling adaptive nitrogen metabolism in response to host-derived nutrient cues. In this study, we found that glnK is upregulated during infection in a mouse pneumonia model. By growing bacteria in mouse bronchoalveolar lavage fluid (BALF), we demonstrated that the expression of glnK is activated by the NtrB-NtrC two-component regulatory system in response to the host nutrient environment. Mutation of glnK impairs bacterial virulence. Transcriptomic analysis revealed downregulation of the type III secretion system (T3SS) genes in the glnK mutant. Further studies revealed a role of GlnK in maintaining the homeostasis of the NtrB-NtrC system through a negative feedback mechanism, which is required for the expression of the T3SS genes. Collectively, these findings reveal a role of GlnK in interconnecting carbon–nitrogen balance and the T3SS in response to the host environment. Full article

Xanthomonas arboricola pv. pruni causes bacterial spot in peaches, a major disease affecting global Prunus production. Despite its economic significance, the virulence mechanisms that enable X. arboricola pv. pruni to colonize peach tissues and induce characteristic necrotic symptoms remain poorly understood. To identify key virulence determinants, a robust and reliable detached-leaf inoculation system was developed, and a genome-wide forward genetic screen of 2400 Tn5 mutants was conducted. A total of 34 mutants with consistently reduced virulence were identified, representing diverse functional categories including secretion systems, nutrient acquisition, primary metabolism, and regulatory pathways. The most prominent findings were the repeated identification of independent mutants in two type III effector genes, xopN and xopX, highlighting these effectors as central and nonredundant contributors to symptom induction. Mutants in the type III secretion system translocon-associated gene hrpF also showed virulence defects. Additional mutants affecting phosphate uptake (pstS), ammonium transport, and vitamin B₆ biosynthesis (pdxA, serC) revealed metabolic requirements essential for in planta fitness. Notably, several mutants reached bacterial population levels comparable to the wild-type isolate but produced little or no symptoms, indicating that bacterial multiplication and symptom development are not necessarily linked. This study provides the first comprehensive genome-wide functional screen of X. arboricola pv. pruni virulence and establishes a framework for dissecting infection mechanisms. The essential factors identified here, particularly XopN, XopX, and key metabolic pathways, represent promising targets for future anti-virulence strategies to manage bacterial spot disease. Characterizing the specific functions of each virulence factor identified in this study will be an important focus of future work. Full article

Background/Objectives: Shigella is the leading bacterial cause of diarrheal disease worldwide. Although multiple vaccine candidates are under development and in clinical trials, no Shigella vaccine is currently available on the market. Shigella comprises four species: S. dysenteriae, S. flexneri, S. boydii, and S. sonnei. S. flexneri has been recognized as the most prevalent species, particularly in low- and middle-income countries (LMICs), and the top serotypes are S. flexneri 2a, 3a and 6. Conversely, S. sonnei has a single serotype and predominates in high-income countries (HICs). Invasion plasmid antigen B (IpaB) is a critical virulence factor of Shigella type III secretion system (T3SS) that is highly conserved across Shigella serotypes. Here, we report the development of a Shigella quadrivalent O-polysaccharide-IpaB conjugate vaccine candidate (IVT Shigella-04). Methods: IVT Shigella-04 contains O-polysaccharides (O-PS) from S. flexneri 2a, 3a, 6, and S. sonnei, each individually conjugated to recombinantly expressed IpaB as the carrier protein using 1-cyano-4-dimethylaminopyridinium tetrafluoroborate (CDAP) chemistry. The immunogenicity of IVT Shigella-04 was evaluated in a rabbit immunization model. Results: Baseline (day 0) IgG concentrations were low for all four Shigella serotypes (<0.5 µg/mL). Following two doses on day 0 and day 28 (2.5 µg of each conjugate per dose; total 10 µg), IgG geometric mean concentrations increased significantly (p < 0.001) by day 42, reaching 67.96 µg/mL (2a), 91.56 µg/mL (3a), 371.31 µg/mL (6), and 11.00 µg/mL (sonnei). Consistently, serum bactericidal activity (SBA) at day 42 increased 13-fold (2a), 34-fold (3a), 63-fold (6), and 224-fold (sonnei) relative to baseline (day 0). Conclusions: IVT Shigella-04 elicited robust serotype-specific humoral and functional immune responses in preclinical models, supporting its further development toward clinical evaluation. Full article

Caffeoyl hexapeptide-9 (CH-9) is a novel cosmetic peptide designed by conjugating hexapeptide-9 (H-9), a known collagen-mimetic peptide with established skin anti-aging activity, with caffeic acid (CA) via an amide bond, leveraging peptide-drug conjugate (PDC) design principles. In ultraviolet (UV)-irradiated cellular and skin models, CH-9 outperformed H-9 in preserving cell viability, restoring collagen types I, III, and IV, and suppressing interleukin-6 and -8 secretion. Additionally, its direct antioxidant activity, absent in H-9, was demonstrated in vitro by scavenging of hydroxyl and peroxyl radicals. Molecular docking indicated CH-9 interacted with the catalytic domain of matrix metalloproteinase 2 (MMP2), a key enzyme in collagen degradation during photoaging, suggesting a potential inhibition of its activity. Molecular dynamics (MD) simulations revealed an improved insertion of CH-9 into a stratum corneum (SC) lipid bilayer compared to H-9, consistent with enhanced skin permeation in vivo. Moreover, CH-9 exhibited improved aqueous and cosmetic serum stability over CA. In a 28-day clinical study, topical application of CH-9 significantly improved skin elasticity and firmness compared to H-9. This work demonstrates that the PDC-based conjugate CH-9 combines enhanced anti-photoaging efficacy with improved transdermal permeation and stability, highlighting a promising strategy for the development of advanced cosmetic ingredients. Full article

Pathogenic bacteria utilize a type III secretion system (T3SS) to inject type III effectors (T3Es) into plant cells, suppressing plant immunity and facilitating colonization. Paracidovorax citrulli, the causal agent of bacterial fruit blotch (BFB) of Cucurbitaceae crops, harbors a functional T3SS like many other plant pathogens. The expression of its T3SS and T3Es is regulated by the two-component system response regulators HrpG and HrpX. Here, we demonstrate that the aspartic acid (Asp) residues at positions 52 and 60 in P. citrulli HrpG are essential for its complete function. Plasmid-mediated complementation of the ΔhrpG mutant with hrpG carrying Asp52→alanine (Ala) or Asp60→Ala mutations failed to restore the ability of P. citrulli to induce a hypersensitive response (HR) in tobacco, whereas the Asp46→Ala mutation fully rescued this phenotype. Furthermore, genomic hrpG point mutations generating strains Aac5 (D52A) and Aac5 (D60A) abolish the activation of hrpX transcription, resulting in decreased HrpX accumulation. Collectively, Asp 52 and Asp 60 in P. citrulli HrpG are essential for transcriptional activation activity of hrpX and HR induction, serving as a potential phosphorylation site (Asp 52) for upstream histidine kinases and a Mg²⁺ coordination site (Asp 60). Given that conserved Asp residues often function as phosphorylation sites in two-component system response regulators, this study provides a foundation for identifying upstream histidine kinases that modulate HrpG activity in P. citrulli. Full article

RNA methyltransferases are key regulators of bacterial physiology, yet their specific roles in virulence remain poorly defined. In this study, we characterize PA3840, a putative RNA methyltransferase in Pseudomonas aeruginosa (P. aeruginosa). Deletion of PA3840 specifically impaired twitching motility without affecting bacterial growth, swimming, or swarming. Notably, PA3840 was found to suppress the expression of Type III Secretion System (T3SS) genes, thereby reducing cytotoxicity and host cell rounding. Consistent with these observations, PA3840 expression attenuated pro-inflammatory cytokine production in epithelial cells by inhibiting NF-κB activation. Mechanistic analysis revealed that PA3840 is translocated into host cells in a Type VI Secretion System (T6SS)-dependent manner. This translocation was reduced by hcp1 deletion and nearly abolished by a double deletion of pscF and hcp3, suggesting the involvement of multiple T6SS components and potential interplay with T3SS machinery. However, direct transfection of PA3840 into host cells failed to suppress cytokine expression, indicating that its immunomodulatory function is mediated by a bacterium-intrinsic mechanism rather than direct intracellular action. Collectively, these findings identify PA3840 as a translocated effector that modulates twitching motility and dampens host inflammation by repressing T3SS and NF-κB signaling, revealing a novel layer of post-transcriptional virulence regulation in P. aeruginosa. Full article

This study evaluated the formulation and stability of a quadrivalent glycoconjugate Shigella vaccine candidate based on four predominant strains (S. flexneri; 2a, 3a, and 6, and S. sonnei) covering ~64% of global Shigella infections. Each glycoconjugate antigen consists of a strain-specific O-polysaccharide (O-PS) covalently linked to the carrier protein IpaB, a component of the Shigella type III secretion system. First, selective competitive ELISAs were developed to measure antigenicity of the four O-PS-IpaB conjugates formulated with different adjuvants (i.e., Alhydrogel^®, AH; Adju-phos^®, AP; and CpG-1018^®, CpG). Next, the monovalent S. sonnei O-PS-IpaB conjugate was studied to elucidate interactions with aluminum salt adjuvants (AH, AP) under different solution conditions. Third, the stability profiles of AH- or AP-adjuvanted S. sonnei O-PS-IpaB conjugate in various formulations (±CpG) were determined at different temperatures. Interestingly, incubation at 25 °C for 2 weeks resulted in increased antigenicity values when the antigen was bound to AP or AH, suggesting increased epitope exposure upon adjuvant binding. When bound to AP adjuvant at pH 5.8, the best glycoconjugate antigen stability was observed at elevated temperatures. The CpG adjuvant under these conditions, however, displayed incompatibility (i.e., material loss), presumably from precipitation due to lack of interaction with AP and presence of the detergent LDAO from the bulk antigen buffer. In contrast, the glycoconjugate antigen and CpG adjuvant were both bound to the AH adjuvant and stable at 2–8 °C, pH 7.0. This AH-CpG formulation of the O-PS-IpaB conjugate antigens was identified as a promising candidate for future animal immunogenicity testing. Full article

Tendon injuries present significant clinical challenges due to limited intrinsic healing and complex pathological mechanisms. Here, we developed a novel 3D bioprinted methacrylated type I collagen (ColMA) scaffold integrated with Growth Differentiation Factor-5 (GDF-5)-loaded Poly (lactic-co-glycolic acid) (PLGA) nanoparticles and dynamically cultured it under perfusion to establish a tenogenic microenvironment in vitro. Pathological human Tendon Stem/Progenitor Cells (hTSPCs) derived from tendinopathic surgical explants were encapsulated to investigate their impaired extracellular matrix (ECM) deposition and associated pro-inflammatory signaling. GDF-5-loaded nanoparticles (average diameter 140 ± 40 nm) were fabricated via microfluidic-assisted nanoprecipitation and homogeneously incorporated within the ColMA synthetic ECM to enable sustained growth factor release. Continuous perfusion culture (1 mL/min) ensured efficient mass transfer and supported cell viability above 70% over 21 days. Pathological hTSPCs exhibited impaired ECM remodeling, characterized by the absence of type I collagen and a 2.56-fold increase in type III collagen at day 7, indicative of a fibrotic-like phenotype. Western blot densitometry demonstrated a 5.31-fold elevation in secreted tenomodulin at day 14, while ECM analysis verified a type III to type I collagen ratio of 4.5. In addition, a markedly pro-inflammatory cytokine profile was observed, with elevated secretion of interleukin-6 (IL-6) and interleukin-8 (IL-8) from day 7 onward, consistent with the chronic inflammatory status of cells derived from pathological tendon tissues. This modular 3D platform represents a robust in vitro model for mechanistic studies and the advancement of personalized regenerative strategies targeting chronic tendon disorders. Full article

One of the hallmarks of Osteogenesis Imperfecta (OI) is phenotypic variability among individuals with the same mutation. The aim of our study is to investigate the under-explored role of osteoblast differentiation in OI phenotypic variability by using human and murine OI osteoblasts. This is the first comparative study of osteoblasts from OI patients vs. healthy pediatric controls. We investigated osteoblasts carrying COL1A1 substitutions Gly352Ser and Gly589Ser, each expressed in two unrelated patients differing in phenotypic severity. Osteoblasts from type III OI patients with both mutations deposited significantly less mineral vs. type IV. RNA-Seq showed osteoblasts from type IV OI patients with different mutations had downregulated mitochondrial pathways, while osteoblasts from type III OI patients showed downregulation of extracellular matrix pathways. Puromycin assay demonstrated osteoblast protein synthesis was significantly upregulated in type III vs. type IV OI patients. UPR PERK and BiP were reduced in osteoblasts with Gly352Ser from type III and IV OI patients and in osteoblasts with Gly589Ser from a type III OI patient, while both proteins were increased in Gly589Ser osteoblasts from the type IV patient. Additionally, in a murine comparative study, Col1a1 Gly349Ser, called Brtl Ser, showed a much more severe skeletal phenotype than Brtl Cys. Brtl Ser calvarial osteoblasts had reduced collagen secretion and folding with abnormal dermal collagen fibrils vs. wildtype. Also, Brtl Ser osteoblasts showed condensed actin filaments but a similar mineral deposition as Brtl Cys. Electron microscopy revealed elongated mitochondria with cristae dropout in patient and mutant murine osteoblasts. Our study yielded novel insights highlighting osteoblast differentiation, mineralization, and a potential role of mitochondria in OI pathology and phenotypic variability. Full article

Irisin is a myokine secreted by muscle in response to exercise. It is derived from a transmembrane protein called fibronectin type III domain-containing protein 5 (FNDC5). The Fndc5 gene is expressed in several tissues, including skeletal muscle, brain, adipose tissue, heart, kidney, and lung. Irisin is cleaved from FNDC5 protein by the enzyme furin and released into circulation. In addition to exercise, several drugs have been shown to increase the production of irisin. Administration of exogenous irisin mimics the beneficial actions of exercise. Irisin can cross the blood–brain barrier and exert neuroprotective actions in the brain. It has been shown to reverse Alzheimer’s pathologies in clinical and animal studies. Irisin also exerts protective effects against obesity, diabetes, and cardiovascular disease, diseases that often coexist with aging AD patients. Multiple approaches have been taken to suggest that exercise may act through irisin. Studies have provided direct evidence linking the two using Fndc5 gene deletion and irisin antibodies. Irisin binds to αVβ1/β5 integrins to mediate the activation of integrin-FAK pathways. While exercise as a lifestyle modification for healthy aging is well recognized, it may present limitations in some aging populations, especially those with disease conditions, including Parkinson’s disease. Administration of exogenous irisin or small molecules that increase the expression of endogenous irisin or facilitate its actions are some alternate approaches that can mimic the beneficial actions of exercise. This review discusses the therapeutic potential of irisin in the treatment of neurodegenerative and other aging-associated diseases. Full article

Group B Streptococcus (GBS) colonization during pregnancy is a major cause of neonatal infection, yet its microbial determinants remain unclear. This pilot study compared the vaginal and gut microbiota of late-pregnancy women with and without GBS colonization to explore potential microbial cues for peripartum risk stratification. Forty-three Japanese pregnant women (GBS-Negative = 34; GBS-Positive = 9) were enrolled at 35–37 weeks of gestation. Vaginal secretions and stool were analyzed by 16S rRNA (V3–V4) sequencing using QIIME 2 with SILVA annotation and community state type (CST) classification. Vaginal communities were mainly Lactobacillus-dominant. GBS-Positive women showed a non-significant tendency toward more L. iners-dominant CST III and fewer L. crispatus-dominant CST I compared with GBS-Negative women. Prevotella, Atopobium, and Gardnerella were significantly enriched in the GBS-Positive group (false discovery rate < 0.05), whereas gut microbial diversity and composition showed no significant differences between groups. Cross-site gut–vagina genus-level correlations were generally weak and non-significant. These findings suggest that, in late pregnancy, GBS colonization is linked to subtle shifts within Lactobacillus-dominant vaginal communities, with more L. iners and bacterial vaginosis-associated genera, rather than global microbiota disruption. The apparent shift from L. crispatus- to L. iners-dominant communities is hypothesis-generating and should be confirmed in larger cohorts. Full article

Cutaneous melanoma remains one of the most aggressive tumors, yet the role innate immunity plays in its progression remains poorly understood. Effector elements with high regulatory potential, capable of both promoting and inhibiting tumor growth—mast cells (MCs), are of particular interest. This includes quantitatively characterizing the interactions between tryptase-positive mast cells (MCs) with atypical Melanin—A⁺ cells and describing their spatial phenotype, in relation to the stage of cutaneous melanoma. A retrospective analysis was carried out on samples retrieved from 128 patients with cutaneous melanoma (AJCC 8th edition: IA–IIID). Histological analysis, histochemistry (toluidine blue, Giemsa), and diplex /multiplex IHC for tryptase and Melan-A were performed; as well as Fluorescence imaging, 3D reconstructions and quantitative mapping in QuPath v 0.6.0. Proximity was assessed by the nucleus-to-nucleus distance: <10 μm (contact), 10–20 μm (paracrine zone), >20 μm (out of interaction). The relative amount of MCs in the intratumoral zone was lower than in the intact dermis, with a simultaneous increase in their absolute density per mm² in the melanoma microenvironment, maximum in the peritumoral area and most pronounced at stage II. Three types of interactions were identified: (i) juxtaposition without secretion, (ii) degranulation of MCs directed to tumor cells, (iii) melanosecretion of Melanin—A⁺ cells directed towards MCs, followed by phagocytosis of melanocores. An inverse intratumoral connection between the number of MCs and the number of Melanin—A⁺ cells was noted; MCs with elongated forms, extensive contacts and polarized tryptase secretion, including granule localization near/at the nuclei of adjacent cells, were frequently observed. The obtained data indicate stage-region-dependent bidirectional cross-talk between melanin and MCs, forming tissue spatial signals, potentially useful as biomarkers and targets for personalized therapy. Full article

Background: Toxoplasma gondii is a globally distributed apicomplexan parasite capable of causing congenital infections and spontaneous abortions in humans. While the parasite-secreted effector proteins TgGRA28 and TgGRA83 are known to mediate virulence or immune modulation, their potential as vaccine targets remains unexplored. Despite its immunomodulatory properties, the role of IL-28B (a type III interferon) in enhancing DNA vaccine efficacy against T. gondii infection remains unclear. Methods: In this study, we constructed eukaryotic expression plasmids pVAX-GRA28, pVAX- GRA83 and pVAX-IL-28B. After transfection into -293-T cell, protein expression encoding TgGRA28 and TgGRA83 was confirmed via indirect immunofluorescence assay (IFA), while IL-28B expression was analyzed by ELISA. Subsequently, C57BL/6J or IFNαR1 knockout mice were immunized with single or dual-antigen DNA vaccines, with or without the molecular adjuvant pVAX-IL-28B. Immune responses were assessed through Toxoplasma-specific antibody levels, cytotoxic T lymphocyte (CTL) activity, cytokine profiling (IFN-γ, IL-2, IL-12p40, IL-12p70), and flow cytometric analysis of lymphocyte subsets and dendritic cells (DCs). Protective efficacy was determined by survival rates and brain cyst burden following challenge with 100 or 10 ME49 T. gondii cysts, respectively. Results: Vaccination with pVAX-GRA28 and pVAX-GRA83 elicited robust humoral immune responses with increased T. gondii-specific IgG levels and also Th1-polarized immunity, characterized by elevated IgG2a/IgG1 ratio, IFN-γ-dominant cytokine responses, and enhanced DCs, CD4+ and CD8+ T-cell activation. The cocktail vaccine conferred superior protection compared to single-antigen formulations, significantly improving survival and reducing cyst formation. Co-administration of pVAX-IL-28B further augmented vaccine-induced immunity, enhancing both cellular and humoral responses. Moreover, these DNA immunization with pVAX-GRA28 and pVAX-GRA83 plus pVAX-IL-28B induced robust protective immunity that was largely independent of type I IFN signaling, consistent with type III IFN biology. Conclusions: Our findings demonstrate that TgGRA28 and TgGRA83 are promising vaccine candidates against toxoplasmosis, capable of inducing protective immunity against acute and chronic infection. Moreover, IL-28B serves as a potent genetic adjuvant, warranting further investigation for its broader application in vaccines targeting apicomplexan parasites. Full article

Enteritis is a common and recurrent disease in shrimp aquaculture, causing significant economic losses and management challenges. However, its specific causative pathogen remains unclear. Here, a pathogen strain, Vibrio parahaemolyticus VSP1, was directly isolated from shrimp with enteritis, and its pathogenicity and genomic characteristics were analyzed. Diseased shrimp exhibited lethargy, empty gut, hepatopancreatic atrophy, and severe intestinal damage. The gut bacterial community of diseased shrimp differed significantly from healthy shrimp (PERMANOVA, p < 0.05), with a 129% increase in Vibrio relative abundance. Nine Vibrio operational taxonomic units (OTUs) were enriched in diseased shrimp, and the dominant OTU1 shared 100% 16S rRNA identity with VSP1. VSP1 grew rapidly, utilized diverse carbon sources, and induced enteritis symptoms in over 90% of challenged shrimp. Genome analysis revealed 98.34% average nucleotide identity with V. parahaemolyticus ATCC 17802 and identified 156 putative virulence-related genes, mainly related to adherence, motility, and secretion systems. Unlike the strain ATCC 17802, VSP1 lacks thermostable direct hemolysin (TDH) and type III secretion system 2 (T3SS2), but contains alternative virulence factors such as Yersinia-like type IV pili and lipooligosaccharides, suggesting a distinct virulence strategy. This study identifies the pathogen responsible for shrimp enteritis and provides a foundation for targeted control strategies in aquaculture. Full article

Pseudomonas aeruginosa is a versatile Gram-negative pathogen that causes various infections in humans. The bacterium possesses a type III secretion system (T3SS) to deliver cytotoxic effector proteins into host cells, which plays an important role in bacterial pathogenesis. The T3SS is regulated by the master regulator ExsA, whose expression is controlled by multiple pathways. Here, we demonstrate that the catabolite repression control protein Crc controls T3SS activity by modulating exsA expression. We find that mutation of crc reduces the intracellular cAMP level by 1.76-fold under T3SS-inducing conditions, leading to approximately 2-fold reduction of the exsA expression. Further investigation reveals that Crc affects the mRNA stability of cyaB, which encodes an adenylate cyclase involved in cAMP synthesis. The cyaB 5′-UTR is identified as a key region through which Crc affects its mRNA stability. Our study elucidates a novel regulatory mechanism by which Crc controls the T3SS through modulating cyaB mRNA stability and subsequent cAMP synthesis under T3SS-inducing conditions. Full article