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Gram-negative bacteria utilize type VI secretion systems (T6SS) for microbial competition and host interaction. While most pathogens rely on the canonical T6SSi, Francisella species uniquely possess T6SSii. The highly virulent human pathogen Francisella tularensis harbors a distinct T6SSii variant that includes pdpC, encoding a putative effector protein. Bioinformatic analysis revealed a conserved amino acid triad in PdpC, homologous to motifs found in Make Caterpillars Floppy family toxins. To investigate the functional relevance of this triad, site-directed mutagenesis was performed in the live vaccine strain of F. tularensis, substituting each residue with alanine. Mutants showed impaired phagosomal escape, reduced intracellular replication, and marked attenuation in the mouse infection model. Equivalent mutations introduced into F. novicida, a model for T6SS-mediated secretion, confirmed the triad’s importance. Mass spectrometry analysis demonstrated that PdpC is secreted in a T6SS-dependent manner. Importantly, the mutations did not affect secretion, and deletion of pdpC did not alter the overall secretion profile. These findings indicate that the conserved triad is essential for PdpC’s effector function but dispensable for its secretion. This study highlights a critical motif required for Francisella virulence and provides new insights into the specialized mechanisms of T6SSii effectors.

Purpose: This study aimed to determine how different strain intensities-including normal, moderately increased, and high strain-influence protein expression profiles and related biological processes in human corneal stromal keratocytes.

Methods: A well-established in vitro model using the Flexcell FX-5000 Tension System, which replicates the natural corneal curvature and enables precise strain application to keratocytes, was used. Keratocytes were exposed to three strain levels: 3% (normal), 6% (moderately increased), and 12% (high). Following strain application, cells were collected for liquid chromatography-tandem mass spectrometry-based proteomic analysis to generate protein expression profiles. Differentially expressed proteins (DEPs) among the three groups were identified and subjected to biological pathway enrichment to reveal strain-dependent biological processes. Western blot analysis was performed to validate the expression of selected DEPs.

Results: Keratocytes exhibited strain intensity-dependent responses. Three percent strain maintained keratocytes in a quiescent phenotype, consistent with our previous findings. Six percent strain activated protective and adaptive programs to preserve tissue homeostasis under stress. In contrast, 12% strain suppressed immune-related processes and induced extracellular matrix (ECM) remodeling. Notably, procollagen-lysine, 2-oxoglutarate 5-dioxygenase 2 (PLOD2) and cathepsin L (CTSL)-two ECM remodeling-related proteins implicated in fibrotic responses-were significantly upregulated under 12% strain, highlighting a potential link between excessive mechanical stress and stromal fibrosis.

Conclusions: These findings demonstrate that corneal strain regulates keratocyte behavior in an intensity-dependent manner and suggest that high mechanical stress may drive pathologic stromal remodeling and fibrotic responses, offering mechanistic insights that may inspire future therapeutic strategies.

Antimicrobial resistance (AMR) in common bacterial pathogens, including methicillin-resistant Staphylococcus aureus (MRSA), is an increasingly dire public health threat, with MRSA accounting for up to 90% of S. aureus infections. To expand the treatment arsenal against MRSA infections, we developed a class of tunable three-dimensional tricyclic 2-pyridones, termed TriPcides, that can kill MRSA resistant to last-resort antibiotics and eliminate MRSA persister cells. No preexisting resistance was detected across hundreds of clinical isolates, and continuous exposure of MRSA to TriPcides did not elicit detectable resistance. Treatment with TriPcides causes a rapid decrease in membrane integrity and increased levels of reactive oxygen species. Last, TriPcides effectively reduce secretion of important virulence factors and result in reduced ulcer size and healing time in S. aureus murine skin and soft tissue infections but do not reduce bacterial burden.

While the excessive use of broad-spectrum antibiotics is a major driver of the global antibiotic resistance crisis, more selective therapies remain unavailable for the majority of bacterial pathogens. This includes the obligate intracellular bacterial pathogens of the genus Chlamydia, which cause millions of urogenital, ocular, and respiratory infections each year. Conducting a comprehensive search of the chemical space for novel antichlamydial activities, we identified over 60 compounds that are chemically diverse, structurally distinct from known antibiotics, non-toxic to human cells, and highly potent in preventing the growth of Chlamydia trachomatis in cell cultures. Some blocked C. trachomatis development reversibly, while others eradicated both established and persistent infections in a bactericidal manner. The top molecules displayed compelling selectivity, yet broad activity against diverse Chlamydia strains and species, including both urogenital and ocular serovars of C. trachomatis, as well as Chlamydia muridarum and Chlamydia caviae. Some compounds also displayed synergies with clinically used antibiotics. Critically, we found the most potent antichlamydial compound to inhibit fatty acid biosynthesis via covalent binding to the active site of Chlamydia FabH, identifying a new mechanism of FabH inhibition and highlighting a possible way to selectively treat Chlamydia infections.

Exercise has been shown to promote wound healing, including tendon repair. Myokines released from the exercised muscles are believed to play a significant role in this process. In our previous study, we used an in vitro coculture and loading model to demonstrate that 2% static loading of myoblasts increased the migration and proliferation of cocultured tenocytes─two crucial aspects of wound healing. IGF-1, released from myoblasts in response to 2% static loading, was identified as a contributor to the increased proliferation. However, the factors responsible for the enhanced migration remained unknown. In the current study, we subjected myoblasts in single culture conditions to 2, 5, and 10% static loading and performed proteomic analysis of the cell supernatants. Gene Ontology (GO) analysis revealed that 2% static loading induced the secretion of NBL1, C5, and EFEMP1, which is associated with cell migration and motility. Further investigation by adding exogenous recombinant proteins to human tenocytes showed that NBL1 increased tenocyte migration but not proliferation. This effect was not observed with treatments using C5 and EFEMP1.

The purpose of this study was to examine the nature of the underlying molecular mechanisms of cell death in human keratocytes treated with nigericin, a known pyroptosis inducer. Human keratocytes were exposed to nigericin, and cell death was assessed through morphological analysis and detection of related molecular markers. Proteomic profiling was performed to identify cell death-related proteins, with key findings validated by western blot. Additionally, organelle disruptions were examined using immunostaining techniques. Pyroptosis-like cell death was observed morphologically in cultured keratocytes. Moreover, an elevated release of IL-1beta was detected, accompanied by a significant loss of mitochondrial membrane potential. However, nigericin treatment induced a form of non-inflammatory cell death characterized by extensive vacuolation, resembling paraptosis. This was accompanied by the absence of caspase-3 activation and endoplasmic reticulum (ER) stress markers, along with increased accumulation of the autophagic marker LC3-II. Proteomic analysis revealed the absence of key components of the canonical pyroptosis pathway, including proteins involved in inflammasome assembly and the gasdermin (GSDM) family. These results were further confirmed by western blot. Significant alterations were also observed in the Golgi apparatus, mitochondria, and lysosomes following nigericin treatment. These findings suggest that nigericin triggers a paraptosis-like cell death in human keratocytes, rather than pyroptosis, as keratocytes lack the canonical executors of pyroptosis. This highlights an alternative mechanism of cell death in the cornea, warranting further exploration to understand its role and potential therapeutic implications.

Light-driven water oxidation by photosystem II sustains life on Earth by providing the electrons and protons for the reduction of CO2 to carbohydrates and the molecular oxygen we breathe. The inorganic core of the oxygen evolving complex is made of the earth-abundant elements manganese, calcium and oxygen (Mn4CaO5 cluster), and is situated in a binding pocket that is connected to the aqueous surrounding via water-filled channels that allow water intake and proton egress. Recent serial crystallography and infrared spectroscopy studies performed with PSII isolated from Thermosynechococcus vestitus (T. vestitus) support that one of these channels, the O1 channel, facilitates water access to the Mn4CaO5 cluster during its S2→S3 and S3→S4→S0 state transitions, while a subsequent CryoEM study concluded that this channel is blocked in the cyanobacterium Synechocystis sp. PCC 6803, questioning the role of the O1 channel in water delivery. Employing site-directed mutagenesis we modified the two O1 channel bottleneck residues D1-E329 and CP43-V410 (T. vestitus numbering) and probed water access and substrate exchange via time resolved membrane inlet mass spectrometry. Our data demonstrates that water reaches the Mn4CaO5 cluster via the O1 channel in both wildtype and mutant PSII. In addition, the detailed analysis provides functional insight into the intricate protein-water-cofactor network near the Mn4CaO5 cluster that includes the pentameric, near planar ‘water wheel’ of the O1 channel.

Eukaryotic ribosome biogenesis proceeds from nucleolus to cytosol assisted by various assembly factors. The process is evolutionarily conserved across eukaryotes but differences between the kingdoms are emerging. Here, we describe how the OPENER (OPNR) protein complex is required for 60S ribosome assembly in the model plant Arabidopsis thaliana. The complex is observed on both nuclear envelope and mitochondria, and contains OPNR, OPENER ASSOCIATED PROTEIN 1 (OAP1), OAP2, Cell Division Cycle 48 D (CDC48D) and Calmodulin-interacting protein 111 (CIP111). Depletion of the OPNR complex components results in reproductive lethality and cytoplasmic retention of assembly factors on 60S ribosomes. Subsequent biochemical analyses and structural modelling suggest that OPNR, OAP1 and OAP2 form a claw-like trimer which grabs the ribosome assembly factor RIBOSOMAL PROTEIN L24C (RPL24C) on the pre-60S ribosome. Our results reveal previously unrecognised subcellular complexity of ribosome biogenesis in plants, and point to mitochondria association as a feature to ensure sufficient translational capacity.

Cytolysin A (ClyA) is a pore-forming protein from a strongly silenced gene in non-pathogenic Escherichia coli, including typical commensal isolates in the intestinal microbiome of healthy mammalian hosts. Upon overproduction, ClyA-expressing bacteria display a cytolytic phenotype. However, it remains unclear whether sublytic amounts of native ClyA play a role in commensal E. coli-host interactions in vivo. Here, we show that sublytic amounts of ClyA are released via outer membrane vesicles (OMVs) and affect host cells in a remarkable manner. OMVs isolated from ClyA+ E. coli were internalised into cultured colon cancer cells. The OMV-associated ClyA caused reduced levels of cancer-activating proteins such as H3K27me3, CXCR4, STAT3 and MDM2 via the EZH2/H3K27me3/microRNA 622/CXCR4 signalling axis. Our results demonstrate that sublytic amounts of ClyA in OMVs from non-pathogenic E. coli can influence the stability of the EZH2 protein, reducing its activity in epigenetic regulation, causing elevated level of the tumour suppressor protein p53.

RNA modification pathways are often mis-regulated in various cancers, with N6-methyladenosine (m6A) having a pivotal role in cancer progression and metastasis. Methyltransferase-like 3 (METTL3), a core component of the m6A methyltransferase complex, functions not only as an m6A writer but also promotes tumorigenesis through m6A-independent mechanisms. Here, we show that METTL3 is mislocalized to the cytoplasm in breast cancer tumors from patients, contributing to the oncogenic phenotype. Cytoplasmic METTL3 interacts with EXOC7, a key regulator of exocytosis, promoting its stabilization. Additionally, METTL3 regulates m6A-dependent alternative splicing of EXOC7. Silencing METTL3 impairs vesicle trafficking and the breast cancer secretome – effects that do not rely on its enzymatic activity but instead involve METTL3-mediated stabilization of EXOC7 and potentially other exocyst components. Furthermore, METTL3 knockdown impairs invadopodia formation, collagen matrix invasion, and focal adhesion morphology in vitro, while inhibition of METTL3 catalytic activity does not. Our findings uncover non-catalytic roles of METTL3 in regulating exocytosis and the cancer secretome.