ABSTRACT Phosphoinositides are low-abundance membrane lipids serving as critical regulators of organelle identity, membrane trafficking, and signal transduction in eukaryotic cells. Live-cell imaging using genetically encoded, protein-based fluorescent probes has revolutionized the study of these lipids. Yet many existing probes were developed and analyzed in isolation across different experimental systems. This study presents a comprehensive evaluation of 37 published protein-based phosphoinositide probes within the budding yeast Saccharomyces cerevisiae . The specificity of candidate probes was tested through the disruption of their corresponding lipid kinase activities. By comparing the performance of multiple probes under uniform conditions, we identified seven constructs based on the following domains that reliably detect the four primary phosphoinositide species in yeast: PX NCF4 and PX Vam7 for PtdIns(3)P; 2PX SnxA for PtdIns(3,5)P 2 ; 2PH Osh2 , PH FAPP1 , and PH OSBP for PtdIns(4)P; and TubbyCT for PtdIns(4,5)P 2 . Furthermore, we systematically mapped the subcellular distribution of phosphoinositides and their producing kinases, providing an updated atlas of their localization. This work offers a validated toolkit and a selection guide for investigating phosphoinositide dynamics in vivo . IMPORTANCE Cells use tiny lipid molecules called phosphoinositides as “zip codes” to organize their internal compartments and direct traffic between them. Because these molecules are scarce and constantly changing, scientists rely on special glowing protein probes to watch them in living cells. However, most of these probes were invented in separate labs under different conditions, making it hard to know which ones truly work. This study tested 37 published probes side-by-side in yeast and found seven reliable “trackers” for the four most important phosphoinositide types. By mapping exactly where these lipids and the enzymes that make them reside, the work provides a standardized toolkit and clear guide that helps researchers confidently study how cells organize their inner life.
ABSTRACT Mycoplasma genitalium ( Mg ) is an important etiological agent of sexually transmitted infections that has emerged as a global health threat. Despite its clinical importance, accurate serodiagnosis remains challenging due to high cross-reactivity caused by non-specific proteins. To address this limitation, we optimized Mg growth in a serum-free minimal medium to characterize the biogenesis and diagnostic applications of Mg -secreted extracellular vesicles (EVs). We demonstrate that Mg generates EVs that retain immunodominant proteins, specifically the key adhesins P140 and P110. From an initial cohort of 1,608 women (12.5% Mg- positive), we selected a longitudinal sub-study population, including 24 women who were PCR-positive at enrollment and a control group of 14 individuals who remained Mg -negative across three visits, each spaced approximately 6 months apart. Comparative ELISA and immunoblotting using sera from Mg -infected and uninfected individuals revealed that the EV-associated antigens provide superior diagnostic discrimination over conventional total-cell lysates and lipid-associated membrane protein extracts. Notably, EV-associated antigens lack common non-specific proteins, such as immunoglobulin-binding protein, MG281. Functional characterization using full-length and truncated P140 variants in cell-based ELISAs with human cervical epithelial cells identified the C-terminal region (P140-C) as the primary host-cell-interacting domain. In addition, this specific region induces an epitope-restricted antibody response in infected individuals. Within the longitudinal clinical cohort, high anti-P140-C antibody titers correlated with a trend toward Mg clearance, suggesting potential prognostic utility despite lacking statistical significance. Collectively, these results establish Mg EVs as a high-fidelity antigenic source and identify P140-C as a promising candidate for the development of next-generation serological assays. IMPORTANCE Serodiagnosis of Mycoplasma genitalium (Mg) faces significant challenges, especially in the context of emerging antimicrobial resistance and asymptomatic infections. This study introduces Mg -derived extracellular vesicles (EVs) as a source of refined antigens for serodiagnosis. By isolating the P140 C-terminal region, we identified a highly specific marker that overcomes antigenic variation, reduces cross-reactivity, and offers a candidate antigen that may warrant evaluation as a marker of infection clearance. By leveraging bacterial EVs as a source of purified diagnostic antigen, our findings offer a framework with broader implications for other antigenically complex pathogens.
ABSTRACT Helicobacter pylori is a primary risk factor for gastric carcinogenesis; however, an in-depth evaluation of its potential role in modulating ferroptosis to influence cancer development remains undetermined. Therefore, this study developed complementary in vitro cell models (using GES-1 and AGS gastric epithelial cells) and C57BL/6 mouse models to systematically evaluate the mechanism by which H. pylori regulates ferroptosis in gastric epithelial cells. The acquired data revealed that H. pylori infection selectively promotes ferroptotic cell death, which elevates lipid peroxidation, impairs the GPX4-GSH antioxidant axis, and causes intracellular iron overload, resulting in characteristic mitochondrial structural damage. H. pylori disrupts iron homeostasis, alters lipid composition, and impairs redox buffering ability, promoting a ferroptosis-prone microenvironment that mediates epithelial cell death via convergent pathways. Furthermore, H. pylori was found to suppress SLC7A11 expression by two distinct mechanisms: ubiquitin-proteasome-mediated protein destabilization and STAT1-driven transcriptional silencing, which disrupt the antioxidant system and promote ferroptosis. Moreover, pharmacological inhibition of ferroptosis significantly increased the proliferation, motility, and invasiveness of H. pylori -infected gastric cells. These results demonstrate a context-dependent dual role of ferroptosis in H. pylori -mediated gastric tumorigenesis: ferroptosis inhibits tumor growth by eliminating damaged epithelial cells, whereas its inhibition promotes malignant progression. This study reveals a multi-faceted mechanism by which H. pylori mediates ferroptosis, indicating the STAT1/SLC7A11 module as a key regulatory hub, and identifies the dichotomous role of ferroptosis in infection-mediated gastric oncogenesis. These results provide new insights into the pathogenic mechanisms of H. pylori -related gastric cancer and highlight the potential diagnostic and therapeutic targets against the condition. IMPORTANCE Helicobacter pylori infection is a major cause of gastric cancer, but how it regulates gastric epithelial ferroptosis to modulate tumor progression remains incompletely understood. This study explores the underlying mechanisms using cellular and animal models. We show that H. pylori disrupts iron homeostasis and impairs the GPX4-GSH antioxidant system, triggering lipid peroxidation and mitochondrial damage to induce ferroptosis. Mechanistically, H. pylori downregulates SLC7A11 through ubiquitin-proteasome-mediated degradation and STAT1-dependent transcriptional repression. Ferroptosis exhibits a dual role in H. pylori -associated tumorigenesis, suppressing early tumorigenesis while its inhibition promotes malignant progression. This work defines the STAT1/SLC7A11 axis as a key regulatory module and provides novel targets for H. pylori -related gastric cancer.
ABSTRACT Conventional drug susceptibility testing may overlook subpopulations that survive above the minimum inhibitory concentration (MIC). Fluconazole (FLC), a fungistatic drug central to cryptococcal meningitis therapy, selects for tolerance (slow-growing cells that persist above the MIC), heteroresistance (~1% of cells growing above the MIC but revert without drug), and subpopulation resistance (emerging genetically stable resistant cells). To measure these drug subpopulation phenotypes simultaneously, we developed ColonyPy, a Python-based image analysis pipeline that quantifies colony number and size within the zone of inhibition (ZOI) in disk diffusion assays. Using ColonyPy, we assessed tolerance and heteroresistance/subpopulation resistance in Cryptococcus neoformans clinical isolates grown on Sabouraud dextrose agar (SDA), yeast peptone dextrose agar (YPD), and Mueller–Hinton agar (MHA) with 2% glucose. Our results show that (i) clinical isolates vary widely in tolerance and heteroresistance/subpopulation resistance; (ii) these phenotypes differ significantly across media types; (iii) YPD supports the highest levels of heteroresistance/subpopulation resistance; and (iv) heteroresistance, but not subpopulation resistance, is observed in all the susceptible clinical isolates tested. Together, these findings demonstrate that tolerance and heteroresistance are independent phenotypes that are influenced by nutrient conditions. Our data show the presence of adaptive subpopulations that are able to grow above MIC fluconazole drug concentrations within clinical C. neoformans isolates classified as sensitive in traditional MIC assays that could influence treatment outcomes. IMPORTANCE Cryptococcus neoformans is the leading cause of fungal meningitis in people living with HIV, and fluconazole remains a cornerstone of treatment. Despite its widespread clinical use, fluconazole treatment failure is poorly understood, and conventional susceptibility testing methods fail to capture the full complexity of growth phenotypes that are not cleared by antifungals, including tolerance, heteroresistance, and subpopulation resistance. This study demonstrates that C. neoformans clinical isolates can simultaneously express two distinct drug-survival phenotypes—tolerance and heteroresistance. The balance between these phenotypes is isolate specific and shaped by environmental nutrient conditions. To enable objective, simultaneous quantification of both antifungal-survival phenotypes, we developed ColonyPy—an automated Python-based image analysis pipeline that classifies and quantifies tolerant and heteroresistant/subpopulation-resistant colonies from disk diffusion assay images. Our findings challenge the adequacy of single-metric susceptibility testing and provide a practical tool for investigating how phenotypic subpopulation heterogeneity contributes to fluconazole treatment failure in cryptococcal meningitis.
ABSTRACT Algicidal bacteria are promising microorganisms for controlling harmful algal blooms (HABs) because of their ability to inhibit algal growth or lyse algal cells in aquatic systems. However, our understanding of how to enhance their performance within complex microbial communities remains limited. In this study, we evaluated the algicidal activity of previously isolated bacterial strains in a constructed cyanobacterial consortium dominated by Microcystis aeruginosa and containing indigenous lake microorganisms. Our results revealed that while individual strains generally showed limited inhibitory effects, certain strain combinations exhibited pronounced algicidal activity. Notably, co-inoculation of strain Chromobacterium sp. MERYL1-35 and strain Paucibacter sp. MERXLGS1 achieved the highest algicidal efficiency among the tested treatments. Growth assays and quantitative proteomic analysis demonstrated that these two strains establish a distinct antagonistic interspecies interaction, where Chromobacterium exerts strong inhibitory effects on Paucibacter , accompanied by significant expression alterations in certain bacterial virulence and interaction-associated proteins. We propose that this collective enhancement in population survivability and antagonistic potential of Chromobacterium substantially contributes to the enhanced algicidal activity in the dual-strain system. These findings provide new insights into the ability-enhancing strategy of algicidal bacteria and their complex interactions with target cyanobacteria. IMPORTANCE Eco-friendly and sustainable control methods of harmful algal blooms (HABs) have been urgently needed in recent years. In this study, we demonstrated that the removal efficiency of cyanobacteria by algicidal bacteria can be substantially enhanced through designed co-inoculation strategies. We found that the enhanced algicidal activity of the combined Chromobacterium and Paucibacter dual-strain inoculations is mediated by a unique antagonistic interaction, which differs from other consortia that more likely arise from cooperative strain interactions. These findings advance our understanding of the complex microbial interplay between algicidal bacteria and phytoplankton and may provide new insights into the development of biological control strategies targeting HABs.
