Maleth, J. | Search bioRxiv

bioRxiv2026

A preclinical resistance framework discovers the virulence risks of antibiotics in development

Several new antibiotics target multidrug-resistant pathogens, yet resistance is still evaluated mainly by drug-susceptibility, leaving consequences for bacterial pathogenicity poorly understood. Here, we develop a framework integrating resistance evolution, genomic surveillance and host-pathogen phenotyping to classify antibiotics by resistance potential and pathogenic consequences. Applying this framework to Klebsiella pneumoniae identified functionally distinct antibiotic candidates associated with elevated virulence risk. Resistance evolution rapidly increased virulence through clinically-relevant mutations, without direct selection for pathogenicity. Despite distinct genetic routes, resistance converged on cell-envelope rewiring. A single resistance mutation increased epithelial adhesion, intracellular colonization, macrophage immune-evasion, and tissue persistence in murine infection models, transforming K. pneumoniae into a more invasive and cytotoxic pathogen. Risk-profile analysis revealed partial decoupling of resistance and pathogenicity, with some low-resistance antibiotics yielding highly-virulent populations. These findings establish resistance-driven virulence as an underappreciated translational hazard and call for incorporating host-pathogen interactions into resistance surveillance and preclinical antibiotic development.

microbiology↗

bioRxiv2025

Cholesteryl esters and high protein-to-lipid ratios distinguish Non-Vesicular Extracellular Particles from Extracellular Vesicles

Extracellular vesicles (EVs) are central to intercellular communication, yet the mechanisms underlying their biogenesis and diversity remain incompletely understood. Here, we integrate meta-analysis, advanced lipidomic, protein-to-lipid profiling, and super-resolution imaging to define the fundamental principles governing EV heterogeneity. Our meta-analysis of published transmission electron microcrographs across kingdoms reveals a highly conserved 110 nm average diameter and 200 nm upper size limit for intraluminal vesicles (ILVs), which are secreted as exosomes. Besides classical EV populations, we also characterize a distinct nanoparticle class: 167000 xg pellet of non-vesicular extracellular particles (167k-NVEPs), which exhibit a significantly higher protein-to-lipid ratio than 14000 xg pellet of large EVs (14k-lEVs) and 100000 xg pellet of small EVs (100k-sEVs), as measured by both biochemical assays and Raman spectroscopy. Lipid profiling demonstrates that 167k-NVEPs exhibit significant enrichment in cholesteryl esters and triacylglycerols, lipids typically associated with lipid droplets and the endosome/lysosome system. Analysis of lipid carbon-chain lengths reveals distinct signatures: 167k-NVEPs show pronounced enrichment at 16 and 18 carbons, while 100k-sEVs display enrichment at 32 and 34 carbons. This divergence indicates a potential connection to flexible biogenesis pathways. Marker heterogeneity across EV populations, confirmed by confocal and super-resolution microscopy, further underscores the limitations of relying on canonical tetraspanins for EV classification. Notably, 167k-NVEPs (likely exomeres) exhibit enrichment of Arf6 and CD63. Together, our findings provide compositional, biophysical, and molecular evidence supporting the formal recognition of 167k-NVEPs as a distinct class of extracellular particles and enabling exploring in disease biology and therapeutic delivery. Significance StatementExtracellular vesicles (EVs) are critical mediators of intercellular communication, yet their classification remains clouded by ambiguity in terms of their composition and biogenesis. This study resolves critical uncertainties through a cross-kingdom meta-analysis, establishing a conserved [~]110nm diameter and [~]200 nm upper size limit for intraluminal vesicles (ILVs), the precursors to exosomes. More significantly, we identify non-vesicular extracellular particles (167k-NVEPs) as a distinct class based on their unique sterol-rich lipidome, enrichment in lipids of 16 and 18 carbon chain length, elevated protein-to-lipid ratio, and functional cargo delivery. These features, alongside evidence of non-canonical origin and functional cargo delivery, establish NVEPs as a discrete class of extracellular particles.

cell biology↗

bioRxiv2020

Decreased calmodulin recruitment triggers PMCA4 dysfunction and pancreatic injury in cystic fibrosis

Exocrine pancreatic damage is a common complication of cystic fibrosis (CF), which can significantly debilitate the quality of life and life expectancy of CF patients. The cystic fibrosis transmembrane conductance regulator (CFTR) has a major role in pancreatic ductal ion secretion, however, it presumably has an influence on intracellular signaling as well. Here we describe in multiple model systems, including iPSC-derived human pancreatic organoids from CF patients, that the activity of PMCA4 is impaired by the decreased expression of CFTR in ductal cells. The regulation of PMCA4, which colocalizes and physically interacts with CFTR on the apical membrane of the ductal cells, is dependent on the calmodulin binding ability of CFTR. Moreover, CFTR seems to be involved in the process of the apical recruitment of calmodulin, which enhances its role in calcium signaling and homeostasis. Sustained intracellular Ca2+ elevation in CFTR KO cells undermined the mitochondrial function and increased apoptosis. Based on these, the prevention of sustained intracellular Ca2+ overload may improve the exocrine pancreatic function and may have a potential therapeutic aspect in CF.

cell biology↗