Jardin, J. | Search bioRxiv

bioRxiv2026

Isolation and characterization of novel filamentous phages from Swiss-type cheeses infecting the Gram-positive bacterium Propionibacterium freudenreichii

Filamentous phages infecting Gram-positive bacteria remain largely unexplored. Notably, only two filamentous phages, B5 and Philemon infecting Propionibacterium freudenreichii, have been described to date in the phage-rich dairy ecosystem. Although both were genomically characterized, only B5 was confirmed to be an infective filamentous single-stranded DNA phage. The aim of this study was to isolate and characterize new filamentous phages from Swiss-type cheese to investigate their diversity, structural features, host specificity, and potential adaptation to the dairy environment. Thirty raw and pasteurized milk cheeses from France were screened for phages infecting P. freudenreichii strains. Eleven phages were isolated, nine of which displayed a filamentous morphology. Named MINOG1 to MINOG9, these filamentous phages exhibited genomic features typical of this morphotype, including small single-stranded DNA genomes with collinear genes organized into functional modules. Comparison with B5 and Philemon revealed sequence divergence ranging from 0.1% to 7%. These phages also exhibited a diverse host range. To further explore phage-P. freudenreichii interactions, we screened the genomes of the strains used in this study, as well as additional genomes retrieved from the NCBI database, for CRISPR spacers predicted to target these filamentous phages. Numerous strains contained CRISPR spacers showing 79 to 100% identity to genomic regions of these phages. Two P. freudenreichii strains displayed markedly different phage resistance levels despite exact spacer-protospacer matches with phages B5, MINOG1, MINOG2, and MINOG8. Conversely, several strains were resistant to nearly all tested phages despite lacking CRISPR spacers targeting them suggesting the presence of additional defense systems in P. freudenreichii. IMPORTANCEFilamentous phages can play important roles in bacterial ecology by modulating host physiology, population dynamics, and bacterial adaptation to specific environments. However, filamentous phages infecting Gram-positive bacteria remain among the least explored bacterial viruses, and their diversity, ecology, and interactions with their hosts are still poorly understood. This knowledge gap is particularly relevant in dairy ecosystems, where phages are abundant and can influence microbial communities and fermentation processes. In characterizing nine new filamentous phages infecting Propionibacterium freudenreichii from Swiss-type cheeses, this study expands the known diversity of filamentous phages associated with Gram-positive bacteria and provides new insights into phage-host interactions and bacterial defense strategies in dairy-associated bacteria.

microbiology↗

bioRxiv2025

Staphylococcus aureus derived extracellular vesicles trigger multiple inflammatory pathways in host cells and deliver their RNA cargo following their internalization and lysis within late endosomes

Staphylococcus aureus is a major opportunistic Gram-positive pathogen whose virulence can be partly mediated by extracellular vesicles (EVs). These nanosized particles carry a diverse cargo and can play key roles in host-pathogen interactions. Here, we investigated the production, composition, and host cell interactions of EVs secreted by the methicillin-resistant S. aureus strain N315, one of the most widely studied clinical isolates. To mimic infection, EVs produced from RPMI supplemented with 10% LB were purified by size exclusion chromatography, and characterized by nanoparticle tracking analysis and electron microscopy. N315 EVs contained a diverse molecular cargo, selectively enriched, and including proteins, lipoproteins, DNA, RNA, lipoteichoic acid, and peptidoglycan. Protection assays and confocal microscopy confirmed the intravesicular localization of RNAs and proteins. N315 EVs were recognized by multiple host pattern recognition receptors (PPRs) in human MG-63 cells, located at the cell surface (TLR1, TLR2, TLR4, TLR6), within endosomes (TLR3, TLR7, TLR4) and in the cytoplasm (NOD2). EV exposure promoted the selective upregulation of these PRRs, activated the NF-{kappa}B and JAK/STAT signaling pathways, and induced the expression of pro-inflammatory cytokines such as IL1{beta}, IL6, and CXCL8. N315 EVs were internalized via a dynamin-dependent endocytosis mechanism and trafficked to late endosomes, where RNA cargo release occurred. Importantly, inhibition of EVs uptake altered cytokine gene expression, confirming that internalization is required for immunomodulation. This work provides a better understanding of the mechanisms of interaction of S. aureus EVs with host cells and sheds light on the fate of EVs and their cargo once internalized by cells. IMPORTANCEAntibiotic resistance, coupled with the limited discovery of new antimicrobial agents, poses a growing threat to public health. Staphylococcus aureus is one of the six highly virulent and antibiotic-resistant bacterial species responsible for both severe hospital- and community-acquired infections. This pathogen demonstrates a remarkable ability to adapt to diverse hosts and environments and to produce a broad arsenal of virulence factors. Gaining deeper insight into its pathogenic mechanisms and host interactions may reveal novel therapeutic targets and alternative treatment strategies. Over the past decade, a key advancement has been the identification of extracellular vesicle production by S. aureus and its critical role as a new secretion system for virulence factors. This study shed light on the immunogenic effect of EVs and their role as a vehicle for the delivery of their contents into host cells. These adjuvant-like properties open new avenues for research and potential intervention.

microbiology↗