Biology subjects

Pagliuso, A.

Publications and source records attributed to Pagliuso, A..

4 recordsLinked to original sources

bioRxiv2025

Large-scale phenotyping and comparative genomics reveal genetic features of Listeria persistence in epithelial cells

During infection in epithelial cells, after invading the cytosol, multiplying, and spreading, Listeria monocytogenes (Lm) ceases to produce ActA and becomes trapped in Listeria-containing vacuoles (LisCVs). These persistence acidic vacuoles harbor bacterial subpopulations that resist to stress in a metabolically dormant state. Although LisCVs have been proposed as a hallmark of Lm persistence in epithelial cells, their prevalence across strains and the bacterial factors underlying their formation remain uncharacterized. Given the significant genetic diversity within the species, it is important to consider this variability when studying persistence phenotype. Therefore, we screened over one hundred Lm isolates spanning two major evolutionary lineages and belonging to 23 clonal complexes from diverse ecological origins. Strikingly, the vast majority of strains, including both clinical and environmental isolates, were capable of forming LisCVs, suggesting that vacuolar persistence is a widespread and conserved feature of Lm pathogenesis. Nevertheless, among the group of hypo-virulent strains mostly associated with food and carrying a truncated InlA, we identified four isolates with an altered persistence phenotype. Two of them showed defects in the early stages of infection and carried mutations in key virulence genes (hly and gshF). The other two, instead, were specifically affected in the persistence by showing a reduced ability to form LisCVs. Comparative genomic analysis revealed that a mutation in the folP gene, required for folate biosynthesis, was responsible for impaired persistence. Live-imaging and microscopy analysis highlighted a reduced bacterial motility and intercellular spreading of the folP mutant, although the level of ActA at the bacterial surface was increased. Together, our work identifies folate biosynthesis as a critical metabolic pathway governing Lm persistence by regulating ActA levels and activity. Downregulation of ActA at the bacterial surface is therefore a crucial event for the establishment of the intracellular persistent niche during long-term infection of epithelial cells. Author SummaryLong regarded as a strictly cytosolic pathogen, Listeria monocytogenes is now revealing a dual lifestyle that includes intracellular vacuoles. Recent evidence shows that in epithelial cells this bacterium can enter a dormant state within acidic vacuoles, termed LisCVs, potentially contributing to silent carriage and antibiotic treatment failure. Here, we reveal that vacuolar persistence is a widespread and conserved feature among diverse Listeria strains. Strikingly, we identify folate metabolism as a key regulator of this phenotypic switch, linking metabolic cues to the shutdown of bacterial motility. Our findings uncover a novel connection between central metabolism and intracellular niche adaptation, shedding new light on how Listeria survives and hides within the host.

microbiology↗

bioRxiv2025

Using the chicken embryo as an in vivo model to revive viable but non-culturable (VBNC) pathogens

The chicken embryo has emerged as a popular in vivo model with increasing application in biomedical research, due to its simplicity, affordability, and adaptability in the study of various biological phenomena. This model has been used to investigate microbial pathogenicity, and is becoming a useful tool to study bacterial dormancy. The viable but non-culturable (VBNC) state is a dormant state in which bacteria become metabolically quiescent and resistant to cultivation to preserve their viability in harsh environments. Under favorable conditions, VBNC bacteria can "wake up" back into a metabolically active and culturable state. Bacterial pathogens that switch to a VBNC state, such as the foodborne listeriosis-causing Listeria monocytogenes, are a public health concern, as they elude detection by conventional growth-dependent methods and can recover their virulence upon revival. This urges a better understanding of the conditions and mechanisms driving the revival of VBNC pathogens. The method presented here showcases the chicken embryo as an efficient in vivo model to revive VBNC L. monocytogenes back into a culturable status. Where in vitro revival attempts, largely based on nutritional replenishing, were unproductive, this protocol succeeds in promoting the reactivation of cell wall-deficient VBNC forms of L. monocytogenes generated by starvation in mineral water. Importantly, our results underline the requirement of the embryo for the revival of VBNC L. monocytogenes, indicating an important role of embryo-associated factors in this process. Other potential uses for this method include the screening and identification of bacterial factors implicated in the mechanisms of VBNC state revival. This model can thus provide insight into the molecular workings of bacterial dormancy, whose knowledge is critical to reduce the public health risks entailed by undetectable pathogens. SUMMARYThis method showcases the chicken embryo as a simple and cost-effective in vivo model to revive the bacterial pathogen L. monocytogenes from a viable but non-culturable (VBNC) state, and with potential further uses in the understanding of bacterial dormancy mechanisms.

microbiology↗

bioRxiv2023

Diving into bacterial dormancy: emergence of osmotically stable wall-less forms in an aquatic environment

Bacteria can respond to environmental stresses by entering a dormant state, called viable but non-culturable (VBNC) state, in which they no longer grow in routine culture media. VBNC pathogens pose thus a significant risk for human and animal health as they are not detected by standard growth-based techniques and can "wake up" back into a vegetative and virulent state. Although hundreds of species were reported to become VBNC in response to different stresses, the molecular mechanisms governing this phenotypic switch remain largely elusive. Here, we characterized the VBNC state transition process in the Gram-positive pathogen Listeria monocytogenes in response to nutritional deprivation. By combining fluorescence microscopy, cryo-electron tomography and analytical biochemistry, we found that starvation in mineral water drives L. monocytogenes into a VBNC state via a mechanism of cell wall (CW) shedding that generates osmotically stable CW-deficient (CWD) coccoid forms. This phenomenon occurs in multiple L. monocytogenes strains and in other Listeria species, suggesting it may be a stress-adapting process transversal to the Listeria genus. Transcriptomic and gene-targeted approaches revealed the stress response regulator SigB and the autolysin NamA as major moderators of CW loss and VBNC state transition. Finally, we show that this CWD dormant state is transient as VBNC Listeria revert back to a walled, vegetative and virulent state after passage in embryonated eggs. Our findings provide unprecedented detail on the mechanisms governing the transition to a VBNC state, and reveal that dormant CWD bacterial forms can naturally arise in aquatic environments without osmotic stabilization. This may represent an alternative strategy for bacterial survival in oligotrophic conditions, which can potentially generate public health-threatening reservoirs of undetectable pathogens.

microbiology↗

bioRxiv2019

An RNA-binding protein secreted by Listeria monocytogenes activates RIG-I signaling

Recent studies have reported on the presence of bacterial RNA within or outside extracellular membrane vesicles, possibly as ribonucleoprotein complexes. Proteins that bind and stabilize bacterial RNAs in the extracellular environment have not been reported. Here, we show that the bacterial pathogen Listeria monocytogenes secretes a small RNA binding protein that we named Zea. We show that Zea binds and stabilizes a subset of L. monocytogenes RNAs causing their accumulation in the extracellular medium. Furthermore, Zea binds RIG-I, the vertebrate non-self-RNA innate immunity sensor and potentiates RIG-I-signaling leading to interferon {beta} production. By performing in vivo infection, we finally show that Zea modulates L. monocytogenes virulence. Together, this study reveals that bacterial extracellular RNAs and RNA binding proteins can affect the host-pathogen crosstalk.

cell biology↗