Biology subjects

Grenga, L.

Publications and source records attributed to Grenga, L..

7 recordsLinked to original sources

bioRxiv2025

Achromobacter xylosoxidans modulates Pseudomonas aeruginosa virulence through a multi-target mechanism of competition

The colonization and persistence of Pseudomonas aeruginosa in chronically diseased lungs are driven by the production of various virulence factors. However, pulmonary infections in cystic fibrosis (CF) patients are predominantly polymicrobial. While Achromobacter xylosoxidans is an opportunistic pathogen in these patients, its impact on P. aeruginosa virulence during co-infection remains largely unknown. This study investigated the interaction between P. aeruginosa and two clonally related A. xylosoxidans strains, Ax 198 and Ax 200, co-isolated from a CF patient sputum. We found that the interaction between P. aeruginosa and co-isolated A. xylosoxidans was strain-dependent, with the Ax 200 strain significantly reducing P. aeruginosa virulence in a zebrafish model, providing the first in vivo evidence of interaction between these two species during co-infection. Proteomic analysis revealed that the P. aeruginosa proteome was differently impacted by the two A. xylosoxidans strains, with Ax 200 altering proteins involved in biofilm formation, swimming motility, iron acquisition, and secretion systems. These proteomic findings were further validated by phenotypic assays, which confirmed that A. xylosoxidans affected major P. aeruginosa virulence phenotypes, including biofilm formation, swimming motility, and siderophore production. Genetic analysis also confirmed that distinct regulatory mechanisms, including mechanisms involved in the iron cycle, may account for the strain-dependent interaction effects of A. xylosoxidans with P. aeruginosa. These findings reveal a novel multi-target competitive mechanism through which A. xylosoxidans significantly disrupts P. aeruginosa virulence. IMPORTANCEPseudomonas aeruginosa, a major human pathogen, is a leading cause of mortality in cystic fibrosis (CF) patients. Understanding its virulence mechanisms is critical for developing effective infection management strategies. Given the polymicrobial nature of CF infections, it is essential to investigate interspecies interactions that may influence bacterial virulence. While A. xylosoxidans is recognized as an opportunistic pathogen in CF, its impact on P. aeruginosa virulence during co-infection remains largely unexplored. Deciphering the molecular basis of P. aeruginosa virulence in polymicrobial settings could reveal novel and specific therapeutic targets to improve treatment strategies.

microbiology↗

bioRxiv2025

Critical Assessment of MetaProteome Investigation 2 (CAMPI-2): Multi-laboratory assessment of sample processing methods to stabilize fecal microbiome for functional analysis

BackgroundFecal samples are widely used as a proxy for studying gut microbiome composition in both human and animal research. Fecal metaproteomics provides valuable insights by tracking changes in the relative abundance of microbial taxa and their protein functions. To ensure reliable results, it is crucial to minimize alterations in the metaproteome occurring from sample collection to protein extraction. Therefore, employing effective stabilization methods is essential to preserve the integrity of the fecal metaproteome from sample collection to laboratory analysis, particularly over long distances or when rapid freezing options are not readily available. In line with these needs, the second edition of the Critical Assessment of MetaProteome Investigation (CAMPI-2) was specifically focused on testing sample stabilization protocols to be applied before metaproteomic analysis. ResultsThis collaborative multicenter study assessed the ability of five different stabilization methods, based on two commercial devices and three specific reagents (acetone, lithium dodecyl sulfate, and an RNAlater-like buffer), respectively, to stabilize the fecal metaproteome during room-temperature storage (14 days) and shipment to mass spectrometry facilities. The five methods were tested simultaneously by eight different laboratories across Europe, using aliquots from the same fecal sample. After protein extraction and digestion, duplicate aliquots of the resulting peptides were analyzed independently by two mass spectrometry facilities at distinct international locations. Analysis of the mass spectrometric data using two different search engines revealed that the fecal metaproteome profile differed considerably depending on the stabilization method used in terms of richness, alpha and beta diversity, reproducibility, and quantitative distribution of main taxa and functions. Although each method showed unique strengths and weaknesses, a commercial swab-based device stood out for its remarkable reproducibility and ranked highest for most of the metrics measured. ConclusionsCAMPI-2 allowed a robust evaluation of five different methods for preserving fecal metaproteome samples. The present investigation provides useful data for the design of metaproteomics and multi-omics studies where fecal sampling cannot be immediately followed by long-term storage at -80{degrees}C. Further optimization of the tested protocols is necessary to improve stabilization efficiency and control bias in the taxonomic and functional profile of the gut microbiome.

microbiology↗

bioRxiv2024

Key gut microbiota components and functions in an aquatic keystone species across diets assessed by metaproteomics

The gut microbiota plays a crucial role in maintaining host fitness and modulating contaminant toxicity-related responses. However, information on how the gut microbiota of sentinel species responds to environmental factors is limited. In this study, we characterized the gut microbial community and its functions under normal, contaminant-free conditions by examining the effects of different diets over a 10-day period (alder leaf, carrot, spinach, and protein-rich granules) on the amphipod Gammarus fossarum, commonly used in bioassays for ecotoxicity assessment of contaminated rivers. Metaproteomic analysis of intestine samples enabled taxonomic characterization of the gut microbiota from this millimetric animal, assignment of biological functions to each microbial entity, and functional analysis of host proteins. The most abundant microbes detected in the gut belong to 37 bacterial and 5 fungal genera. Functional analyses of host and microbial proteins revealed complementary metabolic activities, allowing the degradation of complex polysaccharides such as cellulose and chitin. Diet was found to shape microbial community structure, with foodborne microorganisms strongly influencing structural changes during short-term feeding in amphipods. These microorganisms remained viable post-ingestion and contributed to food digestion. Functional stability was maintained across different diets, although the protein-rich granules diet induced functional shifts in both the host and its microbiota, reflecting their adaptation to a novel nutrient source. Finally, we identified a core microbiota driving key gut functions, less affected by dietary variations. These findings are significant for future ecotoxicological and biomonitoring investigations, leveraging the microbiomes of these sentinel animals as pivotal targets.

molecular biology↗

bioRxiv2020

Proteotyping SARS-CoV-2 virus from nasopharyngeal swabs: a proof-of-concept focused on a 3 min mass spectrometry window

Rapid but yet sensitive, specific and high-throughput detection of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in clinical samples is key to diagnose infected people and to better control the spread of the virus. Alternative methodologies to PCR and immunodiagnostic that would not require specific reagents are worth to investigate not only for fighting the COVID-19 pandemic, but also to detect other emergent pathogenic threats. Here, we propose the use of tandem mass spectrometry to detect SARS-CoV-2 marker peptides in nasopharyngeal swabs. We documented that the signal from the microbiota present in such samples is low and can be overlooked when interpreting shotgun proteomic data acquired on a restricted window of the peptidome landscape. Simili nasopharyngeal swabs spiked with different quantities of purified SARS-CoV-2 viral material were used to develop a nanoLC-MS/MS acquisition method, which was then successfully applied on COVID-19 clinical samples. We argue that peptides ADETQALPQR and GFYAQGSR from the nucleocapsid protein are of utmost interest as their signal is intense and their elution can be obtained within a 3 min window in the tested conditions. These results pave the way for the development of time-efficient viral diagnostic tests based on mass spectrometry.

microbiology↗

bioRxiv2020

Shotgun proteomics of SARS-CoV-2 infected cells and its application to the optimisation of whole viral particle antigen production for vaccines

Severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2) has resulted in a pandemic and continues to spread quickly around the globe. Currently, no effective vaccine is available to prevent COVID-19 and an intense global development activity is in progress. In this context, the different technology platforms face several challenges resulting from the involvement of a new virus still not fully characterised. Finding of the right conditions for virus amplification for the development of vaccines based on inactivated or attenuated whole viral particles is among them. Here, we describe the establishment of a workflow based on shotgun tandem mass spectrometry data to guide the optimisation of the conditions for viral amplification. In parallel, we analysed the dynamic of the host cell proteome following SARS-CoV-2 infection providing a global overview of biological processes modulated by the virus and that could be further explored to identify drug targets to address the pandemic.

microbiology↗

bioRxiv2020

Second messenger control of mRNA translation by dynamic ribosome modification

Control of mRNA translation is a crucial regulatory mechanism used by bacteria to respond to their environment. In the soil bacterium Pseudomonas fluorescens, RimK modifies the C-terminus of ribosomal protein RpsF to influence important aspects of rhizosphere colonisation through proteome remodelling. In this study, we show that RimK activity is itself under complex, multifactorial control by the co-transcribed phosphodiesterase trigger enzyme (RimA) and a polyglutamate-specific protease (RimB). Furthermore, biochemical experimentation and mathematical modelling reveal a role for the nucleotide second messenger cyclic-di-GMP in coordinating these activities. Active ribosome regulation by RimK occurs by two main routes: indirectly, through changes in the abundance of the global translational regulator Hfq and directly, with translation of surface attachment factors, amino acid transporters and key secreted molecules linked specifically to RpsF modification. Our findings show that post-translational ribosomal modification functions as a rapid-response mechanism that tunes global gene translation in response to environmental signals.

microbiology↗

bioRxiv2019

Pan-genome analysis identifies intersecting roles for Pseudomonas specialized metabolites in potato pathogen inhibition

Agricultural soil harbors a diverse microbiome that can form beneficial relationships with plants, including the inhibition of plant pathogens. Pseudomonas spp. are one of the most abundant bacterial genera in the soil and rhizosphere and play important roles in promoting plant health. However, the genetic determinants of this beneficial activity are only partially understood. Here, we genetically and phenotypically characterize the Pseudomonas fluorescens population in a commercial potato field, where we identify strong correlations between specialized metabolite biosynthesis and antagonism of the potato pathogens Streptomyces scabies and Phytophthora infestans. Genetic and chemical analyses identified hydrogen cyanide and cyclic lipopeptides as key specialized metabolites associated with S. scabies inhibition, which was supported by in planta biocontrol experiments. We show that a single potato field contains a hugely diverse and dynamic population of Pseudomonas bacteria, whose capacity to produce specialized metabolites is shaped both by plant colonization and defined environmental inputs.

microbiology↗