Dataset Information

Characterization of F. tularensis LVS outer membrane vesicles

ABSTRACT: Francisella tularensis is a Gram-negative bacteria responsible for tularemia, a disease for which the high prevalence of failure and relapses is a main concern. Directed evolution experiments carried out in presence of antibiotics revealed that acquisition of fluoroquinolones (FQ) resistance was not exclusively related to mutations in DNA gyrase genes which are the first targets of these molecules. Here, using F. tularensis LVS as model, we investigated the role of FupA/B (Fer-Utilization Protein), whose possible contribution to FQ resistance was suggested by genomic analysis of resistant isolates. Using trans-complementation/mutagenesis approaches we definitely connected FupA/B expression to FQ sensitivity. Furthermore, we showed that the virulent strain F. tularensis subsp. tularensis SCHU S4, lacking the homologous FupA protein, exhibited a lower FQ susceptibility. Importantly, the deletion of this lipoprotein promoted an increased secretion of outer membrane vesicles (OMVs). Mass spectrometry-based quantitative proteomic characterization of OMVs from LVS and LVS-∆fupA/B strains led to the identification of 801 proteins among which a subset of 23 proteins exhibited a differential abundance between OMVs from both strains and may therefore contribute to the observed increased FQ susceptibility. We finally demonstrated that OMVs are structural key elements of F. tularensis LVS biofilms providing protection against FQ. Taken together, these results showed that mutations targeting FupA/B contribute to antimicrobial resistance through quantitative and qualitative modulations of OMV biogenesis and biofilm formation, providing new basis for understanding and fighting against Francisella antibiotic resistance and/or persistence.

INSTRUMENT(S): LTQ Orbitrap Velos

ORGANISM(S): Francisella Tularensis Subsp. Holarctica (strain Lvs)

SUBMITTER: Yohann Couté

LAB HEAD: Virginie Brun

PROVIDER: PXD010305 | Pride | 2019-11-12

REPOSITORIES: Pride

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Francisella tularensis: FupA mutation contributes to fluoroquinolone resistance by increasing vesicle secretion and biofilm formation.

Siebert Claire C Lindgren Helena H Ferré Sabrina S Villers Corinne C Boisset Sandrine S Perard Julien J Sjöstedt Anders A Maurin Max M Brochier-Armanet Céline C Couté Yohann Y Renesto Patricia P

Emerging microbes & infections 20190101 1

Francisella tularensis is the causative agent in tularemia for which the high prevalence of treatment failure and relapse is a major concern. Directed-evolution experiments revealed that acquisition of fluoroquinolone (FQ) resistance was linked to factors in addition to mutations in DNA gyrase. Here, using F. tularensis live vaccine strain (LVS) as a model, we demonstrated that FupA/B (Fer-Utilization Protein) expression is linked to FQ susceptibility, and that the virulent strain ...[more]

PMID: 31164053

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Project description:Raghunathan2010 - Genome-scale metabolic network of Francisella tularensis (iRS605) This model is described in the article: Systems approach to investigating host-pathogen interactions in infections with the biothreat agent Francisella. Constraints-based model of Francisella tularensis. Raghunathan A, Shin S, Daefler S. BMC Syst Biol 2010; 4: 118 Abstract: BACKGROUND: Francisella tularensis is a prototypic example of a pathogen for which few experimental datasets exist, but for which copious high-throughout data are becoming available because of its re-emerging significance as biothreat agent. The virulence of Francisella tularensis depends on its growth capabilities within a defined environmental niche of the host cell. RESULTS: We reconstructed the metabolism of Francisella as a stoichiometric matrix. This systems biology approach demonstrated that changes in carbohydrate utilization and amino acid metabolism play a pivotal role in growth, acid resistance, and energy homeostasis during infection with Francisella. We also show how varying the expression of certain metabolic genes in different environments efficiently controls the metabolic capacity of F. tularensis. Selective gene-expression analysis showed modulation of sugar catabolism by switching from oxidative metabolism (TCA cycle) in the initial stages of infection to fatty acid oxidation and gluconeogenesis later on. Computational analysis with constraints derived from experimental data revealed a limited set of metabolic genes that are operational during infection. CONCLUSIONS: This integrated systems approach provides an important tool to understand the pathogenesis of an ill-characterized biothreat agent and to identify potential novel drug targets when rapid target identification is required should such microbes be intentionally released or become epidemic. This model is hosted on BioModels Database and identified by: MODEL1507180003. To cite BioModels Database, please use: BioModels Database: An enhanced, curated and annotated resource for published quantitative kinetic models. To the extent possible under law, all copyright and related or neighbouring rights to this encoded model have been dedicated to the public domain worldwide. Please refer to CC0 Public Domain Dedication for more information.

Project description:Tenacibaculum maritimum, the etiological agent of tenacibaculosis in marine fish, constitutively secretes extracellular products (ECPs), which play a main role in virulence. However, their protein content has not been yet comprehensively studied. In this study a collection of 64 T. maritimum strains belonging to the four serotypes described so far (O1 to O4) was used to analyse the prevalence of extracellular proteolytic and lipolytic activities related to virulence. Results showed the existence of a large heterogeneity of the enzymatic capacity among serotype O4 isolates. Most notably, the ECPs of T. maritimum SP9.1 belonging to serotype O4 contain a large amount of outer membrane vesicles (OMVs), which were characterized by electron microscopy and purified by successive steps of filtration and centrifugation. Total protein content of ECPs, and the proteins associated to OMVs and soluble fraction of the ECPs (S-ECPs) were identified by nLC-TIMS-QTOF. A total of 641 proteins were identified in ECPs including some virulence related factors, which were mainly found in one of the fractions, either in OMVs or S-ECPs. Outer membrane proteins such as TonB-dependent transporters and the T9SS-related proteins PorP, PorT and SprA appeared to be mainly associated with OMVs. By contrast, putative virulence factors such as sialidase SiaA, chondroitinase CslA, sphingomyelinase Sph, ceramidase Cer and collagenase Col were found only in the S-ECPs. These findings demonstrate that T. maritimum release, through surface blebbing, outer membrane vesicles that are enriched specifically in TonB-dependent siderophore transporters and proteins of the type IX secretion system. Interestingly, our results also showed that OMVs could play a role in virulence by promoting surface adhesion, biofilm formation, and maximizing cytotoxic effects of the ECPs. The T. maritimum secretome analysis provides insights into the extracellular products function and can constitute the basis for future studies aimed to elucidate the role of OMVs in the pathogenesis of fish tenacibaculosis.

Project description:The highly infectious bacterium Francisella tularensis is a facultative intracellular pathogen, whose virulence requires proliferation inside host cells, including macrophages. Although some Francisella determinants of intracellular growth have been identified, much remains to be understood about the pathogenesis of this organism. In particular, how Francisella responds to its intracellular environment could provide clues about its intracellular biology and reveal pathogenic determinants based on their intracellular expression profiles. Here we have performed a global transcriptional profiling of the highly virulent F. tularensis subsp. tularensis Schu S4 strain during its intracellular cycle within primary murine macrophages. Phagocytosed bacteria rapidly responded to their intracellular environment and progressively altered their transcriptional profile over time. Differential gene expression profiles were revealed that correlated with specific intracellular locations of the bacteria. Upregulation of general and oxidative stress response genes was a hallmark of the early phagosomal and late endosomal stages, while induction of a subset of transport and metabolic genes characterized the cytosolic replication stages. Expression of the Francisella Pathogenicity Island (FPI) genes, the functions of which are associated with intracellular proliferation, increased during the intracellular cycle. Similarly, 27 chromosomal loci putatively encoding hypothetical, secreted, outer membrane proteins or transcriptional regulators were identified as upregulated during the intracellular cycle. In-frame deletion of FTT0383, the Schu S4 ortholog of fevR, of FTT0369c or FTT1676 abolished the ability of Schu S4 to either survive or proliferate intracellularly, demonstrating that bacterial factors of intracellular pathogenesis can be identified based on their intracellular expression profile. In conclusion, establishing the intracellular transcriptome of Francisella has revealed important aspects of its intracellular biology and identified novel virulence determinants of this pathogen. Keywords: Time series Intracellular cycle within primary murine macrophages using the time series of zero, one, two, four, eight, twelve, sixteen and twenty-four hours

Dataset Information

Characterization of F. tularensis LVS outer membrane vesicles

Publications

<i>Francisella tularensis</i>: FupA mutation contributes to fluoroquinolone resistance by increasing vesicle secretion and biofilm formation.

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