New approaches for microorganisms detection combining proteomics, immuno-enrichment and bioinformatics

Identifier rapidement des microorganismes pathogènes dans des échantillons environnementaux est un enjeu majeur dans le domaine de la biodéfense. Dans ce contexte, la spectrométrie de masse MALDI-TOF peut offrir une réponse simple, rapide et peu coûteuse. L'enjeu de la thèse, dans le cadre du projet ANR franco-allemand GEFREASE, a été de développer des méthodes permettant l'identification des microorganismes pathogènes et notamment de mettre en place des approches ciblées pour la préparation d'échantillon à l'aide d'anticorps en amont de la spectrométrie de masse. Dans un premier temps, l'étude du protéome de la bactérie modèle, Francisella tularensis subsp. holarctica LVS, responsable de la tularémie, a permis d'identifier les protéines et les peptides les plus abondants donnant un signal intense par spectrométrie de masse. Ensuite l'étude protéogénomique de douze protéines cibles a permis de choisir trois biomarqueurs dont le profil des masses par spectrométrie de masse de type MALDI-TOF (approche top-down) est spécifique de l'espèce et de la sous-espèce des bactéries du genre Francisella. Par cette méthode la virulence d'une souche est donc rapidement déterminée puisqu'elle est dépendante de la sous-espèce à laquelle la bactérie appartient. Ce test mis au point présente l'avantage d'être simple et rapide. Dans un deuxième temps, la mise au point d'un protocole d'enrichissement de la bactérie modèle par immunocapture magnétique a permis de montrer qu'il est possible de concentrer des bactéries grâce à des billes magnétiques couplées à des anticorps dirigés contre la bactérie entière. Cette approche a été expérimentée dans le cas de mélanges de bactéries où la bactérie modèle était largement minoritaire et dans des échantillons de matrices alimentaires diverses telles que de l'eau minérale ou du lait. La méthodologie a été validée sur un agent de classe 3, Francisella tularensis subsp. tularensis.The rapid identification of pathogenic microorganisms in environmental samples is a major issue in the biodefense field. MALDI-TOF mass spectrometry can offer a fast, straightforward and inexpensive answer. In the framework of the Franco-German ANR project GEFREASE, the purpose of the thesis was to develop methodologies allowing identification of pathogenic microorganisms and particularly to set up targeted approaches using antibodies for sample preparation beforehand mass spectrometry. First of all, the proteome study of Francisella tularensis subsp. holarctica LVS, responsible for tularemia, allowed us to identify the most abundant proteins and peptides, and for which the most intense signals are observed when using mass spectrometry. The proteogenomic study of twelve of these proteins enable us to choose three biomarkers for which the masses monitored by MALDI-TOF mass spectrometry (top down approach) allow deciphering the Francisella species and subspecies. The interest of this work is being able to conclude on a strain virulence based on the knowledge of the subspecies it belongs. The finalized test is easy and fast. Secondly, the development of a magnetic immunocapture of Francisella tularensis subsp. holarctica LVS allowed us to show that it is possible to concentrate bacteria using magnetic beads coupled to antibodies raised against the entire bacterium. This approach has been experimented in the case of bacterial mixtures where the model bacterium was largely in minority and for samples containing various food matrices such as mineral water or milk. The methodology has been validated on a class 3 agent, Francisella tularensis subsp. tularensis

Nouvelles approches combinant protéomique, immuno-enrichissement et bioinformatique pour la détection de microorganismes

The rapid identification of pathogenic microorganisms in environmental samples is a major issue in the biodefense field. MALDI-TOF mass spectrometry can offer a fast, straightforward and inexpensive answer. In the framework of the Franco-German ANR project GEFREASE, the purpose of the thesis was to develop methodologies allowing identification of pathogenic microorganisms and particularly to set up targeted approaches using antibodies for sample preparation beforehand mass spectrometry. First of all, the proteome study of Francisella tularensis subsp. holarctica LVS, responsible for tularemia, allowed us to identify the most abundant proteins and peptides, and for which the most intense signals are observed when using mass spectrometry. The proteogenomic study of twelve of these proteins enable us to choose three biomarkers for which the masses monitored by MALDI-TOF mass spectrometry (top down approach) allow deciphering the Francisella species and subspecies. The interest of this work is being able to conclude on a strain virulence based on the knowledge of the subspecies it belongs. The finalized test is easy and fast. Secondly, the development of a magnetic immunocapture of Francisella tularensis subsp. holarctica LVS allowed us to show that it is possible to concentrate bacteria using magnetic beads coupled to antibodies raised against the entire bacterium. This approach has been experimented in the case of bacterial mixtures where the model bacterium was largely in minority and for samples containing various food matrices such as mineral water or milk. The methodology has been validated on a class 3 agent, Francisella tularensis subsp. tularensis.Identifier rapidement des microorganismes pathogènes dans des échantillons environnementaux est un enjeu majeur dans le domaine de la biodéfense. Dans ce contexte, la spectrométrie de masse MALDI-TOF peut offrir une réponse simple, rapide et peu coûteuse. L'enjeu de la thèse, dans le cadre du projet ANR franco-allemand GEFREASE, a été de développer des méthodes permettant l'identification des microorganismes pathogènes et notamment de mettre en place des approches ciblées pour la préparation d'échantillon à l'aide d'anticorps en amont de la spectrométrie de masse. Dans un premier temps, l'étude du protéome de la bactérie modèle, Francisella tularensis subsp. holarctica LVS, responsable de la tularémie, a permis d'identifier les protéines et les peptides les plus abondants donnant un signal intense par spectrométrie de masse. Ensuite l'étude protéogénomique de douze protéines cibles a permis de choisir trois biomarqueurs dont le profil des masses par spectrométrie de masse de type MALDI-TOF (approche top-down) est spécifique de l'espèce et de la sous-espèce des bactéries du genre Francisella. Par cette méthode la virulence d'une souche est donc rapidement déterminée puisqu'elle est dépendante de la sous-espèce à laquelle la bactérie appartient. Ce test mis au point présente l'avantage d'être simple et rapide. Dans un deuxième temps, la mise au point d'un protocole d'enrichissement de la bactérie modèle par immunocapture magnétique a permis de montrer qu'il est possible de concentrer des bactéries grâce à des billes magnétiques couplées à des anticorps dirigés contre la bactérie entière. Cette approche a été expérimentée dans le cas de mélanges de bactéries où la bactérie modèle était largement minoritaire et dans des échantillons de matrices alimentaires diverses telles que de l'eau minérale ou du lait. La méthodologie a été validée sur un agent de classe 3, Francisella tularensis subsp. tularensis

Assessing the exoproteome of marine bacteria, lesson from a RTX-Toxin abundantly secreted by Phaeobacter strain DSM 17395

Bacteria from the Roseobacter clade are abundant in surface marine ecosystems as over 10% of bacterial cells in the open ocean and 20% in coastal waters belong to this group. In order to document how these marine bacteria interact with their environment, we analyzed the exoproteome of Phaeobacter strain DSM 17395. We grew the strain in marine medium, collected the exoproteome and catalogued its content with high-throughput nanoLC-MS/MS shotgun proteomics. The major component represented 60% of the total protein content but was refractory to either classical proteomic identification or proteogenomics. We de novo sequenced this abundant protein with high-resolution tandem mass spectra which turned out being the 53 kDa RTX-toxin ZP_02147451. It comprised a peptidase M10 serralysin domain. We explained its recalcitrance to trypsin proteolysis and proteomic identification by its unusual low number of basic residues. We found this is a conserved trait in RTX-toxins from Roseobacter strains which probably explains their persistence in the harsh conditions around bacteria. Comprehensive analysis of exoproteomes from environmental bacteria should take into account this proteolytic recalcitrance

MS/MS spectrum of the semi-tryptic peptide [288–299] belonging to ZP_02147451.

<p>The MS/MS spectrum was acquired with a FT/FT procedure with an LTQ-Orbitrap XL hybrid mass spectrometer. The peptide sequence is shown on the top with the collision-induced fragmentation pattern. The <i>b</i> and <i>y</i> ions are shown in blue and red, respectively. The <i>y₁₁</i> di-charged ion is labeled in green.</p

Occurrence of basic residues in proteins from <i>Phaeobacter strain DSM 17395.</i>

<p>The graph reports the ratio Lysine (K) and Arginine (R) residues per protein length of all the proteins with length above 100 residues encoded by <i>Phaeobacter strain DSM 17395</i>. Proteins are symbolized by a blue triangle. RTX-like proteins and the other exoproteins detected by tandem mass spectrometry are represented by red and yellow squares, respectively. The ZP_02143988.1 (a), ZP_02147451.1 (b), ZP_02144693.1 (c), ZP_02146960.1 (d), ZP_02144235.1 (e), and ZP_02146514.1 (f) RTX-toxins are indicated. Only the ZP_02143988.1 RTX-like protein has been detected by mass spectrometry.</p

ZP_02147451 sequence coverage with non-tryptic, semi-tryptic, and tryptic peptides.

<p>The ZP_02147451 sequence is represented with its peptidase motif (residues 240 to 279) pointed out with purple stars. Peptides identified with the “no-enzyme” Mascot search are symbolized with a line underlining the sequence. Tryptic, semi-tryptic, and non-tryptic peptides are indicated in yellow, blue and red, respectively.</p

SDS-PAGE of the exoproteome of <i>Phaeobacter strain DSM 17395</i>.

<p>Exoproteins were resolved by a long migration on a 10% SDS-PAGE and stained with SimplyBlue SafeStain (Invitrogen). Lane <b>M</b>: SeeBlue Plus2 molecular weight range marker (Invitrogen). Lane <b>E1</b>: <i>Phaeobacter strain DSM 17395</i> exoproteome grown in Marine Broth (20 µg). Lane <b>E2</b>: <i>Phaeobacter strain DSM 17395</i> exoproteome grown in Marine Broth (8 µg). The 55 kDa major component is indicated with an arrow.</p

List of the first ten proteins identified from the 55<i>P. gallaeciensis</i> DSM¹.

1<p>Detected with at least three different peptides.</p><p>*Periplasmic component.</p

List of proteins identified in the 55/FT proteomic procedure¹.

1<p>Detected with at least three different peptides. The values are from a representative experiment.</p><p>*Periplasmic component.</p>$<p>EUF stands for Enzyme Unspecificity Factor, defined as the ratio number of unique peptides (no-enzyme MASCOT search) per number of unique peptides (trypsin MASCOT search).</p

Proteogenomic Biomarkers for Identification of <i>Francisella</i> Species and Subspecies by Matrix-Assisted Laser Desorption Ionization-Time-of-Flight Mass Spectrometry

<i>Francisella tularensis</i> is the causative agent of tularemia. Because some <i>Francisella</i> strains are very virulent, this species is considered by the Centers for Disease Control and Prevention to be a potential category A bioweapon. A mass spectrometry method to quickly and robustly distinguish between virulent and nonvirulent <i>Francisella</i> strains is desirable. A combination of shotgun proteomics and whole-cell matrix-assisted laser desorption ionization-time-of-flight (MALDI-TOF) mass spectrometry on the <i>Francisella tularensis</i> subsp. <i>holarctica</i> LVS defined three protein biomarkers that allow such discrimination: the histone-like protein HU form B, the 10 kDa chaperonin Cpn10, and the 50S ribosomal protein L24. We established that their combined detection by whole-cell MALDI-TOF spectrum could enable (i) the identification of <i>Francisella</i> species, and (ii) the prediction of their virulence level, i.e., gain of a taxonomical level with the identification of <i>Francisella tularensis</i> subspecies. The detection of these biomarkers by MALDI-TOF mass spectrometry is straightforward because of their abundance and the absence of other abundant protein species closely related in terms of <i>m</i>/<i>z</i>. The predicted molecular weights for the three biomarkers and their presence as intense peaks were confirmed with MALDI-TOF/MS spectra acquired on <i>Francisella philomiragia</i> ATCC 25015 and on <i>Francisella tularensis</i> subsp. <i>tularensis</i> CCUG 2112, the most virulent <i>Francisella</i> subspecies