A Flavor Lactone Mimicking AHL Quorum-Sensing Signals Exploits the Broad Affinity of the QsdR Regulator to Stimulate Transcription of the Rhodococcal qsd Operon Involved in Quorum-Quenching and Biocontrol Activities

In many Gram-negative bacteria, virulence, and social behavior are controlled by quorum-sensing (QS) systems based on the synthesis and perception of N-acyl homoserine lactones (AHLs). Quorum-quenching (QQ) is currently used to disrupt bacterial communication, as a biocontrol strategy for plant crop protection. In this context, the Gram-positive bacterium Rhodococcus erythropolis uses a catabolic pathway to control the virulence of soft-rot pathogens by degrading their AHL signals. This QS signal degradation pathway requires the expression of the qsd operon, encoding the key enzyme QsdA, an intracellular lactonase that can hydrolyze a wide range of substrates. QsdR, a TetR-like family regulator, represses the expression of the qsd operon. During AHL degradation, this repression is released by the binding of the γ-butyrolactone ring of the pathogen signaling molecules to QsdR. We show here that a lactone designed to mimic quorum signals, γ-caprolactone, can act as an effector ligand of QsdR, triggering the synthesis of qsd operon-encoded enzymes. Interaction between γ-caprolactone and QsdR was demonstrated indirectly, by quantitative RT-PCR, molecular docking and transcriptional fusion approaches, and directly, in an electrophoretic mobility shift assay. This broad-affinity regulatory system demonstrates that preventive or curative quenching therapies could be triggered artificially and/or managed in a sustainable way by the addition of γ-caprolactone, a compound better known as cheap food additive. The biostimulation of QQ activity could therefore be used to counteract the lack of consistency observed in some large-scale biocontrol assays

Rhodococcus equi's Extreme Resistance to Hydrogen Peroxide Is Mainly Conferred by One of Its Four Catalase Genes

Rhodococcus equi is one of the most widespread causes of disease in foals aged from 1 to 6 months. R. equi possesses antioxidant defense mechanisms to protect it from reactive oxygen metabolites such as hydrogen peroxide (H(2)O(2)) generated during the respiratory burst of phagocytic cells. These defense mechanisms include enzymes such as catalase, which detoxify hydrogen peroxide. Recently, an analysis of the R. equi 103 genome sequence revealed the presence of four potential catalase genes. We first constructed \u394katA-, \u394katB-, \u394katC-and \u394katD-deficient mutants to study the ability of R. equi to survive exposure to H(2)O(2)in vitro and within mouse peritoneal macrophages. Results showed that \u394katA and, to a lesser extent \u394katC, were affected by 80 mM H(2)O(2). Moreover, katA deletion seems to significantly affect the ability of R. equi to survive within murine macrophages. We finally investigated the expression of the four catalases in response to H(2)O(2) assays with a real time PCR technique. Results showed that katA is overexpressed 367.9 times (\ub1122.6) in response to exposure to 50 mM of H(2)O(2) added in the stationary phase, and 3.11 times (\ub10.59) when treatment was administered in the exponential phase. In untreated bacteria, katB, katC and katD were overexpressed from 4.3 to 17.5 times in the stationary compared to the exponential phase. Taken together, our results show that KatA is the major catalase involved in the extreme H(2)O(2) resistance capability of R. equi

N-Acyl Homoserine Lactones in Diverse Pectobacterium and Dickeya Plant Pathogens: Diversity, Abundance, and Involvement in Virulence

Soft-rot bacteria Pectobacterium and Dickeya use N-acyl homoserine lactones (NAHSLs) as diffusible signals for coordinating quorum sensing communication. The production of NAHSLs was investigated in a set of reference strains and recently-collected isolates, which belong to six species and share the ability to infect the potato host plant. All the pathogens produced different NAHSLs, among which the 3-oxo-hexanoyl- and the 3-oxo-octanoyl-l-homoserine lactones represent at least 90% of total produced NAHSL-amounts. The level of NAHSLs varied from 0.6 to 2 pg/cfu. The involvement of NAHSLs in tuber maceration was investigated by electroporating a quorum quenching vector in each of the bacterial pathogen strains. All the NAHSL-lactonase expressing strains produced a lower amount of NAHSLs as compared to those harboring the empty vector. Moreover, all except Dickeya dadantii 3937 induced a lower level of symptoms in potato tuber assay. Noticeably, aggressiveness appeared to be independent of both nature and amount of produced signals. This work highlights that quorum sensing similarly contributed to virulence in most of the tested Pectobacterium and Dickeya, even the strains had been isolated recently or during the past decades. Thus, these key regulatory-molecules appear as credible targets for developing anti-virulence strategies against these plant pathogens

Quorum Sensing Signaling Molecules Produced by Reference and Emerging Soft-Rot Bacteria (Dickeya and Pectobacterium spp.)

International audienceBACKGROUND: Several small diffusible molecules are involved in bacterial quorum sensing and virulence. The production of autoinducers-1 and -2, quinolone, indole and γ-amino butyrate signaling molecules was investigated in a set of soft-rot bacteria belonging to six Dickeya or Pectobacterium species including recent or emerging potato isolates. METHODOLOGY/PRINCIPAL FINDINGS: Using bacterial biosensors, immunoassay, and chromatographic analysis, we showed that soft-rot bacteria have the common ability to produce transiently during their exponential phase of growth the N-3-oxo-hexanoyl- or the N-3-oxo-octanoyl-l-homoserine lactones and a molecule of the autoinducer-2 family. Dickeya spp. produced in addition the indole-3-acetic acid in tryptophan-rich conditions. All these signaling molecules have been identified for the first time in the novel Dickeya solani species. In contrast, quinolone and γ-amino butyrate signals were not identified and the corresponding synthases are not present in the available genomes of soft-rot bacteria. To determine if the variations of signal production according to growth phase could result from expression modifications of the corresponding synthase gene, the respective mRNA levels were estimated by reverse transcriptase-PCR. While the N-acyl-homoserine lactone production is systematically correlated to the synthase expression, that of the autoinducer-2 follows the expression of an enzyme upstream in the activated methyl cycle and providing its precursor, rather than the expression of its own synthase. CONCLUSIONS/SIGNIFICANCE: Despite sharing the S-adenosylmethionine precursor, no strong link was detected between the production kinetics or metabolic pathways of autoinducers-1 and -2. In contrast, the signaling pathway of autoinducer-2 seems to be switched off by the indole-3-acetic acid pathway under tryptophan control. It therefore appears that the two genera of soft-rot bacteria have similarities but also differences in the mechanisms of communication via the diffusible molecules. Our results designate autoinducer-1 lactones as the main targets for a global biocontrol of soft-rot bacteria communications, including those of emerging isolates

Caractérisation des protéines secrétées par Rhodococcus equi et évaluation de leur pouvoir immunogène en vue de la mise au point d'un vaccin chez le poulain

Rhodococcus equi, pathogène ubiquiste intracellulaire facultatif, est l agent de la rhodococcose équine, première cause de mortalité chez les poulains de un à six mois. Un vaccin expérimental développé par l Afssa site de Dozulé, à base de protéines membranaires de R. equi, protège les poulains durant leurs premiers jours de vie (au moins 45 jours), par transfert passif d anticorps maternels anti-R. equi. Une protection couvrant toute la période de sensibilité jusqu à l âge de six mois nécessite donc le développement d un vaccin relais à administrer au poulain, dont la fraction antigénique a été sélectionnée parmi les protéines sécrétées par R. equi. L analyse de ces protéines par gels SDS PAGE et spectrométrie de masse a abouti à l identification de 48 protéines dans le sécrétome de R. equi ATCC 33701. Sur la base d études bibliographique et immunologique à l aide de sérums de poulains morts de rhodococcose, 19 protéines ont été sélectionnées et surexprimées. La caractérisation du pouvoir immunogène des 19 protéines recombinantes par Western blot a permis de réduire le nombre de candidats à neuf. La sélection finale des candidats pour la mise au point du vaccin, basée sur leur propriété immunoprotectrice chez la souris nécessite la mise au point des conditions d infection et d immunisation qui ont été établies au cours de ce travail. Cette optimisation nous a permis i) d obtenir les sérums de souris immunisées utilisées pour la sélection des 9 protéines parmi les 19 et ii) de démontrer l importance d un adjuvant dans la polarisation de la réponse vers la voie cellulaire protectrice. Il reste néanmoins à définir le meilleur adjuvant à employer chez le poulain.Rhodococcus equi is an ubiquitous facultative intracellular pathogen responsible for equine rhodococcosis, the major cause of mortality in foals between one to six months of age. A previously developed vaccine, composed with R. equi membrane proteins and delivered in mares, protects foals through passive transfer of immune maternal antibodies during at least 45 days of age. Thus, a protection during the whole length of the risk phase of six months of age requires an active vaccination. In order to develop such a vaccine, secreted proteins of R. equi were studied. Analysis of these proteins by SDS PAGE and mass spectrometry led to the identification of 48 proteins in the secretome of the ATCC 33701 strain. Based on data from the literature as well as from results obtained with sera from rhodococcosis died foals, 19 proteins were finally selected and overexpressed in E. coli. This number of candidates was reduced to nine proteins after immunological characterization of each recombinant protein by Western blot. The final candidate selection based on immunoprotective character in mice needs the optimization of infection and immunization conditions which were defined in this work. This last study allowed us i) to obtain sera of immunized mice used to select the nine candidates and ii) to demonstrate the essential role of adjuvant to potentiate an immunoprotective cellular response. Nevertheless, the adjuvant for the vaccine in foals remains to be defined.CAEN-BU Sciences et STAPS (141182103) / SudocSudocFranceF

In-depth understanding of plant-microorganisms interactions to develop a new generation of biocontrol products

National audienc

The pathogenic potential of Pseudomonas fluorescens MFN1032 on enterocytes can be modulated by serotonin, substance P and epinephrine

International audienc

Biosensors Used for Epifluorescence and Confocal Laser Scanning Microscopies to Study Dickeya and Pectobacterium Virulence and Biocontrol

International audiencePromoter-probe vectors carrying fluorescent protein-reporter genes are powerful tools used to study microbial ecology, epidemiology, and etiology. In addition, they provide direct visual evidence of molecular interactions related to cell physiology and metabolism. Knowledge and advances carried out thanks to the construction of soft-rot Pectobacteriaceae biosensors, often inoculated in potato Solanum tuberosum, are discussed in this review. Under epifluorescence and confocal laser scanning microscopies, Dickeya and Pectobacterium-tagged strains managed to monitor in situ bacterial viability, microcolony and biofilm formation, and colonization of infected plant organs, as well as disease symptoms, such as cell-wall lysis and their suppression by biocontrol antagonists. The use of dual-colored reporters encoding the first fluorophore expressed from a constitutive promoter as a cell tag, while a second was used as a regulator-based reporter system, was also used to simultaneously visualize bacterial spread and activity. This revealed the chronology of events leading to tuber maceration and quorum-sensing communication, in addition to the disruption of the latter by biocontrol agents. The promising potential of these fluorescent biosensors should make it possible to apprehend other activities, such as subcellular localization of key proteins involved in bacterial virulence in planta, in the near future

Immune response to Rhodococcus equi ATCC 33701-secreted proteins in mice and identification of immunogenic recombinant proteins by dot-blotting

International audienceRhodococcus equi remains a significant pathogen, causing severe pneumonia in foals. The development of vaccines and serologic diagnosis could be greatly facilitated by studying the humoral immune response to this equine pathogen. In this study, a crude extract of R. equi ATCC 33701-secreted proteins combined with the Montanide® ISA70 adjuvant was found to be highly immunogenic in mice with the highest titer of 99,000 on day 42 after the first subcutaneous immunization. This immune response was dependent on the quantity of proteins injected and the presence of adjuvant. By dot-blotting, eight recombinant secreted proteins were identified to react strongly with sera from immunized mice. Of these eight proteins, four were detected as immunogenic only when administered in conjunction with adjuvant. This screening strategy led to the identification of promising new candidates for vaccine development