Impaired maturation of the siderophore pyoverdine chromophore in Pseudomonas fluorescens ATCC 17400 deficient for the cytochrome c biogenesis protein CcmC

AbstractPyoverdines are the main siderophores of fluorescent pseudomonads. They comprise a quinoline chromophore, a peptide chain, and a dicarboxylic acid or a dicarboxylic acid amide side chain. Each Pseudomonas species produces a pyoverdine with a different peptide chain. A cytochrome c biogenesis ΔccmC mutant of Pseudomonas fluorescens ATCC 17400 produces multiple pyoverdine forms, showing differences at the level of the chromophore or the side chain. When grown in the presence of L-cysteine, ΔccmC produces only ferribactin, a non-fluorescent precursor of pyoverdine, while addition of oxidized glutathione improves pyoverdine production. We suggest that the conversion of ferribactin to pyoverdine does not take place in the ΔccmC mutant because of lack of oxidizing power in the periplasm

Adaptation des bactéries pathogènes au stress oxydant

Communication oral

Réponse aux stress de Campylobacter jejuni (rôle du régulateur Cj1000 et mode d'action des molécules pro-oxydantes isothiocyanates)

Campylobacter jejuni est une epsilon-protéobactérie microaérophile responsable d'un grand nombre de gastro-entérites d'origine alimentaire dans les pays occidentaux. Chacune des niches écologiques que C. jejuni peut contaminer a ses propres caractéristiques, stress et contraintes auxquelles C. jejuni doit s'adapter. L'exposition à l'oxygène, le système immunitaire des hôtes et l'activité métabolique peuvent générer un stress oxydant, qui constitue une contrainte majeure en perturbant l équilibre rédox de la cellule. Pour s'adapter à ces contraintes, C. jejuni ne possède que peu de régulateurs connus de l'expression des gènes, dont un seul de type LysR, Cj1000. Nous avons muté le gène cj1000 et démontré son rôle dans la régulation de la réponse au stress oxydant et dans la régulation des voies respiratoires. Nous avons également mis en évidence l importance de Cj1000 dans la colonisation des hôtes de C. jejuni. Pour lutter contre la contamination des aliments par C. jejuni, une alternative possible aux antibiotiques est l'utilisation de molécules naturelles aux propriétés antimicrobiennes et sans danger pour l'Homme. Les isothiocyanates sont des molécules d origine végétale anticancéreuses aux propriétés pro-oxydantes et antibactériennes. Dans cette étude, nous avons évalué la sensibilité de différents isolats de C. jejuni aux isothiocyanates. À partir du profil transcriptomique de C. jejuni après exposition au benzylisothiocyanate et d'expériences complémentaires, nous avons prouvé que les isothiocyanates provoquaient l'agrégation de protéines, induisaient un stress oxydant et perturbaient le métabolisme énergétique et la respiration.Campylobacter jejuni is a microaerophile epsilon-proteobacteria which is responsible for many food-borne gastroenteridis in western countries. C. jejuni must adapt to stressful conditions to colonise various environments. Oxygen exposure, metabolic activity and the host immune system are potential sources of oxidative stress that disrupts the redox balance. Transcriptional regulation mediates adaptation of pathogens to environmental conditions and stress. C. jejuni possess only few regulators, including the sole LysR-type regulator Cj1000. We constructed a cj1000 mutant strain and we demonstrated the role of Cj1000 in the regulation of the oxidative stress response and respiratory pathways. Additionally, we highlighted the crucial role of Cj1000 in host colonisation. Natural, safe antibacterial molecules are possible alternatives to antibiotics for food preservation. Isothiocyanates are plant-derived anticancerous and antibacterial molecules that display pro-oxidant properties. In this study, we assayed the sensitivity of several C. jejuni isolates to isothiocyanates. The transcriptomic profile of C. jejuni exposed to benzylisothiocyanate and additional experiments demonstrated that isothiocyanates induced protein aggregation, generated an oxidative stress and disrupted energy metabolism and respiration.RENNES1-BU Sciences Philo (352382102) / SudocSudocFranceF

The antibacterial properties of isothiocyanates.

International audienc

Pseudomonas aeruginosa Soluble Pyocins as Antibacterial Weapons

International audiencePseudomonas aeruginosa is an opportunistic pathogen causing nosocomial infections and associated with lung infections in cystic fibrosis (CF) patients (Lyczak et al., Microbes Infect 2:1051-1060, 2000). Multiple drug-resistant P. aeruginosa strains pose a serious problem because of antibiotic treatment failure. There is therefore a need for alternative anti-Pseudomonas molecules. Soluble pyocins (S-pyocins) are bacteriocins produced by P. aeruginosa strains that kill sensitive strains of the same species. These bacteriocins and their immunity gene are easily cloned and expressed in E. coli and their activity spectrum against different P. aeruginosa strains can be tested. In this chapter, we describe the procedures for cloning, expression, and sensitivity testing of two different S-pyocins. We also describe how to identify their receptor binding domain in sensitive strains, how to construct chimeric pyocins with extended activity spectra, and how to identify new pyocins in genomes by multiplex PCR

Iron metabolism: a promising target for antibacterial strategies.

International audienceIn the fight against pathogenic and opportunistic bacteria, development and spreading of resistance to antibiotics is an increasing public health problem. The available antibacterial treatments are becoming less and less effective, making urgent the discovery of new active molecules. One strategy that has been explored to bypass the bacterial adaptation to drugs is to target the iron metabolism of bacteria, since iron is critical for all bacteria to grow. To date, three major ways have been assessed to exploit weaknesses in the bacterial iron metabolism: the "Trojan Horse strategy" which takes advantages of natural iron-uptake systems to deliver antimicrobial compounds inside the cells; the use of iron-antagonists and iron-chelators in order to reduce iron availability and the inhibition of enzymatic steps of iron metabolism via chemical compounds. This review discusses these antibacterial strategies interfering with several levels of the bacterial iron metabolism, with a special emphasis on recently published and/or patented discoveries

Multiple phenotypic alterations caused by a c-type cytochrome maturation ccmC gene mutation in Pseudomonas aeruginosa.

International audienceIn some Proteobacteria biogenesis of c-type cytochromes depends on the products of the ccmABCDEFG(H) genes, which encode inner-membrane proteins. Inactivation of some ccm genes, in particular ccmC, has an impact on other processes as well, including siderophore production and utilization. Non-polar insertions were generated in the Pseudomonas aeruginosa ccmA, ccmC, ccmE, ccmF and ccmH genes, and their impacts on different phenotypes were compared. Only in the case of the ccmC mutant was cytochrome c production totally abrogated. The ccmC mutant, and to a lesser extent the ccmF mutant, showed a range of other phenotypic changes. The production of the siderophore pyoverdine was very low and growth under the condition of iron limitation was severely restricted, but production of the second siderophore, pyochelin, was increased. Interestingly, other traits were also strongly affected by the ccmC mutation, including the production of pyocyanin, swarming and twitching motility, and rhamnolipid production. The production of N-acyl homoserine lactones or the Pseudomonas quinolone signal (PQS) was, however, not affected in the ccmC and ccmF mutants. The ccmC mutant was also found to accumulate porphyrins, and catalase production was undetectable, consistent with the increased sensitivity to hydrogen peroxide. Finally, reduction in the content of [Fe-S] clusters was evidenced in both ccmC and ccmF mutants. Wild-type phenotypes were restored by complementation with a ccmC gene from Pseudomonas fluorescens ATCC 17400. In conclusion, we have demonstrated that CcmC is a key determinant for cytochrome c biogenesis, pyoverdine maturation, and expression of some quorum sensing-regulated traits

Elucidating mechanisms of resistance by Pseudomonas aeruginosa to the cationic peptide, colistin.

Poste

Method for screening antimicrobial gels against multi-species oral biofilms

International audienceWe described a microtiter plate-based method that was effectively tailored for testing gel formulations against oral multispecies biofilms established on peg-lids. This method lifts the limitations imposed mainly by the anaerobic nature of the targeted bacterial species and the viscous properties of the targeted treatments

The post-transcriptional regulator CsrA plays a central role in the adaptation of bacterial pathogens to different stages of infection in animal hosts.

International audienceThe importance of Csr post-transcriptional systems is gradually emerging; these systems control a variety of virulence-linked physiological traits in many pathogenic bacteria. This review focuses on the central role that Csr systems play in the pathogenesis of certain bacteria and in the establishment of successful infections in animal hosts. Csr systems appear to control the 'switch' between different physiological states in the infection process; for example switching pathogens from a colonization state to a persistence state. Csr systems are controlled by two-component sensor/regulator systems and by non-coding RNAs. In addition, recent findings suggest that the RNA chaperone Hfq may play an integral role in Csr-mediated bacterial adaptation to the host environment